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Does early exercise affect the midcarpal joint of young Thoroughbreds?

An investigation into the influence of exercise on lesion prevalence, matrix swelling & histomorphometric changes in the cartilage and subchondral bone of the equine midcarpal joint

Woong Kim

PhD in Chemical and Materials Engineering The University of Auckland 30th April, 2010

This thesis was submitted in partial fulfilment of the requirements for the degree of PhD in Chemical and Materials Engineering, The University of Auckland, 2010.

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ABSTRACT

The aim of this investigation was to investigate the possible influence of early conditioning exercise on the 18-months old Thoroughbreds in terms of lesion development, extracellular matrix and histomorphometric changes in the cartilage and subchondral bone of the midcarpal joint. 12 Thoroughbreds were divided equally into two groups; a treatment (CONDEX) and a control group (PASTEX) at birth. Both were raised in free pasture conditions from birth for 18 months before euthanasia with the CONDEX only receiving the additional imposed exercise from birth. At the end of the trial, both the CONDEX and PASTEX horses were euthanized and their midcarpal joints were harvested. The investigation involved i) mapping and characterising the gross lesions in the midcarpal joint with respect to known sites of high stress and vulnerability, ii) measuring swelling strains in the cartilage matrix with respect to depth from different topographical regions, iii) histomorphometrically quantifying the area and thickness of hyaline and calcified cartilage and vasculatures, while also measuring the preferential angles of the collagen fibrils in these zones, and counting the number of vascular channels proximal to the cement line as well as measuring the cement line irregularity itself. Both the CONDEX and PASTEX were found to contain five different categories of lesions with similar levels in each category. However, the number of sites affected by mild traumatic osteochondrosis was lower in the CONDEX. There was no detectable exercise effect in the extracellular matrix swelling behaviour (P = 0.795) and neither was the overall histomorphometry (P = 0.4271). Conversely, in the CONDEX, the vascular channel area (VCa) showed a significant exercise effect (P = 0.0461) while the histomorphometric variables were found to be highly correlated to each other (P = 0.052). These findings suggest that the imposed early exercise may have resulted in a lower number of mild osteochondrosis lesions in the dorsal region of third carpal bone while significantly increasing the overall subchondral vascular activity and the development of a coherent tissue response between the hyaline cartilage, calcified cartilage and the vascularity. In conclusion, the findings of this study together with those of others [1-3] suggest that early exercise induced a positive response in the CONDEX without increasing deleterious changes in the midcarpal joint when compared with the PASTEX. It is thus recommended that potential benefits of early exercise should be further explored.

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PREFACE

Conditioning is an optimization process of a material with a specific purpose in mind, in which one attempts to bring out its full potential. This requires an in-depth understanding of two essential aspects; first is the material itself and the second is its operational environment. This concept can be readily applied beyond the scope of traditional engineering applications to the field of physiology and veterinary such as the cartilage and bone of the joint. Cartilage is a biological load-bearing material of the musculoskeletal system operating under a wide range of loading environment in vivo. It is susceptible to osteoarthritis, a joint disease which results in the deterioration of cartilage and bone; also one of the most important causes of wastage in the horse through the lameness. But thankfully, cartilage can be conditioned through moderate levels of exercise that can subsequently increase its physiologic threshold of safe function. The purpose of this study was to find whether a mild intensity early exercise could make positive (or negative) differences in the cartilage of the midcarpal joint of young Thoroughbreds. If a positive effect can be demonstrated successfully, the results could be used to design an effective exercise program that is able to reduce the joint injuries and lameness in the horse. This thesis was written for the readers who may be unfamiliar to the clinical equine veterinary in mind. The Background, which spans into three chapters; (i) Thoroughbred horse, (ii) Articular cartilage and (iii) Collagen, will equip the reader in the subjects of complex cartilage and subchondral bone tissue responses to the exercise and osteoarthritis. My utmost attention was to make this thesis an engaging and enjoyable reading experience while not burdening the reader with non-essential details. Furthermore, attempts were made to use fewer complex medical terms and phrases where possible, and if used, many of these terms have been predefined in the Glossary section.

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Readers who wish to quickly browse the overall findings, I recommend you to read the chapter Summary of each finding on page 202 first. This chapter was written to brief you on what this study has found while the corresponding in-depth explanations can be found in the main Discussion sections located within each analysis. This project was only made possible because of the people who had visions to design, passion to execute, persevered to complete the project, all of which was supported by funding bodies that truly believed their inputs would overall benefit the welfare of the Thoroughbreds worldwide. I would like to thank my supervisor Professor. N. D. Broom for his kind mentorship and profound patience. Also Professor. E. Firth and Dr. C. Rogers of Massey University for the project as well as the correspondence. Dr. C. W. McIlwraith and Dr. C. Kawcak of Colorado State University for their support in this project as well as providing the funding. I would like to also thank Dr. B. McArdle of the University of Auckland, who had carried the burden of much of the statistical analysis with uncanny enthusiasm and professionalism. Lastly I would like to show my gratitude for these funding bodies for supporting this project; the Grayson Foundation, the Colorado State University Equine Orthopaedic Center and the University of Auckland.

Woong Kim

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1.

TABLE OF CONTENTS

ABSTRACT .............................................................................................................................................................. II PREFACE ...............................................................................................................................................................VI TABLE OF CONTENTS...........................................................................................................................................VIII GLOSSARY ............................................................................................................................................................. XI THOROUGHBREDS ............................................................................................................................................... 14 1.1 INTRODUCTION ............................................................................................................................................... 14 1.2 ECONOMY OF THOROUGHBRED BREEDING, TRAINING AND RACING........................................................................... 16 1.3 COMMON HEALTH ISSUES AND MAL-PRACTICES ..................................................................................................... 17 1.4 ANATOMY OF CARPAL JOINT AND COMMON PROBLEMS .......................................................................................... 18 1.4.1 Anatomic Nomenclature and Usage ................................................................................................... 19 1.4.2 Carpus - knee joint of thoracic limb .................................................................................................... 20 ARTICULAR CARTILAGE ................................................................................................................................ 28 2.1 INTRODUCTION ............................................................................................................................................... 28 2.2 STRUCTURE AND MAKE UP OF ARTICULAR CARTILAGE ............................................................................................. 29 2.2.1 Superficial zone (zone I) ...................................................................................................................... 31 2.2.2 Transitional zone (zone II) ................................................................................................................... 31 2.2.3 Deep zone (zone III) ............................................................................................................................. 31 2.2.4 Calcified cartilage (zone IV)................................................................................................................. 32 2.2.5 Subchondral bone ............................................................................................................................... 34 2.2.6 Vascular channels ............................................................................................................................... 35 2.2.7 Chondrocytes ...................................................................................................................................... 39 2.2.8 Collagen .............................................................................................................................................. 41 2.2.9 Proteoglycans ...................................................................................................................................... 42 2.3 CARTILAGE MATRIX TURNOVER .......................................................................................................................... 44 2.3.1 Cytokines ............................................................................................................................................. 44 2.3.2 Extracellular Proteinases ..................................................................................................................... 46 2.4 MATERIAL PROPERTIES OF CARTILAGE MATRIX...................................................................................................... 48 2.4.1 Three mathematical interpretations of cartilage ................................................................................ 49

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5. 4. 3.

2.5 HYALINE CARTILAGE - RESPONSE TO LOADING, EXERCISE AND IMMOBILIZATION ........................................................... 50 2.6 AGING OF ARTICULAR CARTILAGE ....................................................................................................................... 51 2.7 OSTEOARTHRITIS ............................................................................................................................................. 52 2.7.1 How OA affects cartilage and bone .................................................................................................... 53 COLLAGEN ................................................................................................................................................... 56 3.1 INTRODUCTION ............................................................................................................................................... 56 3.2 THE BASIC STRUCTURE AND DOMAINS IN COLLAGEN ............................................................................................... 57 3.3 COLLAGEN TYPES............................................................................................................................................. 60 3.3.1 Fibrillar Collagens - Type I, II, III, V and XI ........................................................................................... 61 3.3.2 Non-fibrillar Collagens ........................................................................................................................ 63 3.4 BIOSYNTHESIS OF COLLAGENS............................................................................................................................. 65 3.4.1 Transcription and translation .............................................................................................................. 67 3.4.2 Post-translational modifications ......................................................................................................... 68 3.4.3 Extracellular processing and modification .......................................................................................... 69 3.5 COLLAGEN CROSS-LINKS IN ARTICULAR CARTILAGE .................................................................................................. 70 3.5.1 Enzymatic intermediate cross-links ..................................................................................................... 71 3.5.2 Mature non-reducible cross-links ........................................................................................................ 73 3.5.3 How collagen types II, XI and IX are assembled .................................................................................. 74 3.5.4 Non-enzymatic cross-linking via glycation .......................................................................................... 77 3.5.5 Cartilage cross-links and Equine exercise ............................................................................................ 78 3.6 BIOMARKERS OF CARTILAGE DEGRADATION ........................................................................................................... 80 3.7 SUMMARY ..................................................................................................................................................... 82 ANALYSES .................................................................................................................................................... 84 TEST SUBJECTS, EXERCISE PROGRAM AND EUTHANASIA.................................................................................................. 85 4.1 ANALYSIS 1 - CARTILAGE LESION MAPPING ON THE MIDCARPAL JOINT ........................................................................ 87 4.1.1 Methods .............................................................................................................................................. 87 4.1.2 Results ................................................................................................................................................. 88 4.1.3 Discussion .......................................................................................................................................... 120 4.2 ANALYSIS 2 INFLUENCE OF EXERCISE ON EXTRACELLULAR MATRIX SWELLING BEHAVIOUR ........................................... 134 4.2.1 Methods ............................................................................................................................................ 134 4.2.2 Statistical analysis and results .......................................................................................................... 137 4.2.3 Discussion .......................................................................................................................................... 143 4.3 ANALYSIS 3 - HISTOMORPHOMETRIC ANALYSIS .................................................................................................... 147 4.3.1 Methods ............................................................................................................................................ 147 4.3.2 Statistical analysis and results .......................................................................................................... 152 4.3.3 Discussion .......................................................................................................................................... 168 SUMMARY OF EACH FINDING .................................................................................................................... 202

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6. 7. 8.

SELF-ASSESSMENT ..................................................................................................................................... 222 FURTHER WORK ........................................................................................................................................ 226 CONCLUSIONS ........................................................................................................................................... 228

APPENDICES....................................................................................................................................................... 230 8.1 GEXA TEST SUBJECTS (18-MONTHS AND 36-MONTHS) ........................................................................................ 232 8.2 ANALYSIS 2 - SAS SOURCE CODES AND OUTPUT .................................................................................................. 233 8.2.1 Linear mixed modelling code ............................................................................................................ 233 8.2.2 Output ............................................................................................................................................... 234 8.3 ANALYSIS 2 - MATRIX SLICE SWELLING STRAIN DATA ............................................................................................. 236 8.4 ANALYSIS 2 - MEAN SWELLING STRAIN DATA (SAS) .............................................................................................. 268 8.5 ANALYSIS 2 - COHORT MEAN SWELLING STRAIN DATA (SAS) .................................................................................. 277 8.6 ANALYSIS 3 - SAS SOURCE CODES AND OUTPUT .................................................................................................. 278 8.6.1 Multivariate ANOVA (MANOVA) code .............................................................................................. 278 8.6.2 Output ............................................................................................................................................... 279 8.6.3 Univariate mixed model analyses (ANOVA) ...................................................................................... 282 8.6.4 Output ............................................................................................................................................... 282 8.6.1 Structure equality modelling / Path analysis Source Code (R) .......................................................... 284 8.7 ANALYSIS 3 - OSTEOCHONDRAL HISTOMORPHOMETRIC DATA ................................................................................. 286 8.8 ANALYSIS 3 - MEAN HISTOMORPHOMETRIC DATA (SAS) ....................................................................................... 295 8.9 ANALYSIS 3 - HISTOMORPHOMETRIC INDICES ...................................................................................................... 297 REFERENCES....................................................................................................................................................... 389

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Autocrine - A form of signalling in which a cell secretes a hormone or chemical messenger. Alymphatic - Lack of a lymphatic system. Aneural - Lack of nerve cells or system.

GLOSSARY

Angiogenesis - A physiological process involving the growth of new blood vessels from pre-existing vessels. Apoptosis - Death of cells which occurs as a normal part of an organism's development. Avascular - Lack of blood vessels. Bone cyst - Usually cavitary lesion of bone. May result from pressure or stress on articular cartilage and subchondral bone, leading to synovial fluid intrusion or bone contusion. Bone cysts frequently occur in association with joint space loss and bone eburnation; communication with the joint cavity may or may not be present. Bone cysts may occur after trauma and in osteoarthritis. Cartilage oligomeric matrix protein (COMP) - May have a role in stabilizing the collagen network and /or in promoting the collagen fibril assembly. Predominantly present in the interterritorial compartment in adult articular cartilage at a distance from the chondrocytes. Chondro- - Prefix, meaning of cartilage Chondroclast - A cell that is concerned with the absorption of cartilage. Chondroitin sulphate - A protein with a sugar component that forms an important constituent of articular cartilage, bone and other connective tissues. Chondroitin sulphate is available in combination with glucosamine as a supplement to aid joint mobility and to reduce pain in patients with osteoarthritis. Chondromalacia - Softening, inflammation, and degeneration of cartilage at a joint affecting the under surface of the kneecap, resulting in pain in the front of the knee and grating, which is made worse by kneeling, squatting, or climbing stairs.

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Chondron - Chondrocyte division within a lacuna (see image on the left). Condyle - A knob of bone, round or ellipsoid in shape that fits into a socket of an adjacent bone to form a joint. Contralateral - Means on the other side. The contralateral breast is the breast on the other breast. by as on by

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Cytokines - Protein molecules, released by cells when activated antigen, that are involved in cell-to-cell communications, acting enhancing mediators for immune responses through interaction with specific cell-surface receptors leucocytes. Kinds of cytokines include interleukins (produced by leucocytes), lymphokines (produced lymphocytes), interferons and tumour necrosis factor.

Extracellular matrix (ECM) - A part of animal tissue that usually provides structural support to the animal cells.

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Edema - Accumulation of fluid in the tissues, as a result of venous obstruction or fluid-electrolyte imbalance, e.g., in renal failure, impaired peripheral circulation as in cardiac decompensation, etc. Endothelium - Layer of cells lining the blood vessels, heart, and other organs and cavities of the body, and formed from the embryonic mesoderm. Growth factor - Any of various chemicals, particularly polypeptides, that have a variety of important roles in the stimulation of new cell growth and cell maintenance. Hypocellular - Lack of cells. Hypoxia - A deficiency of oxygen in body tissues, which can result from living in an oxygen-deficient environment, inadequate inspiration, or deficiency of red blood cells or haemoglobin (required for oxygen transport). Interleukins - A group of at least 15 soluble proteins, secreted by leukocytes that function to promote the growth and differentiation of cells of the immune system. The different interleukins are designated IL1, IL2, etc., in order of their discovery. Matrix metalloproteinase (MMP) - Any one of a group of zinccontaining proteases capable of digesting the extracellular tissue matrix. These enzymes play an important role in cell division, cell migration, inflammation, neoplastic invasion, and angiogenesis. In cartilage, they are responsible for catabolism and turnover of the matrix and are induced during the process of chondrocyte hypertrophy. MMPs are critical for angiogenesis in the growth plate and thus are necessary for normal calcification and bone formation. Osteo- - Prefix, meaning of bone Osteophytes - Also known as bone spurs; bony projections that form along joints. Bone spurs form due to the body's increase of a damaged (usually due to arthritis) joint's surface area. However, they usually limit joint movement and typically cause pain. Paracrine - A form of cell signalling in which the target cell is near. Postnatal - Following childbirth or the birth of young. Primary OA - Osteoarthritis (OA) with a cause unidentified and was typified by the insidiously developing disease of old people. Prostaglandin - Any of a group of organic compounds derived from essential fatty acids and causing a range of physiological effects in animals. Proteolysis - Breakdown of proteins or peptides into amino acids by the action of enzymes. Secondary OA - Osteoarthritis with an etiologic factor could be demonstrated. Subchondral bone cyst - A prominent feature of osteoarthritis. Occasionally such cysts form as a sequela of bone injury. Radiographically the lesions are multiple and radiolucent, with a surrounding sclerotic margin. Joint-space narrowing and bone sclerosis are accompanying features.

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Vascular endothelial growth factor (VEGF) - A mediator of angiogenesis expressed by hypertrophic chondrocytes in the growth plate during limb development. Zone of calcified cartilage (ZCC) - Zone below a hyaline cartilage layer where the calcium salts are deposited in the matrix.

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THOROUGHBREDS

1.1 INTRODUCTION
Thoroughbred is a common racehorse breed with a pedigree of unmixed lineage, also known for their agility, speed and temperament. All modern Thoroughbreds can trace their pedigrees back to three ancestral Arabian stallions; the Byerley Turk, the Darley Arabian, and the Godolphin Arabian [4, 5], which were crossbred with English mares during late 17th and early 18th centuries in England. A recent study has shown that using DNA techniques, 95% of all modern male Thoroughbreds can trace their linage to the Darley Arabian [6]. There are millions of Thoroughbreds exist today and 118,000 foals are registered each year worldwide [7]. Thoroughbred can be also bred for other riding disciplines such as show jumping, combined training, dressage, polo, and fox hunting and commonly cross-bred with other breeds to create new breeds or to improve existing ones.

Table 1 - Body weight (kg) 95% & Wither height (cm) of Thoroughbreds Worldwide. [8]

7 days Weight (Kg) Wither height 66.9 106.1

1 mo. 98.6 112.0

6 mo. 247.1 135.0

12 mo. 350.7 147.1

18 mo. 444.9 153.8

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A typical height for a mature Thoroughbred can range between 157 to 173 cm, with an average height of 163 cm and weight of 450kg (Table 1) [8]. After centuries of selective breeding for bigger and faster horses, the modern Thoroughbreds are now more muscular, larger and taller than their ancestors, for example, in 1700s, the average height of a Thoroughbred was about 133cm, whereas the current average height is 30cm now more.

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A typical Thoroughbred weighing 450kg running approximately 50km/hr can generate a large amount of impact force in the joint, and it is always the case that this enormous force is transmitted through only one of two frontal legs in a hyper-extended position (Figure 1) [9]. Such large forces over several years can be stressful to the joint and may cause microcracks to catastrophic fractures in the bone. Recently, there has been an unprecedented frequency of catastrophic fractures in the front legs during the racing and training. It is estimated in the U.S. alone 704 horses died while racing in 2005, an alarming rate of 2 deaths per day, which does not include fatalities during the training sessions [10].

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Figure 1 - A photograph of a racing horse with its left forelimb bent at an unnatural angle in a hyperextend position indicated by a red line. The fatigue in the leg combined during the high speed galloping often causes delayed reaction of the appropriate muscles that firmly align the leg and can induce unnatural bending of the leg. Courtesy of W. Berkeley [11].

The recent high profile breakdown cases such as a death of a $US 13 million Thoroughbred Babaro on the race track have brought acute attention from the public. As for the trainers, they blame the race track, however the construction and maintenance of racetracks of today are much more sophisticated than it was decades ago when breakdowns were even rarer than today [12]. It seems that these fatal injuries are caused by prolonged and excessive stresses on naturally fragile skeletal frameworks of Thoroughbreds that have been resulted from 1) poor selective breeding practice, which only aims for higher muscle mass without skeletal infrastructure considerations 2)

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higher number of race runs per annum with shorter resting periods and 3) early start of racing career on immature horses. The racing industry has been criticized for unethical management of horses, which is largely influenced by the market demand and harsh economic decisions, in which many immature horses with their fragile bone are entered into racing early in order to recoup owners investments quicker, ultimately exposing them to higher risk of injury and death [9, 12, 13]. As for the equine research, traditionally much of the focus has been directed at improving the performance, stamina and reduction in injuries of the muscle, tendon and heart. However, a recent increase in the fatal race track breakdowns has now shifted the research focus to finding an appropriate level and duration of an exercise regime, so that the bone and cartilage may become more resistant to these injuries through the conditioning exercise.

1.2 ECONOMY OF THOROUGHBRED BREEDING, TRAINING AND RACING

Thoroughbred is a huge industry worldwide with more than 195,000 active broodmares and 118,000 newly registered foals [14]. Financially, the industry generates around $US 34 billion in revenue [15], providing about 470,000 jobs through a network of farms, training centres and race tracks in the United States [15]. There are about 37,000 Thoroughbred foals registered each year in North America [16], and Australia is the second largest producer of Thoroughbreds in the world, where 30,000 broodmares (females being used for breeding) produce about 18,250 foals [17]. As for Britain, approximately 5,000 foals are born each year [18]. The price of a Thoroughbred yearling (one year old) varies greatly from $US 1,000 to over $US 1,000,000 apiece [19], which depends on age, pedigree, conformation, and other market factors [20]. For example, in 2007 at the Fall Yearling sale at Keeneland (U.S.), there were approximately 4000 young horses being traded generating a total of $US 385 million which averages about $US 101,347 per horse [21]. However, the profitability of a race horse is very slim. It costs about $US 10,000 a year to train and upkeep a horse, but the majority of stocks make losses most of the time. Even if the horse had a moderately successful career, the winnings will usually only breakeven the total cost. Of all 37,000

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Thoroughbred foals registered each year in the U.S., less than 1% ever win a stakes race such as the Kentucky Derby or the Epsom Derby [22].

1.3 COMMON HEALTH ISSUES AND MAL-PRACTICES

Thoroughbreds have a very high rate of accidents and are susceptible to numerous health problems. It is reported that one tenth of all Thoroughbreds suffer orthopaedic problems, including fractures [23] with a high 1.5 career-ending breakdown for every 1,000 horses, an average of two careerending breakdowns per day in the U.S. alone [24]. On the contrary, the rate is lower in the United Kingdom, 0.9 injuries/1000 starts and Australia has 0.44 injuries/1000 starts [25]. There are other common health problems in Thoroughbreds where many suffer exercise induced bleeding from the lungs (Exercise Induced Pulmonary Haemorrhage) [23], and chronic acute lameness from the joint pain, fracture and foot soreness [26]. It is thought that decades of selective inbreeding for the speed is the culprit [23]. Although the modern horses are more muscular and faster, their faster track records do not necessarily mean they are studier animals. It has been pointed out that the current racing regimes are potentially damaging to the anatomy of the Thoroughbred, in which the skeletal structures cannot cope with the impact from the speed that muscles generate [27]. Some blame the poor and unethical breeding practices. Correct conformation is critical in racing horse. However, because horses are treated as commodities, sometimes young horses with misaligned legs are straightened through correction procedures and then sold as a healthy animal for a higher price [25]. Due to the inherent and invisible conformational fault in the leg, the animal will be likely to develop joint diseases in the future and perform poorly in the racing; furthermore, when mated, the faulty gene is likely to be passed on to the offspring. Many of the immature and young 2-year olds are entered into the racing in the U.S. where the race track is shorter and more intense unlike the rest of the world. These young horses look muscular and may appear fully grown, but their bones are often not fully formed and fragile [27]; and these factors have been criticized as one of the main factors contributing to higher injury rate in the U.S. in comparison to the United Kingdom and Australia [28]. Other causes for the injuries include the track surface quality [29], toe grab horseshoes [26], over medication [30] and high-intensity racing schedules [31].

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1.4 ANATOMY OF CARPAL JOINT AND COMMON PROBLEMS

Thoroughbreds are fast animals with well developed limbs adapted to maximize locomotor efficiency at an early age [32]. The number of bones and the amount of muscles, which do not directly contribute to the locomotion, are minimized, thus reducing the overall body weight. In the distal end of the fore and hind limbs, the load is shared by the third metacarpal (Mc3) and third metatarsal (Mt3) bones as the main supporting bones. Growth and development are rapid in Thoroughbreds where the foals mature early and can walk within few hours after birth [32]. The Thoroughbred foal quickly doubles its weight in six weeks and reaches a near-full maturity in 18 months [33]. This speedy growth is completed around its fourth year [33]. Thoroughbreds are agile and strong animals, but they are also plagued by frequent lameness caused by joint injuries and diseases [34], causing the most significant portion of wastage in racing and athletic horses [34-36]. Of these, it is thought that osteoarthritis (OA) accounts for approximately 60% of all lameness [37] with the cost in the USA reported as high as $1 billion per annum [38]. Osteoarthritis is a group of joint disorders, which progressively deteriorates articular cartilage, bone and soft tissues of the joint [39]. It is conventionally classified either into primary and secondary depending whether the cause is known or not. However, for the short-life spanned Thoroughbreds, the causes can be pinpointed to both acute and repetitious traumas, abnormal joint conformity and even from inherited factors [40]. Equine osteoarthritis can be commonly found in the hock (ankle), fetlock, pastern and coffin joints as well as less commonly in the stifle (knee) joint and spine (neck and back). Although OA is less common, the osteochondral fractures in the carpus are common in Thoroughbreds, and they are one of leading cause of lameness in racing horses [39]. Repetitious concussions on the carpus during a high speed gallop can generate tremendous stresses on acute regions of the interlocking joint surfaces. These stresses, when applied for a prolonged period, can generate localised degenerations in the cartilage which may progress to OA together with subchondral sclerosis and necrosis of the underlying bone that in turn might cause sudden fracture in the joint, ultimately resulting in the end to the racing carrier for the horse.

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1.4.1 ANATOMIC NOMENCLATURE AND USAGE

433 434 435 436 437 438 439 440 441 442 443 444 445 A NTERIOR & P OSTERIOR Anterior refers to "front" whereas posterior means "back" of the subject. Although it is easy to apply these terms for biped like humans, where the front and back of the subject is intuitive, it is a major source of confusion for quadrupeds that have four legs like horses. Terms anterior and posterior are used interchangeable with terms like caudal, rostral and dorsal where appropriate. L ATERAL & M EDIAL Since many animals are symmetrical along the left and right axis, common side terms right and left are not often used to describe the relative position of an object. Instead, both left and right are thought as sides and yield a directional term lateral (Latin word lateralis means "to the side") to describe whatever that is not the centre to the torso. The opposite to lateral is called medial [19]
Figure 2 - Positional and directional terms in the horse. From R. A. Kainer [41]

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(Latin word medius means "middle") which is used to define a point in the centre of the organism where the left-right axis intersects. P ROXIMAL & D ISTAL Proximal is coined from a Latin word proximus which means nearest where as distal is from distare which means to stand away from. Both proximal and distal are terms used to describe the relative position of an anatomical site along the appendage (e.g. leg or arm) which forms a natural proximodistal axis. For example, carpus (knee) is proximal (i.e. near to the torso than) to the hoof but distal (i.e. further away from the torso than) to the shoulder (Figure 2). D ORSAL & P ALMAR Both dorsal and palmar are directional terms that are relative to hand. The word palmar is derived from Latin word palma which means palm of the hand to describe the anterior (front side) of the hand whereas the word dorsal describes the back of the hand. Since equine carpus, although it is equivalent knee in horse, it is still a part of hand, thus the dorsal and palmar are used to describe the relative locations of the joint (Figure 2). C RANIAL & C AUDAL Cranial and caudal are used to describe the upper half of the thoracic limb, the regions proximal to the carpus (the wrist, in the forelimb) and the tarsus (the ankle in the hind limb). The objects and surfaces closer to or facing towards the head are cranial and if these are facing away or further from the head are caudal.

1.4.2 CARPUS - KNEE JOINT OF THORACIC LIMB

Horse limbs are well-developed for running fast with long legs that are capable of extended strides. However, body mass is unevenly distributed between front and hind limbs, where the front carries 55~60% of the total body weight [42]. During a fast gallop, this uneven mass is further imposed upon on the fore limbs from the forward momentum generated by the hind limbs [43, 44]. Additionally, in every stride for a split second, the uneven mass is further accentuated by having one of the two fore limbs to experience the initial landing impact on the ground. The high stresses experienced in the fore limbs are minimized by the carpus, the surrounding muscles and ligaments. The carpus

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(equivalent to wrist in human) has four metacarpal bones, with no 1st and 5th digits and vastly reduced 2nd and 4th digits, only leaving 3rd digit to be the main distal limb (Figure 4).

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Figure 3 - Equine skeletal system. From C. Hill [45]

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Figure 4 - Skeletal structure of left equine thoracic limb in side view. From C. Hill [41]

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Carpus is comprised of 11 bones in total and grouped into three rows of separate joints; proximal antebrachio-carpal (radiocarpal), middle carpal (midcarpal) and distal carpometacarpal joints (Figure 5 and Figure 6). The antebrachio-carpal joint consists of the radius and the proximal row of radial (Cr), intermediate (Ci), and ulnar (Cu) carpal bones. The midcarpal joint is consisted of the proximal row of the same Cr, Ci and Cu plus the distal row of the second (C2), third (C3), and fourth (C4) carpal bones. Finally, the distal carpometacarpal joint is made up of the same distal row of carpal bones, C2, C3 and C4, plus the second (Mc2), third (Mc3), and fourth metacarpal (Mc4) bones. The radius and ulnar carpal (Cu) bone articulate with the accessory carpal (Ca) bone, whereas the accessory carpal bone does not bear direct weight.

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Figure 5 - Palmaromedial view of exposed left carpal synovial sheath and relationships of major tendons and ligaments. Flexor retinaculum cut and reflected. SDFT = superficial digital flexor tendon; JDFT = deep digital flexor tendon. From R.A. Kainer [41]

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Figure 6 - Dissection of carpal ligaments. Lateral view (left) and medial view (right). From R.A. Kainer [41]

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Figure 7 - Lateral expansion mechanism of carpus under an axial load. Left carpus showing individual carpal bones are wedged against each other adjoined by extensive inter-carpal ligaments. It is thought that when a sudden axial force is applied, these carpal bones push against each other and transfer the compression into elastic potential energy via stretching the inter-carpal ligaments. From N.J. Deane and A.S. Davies [46]

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In the carpus, the six carpal bones (Cr, Ci, Cu to C2, C3, C4) form an interlocking wedge arrangement, where each bone is slant-wedged against the other and tightly packed by extensive inter-carpal ligaments (Figure 6 and Figure 7). When a sudden axial stress is applied to the joint, the boneligament arrangement converts the axial impact to more moderate tensile force by elastically stretching the inter-carpal ligaments as the interlocking bones push against each other, briefly expanding the joint laterally. By this mechanism, this acute potentially traumatic stress might be safely dissipated through more moderate slow elastic tension in the ligaments [47]. However, continuous cyclic impact in the carpus can still cause intraarticular fractures in the carpal bones and the distal end of the radius leading to lameness.

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I NTRAARTICULAR FRACTURES OF CARPUS

Intraarticular fractures are common in racing horses as well as in hunting, jumping and other actively performing horses. In the racehorse, the most common type of intraarticular fracture occurs is at the carpus with a high incidence rate is reported in horses of 2 to 4-years of age [11]. This age range is exposed to many dangerous predisposing factors for chip or slab fractures (Figure 8); immaturity, high gallop speeds, long limb length and the running distances which can generate high levels of concussive forces that focus on the narrow dorsal surface of the carpal bones.

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Figure 8 - Third carpal bone's slab fracture and its surgical reattachment using a screw to create interfragmentary compression. From C. McIlwraith [11].

A chip fracture is defined as a detached osteochondral fragment of the bone due to traumatic impact, which varies in size and location. It can be firmly attached to the bone, or loose, freely [25]

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floating in the synovial space. Chip fracture occurs commonly in the radial (Cr), third (C3), and intermediate (Ci) carpal bones and the distal end of the radius. The slap fracture is also another type of osteochondral fragment that involves the full thickness of bone with both proximal and distal joint surfaces (Figure 8). It is also most commonly formed in the third (C3), intermediate (Ci) and radial carpal (Cr) bones but not in the radius. For the racing Thoroughbreds and Standardbreds, a high number of fractures occur in the third carpal bone in the carpus [48-50]. In the Thoroughbreds, the most common sites of carpal fracture were the distal radius (35%), the third carpal bone (35%), and the radiocarpal bone (29%); and in all of these, a total of 87% occurred as chip fractures on the dorsal surface of the bone [51].

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C AUSES OF FRACTURES
Through the proximodistal axis in carpus, the force from ground impact is tranmitted through the large metacarpal to the radius [52]. Despite the high magnitude impact, the carpus is supported by the surrounding soft tissue structures (i.e. muscles, tendons and ligaments) which guide a natural articulation. However, as the soft tissue becomes fatigued and there is a danger of uncoordinated, compensatory over-striding and this may lead to trauma in the joint, resulting in possible intraarticular injuries including fractures in the carpus. During a fast gallop, greater axial loading is directed towards the dorsal surface of the carpal bones [53] plus fatigue in the leg also creates additional abnormal compression on the dorsal surface of the carpal bones in two ways. Firstly, when the foot is not in full extension as it impacts the ground, the leg may experience a backward, snapping, or slamming effect of the carpus rather than a smooth interlocking of the carpal bones [54, 55]. Secondly, the caudal support to the carpus is reduced during maximal loading as the flexor muscles begin to fatigue [11]. Furthermore, the joint congruity in the carpus could be reduced by fatigue as well as the extreme speed, poor racing surfaces, an improper landing angle to the race track, and improper trimming [54-56]. All of these factors increase the chance of asynchronous stress distribution in the carpus from the less-than-perfect congruity resulting in possible trauma to the joint and therefore, inducing a higher chance of intraarticular fracture.

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L OCAL REMODELLING OF CARPAL BONES

The cartilage and the subchondral bone of the carpal bones undergo a process of remodelling in response to the compressive and shear stresses [11]. There is a constant level of intermediate compression in the middle and palmar areas in the carpal bones. However, at high loads, the dorsal surfaces of the bones are intermittently exposed to high compressive loads [53]. The dorsal region in the carpal cartilage in comparison to the palmar region, there is an increased production of smaller aggregating proteoglycan molecules, which results in a lower stiffness in the cartilage (i.e. lower aggregate modulus) [57-59]. The stiffness of the subchondral bone decreases as the bone resorption precedes the deposition activities during the initial remodelling phase. Later, this is then followed by an increase in bone mineral density in the region [59] which results in a higher bone stiffness [60] causing more cartilage damage from the higher impact stresses [61].

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H ANDEDNESS IN CHIP FRACTURE

It has been recorded that the occurrence of chip fracture on the right is slightly higher than on the left leg in Thoroughbreds. In Thoroughbreds, 3-year-olds were most commonly affected with chip fractures in the right limb (56%~59%) [50, 62]. This is because racehorses frequently shift to the right lead on the straightaways and at the end of races, placing greater stress on the limb as the horse becomes fatigued.

576

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1. ARTICULAR CARTILAGE

2.1 INTRODUCTION
Articular cartilage is a compliant load-bearing connective tissue, which covers the articulating surface of all diarthrodial joints [63, 64]. The tissue has four known functions; a) protects the bone from abrasion and other damage, b) transmits and distributes high compressive and shearing forces to the subchondral bone, c) provides for joint congruity and maintenance of low contact stress and d) provides smooth, lubricated articulating surfaces [63-66]. Normal articular cartilage has a smooth, glistening, pearly white-glasslike appearance because of its high water content (70% by weight in mature healthy cartilage) and varies in transluscency and thickness from area to area within one joint, and from joint to joint [64]. The tissue is unusual because it is hypocellular, aneural, alymphatic, and avascular requiring its nutrition and metabolic wastes to be exchanged via diffusion, and thus the metabolic and repair activities are vastly slower than other well vascularised tissues. By dry volume, cartilage consists of about 50% collagen, 35% proteoglycan, 10% glycoproteins (e.g. proteinases and their inhibitors, growth factors, lysozyme, fibronectin, chondronectin, cartilage oligomeric protein, etc.), 3% mineral, and 1% lipid; with only 1 to 12% chondrocytes [67]. The sparse population of chondrocytes is responsible for the production, organization, and maintenance of the extracellular matrix [68]. The chondrocytes vary in number, size, and shape depending on the layer of the cartilage zone [69], and each zone is marked by changes in chondrocyte morphology, biochemical composition, macromolecular architecture, which bring forth a range of material properties (e.g. tensile strength and elasticity) unique to their respective zones (see Table 2 and Figure 9 in page 30). Chondrocytes sense mechanical loads and biochemical changes in the extracellular matrix. This information is then incorporated into the remodelling process of the extracellular matrix by finely tuning both the anabolic and catabolic activities. As a result, the biological and mechanical [28]

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properties of articular cartilage are ultimately driven by the interaction between the chondrocytes and the extracellular matrix [70]. However, the fine balance in the metabolic system can be disrupted through many factors including aging, and both traumatic and prolonged cyclic loadings to the joint. Many of these imbalances can lead to degeneration of the joint and result in joint stiffness, immobility, swelling and pain; all these conditions are common symptoms of osteoarthritis.

2.2 STRUCTURE AND MAKE UP OF ARTICULAR CARTILAGE

Articular cartilage is primarily made up of the extracellular matrix (ECM) with sparsely embedded chondrocytes and matrix fluid. The living chondrocytes occupy only 2-5% of the tissue volume [71]. The remainder is the extracellular matrix which largely determines the biomechanical properties of the tissue. The ECM is composed of a highly organised assembly of several macromolecules such as collagens, proteoglycans, non-collagenous proteins and tissue fluid. Although generally all articular cartilage shares a common composition, the makeup and organisation of its major components differ depending on the depth, site, joint, sex and species [72, 73]. The material properties of cartilage depend on the interactions between the fluid and the matrix macromolecules within the ECM. Variations in thickness, cell morphology, and biochemical composition affect the overall matrix characteristics, allowing the tissue to fulfil the site-specific load requirements for the joint [74]. While the depth variations in cartilage are not clearly demarcated, the tissue can be subdivided according to the collagen orientation, proteoglycan distribution and the organisation and morphology of chondrocytes; and hence the four zones are called the superficial (zone I), transitional (zone II), deep (zone III), zone of calcified cartilage (zone IV) and lastly subchondral bone plate, which supports all the above (Table 2, Figure 9).

[29]

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Table 2 - Summary of depth-related variations in articular cartilage. The relative strength of each category is explained by a use of dots []. Modified from M. Aydelotte and K. Kuettner [75].

Superficial Zone I Chondrocytes Density Shape Orientation Matrix Composition Water Aggrecan Keratan SO4 Hyaluronan/PG ratio Fixed Charge Density Collagen Fibre diameter Orientation Mechanical Properties Tensile stiffness Resiliency Discoid Tangential Fine Tangential

Transitional Zone II Spherical Random Medium Oblique

Deep Zone III Ellipsoid Radial Coarse Radial

634

635 636 637 638 639

Figure 9 - Zonal division of articular cartilage and subchondral bone showing four zones of the matrix, tidemark, cement line and subchondral bone. The division of cartilage is based on the overall morphology of the collagen orientation, proteoglycan distribution and the organisation and morphology of chondrocytes. From A. Poole [76].

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2.2.1 SUPERFICIAL ZONE (ZONE I)

The uppermost surface layer of cartilage is also called the tangential zone or zone I, the thinnest zone occupying 5-10% of the matrix volume. The chondrocytes in this region are flattened discoid shape, aligned parallel to the articular surface. The layer boasts its highest concentration of collagen and water while it has relatively low proteoglycan content. The dense collagen fibres which are aligned tangentially are also small in diameter [77]. These collagen fibres may also be preferentially aligned in the direction of stress caused by movement at the joint surface [78] resulting in split-line patterns of the dominant fibrillar orientation when pricked with a pin [79]. The dense collagen network has a high tensile stiffness and reduces focal stress from external loads [80, 81] and act as a "skin" resisting the swelling pressure exerted by the proteoglycans in the layers beneath [78].

2.2.2 TRANSITIONAL ZONE (ZONE II)

The transitional zone (also called the intermediate, middle zone or zone II) is sandwiched between the superficial zone and the deep zone occupying approximately 40-45% of the matrix volume. In this zone, the collagen fibres are amorphously arranged and form a gradual transition from the fine collagen fibrils in the superficial zone to thicker collagen fibres. There is a significant increase in proteoglycan content and this enables the tissue to bear compressive loads (see Material Properties of Cartilage Matrix in page 48) [82, 83]. The chondrocytes are shaped spherical, arranged singly or in small groups dispersed randomly through the matrix. The plump cells have well-developed endoplasmic reticulum and Golgi apparatus, secretory vacuoles, lysosomes, and mitochondria relative to the cells of the superficial zone.

2.2.3 DEEP ZONE (ZONE III)

The deep zone (also called the radial zone or zone III) occupies 40-45% of the total matrix volume. Its water concentration is the lowest in the cartilage but the proteoglycan content is high, which enables the tissue to bear a large amount of compressive loads [82, 83].

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The chondrocytes in this region are predominantly ellipsoid and mainly grouped in radial columns of two to six cells, perpendicular to the articular surface. The cells in the radial zone are characterised by an increased content of organelles associated with active matrix formation, rough endoplasmic reticulum, Golgi complex, and mitochondria. The collagen fibres in the deep zone run in a radial direction aligned with the columns of chondrocytes and these radial fibres extend without interruption into the calcified matrix [84]. The fibres become gradually coarser at increasing depth and form large bundles of approximately 55m in diameter [85].

2.2.4 CALCIFIED CARTILAGE (ZONE IV)

The calcified cartilage layer consists of 5-10% of the total matrix volume with high concentrations of calcium salts but no proteoglycans. The rounded chondrocytes are encased within a calcified cocoon where they are sparsely distributed and where some may be necrotic. The collagen fibres in this region are an extension from the radial zone of the hyaline cartilage and these collagen fibres establish strong connections between the hyaline cartilage and calcified cartilage layers. Furthermore, the highly irregular, inter-digitating contours provide strong anchorages [86, 87]. Beneath the calcified zone, there is a thin layer of subchondral bone [88] separated by the cement line which is then followed by the trabecular bone. The calcified cartilage layer acts as an intermediate layer between the uncalcified cartilage to the subchondral-cortex region allowing smooth translation of dynamic forces [89-91]. The overall stiffness of calcified cartilage is thought to be related to the adjacent subchondral bone density; for example, a high volume fraction subchondral bone is found with calcified cartilage that is also high in volume fraction and yet poorly mineralised, whereas for low volume fraction bone was found with thin and highly mineralised tissue [92, 93]. The thickness of the calcified cartilage layer is determined by the advancement of the tidemark into the non-calcified hyaline cartilage above and subsequent replacement by bone via resorption at the cement line [94]. The cement line forms once the chondroclastic resorption has ceased and the bone opposition has occurred. The resorption process is driven by tartrate-resistant acid phosphatase-positive chondroclasts [95, 96] while the calcification is driven by ECM embedded chondrocytes. This cell-mediated remodelling is similar to the remodelling process which occurs in

[32]

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the subchondral bone, and is increased in arthritis and events of traumatic injuries to the joint. [97, 98] While calcified cartilage thickness in general is approximately 5% of the total cartilage thickness [99], others have also reported that the thickness depends upon mechanical factors [100-103], age [104-106], repetitious loading [97, 107] and joint immobilisation [108]. It has been suggested that mechanical sensitivity of chondrocytes and chondroclasts at the tidemark and the cement line are the main sources of the calcified cartilage thickness [109]. A high compressive load was found to trigger an increase in the subchondral bone volume fraction which, in turn, induced softer less mineralised calcified cartilage. And the thickening of the calcified layer is usually accompanied with multiple tidemarks and increased vascular remodelling in the subchondral bone [97, 110].

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T IDEMARK
The tidemark is a basophilic histological feature in the calcified zone [111] represented by a band of irregular wavy lines. The continuity of collagen fibres through the tidemark [84, 112-115] provide anchorage to the calcified zone and also smooth out the stress from the compliant articular cartilage to the rigid calcified matrices beneath [114]. At the tidemark, calcium, phosphate, lipids and enzymes such as alkaline phosphatase and ATPase are present but proteoglycan is not found [112]. The numerous duplications of tidemarks together with thickening of the calcified zone are believed to be a hallmark feature of osteoarthritis [116-118] as the tidemark may be reactivated by trauma or osteoarthritis, leading to cartilage thinning [119-121]. Some have proposed that the tidemark changes are the secondary effect of cartilage fibrillation and elevated subchondral bone turnover [116, 117], which may exert primary influence on the pathogenesis of OA [122] and increase the stress concentration across the uncalcified cartilage leading to increased stress to the joint [123]. Tidemark is the first site to fail in a single incident of experimental trauma to the joint [124] and microcracks in the calcified cartilage have been demonstrated [125, 126]. Tidemark may also stimulate adaptive changes or remodelling the joint that promote thickening of the subchondral bone [97, 127]. But the tidemarks are also common features in both young and elderly adults [104, 118, 128].

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Advancement of the tidemark [101, 129] might be another indicator of renewed growth and tissue formation, although the underlying mechanism is still uncertain [130]. However, it is certain that tidemark advancement is closely linked with the magnitude and type of loading environment as well as age of the subject. Unloading a mobile joint accelerates tidemark advancement [131] while the advancement occurs more slowly in weight-bearing regions of a joint [105, 132]. It was also found that the level of tidemark irregularity may be positively correlated to the lower weight-bearing region [133] but others have observed that there was no clear difference [134-136]. Another study, employing unloading with cast immobilization, found that this promoted thinning of the calcified cartilage layer in the weight-bearing regions of the canine knee joint without a concurrent thinning in the hyaline cartilage [137]. Tidemark advancement may cease for long periods in mature joints [98, 113, 138] and then may accelerate after the age of 60 in human and may contribute to age-related increases in joint congruity. [119]

2.2.5 SUBCHONDRAL BONE

The subchondral bone plate is an integral and dynamic component of the joint, and its primary function is to provide support for the overlying articular cartilage [111]. The subchondral plate of the convex joint is relatively uniform in thickness, whereas in the concave counterpart, the plate is usually much thicker at its centre than at the periphery [139]. The subchondral bone contains extensive underlying vascular channels [66, 111] which shares its space with neural networks and other bone-related cells [111, 140]. Because of these extensive neuro-vascular networks, there is a high chance of the subchondral plate participating in activities other than mechanical support, such as providing a route for the exchange of nutrients between the cartilage and the bone-marrow compartments [111].

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S UBCHONDRAL BONE ADVANCEMENT AND OA

Similar to the calcified cartilage and tidemarks, the subchondral bone also advances; first by initiating the chondroclastic resorption of the cartilaginous layer followed by osteoblast proliferation and bone deposition [140]. It has been found that reducing the load on a mobile joint activates resorption at the chondro-osseous interface which then promotes subchondral bone and tidemark advancement, vascular invasion and erosion of articular cartilage [108, 130]. [34]

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The advancement of the subchondral plate is thought to be inhibited by intermittent hydrostatic stress generated in the deep layers of cartilage during cyclic loading from the weight bearing and muscular contractions [141-143]. And several authors have speculated that tidemark and chondroosseous interfaces are coupled since they are both affected by a common factor in promoting their advancement; i.e. the lack of intermittent and static pressure on the joint [94, 120, 132, 144, 145]. Others have also suggested that the subchondral bone may have a role in the initiation and progression of OA [146] since all OA patients exhibit bone turnover increase [147] and the thickening or sclerosis of the subchondral bone [148]. On the contrary, it was also shown that the subchondral bone loss can occur in the early stage of OA [149] and bone sclerosis in the late stage of OA [150].

2.2.6 VASCULAR CHANNELS

In the subchondral bone plate (and sometimes in the calcified cartilage region), three types of cavities are found; 1) marrow space, 2) Haversian canal and 3) vascular channel (Figure 10). The extensions of the marrow space from beneath, branch into the subchondral bone and the spaces are more than 100 m wide, irregular in shape and contain fat or marrow cells lined with flat endosteal cells.

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Figure 10 - Vascular channels in the subchondral bone. (Left): The vascular channel (VC) is connecting the bone marrow cavity in the subchondral bone (CB) to both calcified and hyaline cartilage region through the tidemark (TM). (Right) A SEM image of vascular channels on the subchondral plate surface. From H.Duncan et al. [111].

The Haversian canals are extensions of larger marrow spaces, forming cylindrical channels 30-70 m in diameter, which passed through the bone in all directions and contained marrow cells and,

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occasionally, a blood vessel. The canals branch up towards the calcified cartilage where each canal is encapsulated by multiple concentric layers of oppositional lamellar bone, but does not appear to have an endosteal layer [140]. The canals, as they branch out towards the hyaline cartilage, become finer and are reduced to approximately 10-30 m in diameter (human, dogs and rabbits [140]), usually without fully penetrating the calcified cartilage layer; these canals are called the vascular capillaries/channels, also known as vascular canals, capillary invasions or cutting-cones. The channels have been shown to contain 1 or 2 thin walled blood vessel(s) without forming end-loops, together with various types of cells (e.g. osteocytes, osteoblasts, osteoclasts, endothelium cells, erythrocytes, fat cells) [111, 140] and sometimes nerve fibres as well [151]. Vascular channels can be classified either closing (Figure 11) or open cone (Figure 12) depending on whether or not the tip of the vascular channel is blocked with lamellar bony cap [140].

100um

10um

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Figure 11 - SEM images of a closing cone vascular channel (A and B). The vascular channel (V) is surrounded by multiple layers of lamellar bone (B) where the channel is invading into the zone of calcified cartilage from subchondral bone (scb). Modified from J.M. Clark [140].

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Figure 12 - Histological section of the cartilage, showing an open cone vascular channel where it has successfully penetrated into the hyaline cartilage layer. The channel contains numerous erythrocytes (arrow). Left from H.Duncan et al. [111] and right from J.M. Clark [140].

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801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 The role of vascular channels is uncertain but it is thought that they may be involved in providing nutrition to the cartilage [152, 153], a biochemical signal pathway [154], and mineralization in the deep zone of the cartilage [91, 94]. As for the nutrient supply channel hypothesis [152, 153], Clark [140] argued that there are usually too few vascular channels, which penetrate the diffusion limiting calcified cartilage layer to provide enough nutrition to the deep zone of cartilage; and for this reason it is still widely accepted that the synovium still acts as the major nutrient supply [155]. Boyde and Firth using SEM [154] proposed that the channels are used as the biochemical signal pathway between bone and cartilage as they observed the channels were connected to larger marrow spaces in the deeper trabecular bone. On the other hand, Oegema and Bullough et al. [91, 94] proposed the vascular channels act as mineralization initiators to the hyaline cartilage and thus leading to subsequent advancement of the tidemark. Avascularity in articular cartilage is critical as the presence of vascular channels in the tissue will affect the biomechanical properties and may disrupt the delicate metabolic activities in the extracellular matrix; and normal cartilage actively repels vascular invasion via producing antiangiogenic factors within [156]. In a normal joint, vascularity is highly organized and well defined [153] without hyperactive angiogenic process [157]. However, in osteoarthritis, angiogenesis is re-activated and OA cartilage (in vitro) loses its resistance to repel the invasive vascular channels [158]. But, as this area of research is relatively new [159-161] and there is only a small number of parallel studies [156], more studies are required to form an accurate picture of angiogenesis in relation to OA. The process of cartilage angiogenesis probably involves many complex metabolic processes; e.g. changes in the cartilage mineralization rate, and stimulation and inhibition of proteases etc. [162164]. The number of vascular channels (under an assumption that the prevalence of channel is proportional to the angiogenic activity) has been studied in terms of age, stress distribution, trauma, exercise and osteoarthritis recently [111, 154, 165-167]. In one study, the number of vascular channels in human femoral heads was found to be constant with increasing age [165], but another reported that there was a gradual reduction in the number of channels until the age of seventy years, after which there was an increase [166]. [37]

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In terms of weight-bearing, more vascular channels were found in high loaded regions of the human tibial plateaus and femoral heads, where the cartilage thickness was also higher [111, 165]. However another study reported a higher number of vascular channels in low-loaded regions of the third metacarpal bone of race horses [154]. With respect to exercise, mildly exercised canines exhibited fewer vascular channels that touched the tidemark [167] but another study showed that there was no significant difference (P > 0.05) between the trained and control groups [154]. Trueta [168] reported that trauma in the hip joint produces microfractures, which promoted increased vascularity; but to authors knowledge there has been no subsequent reporting of studies linking direct trauma to changes in vascularity. In this thesis, we address whether traumatic event on the C3 and Cr bones in the midcarpal joint induced increased vascularity.

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V ASCULARITY AND OSTEOARTHRITIS

Osteoarthritis (OA) is an intriguing disease because it exhibits not only destructive and degenerative tissue responses, but it also includes a wide range of hyperactive and regenerative symptoms as well. OA studies as early as 1953 have demonstrated a positive correlation between OA and the vascularisation, where the vascular channels from the subchondral bone were frequently observed invading the zones of hyaline and calcified cartilage [159, 169]. In osteoarthritis, new vessels invade the cartilage from the underlying bone, probably in response to disruptions in the balance between the angiogenic and anti-angiogenic factors [170-173]. Increased vascular remodelling and calcified cartilage thickening with multiple tidemarks are characteristics of OA [97, 110]. Subchondral bone formation is evident with an increased bone fraction along with visible radiological subchondral sclerosis and radiographic 'hot spots' [174]. Angiogenesis activity is increased in the plasma of OA patients, suggesting that the angiogenesis may play an important role in OA [163, 175]. Even in an artificially induced OA model in rat (anterior cruciate ligament transection), Hayami et al. found there is an increase in the vascular invasion compared to the control group [176, 177]. One interesting finding is that the vascular invasion occurs most frequently in a cartilage matrix from which proteoglycan contents had been reduced greatly [156] but there is no clear relationship with gross cartilage changes to the vascularity [140].

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The close relationship between OA and vascularity is certain but whether changes in the vasculature precede or result from the clinical arthritis remains uncertain [153]. The vascular pathology may play a important role in the initiation and progression of OA [178] and this could shift the main focus of the OA research from the cartilage to the vasculariture in the subchondral bone [153].

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S TRESS INDUCED BONE -OA AND VASCULARISATION

Normal cartilage is full of angiogenic inhibitors (e.g. troponin and chondromodulin-I) synthesized by chondrocytes, which naturally repel the vascular invasion from the subchondral bone and maintain the avascularity in the extracellular matrix [179, 180]. However excessive mechanical stress has shown to induce pro-angiogenic VEGF (vascular endothelial growth factor) [181, 182] along with other catabolic factors, including MMPs (matrix metalloproteinase) which induce deterioration in the cartilage matrix [183-186]. The members of the VEGF family are known to be important angiogenesis mediators, expressed by hypertrophic chondrocytes in the growth plate during the limb development, but absent in the resting and proliferating chondrocytes [187-190]. The proteins target vascular endothelial cells and stimulate their proliferation and migration, ultimately participating in the formation of blood vessels [187, 191, 192]. The VEGF-mediated capillary invasion is an essential event that regulates growth plate morphogenesis and triggers cartilage remodelling [193] coupled with cartilage resorption to induce bone formation [190]. Low oxygen tension, or hypoxia, in inflamed synovial joints, like in OA, appears to evoke activation of HIF-1a (Hypoxia-inducible factor 1), followed by expression of downstream molecules including the VEGF that collectively alters the chondrocyte metabolism, possibly toward a catabolic response, hence inducing the joint diseases [157]. Furthermore, the vascularisation may play a role in the osteophyte formation, advancement of underlying subchondral bone, and the development of fibrocartilage [163], all of which are common features of OA.

2.2.7 CHONDROCYTES
The chondrocytes are cartilage specific multipotent stem cells, which are responsible for the production and organization of the extracellular matrix and its components, i.e. collagens, proteoglycans, and noncollagenous proteins. In adult human articular cartilage, they account for approximately 1% of the tissue volume [194]. [39]

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Chondrocytes receive mechanical, electrical, and physico-chemical signals transmitted by the extracellular matrix, which in turn affect the regulation of the cell metabolic activities. They are nonuniform in shape and vary in numbers according to their matrix zones and in which, their metabolic activities also differ. For example, the levels of aggrecan synthesis and non-aggregating proteoglycans differ for the cells in the superficial zone, versus those in the deeper layer [195]. Since the articular cartilage is avascular, it is believed that the articular cartilage receives most of its nutrients from the synovial fluid and export its wastage product via diffusion [196]. Thus, nutrition concentration along the radial depth of the tissue is likely to be non-uniform, and this may affect the chondrocyte metabolic rate in each zone. It is not known to what extent this might occur, but it has been proposed that the decreasing concentration may have resulted in a higher cell density near the articular surface than in the deep zone, establishing a depth-specific cell density in the tissue [71, 197].

903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919

ECM REGIONS
The matrix can be subdivided into 1) pericellular, 2) territorial and 3) interterritorial regions around each chondrocyte defined by structural differences and characteristic distributions of proteoglycan, link protein, and hyaluronic acid (hyaluronan) [198]. Each matrix region functions synergistically to produce an integrated, hydroelastic suspension system capable of resisting traumatic compression [199]. The thinnest matrix is the pericellular matrix which surrounds each chondrocyte providing a hydrodynamic protection for the chondrocyte during the physiological loading and plays a metabolic role in pericellular retention of the cell. A chondron is defined as a singular unit of one chondrocyte in its pericellular micro-environment [200], but sometimes it is used for describing the hypertrophic multi cellular chondrocytes in a developing and OA cartilage. In a chondron, a chondrocyte is linked to a high pericellular concentration of sulphated proteoglycans, hyaluronan, biglycans [201] and a range of matrix glycoproteins. The territorial matrix region is well defined in the superficial, transitional, and deep zones, and it is distinct from the smaller pericellular and larger interterritorial regions. It contains collagen fibrils with a larger diameter and arranged in radial bundles [202] which is easily seen in the middle and deep zone of the cartilage. In the territorial region, the proteoglycan concentration is higher [203].

[40]

920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947

The interterritorial region is again distinct from the territorial region [199]. In here, there is a higher concentration of proteoglycans rich in keratan sulphate [203], where the largest diameter collagen fibres are also found.

2.2.8 COLLAGEN
For detailed review of the collagen and collagen cross-link, please refer to the section Chapter 3 Collagen in page 28. The collagen fibrillar network forms the framework in the cartilage matrix and type II collagen is the predominant fibrous component (80% to 90% of the total collagen contents) along with several minor collagens; types IX 1-2% and XI collagen 3% [204, 205]. The type II collagen provides the basic architecture of cartilage [206] and other minor collagens adhere to the type II collagen to form a much stronger collagen network. Type IX collagen is a short fibrillar collagen and is located on the surface of the dominant type II fibril assemblies. It is also considered a proteoglycan because of its single glycosaminoglycan chain. Type IX is covalently bound by hydroxypyridinium (HP, see page73) cross-links to type II collagen, and it is thought to play an important role in the integrity of the collagen network [207, 208]. Type XI collagen is located within the fibrils [209] to partly control their lateral growth during matrix formation by pulling the outer fibrils from inside the assembly [210]. Type X collagen is only synthesized specifically by hypertrophic chondrocytes during early cartilage development and OA, and it is confined to the calcified zone of normal articular cartilage [211]; however, its function is not fully understood [212]. Type VI collagen is present in small quantities in the territorial matrix of isolated chondrons [213] and in osteoarthritic cartilage [214] and is thought to have a role in maintaining the tissue integrity [215, 216]. Types II, IX, and XI are cartilage-specific collagens, which are extensively cross-linked together in a highly organised network, which provides the tissue its form, tensile stiffness and strength [217].

[41]

948 949 950 951 952 953 954 955 956 957 958 959 960 961 962

2.2.9 PROTEOGLYCANS
Proteoglycans are glycoproteins that are heavily glycosylated, made up of a core protein (proteo-) with numerous attachments of glycosaminoglycan (GAG, -glycans) chains covalently bound to the core protein (Figure 13). High levels of proteoglycans are present not only in the cartilage but in many adult tissues including blood vessels and skin, and they have a great affinity for water and able to swell many times their molecular weight. Heinegard et al. classified proteoglycans into classes of large and small proteoglycans, the aggregating and non-aggregating, and then further subdivided these based on the type of the glycosaminoglycan chains [218]. Most widely used nomenclatures are based on their morphology (e.g. aggrecan, which aggregates with hyaluronan), or by the feature (e.g. decorin, which decorates the surface of type I collagen fibrils), or by its primary structure (e.g. biglycan, which has two glycosaminoglycan chains, see Table 3). Other forms of proteoglycan also exist such as the hyaluronan (hyaluronic acid) and link proteins [219-223].

Table 3 - Proteoglycans found in cartilage. From P. Neame [224].

Glycosaminoglycan Chondroitin sulphate Dermatan sulphate

Primary core protein Aggrecan Decorin Biglycan

Wet weight of Cartilage (%)* 5-10 0.03-0.12 0.06-0.24 0.1-0.3 0.05-0.25

Wet weight (nmol/gm)* 1-10 0.3-0.6 0.25-0.5 1.5-5 0.03-0.8

Other names PG-LA1 PG-S2 PGII PG-S1 PG-LA1 59-kDa protein Hyaluronic acid

Heparan sulphate Keratan sulphate

Aggrecan Fibromodulin

Hyaluronan

963 964

*Approximated figures which may vary up to 50%

965 966 967 968 969

A GGRECANS
Aggrecans are large aggregating proteoglycans representing 90% of the total proteoglycan mass in cartilage, and it is the most studied of all the proteoglycans as it can be isolated in gram quantities with relative ease. Aggrecan consists of a protein core, keratan sulphate, chondroitin sulphate and glycosaminoglycan chains. Aggrecan forms large aggregates consisting of many aggrecan monomers [42]

970 971 972 973 974 975 976

attached non-covalently to hyaluronan [225]. As many as 100 aggrecan monomers can be attached onto a single hyaluronan chain. The numerous carboxyl and sulphate groups on the aggrecan chains then bind with the cationic interstitial fluid, increasing the overall volume of the wet proteoglycan compounds. Because the swelling is resisted with tension from the highly organised collagen network, the overall internal pressure of the matrix increase dramatically, giving rise to the stiffness and viscoelastic properties of the cartilage [226].

Core protein

Chondroitin sulphate Keratan sulphate

Hya

Link protein

luro

nic

acid

977 978 979 980 981 982 983 984 985 986 987
Figure 13 - A proteoglycan (aggrecan) model. It is found in cartilage where both chondroitin sulphate and keratan sulphate are present on core protein. As many as 100 hydrophilic aggrecan monomers can attach onto a single hyaluronan chain which enables exponential swelling-up of the extracellular matrix via the mechanisms of cationic interstitial binding.

H YALURONAN
Hyaluronic acid (hyaluronan) binds to the collagen fibrillar network and its main function is to assist in retaining the aggrecan within the extracellular matrix in cartilage [198]. In synovial fluid,

hyaluronan also acts as a lubricant. It is synthesized and secreted from the chondrocytes largely in the transitional zone. The molecule is unique among the proteoglycan family because it neither has a core protein nor is it sulphated like other types of proteoglycan. Its molecular mass is relatively

[43]

988 989

large, ranging from 3 X 102 to 2 X 103 kDa, and it contributes 0.5 to 2.5% of the wet weight of hyaline cartilage [227].

990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016

S MALL PROTEOGLYCANS AND N ON - COLLAGENOUS PROTEINS

Other smaller and non-aggregating proteoglycans include decorin, biglycan, and fibromodulin. They contribute approximately 3% of the total proteoglycan mass, and they also bind to matrix macromolecules to assist in stabilising the matrix. Their roles are not fully understood, but most in vitro studies suggest that decorin and fibromodulin interact with the collagen, and may regulate the size of the collagen fibrils. They may also play a role in cell adhesion, multiplication, differentiation, and migration. Link protein binds non-covalently to a specific site near the N terminus of the aggrecan core protein and to hyaluronan in the assembly of aggrecan [228, 229]. Anchorin CII appears to help anchor chondrocytes to the collagen fibrils [230] while tenascin and fibronectin influence the interactions between the chondrocytes and the matrix.

2.3 CARTILAGE MATRIX TURNOVER

Cartilage matrix continuously remodels itself by balancing both synthesis and degradation to continually renew matrix components [231]. However, if there is an imbalance of the catabolic and anabolic regulations, this can lead to degeneration in the cartilage. Although most of the anabolic and catabolic activities are driven by chondrocyte responding to its external stimuli, cartilage metabolism is complex and not fully understood. The participating molecules can be largely categorised into cytokines, hormones, proteolytic enzymes and tissue inhibitors of

metalloproteinase (TIMP) plus other minor molecules such as prostaglandins and plasminogen activators, etc (Figure 14).

2.3.1 CYTOKINES
Cytokines are the main player in the matrix regulation. They are protein mediators which facilitate communication between cells (paracrine regulation) or within the cell (autocrine regulation). Once activated, they function by attaching to the designated receptors on the target cell. Interleukins (IL), interferons (IF), tumor necrosis factors (TNF), and growth factors (GF) are all cytokines that trigger the chondrocytes to initiate catabolic and anabolic sequences [232, 233]. [44]

1017 1018 1019 1020 1021 1022 1023 1024 1025

Via the cytokines, the chondrocytes can enhance or retard cell division, promote or inhibit matrix synthesis and degradation, amplify and suppress their mutual effects synergistically or additively [234]. Consider for example the degradation of collagen IX (Figure 14): upon receiving an external stimulus, the chondrocyte releases a cytokine called interleukin-1. The interleukin-1 then attaches to a chondrocyte receptor which induces the expression of matrix proteinases such a matrix metalloproteinase (MMP) and also the proteolytic enzyme called stromylesin. The released stromylesin degrades the aggrecan and type IX collagens [206, 235, 236] by cleaving the native type IX collagen molecules from the surface of the type II-XI-IX collagen fibrillar assembly.

Matrix synthesis
Hormones Hormones

o oc luc G

Inhibition

s oid rtic
Cytokines

m Ga

a ph -a l N F ro n T rfe nte a -i ins m

Cytokines (growth factors)

Int

er

k lue

u Stim

latio

Inert matrix
Cytokines Free O2 Radicals

Gr o Ins wth h ulin o rm Ca on e lcit on An in d ro ge Pla ns tel Fib et-d e ro b las rived Ins tG GF ulin F Tra GF ns for mi ng GF be ta

Stimulation

m Ga

Prostaglandins

Inhibition

ma

e ro e rf int

n a

Stimulation

ph -a l NF T

TIMP family
Kallikreins

Stim u

latio

an ro t op ll se eta MP) eina i x M (M p ro t s tr e se Ma ri n na Se tei ro rp the O

e as

Extracellular Proteinases

i Inh

biti

on
Plasminogen activators

Matrix degradation

1026 1027 1028 1029

Figure 14 - Cartilage matrix metabolic regulation. The state of the inert matrix is carefully balanced by the matrix synthesis and degradation activities. TIMP (tissue inhibitor of metalloproteinase), GF (growth factor), TNF (tumor necrosis factor).

[45]

1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 The interleukin family is special because while the interleukin-1 promotes matrix synthesis, interleukin-6 induces matrix synthesis inhibition by activating the TIMP (tissue inhibitors of metalloproteinases). This particular example demonstrates that one type of matrix molecules can regulate both catabolic and anabolic activities [237]. Also, some cytokines such as tumor necrosis factor- (TNF-) and -interferon, have a dual action whereby they can both inhibit matrix synthesis and stimulate the degradation of proteoglycans and collagens [238]. Anabolic regulations are driven by the growth factors (e.g. platelet-derived growth factor, fibroblast growth factor, insulin growth factor, and transforming growth factor-), which stimulate DNA and protein synthesis while opposing the catabolic activities [239, 240]. Hormones also participate actively in both inhibition and stimulation of matrix synthesis. The growth hormones, insulin, calcitonin and androgens stimulate the chondrocyte proliferation and synthesis of matrix macromolecules while the glucocorticoids inhibit the matrix synthesis [241].

2.3.2 EXTRACELLULAR PROTEINASES

Proteinases are a group of enzymes that breakdown specific proteins when stimulated by cytokines at a given pH environment. There are five main classes of proteinase categorised according to the chemical group in the hydrolysis of peptide bonds [242] (Figure 15). The serine and metalloproteinases are active at neutral pH and function extracellularly while the cysteine, aspartate and threonine proteinases are predominantly active at an acidic pH and function intracellularly. Some proteinases are not directly involved in the cleavage of matrix proteins, but act indirectly by activating proenzymes which, in turn, degrade the matrix individually or in combination with another proteinase, e.g. prostromelysin and procollagenase [243].

[46]

Intracellular
Aspartic Cysteine
Cathepsin B Cathepsin K Cathepsin L Cathepsin S

Extracellular
Threonine Serine
Elastase Cathepsin G Plasmin PAs Furin

MetalloCollagenases Stromelysins Gelatinases

MT1~6 MMP ADAMs ADAMTSs

Cathepsin D

Proteasome

1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077

Low pH

Proteinases

Neutral pH

Figure 15 - Categorisation of proteinases. Proteinases can be largely divided into five categories (Aspartic, Cystein, Threonine, Serine and Metallo-), where they can be broadly grouped either intracellularly vs. extracellularly or low pH vs. high pH acting. Modified from T.E. Cawston and A.J. Wilson [244].

M ATRIX M ETALLOPROTEINASES
Matrix metalloproteinases (MMPs) form a sub-group within the metalloproteinase family. They all have a highly conserved motif containing three histidine residues that bind zinc at the catalytic site [244]. The metalloproteinases can be divided into five multigene families; the serralysins, the astacins, the ADAMs (ADisintegrin And Metalloproteinase), the adamalysins, the pappalysins [245] and lastly the MMPs [246]. Traditionally MMPs were divided according to their substrates (a molecule upon which an enzyme acts) and the mostly widely known MMPs are the stromelysins, collagenases and gelatinises [247]. MMPs are known for their ability to cleave components of the extracellular matrix and can also cleave other proteinases, proteinase inhibitors, latent growth factors, chemotactic molecules, growth factor binding proteins, cell surface receptors and cell-cell adhesion molecules [248]. The catabolic inhibition of MMPs is enforced by the TIMPs (tissue inhibitors of metalloproteinases) [249, 250], which bind tightly to the MMPs in a 1:1 ratio; thus if the TIMP levels exceed those of an active enzyme, connective tissue turnover is prevented. In normal articular cartilage, the catabolic expressions are suppressed as there is a slight excess in the TIMP concentration over the MMPs [251]. MMP-1, MMP-8 and MMP-13 (collagenase-1, -2 and -3) are able cleave fibrillar collagens at a specific site, producing characteristic and sized fragments. The MMP-13 prefers to cleave type II collagen, whereas the MMP-1 and MMP-8 prefer type III and I, respectively. All three collagenases

[47]

1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107

are responsible for matrix degradation, which are present in both rheumatoid and osteoarthritis [252]. Collagenases MMP-1 and MMP-13 are synthesized by macrophages, fibroblasts and chondrocytes when stimulated with inflammatory mediators such as interleukin-1 (IL-1 ) and tumor necrosis factor- (TNF-). MMP-3 and MMP-10 are also called stromelysin-1 and -2, which degrade several cartilage matrix components including aggrecan, link protein, [253] and collagen types II, IX, X, and XL [206, 235, 236, 254]. Thus the stromelysins are present in tissues in which active remodelling is taking place, such as in the rheumatoid or osteoarthritic cartilage and synovium [255-257]. MMP-2 and MMP-9 (gelatinase A, B) cleave denatured collagen (gelatin), type IV and V collagen and elastin. Their levels are elevated in synovial fluids and tissues of rheumatoid arthritis [258-260].

2.4 MATERIAL PROPERTIES OF CARTILAGE MATRIX

Articular cartilage is a permeable, viscoelastic material composed of proteoglycan aggregates and extracellular fluid in a dense collagen fibrillar network [261-264]. While collagen fibrils dominates the matrix tensile behaviour [265], the osmotic properties of the proteoglycans embedded into its collagen network provide its resistance to volumetric compression and shear [266]; furthermore, it is able to fully regain its original volume by hydrating during periods of rest. Proteoglycan concentration in articular cartilage is unusually high, 5 to 10 times higher than other connective tissues, which results in a high osmotic pressure of 0.1 to 0.2 MPa [267, 268]. The negatively charged sulphate, glycosaminoglycans carboxyl groups and the associated cations contribute 75% to 85% of this total osmotic pressure via the Gibbs-Donnan effect [269]. This high osmotic pressure may contribute up to 50% of the compressive stiffness of the articular cartilage [270, 271] and it is balanced by the tension from the collagen network. During the resting period, the cartilage matrix is under negative pressure as the proteoglycans are only hydrated to around 20% of their domain capacity while the collagen network limits further expansion [220]. Once a load is applied, due to incompressibility of the proteoglycan-water compound trapped in the collagen network, the matrix initially undergoes an elastic deformation. However, the deformation is then followed by a "creep" as the interstitial water is slowly forced from the hydrated proteoglycan domains [81] until the osmotic potential of the hydrated proteoglycans is equal to the applied pressure (i.e. its equilibrium state) [272-274]. [48]

1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136

The osmotic pressure is dependent on the proteoglycan charge density, distribution and its molecular conformation, where there is a strong, positive correlation between the tensile modulus of the extracellular matrix and the collagen and proteoglycan concentrations [265, 275]. Articular cartilage exhibits topographical variations in the matrix molecular organisation and composition, which are correlated with loading pattern differences across the joint surface [83, 276279]. The high-weight-bearing regions are richer in proteoglycans than the low-weight-bearing regions [278, 280, 281]. In the superoanterior region of the human femoral head, which is a site of high contact stress, the cartilage is thicker, the fixed charge density higher, and water content is lower than in the region below the fovea which is a lower contact stress site [278]; the collagen content is also higher in the higher stress sites [83, 281].

2.4.1 THREE MATHEMATICAL INTERPRETATIONS OF CARTILAGE

Mathematical models have been developed in order to describe and predict cartilage behaviour into three broad categories; the elastic, viscoelastic and poroelastic models, increasing the level of sophistication respectively. The elastic model is the simplest mathematical description of cartilage behaviour. It is used for simple kinematic studies of a joint such as rapid cyclic load studies. The model assumes the cartilage is an elastic incompressible solid and its prediction neglects the time-dependent aspects of the cartilage behaviour. Such a model is useful for estimating the contact areas and joint conformity. The viscoelastic model encompasses time-dependent response, which is defined by a set of equations that match observed load-motion behaviours without explaining their physical origins. It can provide a useful description of experimental behaviour under given specific conditions, and the model is useful for comparative studies [275]. The poroelastic model was initially developed to describe the consolidation of fluid-saturated soils under geomechanical loads and it is a well established model. Variations of the poroelastic model exist, such as the biphasic and triphasic models [261, 270], and it includes time-dependent behaviour. The basic modelling approach is that the load is shared between an incompressible fluid phase and a porous elastic solid phase [275]. One can integrate and modify the model with optional components of directionality, electrochemical interaction, and nonlinear permeability, etc. Another

[49]

1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165

variation of the poroelastic model, poro-visco-hyperelastic model also exists which utilises porepressure concept [282, 283].

2.5 HYALINE CARTILAGE - RESPONSE TO LOADING, EXERCISE AND IMMOBILIZATION

One of the single significant factors affecting the cartilage and bone metabolism is the mechanical stimuli from joint motion, impact and weight bearing. The continual loading within the physiological limit (and frequency) is essential for maintaining a healthy and readily adaptable extracellular matrix that is able to function under the ever changing loading environment. The chondrocyte metabolic response under a static load condition differs from a dynamic loading condition. Static loading on cartilage generally inhibits biosynthetic activity [284] while the dynamic loading appears to have a more complex effect, all depending on multiple factors such as the load direction, intensity, frequency, duty cycle, and time in culture [285-288] and also the anatomic site [289, 290]. A moderate cyclic load encourages proteoglycan and protein synthesis [291, 292] but a heavier or prolonged load appears to inhibit the synthesis [285, 286, 293], particularly in cartilage from anatomic sites that are not usually subjected to heavy loads in vivo [67]. The habitually loaded sites have a higher uronic acid content (a comparative measure of chondroitin sulphate and hyaluronan) than habitually unloaded sites [279]. Increasing weight bearing induces an increase in proteoglycan content mimicking maturation and aging [294]. The volume fraction of the chondrocytes in the superficial zone of regions bearing a high physiologic load has been reported to be less than half that in the low weight-bearing regions, possibly due to cell necrosis due to the high load [295]. The effects of immobilization, achieved by placing the joint in a plaster cast or by bridging with external fixators [296, 297], have resulted in osteoarthritic-like abnormalities such as joint space narrowing, osteophytosis and subchondral sclerosis. Gross changes also included articular cartilage fibrillation, ulceration and capsular thickening, and at the biomolecular level, there was a reduction in proteoglycan content, synthesis, extractability, and ability to aggregate in the presence of hyaluronan [296-298].

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The cartilage becomes thicker when subjected to moderate increases in activity during adolescence [299, 300]. The thickness is also determined by atrophy or degeneration in response to a decrease in loading associated with amputation [298, 301], paralysis [302-304] or immobilization [305-309]. In short, the thickness, at least in part, is affected by the mechanical factors associated with joint loading and motion while the thinning is associated with a reduction in weight bearing [130]. The cyclic hydrostatic forces generated through mechanical loading is thought to inhibit tidemark and subchondral advancement [141-143], both of which encourage the retention of cartilage thickness or its thickening [105, 132]. In conclusion, cartilage requires mechanical stimuli to maintain and adjust its metabolic activities and retain its normal extracellular matrix properties in relation to the external loading environment. Whilst static loading is essential for maintaining the cartilage health, dynamic cyclic loading (within the physiologic level) has the greater influence, as it is responsible for suppressing the tidemark advancement process which, in turn, regulates the overall cartilage thickness.

2.6 AGING OF ARTICULAR CARTILAGE

Aging alters the matrix composition and retards chondrocyte activity. The chondrocytes sensitivity to growth factors [310] and regulative cytokines (e.g. interleukin-1 and tumor necrosis factor-) [311] is decreased, leading to initiation and progression to osteoarthritis-like symptoms [310]. The size of aggrecan is progressively reduced, where intact molecules are less common, and the number of smaller keratan sulphate-rich molecules increases [312] and the hyaluronan chains become shorter [227]. In general, both matrix turnover and proteoglycan degradation increases and thus affects the osmotic capacity, leading to tissue weakness [311, 313]. Although the hydroxypyridinium (HP) crosslinks in the collagen network remain steady throughout adult life [314], the inevitable degradation processes result in unwinding of the collagen triple helix [315] and in the end, type II collagen concentration decreases [316, 317]. The weakened matrix cannot withstand the osmotic swelling pressure of the hydrophilic proteoglycans; this leads to localized swelling, and softening of the tissue. The soften tissue further accelerates the degradation process when exposed to physiological levels of loads. As a result of proteoglycan and collagen loss, the pericellular micro-environment expands

[51]

1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224

to occupy the depleted pericellular regions. The chondrocytes in the region attempt matrix repair by initiating proliferation while synthesizing new matrix. However, the degradative enzymes overwhelm the synthesis activity and the repair fails. Hypertrophic chondrons are formed from cell division and proliferation, where they remain in clumps bound together by sheets of type VI collagen. This type of chondrocyte expansion has been viewed by some as one of the earliest matrix changes associated with cartilage catabolism [200, 318].

2.7 OSTEOARTHRITIS
Osteoarthritis (OA) is a progressive degenerative joint disorder with diverse aetiologies. Both mechanical and biologic events are thought to destabilise the normal coupling of synthesis and degradation in the joint tissues. Until recently, OA has been thought of as a symptomatic chronic disorder, a part of the natural aging process [319]. However, it is now defined as a group of overlapping distinct diseases [320], not only affecting the articular cartilage, but involves the subchondral bone, ligament, capsule, synovial membrane, and periarticular muscles. Although the aetiologies are diverse, they eventually lead to self-destruction of the cartilage, diminished strength and mobility of the joint, accompanied by chronic and intense joint pain, tenderness and inflammation. At a superficial level OA involves both generative and degenerative processes. The problem is that new tissue is being formed in the wrong place and at the wrong time [321] resulting in a loss of proper function of the joint. The OA joint suffers from a loss of cartilage, subchondral bone sclerosis, cysts, marginal osteophytes, increased metaphyseal blood flow, and variable synovial inflammation leading to joint pain, limitation of movement, crepitus and occasional effusion. Although not lifethreatening, mainly due to increasing life spans in the developed countries, the prevalence of OA is increasing and has a significant health impact demographically [322-324]. The economic cost of OA is estimated to be approximately $180 billion in the U.S. annually [325] surmounted by the morbidity, diminished income, and medical costs [326]. The disorder is largely irreversible and the widely prescribed treatment methods involve pain management and related therapies. Only in the most severe cases, invasive cartilage or joint replacement procedures are prescribed.

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1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254

Difficulty in OA treatment arises due to the complex multi-faceted nature of the disease as well as the not having a full understanding of the synthesis and regulation of the joint. The current focus of OA research ranges from tissue engineering, animal model, gene therapies to stem cell augmentation.

2.7.1 HOW OA AFFECTS CARTILAGE AND BONE

OA weakens cartilage, reducing its ability to resist tensile, compressive, and shear forces [83, 327]. There is also a loss of hydraulic permeability of the cartilage matrix [328] due to weakening of the collagen network [329] and altered composition and concentration of proteoglycan content [330333]. In OA, the cartilage experiences the fragmentation of the surface, fissures, cloning of the hypertrophic chondrocytes, while the underlying bone exhibits uneven crystal deposition, sclerosis, and focal osteo-necrosis, and invasion of the tidemark by the blood vessels. The joint will attempt self-repair in response to degeneration but the previous normal joint capacity is never recovered and degenerative changes continue. Under arthroscopic or radiographic examination it is not easy to determine whether many of the gross level lesions observed on the cartilage are from mechanical erosion or mild osteoarthritis. Some cartilage lesions are of questionable clinical significance and may not cause pain or inflammation in the joint. Conversely, other lesions may progress to full blown OA [40]. While cartilage lesions are the gross criteria for OA, they may not be the centrally important cause of clinical disease [334]. Upon gross inspection, mild OA cartilage first loses its normal lustre and consistency, becomes yellow, soft and forms blisters [335], which then leads to pitting and superficial fraying of the cartilage. OA cartilage exhibits various stages of gross changes that can be regarded as progressive where one type of gross change (lesions) is followed by another, or exist in combination [334]. Wear is the least severe form of OA and it is defined as the partial-thickness loss of cartilage [336]. It is then followed by ulceration, a localised form of lesion in the cartilage [334]. Erosion is the next stage of cartilage loss and can lead to a full-thickness loss [336, 337]. A more severe form of lesion is eburnation where the underlying bony surface is now well exposed and polished [338]. Sometimes there are multiple parallel lines on the cartilage surface in the direction of joint motion which are

[53]

1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275

called wear lines [335-337, 339]. The clinical significance and the cause of these wear lines are unknown. Other forms of mild gross cartilage change include flaking [340] or early fibrillation[338]. These disruptions are confined to the tangential layer of the matrix [340, 341]. They are only visible when rubbed with India ink or when histologically prepared. In the fibrillated cartilage, a variable degree of chondrocyte necrosis occurs while chondron formation has been described as part of the repair response [342, 343]. Osteophytes (or bone spurs) are formed at the joint margin, which are thought to be an attempt to improve congruity following degeneration of articular cartilage while the subchondral plate suffers from bone sclerosis [335, 337]. The rapidly growing cartilage on the surface of the osteophyte resembles an immature cartilage in the nature of its proteoglycans and very low content of keratan sulphate [344]. At the calcified zone, the tidemark duplication can be observed while it is advancing towards to the articular surface [98, 345] (see page 33 for OA tidemark changes). In the OA joint, cartilage and bone metabolism are increased [346-348], where the cancellous bone volume is increased, which is driven by both osteoblasts and osteoclasts [321, 349, 350]. For example, increased amounts of bone volume were found in the iliac crest of patients with OA in the hand [351, 352] along with higher levels of growth factors [353]. There is also an increase in the synthesis of various cytokines in OA subchondral bone, including IL-8, IL-10 and TNF- at the sites of sclerosis compared with non-sclerotic regions [354, 355]. Sclerosis has traditionally been regarded as a reaction to both cartilage loss and increased joint loading. Similar bone changes are evident with cartilage fibrillation [150] and sometimes from radiographical evidences [347, 356].

1276 1277 1278 1279 1280 1281 1282 1283

OA CHANGES IN CARTILAGE AT THE MOLECULAR LEVEL

In OA, there is a significant increase in the degradation and subsequent synthesis of the collagen type II and aggrecan [357, 358], affecting their concentrations, molecular sizes and aggregation levels driven by MMPs, such as prostaglandins and other inflammatory factors, IL-1 and TNF- [357, 359]. The collagenases (MMP-1, -8, -13) cleave the type II collagen triple helix fibrils, results in unwinding and fragmentation of the collagen fibrillar assembly [320], while type IX or type XI collagens are untouched. The aggrecan and keratan sulphate concentrations are lowered and the aggregation size

[54]

1284 1285 1286 1287 1288 1289

of aggrecan is reduced. However, the chondroitin-4-sulfate content is increased relative to chondroitin 6-sulfate [360]; whereas the decorin and biglycan contents undergo little changes [361]. The matrix fragments from the collagen and proteoglycan proteolysis are diffused out of the cartilage into the synovial fluid, which may enter the lymphatics and ultimately reach the blood circulation [362, 363]. The reduced collagen integrity allows higher water content in the matrix which results in an excessive swelling, further reducing the stiffness of the matrix [172, 276].

1290

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3. COLLAGEN

3.1 INTRODUCTION
Collagen is one of the most abundant proteins in the extracellular matrix in the mammal, and it is found as the main protein in connective tissues such as epithelial, muscle, nerve, cartilage, tendon, etc. The primary function of collagen is to provide stability to the tissue and the structural integrity to the organ. The composition and fibrillar architecture of collagen are tissue specific, best suited to the mechanical and biochemical roles of the tissue, and often regulated by the matrix resident cells which are also responsible for the matrix synthesis and maintenance. Collagen molecular size, function and its tissue distribution vary considerably depending on the type of collagen. There are currently 29 genetically distinct collagen types that have been identified [364370] and this number is likely to increase in the future. Collagens are grouped based on their structure, splice variants, the presence of additional nonhelical domains, assembly and also according to their functional roles. All collagens are loosely grouped into one of the following classes; the fibril-forming, the fibril-associated, the network forming, the anchoring fibril, the transmembrane and the basement membrane collagens. However numerous they are, all collagen molecules share a common characteristic structural motif, where every single collagen molecule (tropocollagen) is made up of three polypeptide strands (peptides) characterized by the repeating amino acid motif (Gly-X-Y). In here, X and Y can be other collagen-specific amino acids (Figure 16), which in turn further define the overall collagen characteristics as well as the final architecture of the matrix.

[56]

1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 While some genetically based diseases (e.g. Pagets syndrome) are caused by the errors in the collagen synthesis and degradation, collagen also holds much potential as a delivery medium as it has both excellent binding capacity and biodegradability in the human body. It is currently being studied as delivery systems for drugs, growth factors or cells [372-374].
Figure 16 - Gly-X-Y amino acid sequence and collagen fibril. Every collagen fibril is made up of Gly-X-Y amino acid sequence where X and Y can be any additional collagen protein domains, which results in different collagen type. Diagram from J. Koolman, K. Roehm, and J. Wirth [371].

3.2 THE BASIC STRUCTURE AND DOMAINS IN COLLAGEN

Every collagen, regardless of its type, carries a homogenous characteristic of two structural features; (1) each collagen molecule is formed from three -chains in a right-handed triple helix conformation [365, 375] and (2) each -chain is made up of either Gly-Pro-Y or Gly-X-Hyp in n repeating units (as noted as (Gly-X-Y)n) in left handed helix conformation. When three identical -chains are twisted into a tropocollagen (Figure 18), it is called a homotrimer (e.g. collagen types II, III, VII, VIII, X), whereas if two or more different -chains are used, then it is called a heterotrimer (e.g. collagen types I, IV, V, VI, IX, and XI).

[57]

1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 Glycine (Gly), proline (Pro) and hydroxyproline (Hyp) are three of the 20 -amino acids. Glycine is a precursor of many proteins and is one of the smallest in the amino acid family, whereas both proline and hydroxyproline increase the collagen stability through quantum mechanical stereoelectronic effects [377]. The three precursor -chains form a triple helix tropocollagen (or a procollagen) in the cell nucleus before exported to the extracellular space. When the tropocollagen is exported from the nucleus, it is clipped by procollagenase at the both telopeptide regions of non-collagenous domains, i.e. the Npropeptide and C-propeptide domains (Figure 17). The N and C-propeptide ends are removed from the molecule; this action allows the triple helical part of the molecule available for the fibrillar aggregation into a group of collagen fibrils (Figure 18). The non-collagenous domains (N- and C-propeptides) are important structural components. The Cpropeptide is thought to play a fundamental role in the initiation of triple helix formation, whereas the N-propeptide is thought to play a role in the regulation of primary fibril diameters [378]. The two short non-helical telopeptide regions between the N and C domains are thought to be involved in the covalent cross-linking to other collagen molecules in the fibrillar assembly and also may allow linkage to other molecules of the matrix [379].
Figure 17 - Molecular structure of fibrillar Type I and II collagen molecule with the various subdomains as well as the cleavage sites for N- and C-procollagenases. From K. Gelse, E. Poschl, and T. Aigner [376].

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1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373
Figure 18 - Three chains form a triple helix procollagen molecule with two endings called N-propeptide and Cpropeptides. The N- and C- propeptides are clipped off by proteinases and the segment is assembled into the fibrillar aggregate through intermolecular cross-links (x). Since all molecules are identical in length approximately 300nm, this assembly results in banding pattern of D=67nm. From D. Hulmes [380]

The above description of collagen synthesis is based on the fibril-forming collagens. The structural features for the non-fibrillar collagens are slightly different. The non-fibrillar collagens have unusual non-helix interruptions in the molecular structure, which is a normally continuous helical domain in the fibrillar collagen molecule. These interruptions are thought to affect the intra-molecular flexibility and allow specific proteolytic cleavage to occur with another collagen molecule. The so-called FACIT collagens (Fibril-Associated Collagens with Interrupted Triple, e.g. type IX, XII, XIV, XIX, XX, XXI), are characterized by several non-collagenous domains interrupting the triple helices and it has been suggested that these non-collagenous domains may function as flexible hinging regions [381]. Conversely, the non-collagenous domains of non-fibrillar collagens (type IV, VI, VII, VIII, X) are involved in network formation and aggregation. The length of the triple helical part varies considerably between different collagen types. The fibrilforming collagens (type I, II, III) have triple helical domains approximately 300 nm in length, i.e. about 1000 amino acids [364, 378], whereas other collagen types have much shorter or contain nontriple helical interruptions, e.g. collagen VI or X are about 200 or 460 amino acids long respectively [364].

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1374 1375 1376 1377 1378 1379 1380 1381

3.3 COLLAGEN TYPES

The 29 collagens identified to date can be broadly divided either into fibrillar or non-fibrillar. The fibrillar collagen family consists of five collagen types (I, II, III, V, XI) and the non-fibrillar collagen family is further divided into six sub-groups; the FACIT collagens (IX, XII, XIV, XIX, XX, XXI), the basement membrane collagens (IV), the transmembrane collagens (XIII, XVII), the microfibrillar collagens (VI), the anchoring collagens (VII, VIII, X) and the multiplexins (XV, XVI, XVIII, see Table 4).

Table 4 - Various collagen types. Adapted from K. Gelse, E. Poschl, and T. Aigner [376].

Type Fibril-forming collagens I II III V XI Basement membrane collagens IV Microfibrillar collagen VI Anchoring fibrils VII VIII X FACIT collagens IX XII XIV XIX XX XXI Transmembrane collagens XIII XVII Multiplexins XV XVI XVIII

Molecular composition [1(I)]22(I) [1(II)]3 [1(III)]3 1(V), 2(V), 3(V) 1(XI)2(XI)3(XI) [1(IV)]22(IV); 1-6 1(VI), 2(VI), 3(VI)

Tissue distribution Bone, dermis, tendon, ligaments, cornea Cartilage, vitreous body, nucleus pulposus Skin, vessel wall, reticular fibres of most tissues (lungs, liver, spleen) Lung, cornea, bone, fetal membranes; together with type I collagen Cartilage, vitreous body Basement membranes Widespread: dermis, cartilage, placenta, lungs, vessel wall, intervertebral disc Skin, dermal -epidermal junctions; oral mucosa, cervix, Endothelial cells, Descemet's membrane Hypertrophic cartilage Cartilage, vitreous humor, cornea Perichondrium, ligaments, tendon Dermis, tendon, vessel wall, placenta, lungs, liver Human rhabdomyosarcoma Corneal epithelium, embryonic skin, sternal cartilage, tendon blood vessel wall Epidermis, hair follicle, endomysium, intestine, chondrocytes, lungs, liver Dermal-epidermal junctions Fibroblasts, smooth muscle cells, kidney, pancreas, Fibroblasts, amnion, keratinocytes Lungs, liver

[1(VII)]3 [1(VIII)]22(VIII) [3(X)]3 1(IX)2(IX)3(IX) [1(XII)]3 [1(XIV)]3 [1(XIX)]3 [1(XX)]3 [1(XXI)]3 [1(XIII)]3 [1(XVII)]3 [1(XV)]3 [1(XVI)]3 [1(XVIII)]3

1382 [60]

1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394

3.3.1 FIBRILLAR COLLAGENS - TYPE I, II, III, V AND XI

The fibril-forming collagens are the most abundant and widespread family of collagens, approximately 90% of the total collagen found in nature. They are responsible for torsional stability and tensile strength in the tissues [204, 364, 382]. Types I and V collagens are widely found as a structural framework in bone, while types II and XI collagens predominantly contribute to the fibrillar matrix of articular cartilage. The mechanical strength of the fibrillar collagen comes from its ability to assemble into highly orientated aggregates with a characteristic supra-structure (Figure 18). The collagen monomers covalently bond to other monomers with a typical quarter-staggered arrangement [383], forming a tightly knitted conformation. Under the electron microscope, this formation can be visualised with a clear dark-light pattern of 6467 nm D periodicity [384, 385] (Figure 18). The typical diameters of fibrillar collagens range from 25 to 400 nm.

1395 1396 1397 1398 1399 1400 1401 1402

T YPE I
Type I collagen is the most abundant and the most studied collagen in the collagen family. It forms more than 90% of the organic mass in the bone and contributes to the major collagen contents of tendons, skin, ligaments, cornea, and many interstitial connective tissues. It provides tensile stiffness and is responsible for the tensile and torsional strength in bone following calcification. It is a heterotrimer triple helix with two identical 1(I)-chains and one 2(I)-chain. Type I is mostly incorporated into a composite containing either type III collagen in skin and reticular fibres [386] or type V collagen in bone, tendon and cornea [387].

1403 1404 1405 1406 1407 1408 1409 1410 1411

T YPE II
Type II collagen is the characteristic and predominant component of hyaline cartilage. It counts for approximately 80% of the total collagen content, and it is found in the vitreous body, the corneal epithelium, the notochord, the nucleus pulposus of intervertebral discs, and in embryonic epithelial mesenchymal transitions [364]. It is composed of three identical 1(II)-chains forming a homotrimeric collagen. However, it is also similar to the type I collagen in terms of size and its biomechanical properties [388]. As the 1(II)chains are secreted into the extracellular matrix, the N-propeptide is removed by a procollagen-Nprotease (ADAMTS-3[389]) and the C-propeptide is removed by procollagen-C-protease (BMP1) [61]

1412 1413 1414 1415 1416 1417 1418 1419 1420

[390]. The collagen fibrils are assembled by lateral associations of collagen molecules in a quarter staggered arrays through intermolecular cross-linking (see Collagen cross-links in articular cartilage on page 70). Collagen fibrils in cartilage form heterofibrils with type XI and IX collagens and even with a minor amount of type X collagen in hypertrophic cartilage. These heterofibrils have the function of limiting the overall fibril diameter to 1550 nm [209]. The type II-XI-IX heterofibrils have higher contents of hydroxylysine as well as glucosyl and galactosyl residues compared to type I collagen, and these higher contents provide interaction sites for the proteoglycans [204]. This proteoglycan interaction is important for the development of a high swelling pressure within the articular cartilage matrix.

1421 1422 1423 1424 1425

T YPE III
Type III is a homotrimer collagen consisting of three 1(III) chains. It is widely distributed in collagen I-containing tissues except bone [379]. The collagen type III is an important component of reticular fibres in the interstitial tissue of the lungs, liver, dermis, spleen, and vessels, in which there is also an abundance of elastic tissues [391] .

1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439

T YPES V AND XI
Type V and XI are heterotrimers and consist of three different -chains (1, 2, 3). The 3-chain of type XI collagen is encoded by the same gene as the 1-chain of type II collagen but the extent of glycosylation and hydroxylation differs from that of the 1(II) chain [364]. Type V and XI collagens form a subfamily within the fibril-forming collagens. The type V forms heterofibrils with the types I and III collagens and contributes to the organic bone matrix, corneal stroma and the interstitial matrix of muscles, liver, lungs, and placenta [382]. The type XI codistributes largely in articular cartilage with type II collagen [204, 364] as the core structure of the heterofibrillar assembly [378]. It is suggested that the type V collagen also may function as the core structure of fibrils with types I and III collagens polymerizing around this central axis. The high content of tyrosine-sulphate in the N-terminal domains of 1(V)- and 2(V)-chains in the type XI collagen is thought to provide the strong interaction with the triple helix and may stabilize the fibrillar complex [392].

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1440

3.3.2 NON-FIBRILLAR COLLAGENS

1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464

FACIT COLLAGENS - T YPE IX, XII, XIV, XVI, XIX, AND XX

The FACIT is an acronym for the Fibril-Associated Collagens with Interrupted Triple helices. As the name suggests, the FACIT collagens are trimeric molecules that are associated with the surfaces of various fibrillar collagens and are characterized by the collagenous domains which are interrupted by the short non-helical domains. There are several FACIT collagens (types IX, XII, XIV, XVI, XIX, and XX) and these play a unified role of regulating the diameter of associated collagen fibrils. Because they are too numerous to cover in this chapter, only type IX, which is directly associated with the cartilage type II collagen, is introduced here. Collagen type IX is an important component of cartilage collagen fibrils, along with collagens II and XI. It does not self-assemble into fibrils like type II collagen but coats the surface of collagen II/XI cartilage fibrils through covalent cross-linking in an antiparallel direction [393]. Type IX collagen is a heterotrimeric molecule with three different -chains (1(IX), 2(IX), and 3(IX)), and has a relatively short collagenous and four non-collagenous regions. The 2(IX) chain sometimes carries a chondroitin and dermatan sulphate glycosaminoglycan chain, and thus can also be regarded as a proteoglycan [394]. The collagen molecule is stabilized by the covalent lysine-derived cross-links to the type II Ntelopeptide, which in turn enable the large and highly cationic globular N-terminal domain to contact and presumably interact with proteoglycans or other matrix components [204, 217]. The domain may also be involved in the linkage of various collagen fibres and interact with other molecules in the extracellular matrix. Type IX collagen is very similar to a subgroup of FACIT collagens types XVI, XIX and XXII in molecular structure and can be considered as the founding member of this subgroup [395]. The collagen type XVI is also found in hyaline cartilage and skin associated with type II collagens [396].

1465 1466 1467

M ICROFIBRILLAR COLLAGEN - T YPE VI

The type VI collagen is a microfibrillar collagen, appearing as fine filaments, microfibrils or segments with a faint crossbanding of 110-nm periodicity [397-402]. It is a relatively widely distributed [63]

1468 1469 1470 1471 1472 1473

collagen type [380] and forms an independent microfibrillar network in virtually all connective tissues, except in bone [397, 403, 404]. Its major function is thought to be that of maintaining tissue integrity [215, 216]. Type VI is a heterotrimer consisting of three different a-chains (1, 2, 3) and has a high density of disulfide cross-links which assist in establishing the network of beaded filaments interwoven with other collagen fibrils [403]

1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488

S HORT CHAIN H EXAGONAL NETWORK - FORMING COLLAGENS - T YPES VIII AND X

The overall functions of types VIII and X are to assist in the formation of hexagonal supramolecular networks. Type VIII is found in the Descemets membrane of the cornea and in the underlying endothelial cells, while type X collagen is found in the hypertrophic zone of cartilage during the endochondral ossification and in the calcifying cartilage [405]. Type VIII collagen consists of 1(VIII) and 2(VIII) chains resulting in both homotrimers and heterotrimers. Although type VIII is similar to type X in structure, both are distributed differently and thus probably have different functions [406]. Type VIII is produced by endothelial cells and assembles in hexagonal lattices [407]. Type X collagen is the characteristic component of hypertrophic cartilage in the fetal and juvenile growth plate and in the ribs and vertebrae [365]. It is a homotrimeric collagen with large C-terminal and short N-terminal domains, and assembles in the form of an hexagonal network [408]. Although its function is not completely understood, type X is thought to be involved in the calcification process in the lower hypertrophic zone of the cartilage [408-411]

1489 1490 1491 1492 1493 1494 1495

B ASEMENT M EMBRANE & A SSOCIATED C OLLAGENS - T YPES IV, VII, XV AND XVIII
Collagen type IV is the most important structural component of basement membranes [412] found in the underlying epithelial, endothelial, fat, muscle and nerve cells, where it is integrated as a twodimensional and highly stable supra-molecular aggregate. The collagen molecule is made up of six subunit chains from 1(IV) to 6(IV), and assembled into three distinct heterotrimeric molecules with a predominant form of 1(IV)22(IV) heterotrimer. Type IV consists of three domains: an N-terminal 7S domain, a C-terminal globular domain (NC1), and a

[64]

1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523

central triple helical part with short interruptions of the Gly-X-Y repeats. Because the collagen contains several discontinuities in the (Gly-X-Y) repeat, the collagen is flexible and it is found where the tissue resilience and flexibility are required [413, 414]. Collagen type VII has the longest triple-helical region among the vertebrate collagens - about 420nm in length and found in the underlying basement membrane at the dermalepidermal junction as a form of anchoring filament. The bundles of collagen VII molecules are arranged tail to tail with a short C-terminal overlapping adjacent to another. Collagens XV and XVIII are made up of several collagenous domains, often referred to as multiplexins (acronym for multiple triple he(x)lices with interruptions). The multiplexins are also associated with basement membranes and carry covalently linked glycosaminoglycan chains, and are therefore also referred as proteoglycans. The collagen molecule contains a C-terminal fragment (endostatin), which is proteolytically cleaved from the collagen molecule and it has anti-angiogenic properties [395].

3.4 BIOSYNTHESIS OF COLLAGENS

The collagen biosynthesis process involves complex and numerous intracellular and extracellular steps, where each step is a vital determinant in the final biochemical and physical characteristics of the collagen fibril and matrix. In this section, the fibrillar collagen type I will be used to explain the basic mechanisms of triple helix formation and processing and matrix assembly, since type I is the most studied and understood collagen type. From gene transcription to the aggregation of collagen fibrils, the process of collagen synthesis can be divided into four main steps; (1) the transcription and translation of the collagen genes (2) the post-translational modification of the collagen molecules, (3) the secretion of collagen fibril into the extracellular space and (4) the extracellular processing and modification of the collagen fibrils and their integration into the matrix. The first two steps (1) and (2) occur within the cell and the steps (3) and (4) occur extracellularly (Figure 19).

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1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 The basic building blocks of the collagen fibre, i.e. the -chains, are first synthesized in the ribosome and then exported into the rough endoplasmic reticulum in the chondrocyte. In here, the chains undergo a series of post-translational modifications, which results in the assembly of procollagen molecules [416]. The process of post-translational modification of the procollagen molecule is highly complex but can be divided into five basic steps; (i) modification of pro-line residues to hydroxyprolines, (ii) modification of lysines to hydroxylysines, (iii) N- and O-linked glycosylation, trimerisation and disulphide bonding, (iv) prolyl cistrans isomerisation and finally (v) folding of the triple helix. The modified procollagen molecules are then transported into the Golgi network where they are packaged in the secretory vesicles before being exported into the extracellular matrix. In the extracellular space, the procollagen molecule is clipped at both the N- and C- terminals and then assembled into a larger collagen molecule via covalent cross-linking. The collagen molecule then aggregates with other nearby molecules to form a strand of collagen fibril, which is then integrated into the existing matrix with other collagen fibrils.
Figure 19 - Overall assembly sequence of a collagen fibre from precursor chains. Each collagen fibre is formed from numerous collagen precursors called -chains. (1) Three chains form a procollagen (tropocollagen) molecule in a triple helix. (2) The endings (called N- and C-propeptides) are clipped off by proteinases and (3) the newly created collagen molecule is then (4) assembled into collagen fibrils with characteristic 67nm banding intervals. (4) These fibrils are again assembled into a much larger aggregate called collagen fibre. From Pearson Education Inc [415].

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1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560
Figure 20 - Collagen synthesis sequence. Collagen synthesis begins at the nucleus where the responsible gene is first read (transcribed) from the DNA into mRNA and then translated into a polypeptide chain, which is then folded to become a collagen protein unit. The synthesis steps in details are as follows; (1) removal of the propeptide (2) hydroxylation of Pro and Lys residues (3) oxidation of Cys in propeptides (5) assemblage to form triple helix (6) removal of the propeptide (7) staggered deposition to form fibrils (8) oxidation of Lys and Hyl to aldehydes (9) cross-linking to form supramolecules. From J. Koolman, K. Roehm, and J. Wirth [371]

3.4.1 TRANSCRIPTION AND TRANSLATION

Instructions for the -chain synthesis are contained in the DNA in the cell nucleus (Figure 20). Initially, a small segment of DNA responsible for the collagen amino acid synthesis (e.g. COL###) is spliced and duplicated into an RNA (i.e. transcription), which is then transported outside the nucleus and translated at the ribosome into a polypeptide chain (i.e. translation). The regulation of the transcriptional activities depends largely on the cell type and is controlled by numerous growth factors and cytokines such as the members of the TGF--superfamily, insulin-likegrowth factors, fibroblast growth-factors and other agents. [67]

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The gene responsible for collagen type I, COL1A1 and COL1A2 (i.e. variations of COL#A#), is transcribed to mRNA via an alternative splicing method, in which a single gene can code multiple proteins [417]. The mRNA is then translated into pre-procollagen molecules in the nucleus which are then exported into the lumen of the rough endoplasmic reticulum (RER) with the help of a signal recognition domain recognized by the corresponding receptors. The signal peptide on the procollagen molecule is removed by a signal peptidase and the molecule then undergoes multiple steps of post-translational modification in the RER.

3.4.2 POST-TRANSLATIONAL MODIFICATIONS

In the RER, the procollagen molecules undergo hydroxylation of proline and lysine residues by adding OH groups which result in hydroproline and hydrolysine. The hydroxylysine residues are important in stabilising the helix formation by providing the stable intermolecular cross-linking of the collagen molecules in the fibrils and additionally provide the binding sites for the carbohydrate attachments. The extent of lysine hydroxylation also varies between tissues and collagen types [418]. This results in different types of collagen due to the extent of lysine hydroxylation.

The presence of 4-hydroxyproline is essential for the formation of intramolecular hydrogen bonds which contribute to both the thermal stability of the triple helical domain and the integrity of the monomer and collagen fibril. Three -chains first align themselves at the C-terminal domains. From here they undergo a winding process to form the triple helix all the way to the N-terminus, thus resulting in a procollagen molecule. During the helix assembly, several other enzymes are involved in order to bring about the efficient formation and folding of the procollagen chains; e.g. PPI (peptidyl-prolyl cis-trans-isomerase) [419], collagen-specific chaperones HSP47 [420] and the enzyme protein disulphide isomerase PDI [421, 422]. After the helix assembly is completed, the triple-helical molecules are packaged within the Golgi compartment, transported into the secretory vesicles and then released into the extracellular space.

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3.4.3 EXTRACELLULAR PROCESSING AND MODIFICATION

The collagen fibril assembly is a complex process and our understanding is mostly based on in vitro experiments. After the secretion from the Golgi compartments, the procollagen trimers are processed depending on their collagen type. Firstly, the large N- and C-terminal propeptide domains at the two ends of the procollagen molecules are cleaved off by the N-proteinase and the Cproteinase, leaving the short non-helical N- and C-telopeptides exposed (Figure 21). Once cleaved, the fibril-forming collagens I, II, III, V, XI spontaneously aggregate into ordered fibrillar structures as their self-assembly capability is encoded in the structure of the collagens, i.e. similar to a crystallization process [379, 423]. The exposed N- and C-propeptide ends are thought to be an essential step in regulating fibril formation. Although cleavage of the N-terminal propeptides is not fully understood, it is thought that the process regulates the diameter of the forming fibrils and influences the fibrillar morphology [378, 379]. Each procollagen molecule is then stacked against another molecule in a quarterstaggered arrangement by hydrophobic and electrostatic interactions. This arrangement is additionally stabilized by the formation of covalent cross-links which provide the mechanical resilience of the collagen fibrils. The covalent cross-link between each collagen molecule is largely dependent on the extent of the hydroxylation state of telopeptide lysine residues.

1607 1608 1609 1610 1611 1612

Figure 21 - Assembly sequence of a collagen molecule in the extracellular matrix space. A newly secreted collagen molecule from the cell is now in the extracellular matrix space (i.e. outside the cell) and both N- and C-propeptides are clipped off by the collagenases making the both ending of the molecule available for cross-linking reaction. Once clipping is finished, the molecule automatically assembles into the adjacent collagen molecular aggregate. From K. Gelse., E. Poschl, and T. Aigner [376].

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Each collagen molecule is approximately 300 nm long and stacked against each other with a 40 nm offset with five-strands in each stacking set; this results in a light-dark periodicity of approximately 65 nm, which is readily visible under the electron microscope (Figure 20). Such periodicity is an ultrastructural hallmark of fibrillar collagen [378, 423, 424]. In tendons, type I collagen fibrils are aligned parallel to each other to form large bundles for maximum tensile strength, whereas in skin, they are more randomly orientated in a complex network of interlaced fibrils for multidirectional flexibility [379]. In articular cartilage, the collagen fibrils are tangentially arranged in a random-sheet formation at the surface layer, whereas beneath the surface, they are radially arranged with respect to the subchondral bone. Such a structural arrangement is able to resist both compression and shear forces effectively.

3.5 COLLAGEN CROSS-LINKS IN ARTICULAR CARTILAGE

Articular cartilage (Table 5) contains the fibrillar collagen types II, XI, the FACIT collagens type IX and the non-fibrillar network collagens type VI and X. Type II collagen forms the majority of the collagen content (80% net dried weight) in the articular cartilage [364] while type XI (3%) [425] is embedded within the type II fibrillar core with N-propeptides branching out and FACIT IX collagen (1%) decorating the surface of this assembly [425]. The non fibrillar collagen type VI (1%)[425] is found in the pericellular matrix surrounding the chondrocytes [426, 427] while type X collagen is found only in the hypertrophic zone of the growth plate or in osteoarthritic cartilage [428].

Table 5 - Collagen Types in articular cartilage (% of total collagen). From D. Eyre, M. Weis, and J.-J. Wu [429]

Collagen type Type II Type III Type IX Type X Type XI Type VI Type XII/XIV Type XIII

Overall contents and locations 75% foetal, >90% adult >10% in adult human articular Covalently fibril-associated collagen, 10% foetal, 1% adult Hypertrophic cartilage only Fibril template, 10% foetal, 3% adult Chondron basket, microfilaments <1% Non-covalently fibril-associated collagens Transmembrane

1634

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1635 1636 1637 1638 1639 1640 1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660

Although a procollagen molecule has a high tensile strength, without the intermolecular cross-links to maintain the integrity of the collagen aggregates, they have little strength, e.g. gelatine. A collagen cross-link is a covalent chemical bond which binds in between the multiple collagen molecules of same and different kinds (e.g. type II-II or II-XI etc). A divalent cross-link binds two collagen molecules, whereas the trivalent cross-link binds three. Many different types and levels of collagen cross-linking are found in the fibril-forming types I, II, III, V and XI and the fibril-associated type IX collagens of the higher vertebrates [430]. The cross-links provide the tissue both structural integrity and functional characteristics [370].

3.5.1 ENZYMATIC INTERMEDIATE CROSS-LINKS

Under various loading environments, cross-links play a pivotal role in preventing slippage between the collagen molecules and fibrils that are stacked together. Cross-linking is therefore closely related to the solubility, thermal stability and mechanical strength of the collagen. Initially, all cross-links in articular cartilage (as well as in bone, tendon, skin, etc.) are formed enzymatically by a single enzyme called lysyl oxidase immediately after the secretion from the cell. The cross-link is established between two collagen molecules of the same or different type (collagen types II, IX and XI type III [431]) at the precisely defined sites along the collagen triple helix. This reaction is based on the oxidative deamination of the -amino groups (-NH3) with the lysine or hydroxylysine residues in the non-helical telopeptides portions of the collagen molecule. The relative concentration of the lysine or hydroxylysine in the telopeptide in the collagen molecule determines which cross-linking pathway the reaction will take. This therefore will have a profound effect on the mechanical and thermal stability on the collagenous tissue (Figure 22). In lysine-rich collagen tissues with little or no hydroxylation (e.g. skin and tendon), the final intermolecular bonds are aldimines (Schiff bases), an organic derivative from aldehyde (R1-CH=N-R2). Although in articular cartilage the hydroxylysine-pathway dominates over the lysine-pathway, both are discussed in this section for the purposes of comparison.

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Figure 22 - Collagen cross-linking process via allysine and the hydroxyallysine pathways. Collagen cross-linking is tissue specific. Intracellular hydroxylation of lysine in the telopeptide gives rise to two pathways of extracellular cross-linking, the lysine aldehyde (allysine) pathway and the hydroxyallysine pathway; the latter results in mainly pyridinium and pyrrolic cross-links in bone and cartilage. Adapted from S.P. Robins [432].

However, the hydroxylysine-rich type II and XI collagens in cartilage (and bone) initially start using the lysine-pathway forming temporary aldimines (i.e. dehydro-hydroxylysinonorleucine) [433], but through subsequent Adomori arrangements, keto-imines (i.e. hydroxylysino-ketonorleucine) are finally formed; which are double-double bonded ketones containing an amine group (Figure 23). Since the keto-imine is a very stable molecule with respect to acid and heat, it is highly insoluble even at the immature fetal stage, which is a preferential characteristic for the skeletal framework tissues such as bone and cartilage. This kind of cross-link is called a divalent reducible intermediate cross-link as it is soon replaced with a more stable form of cross-link, in which the original divalent bond is transformed into a trivalent bond (Figure 23). Lysyl oxidase mediated keto-imine cross-links are found in the homo-type collagen assembly in collagen types II, IX and XI as well as in the cross-link between types II-XI and II-IX collagen fibrils [370]. None of the non-fibrillar collagen types VI, XIV, XII or X have been reported with such crosslinking residues.

[72]

Immature cross-link
Collagen 1 Collagen 1

Mature cross-link
Collagen 1 Collagen 3

Aldimine

Ketoamine

Hydroxylysinopyridioline (HP)

Collagen 2 Collagen 2

Collagen 2

1681 1682 1683 1684 1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706
Figure 23 - Reaction of telopeptide lysine-aldehyde with triple helical hydroxylysine to form the divalent aldimine crosslink. This reaction of telopeptide hydroxylysine-aldehyde with triple helical hydroxylysine to form an aldimine is spontaneously rearranged to form a divalent keto-amine cross-link. Further reaction occurs with a telopeptide hydroxylysine-aldehyde to form a trivalent hydroxylysino-pyridioline cross-link, most stable form of a mature cross-link in cartilage. Adapted from N. Avery and A. Bailey [434]

3.5.2 MATURE NON-REDUCIBLE CROSS-LINKS

The newly formed intermediate reducible keto-imine cross-link is spontaneously matured into the non-reducible pyridinoline trivalent cross-link. This further increases the collagen stability by linking three collagen molecules, ultimately adding an extra collagen fibril to the bonding site [435]. Two possible routes of pyridinoline formation have been proposed so far. Eyre et al. [436] suggested that the cross-link is formed from two keto-imines with the release of hydroxylysine thereby linking three collagen molecules, whereas Robin et al. [437] proposed that a condensation reaction occurred between the keto-amine and an additional hydroxylysine-aldehyde within the same molecules, thereby forming a linkage between two type II collagen molecules. However, the precise mechanism of pyridinoline formation in cartilage is still debatable [434]. When the pyridinoline is derived from a lysine residue from the helix, it is termed lysyl-pyridinoline (LP) whereas if the residue used is from the helical hydroxylysine, then it is termed hydroxylysylpyridinoline (HP). The HP pathway is the common reaction pathway in cartilage and bone [438]. The HP process was first identified by Fujimoto et al. [439], whereas the LP process was identified by Ogawa et al. [440]. There is another mature non-reducible trivalent cross-link often mentioned in the literature termed the pyrrolic cross-link. It is similar to the pyridinoline cross-link and also derived from keto-imine, and readily found in bone and tendon. However, this is not covered in the current survey since such a cross-link does not occur in cartilage [441]. [73]

1707 1708 1709 1710 1711 1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725

According to Eyre et al. [393, 442], the hydroxylysyl-pyridinoline (HP) cross-links are often found in the homofibrillar collagen assembly of type II and IX but not in type XI nor type III. On the contrary, Greenwald reported that HP cross-links exist in appreciable quantities in type XI [435]. However, there is very little available literature describing the types and degree of cross-linking in cartilage. This may be due to the technical difficulties in identifying the processes of cross-linking in vivo where the collagen content is also often greatly affected by the tissue, gender and species. Both the reducible intermediate keto-imine and the non-reducible mature hydroxylysyl-pyridinoline crosslinks exist together but their relative ratio changes with the level of tissue maturation. The keto-imines are found predominant in newly synthesized collagens or fetal tissues, whereas the HP cross-links are dominant in more mature tissues. Besides the enzymatically formed keto-imine and HP, there is another type of cross-link called glycation. The glycation crosslink is formed adventitiously and driven by non-enzymatic random processes. It occurs as a part of tissue aging, which further strengthens the collagen fibrils at the cost of reduced flexibility and increased fragility in the tissue. The glycation will be discussed in the section dealing with Non-enzymatic cross-linking via glycation on page 77.

3.5.3 HOW COLLAGEN TYPES II, XI AND IX ARE ASSEMBLED

The assembly of articular cartilage-specific collagen fibrils is an area of on-going research. The following is a brief overview of the most current research in this area.
HP Keto-imine HP

HP Keto-imine

toKe ine im

Type II

Keto-imine

Ke to

-i m

ine

Type XI
1726 1727 1728 1729

Type IX

Figure 24 - A proposed model of inter- and intra-cross-linking relationship between different collagen types found in articular cartilage. All collagen types are benefited from both keto-imine (amine) type and hydroxylysil-pryridinoline (HP) cross-links. However, only keto-imine cross-links are thought to form between different types of collagens.

[74]

1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759 1760

In articular cartilage, the fibrillar collagen types II, III, XI and the decorating FACIT IX are extensively cross-linked. Together, keto-imine and hydroxylysil-pryridinoline (HP) form the intrafibrillar links cross-links whereas as keto-imine forms the inter-fibrillar cross-links (Figure 24). In all of these, the fibrillar type II collagen is the central structure in the collagen assembly to which all other collagens, types III, XI and IX are cross-linked through lysyl oxidase mediated divalent ketoimine cross-links [370, 429, 431, 443, 444]. And some of these collagens, both types II and IX, also form the mature type trivalent hydroxylysil-pryridinoline (HP) cross-links to stabilise within their own fibrillar architecture as well as their relationship to each other [207, 429]. It seems that the type XI of articular cartilage (bovine) also contains a minute amount of HP cross-linking, but it is considered negligible compared to the keto-imine residue level [393, 442]. Figure 25 provides a three-dimensional conceptual assembly of articular cartilage collagen involving collagen types II, XI and IX published by Eyre et al. [445] and based on the proposed model of Holmes and Kadler [446]. Although the type II collagen (blue in Figure 25) is universally cross-linked to the collagen types III, XI and IX, and it appears to be the most predominant feature in the collagen assembly, the type XI (yellow in Figure 25) assumed to provide the main architectural framework. y The collagen type XI fibrils, acting as a growth template, form the central core within the assembly which is then surrounded by multiple layers of type II fibrils. The branches of N-propeptide domains on the collagen XI are then thought to inhibit further increase in lateral growth [447]. The type XI collagen is therefore thought to function as both the assembly template as well as radial growth inhibitor [448]. The FACIT type IX collagen (pink in Figure 25) always co-exists with the type II collagen by decorating the surface of type II fibrils through covalent bonding via a lysyl oxidase mechanism [443, 444]. The function of type IX in the assembly is thought to interact with proteoglycans or other matrix components [204, 217] as well as other molecules of the extracellular matrix. Type III is found in soft and scar tissues, and it is not normally considered as an articular cartilage collagen [449]. However, recently Wu et al. reported a significant amount of type III being covalently linked to type II collagen in both adult human and bovine articular cartilage [431].

[75]

1761 1762

1763 1764 1765 1766 1767 1768 1769 1770 1771

Figure 25 - Holmes and Kadlers proposed model of hierarchical depiction of a heterotypic collagen fibril, emphasizing the internal axial relationships required for mature cross-link formation. Upper: Three-dimensional concept of the type II/IX/XI heterotypic fibril of developing cartilage matrix. Middle: Detailed illustration of the nearest neighbour axial relationships required for the trifunctional intermolecular cross-links to form in the collagens of cartilage, bone, and other high-tensile strength tissue matrices. The exact 3D spatial pattern of cross-linking bonds is still unclear. Lower: Detailed illustration of the axial stagger of the individual collagen molecules required for pyridinoline type cross-linking. From D.R. Eyre, M.A. Weis, and J.J. Wu [445]

[76]

1772 1773 1774 1775 1776 1777

3.5.4 NON-ENZYMATIC CROSS-LINKING VIA GLYCATION

Glycation is an end-stage cross-link formed after the intermediate enzymic divalent (keto-imine) and mature trivalent cross-links (HP, Figure 26). It occurs through adventitious reaction with glucose and its products form additional intermolecular cross-links through the linking of lysine and arginine residues in the triple helical region of the collagen molecule [430, 450].

(a)
Adimine/Ketoamine Immature Divalent Cross-link formation

(b)
Hydroxylysino-pyridioline (HP) Mature Trivalent Cross-link formation

(c)
Glycation Cross-link formation

1778 1779 1780 1781 1782 1783 1784 1785 1786 1787 1788 1789 1790 1791
Figure 26 - Avery and Baileys proposed locations of cross-links in collagen fibres; (a) divalent immature cross-linking within the fibre [I], (b) trivalent mature cross-links linking adjacent fibres [)] and (c) glycation cross-linking within and between fibres [*]. Chemical cross-linking would also be located within and between collagen fibres as indicated for the glycation cross-linking. Adapted from N.C. Avery and A.J. Bailey [451]

The formation of glycation cross-links, or advanced glycation end products (AGEs), accumulates with age, resulting in a stiffer and more brittle collagen network, thus decreasing the optimal efficiency of the fibre. Whereas the addition of the enzymic cross-links is regarded as a stabilising process, the glycation cross-links can be regarded as a deleterious aspect of the ageing process [450]. Currently, the list of AGEs include Ne-(carboxymethyl)lysine (CML) [452-454] and Ne-(carboxyethyl)-lysine (CEL) [455], fluorescent cross-links, such as pentosidine formed between lysine and arginine residues [456, 457], and also some chemically unidentified compounds. Of these, pentosidine is one of the best characterized cross-links formed between fructose-lysine and arginine [456]. [77]

1792 1793 1794 1795 1796 1797 1798 1799 1800 1801 1802 1803 1804 1805 1806 1807 1808 1809 1810 1811 1812 1813 1814 1815 1816 1817 1818 1819 1820 1821 1822

Cartilage contains a relatively large number of pentosidine cross-links [456, 458]. The level of pentosidine in articular cartilage increase linearly with age [459-461] and it is also present in the proteoglycans [462], thus measurement of the pentosidine level may indicate the degree of ageing of the cartilage matrix. The pentosidine level has been positively correlated to the stiffness of the collagen network [450] and therefore the level is thought to negatively influence the cartilage deformation characteristics under load, and thus may accelerate the development of OA [463-465]. Pentosidine levels have been used to indirectly measure the biological age of cartilage tissue in conjunction to the exercise regime applied to horse [466, 467]. However, some have pointed out that the pentosidine level alone is insufficient to measure the age of the cartilage as the number of pentosidine cross-links exist approximately 1 per 20 collagen molecules [460, 461], thus considered too few to provide an accurate assessment of cartilage characteristics.

3.5.5 CARTILAGE CROSS-LINKS AND EQUINE EXERCISE

Because of the difficulties in acquiring sufficient numbers of homogenous live animals subjected to a well-controlled training regime, there is a scarcity of data relating to the influence of exercise on the cartilage matrix [32]. The situation is further complicated by our incomplete understanding of the collagen cross-linking process and actual collagen architecture. However, some recent equine studies of the exercise effects have cautiously provided some important preliminary conclusions based on the measurement of cartilage material properties and levels of cross-link residues (HP, pentosidine), GAGs and the non-collagenous proteins like COMP (cartilage oligomeric matrix protein). The consensus view arising these equine studies [32, 466, 468-471] is that significant collagen development in the articular cartilage can only occur during the early growth phase before the horse reaches their maturation [466, 471]. After the maturation, the cartilage collagen framework is likely to resist further significant improvements and degradation [472], probably due to the combined effects arising from the resilient nature of collagen as well as extensive cross-linking in the hetropolymeric assembly. These findings suggest that exercise induced cartilage conditioning can only be effective during the active development and growth phase before the maturation is reached. However, even after the maturation age, proteoglycans readily respond to changes in the intensity and duration of exercise, and are often restored to healthy levels following prolonged rest periods [473]. [78]

1823 1824 1825 1826 1827 1828 1829 1830 1831 1832 1833 1834 1835 1836 1837 1838 1839 1840 1841 1842 1843 1844 1845

Exercise can induce both positive and negative changes in equine cartilage thus resulting in sitespecific cartilage alterations from the neutral post-natal tissue state. Equine studies, in which the intensity of exercise is has been controlled by speed of walking and running, have shown that there is a minimal level of exercise intensity at which a positive adaptive response is triggered in the cartilage [474-477]. However, if the intensity of exercise is too high, it can also cause negative changes in the cartilage [478-480]. Equine and canine studies have shown that moderate levels of sustained exercise had little influence on neither the level of HP cross-links nor the overall collagen concentration in the cartilage [473, 481]. Conversely high intensity exercise can increase the frequency of cartilage lesions and decrease the level of HP cross-linking, leading to with increased water content and a loose collagen network [482]. Another comparative study between untrained and trained horses [483] employing high intensity exercise showed that exercise can also result in thinner but more densely packed collagen fibrils while a lowering of the COMP level. However, the significance of the reported in changes in COMP level was not clear. The exact threshold level of exercise required to induce a beneficial effect is still uncertain but overall, it is well established that exercise plays a significant role in the development and adaptive response of cartilage. However, it needs to be emphasized that a given type and level of exercise intensity will induce different levels of changes in different joints or different sites in the same joint. The detection of these changes is further complicated by the limitations in accuracy of the measurement methods and the simplifying assumptions used.

[79]

1846 1847 1848 1849 1850 1851 1852 1853 1854 1855 1856 1857 1858 1859 1860 1861 1862 1863 1864 1865 1866 1867 1868 1869 1870 1871 1872 1873 1874

3.6 BIOMARKERS OF CARTILAGE DEGRADATION

Biomarkers are naturally occurring biochemical molecules from various stages of both normal and pathological processes. Because osteoarthritis is a disease which alters normal metabolic processes in the joint tissues (i.e. bone, cartilage, and synovial membrane), the disease can influence the levels of the joint-related biochemical molecules present in the body. By monitoring these levels, the information can be used to judge the state and the progression of the disease and can also assess the effectiveness of the drugs prescribed. Furthermore, the biomarkers can provide continuous supply of quantifiable data relatively easily and therefore are more advantageous than traditional joint disease assessment methods such as scoring and radiographic examination. The current and most widely used scoring system relies on the pain and mobility indices but it remains difficult to quantify them since it is very subjective to the person. The plain radiography, such as x-ray and MRI can provide highly visual information but lack sensitivity and a series of examinations over a period must be taken for the purpose of monitoring OA progression. The potential usefulness of biomarkers has gained much attention in the research community and the U.S. National Institute of Health has introduced biomarkers as one of its long term research initiatives [484]. However, biomarkers are not without shortcomings. Firstly, due to the relative infancy of biomarker research, there is as yet an insufficient amount of reference data from patients with OA [485]. This issue will no doubt be resolved as more data is gathered. Secondly, the biomarkers can be collected from blood, urine and synovial fluid; among these, the synovial fluid represents most accurate state of the joint. However, the collection of synovial fluid is difficult due to many joints are either too small (e.g. fingers) or there are ease of access issues (e.g. hip and vertebrae), the one exception being the knee joint. Lastly, the biomarkers are affected by a wide range of genetic and metabolic factors such as gender, age, body mass index, and hormone replacement therapy as well as even circadian variations [485]. Despite these difficulties, the OA-joint specific biomarkers such as serum COMP [486], urinary CTX-II [487], and chondroitin sulphates [488] have been successfully correlated with OA.

[80]

1875 1876 1877 1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901

Although there are numerous biomarkers for other joint tissues, such as bone and synovium, in this section we will discuss cartilage-related biomarkers only. There are two types of biomarker candidates which can be used to detect the state of the cartilage collagen; one is the natural by-products created during collagen synthesis and the other is the collagen fragments arising from the collagen degradation processes. The level of cartilage synthesis can be evaluated by assaying epitopes located on the chondroitin sulphate chains of the aggrecan (846 epitope) propeptides of type II collagen (C- propeptide of type II procollagen [PIICP] and N-propeptide of type II procollagen [PIINP]), or other proteins such as YKL40 and cartilage-derived retinoic-acid sensitive protein. Conversely, breakdown processes in cartilage can be monitored by measuring the fragments of the aggrecan protein moiety, keratan sulphate epitopes (e.g., epitope 5D4), type II collagen fragments, or noncollagenous proteins such as COMP. Hydroxyproline residues (HP) have, for some time, been used as the standard marker for collagen degradation. However, it has been pointed out by Uebelhart et al. that using HP level alone to assess the extent cartilage degradation can be misleading since the HP can be produced by the liver as well as the bone, tissues in which their levels are easily influenced by diet [489]. COMP is the most extensively studied cartilage marker to date and many studies [490-498] have been published regarding its role in joint inflammation, and rheumatoid and osteoarthritic disease. Investigators have reported that COMP may have a role in matrix assembly [490-494] and fibrillogenesis [491, 498]. Also, COMP levels might be positively correlated to the progression in OA [496] and the severity of cartilage lesions in horses [497]. However, the biologic significance of COMP levels in cartilage remains unclear because COMP is produced not only by the chondrocytes, but also by the synovial cells, tendon fibroblasts, and osteoblasts [499] where the high-circulation COMP levels may indicate an increased level of cartilage breakdown as well as inflammation in the synovial membrane.

[81]

1902 1903 1904 1905 1906 1907 1908 1909 1910 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920 1921 1922 1923 1924 1925 1926 1927 1928 1929 1930

3.7 SUMMARY
Collagen is a structural propeptide protein and a key component of the extracellular matrix found in all major types of connective tissues and organs. The extracellular matrix provides an appropriate environment for the resident cells and a robust medium through which mechanical forces can be transmitted. The matrix also has the capacity to undergo continuous remodelling and development to suit the bio-mechanical criteria of the loading environment. There are about 30 different varieties of collagen that have been identified to date and these are grouped as either fibrillar or non-fibrillar collagens. These are then further classified into (1) fibrilforming, (2) fibril-associated, (3) network forming, (4) anchoring fibrils, (5) transmembrane and (6) basement membrane collagens. In articular cartilage, the majority of its collagen contents are from the fibrillar collagen types II, XI and IX with a much smaller amount of the non-fibrillar network collagens type VI and sometimes type X. These collagens are intricately interwoven with the hydrophilic proteoglycans, where the arrangement creates internal pressure within the matrix allowing the cartilage to viscoelastically deform in response to applied loads. The mechanical strength of the cartilage matrix results from the multiple levels of cross-linking between the collagen molecules. However the glycation type cross-links, a naturally occurring aging process, is a degenerative process where it is thought to increase the susceptibility to OA by reducing the inherent flexibility in the cartilage. Osteoarthritis, a metabolic disease of the joint, can also diminish the integrity of the cartilage matrix by increasing the degradation collagen and its cross-links. The degradation residues from the destruction of the cross-linking system can be detected from the urine, blood and synovial fluid, and these chemical signatures can be utilised as biomarkers. By monitoring the levels of these chemicals, the information can be used to judge the state and the progression of the disease and can also be used to assess the effectiveness of the drugs prescribed. Thus biomarkers are promising over other traditional forms of disease assessment techniques such as scoring and radiographic methods because they can produce the quantifiable data less invasively, more frequently and economically.

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1932

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1933 1934 1935 1936 1937 1938 1939 1940 1941 1942 1943 1944

4.

ANALYSES

The analysis which forms the basis of this thesis consisted of three parts designed to identify whether there was an effect of early exercise on the 18-months old Thoroughbreds. These were 1) mapping the lesion pattern and frequency on the cartilage surface with microscopy, 2) measuring the cartilage matrix swelling involving dorsal and palmar regions of C3 and Cr facets where high incidence of damages are known to occur, and 3) conducting histomorphometric analysis of the osteochondral tissues (Figure 27).

Exercised (CONDEX)
#1 Cartilage lesion mapping
Macro Level analysis

Control (PASTEX)

#2 ECM Matrix
swelling Micro level analysis

#3 Histomorphometric
Micro level analysis

1945 1946 1947 1948 1949 1950 1951

Figure 27 - The overall analysis consists of three parts; 1) gross cartilage lesion mapping, 2) matrix swelling behaviour and 3) histomorphometric analysis of various cartilage and subchondral bone features. The main aim of the three-part analysis was to determine if there was any effect of early exercise in the exercised horses (CONDEX) compared to the control horses (PASTEX).

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1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963

TEST SUBJECTS, EXERCISE PROGRAM AND EUTHANASIA

33 Thoroughbred foals were raised as part of the GEXA project, an international collaboration study led by GERA1. For the studies reported in this thesis, tissue samples from 12 of the 33 horses euthanized at 18-months were used for the analysis (see page 232 for the complete list of the horses). The twelve Thoroughbred foals were blocked on the basis of sex and sire and separated into 2 groups; a control group PASTEX and an early exercised group CONDEX (Table 6). The horses were housed at a custom built facility at Massey University, Palmerston North, New Zealand and the study and its procedures were approved by the Massey University Animal Ethics Committee.

Table 6 - A list of 18-month Thoroughbreds participated in the GEXA project. The horses were classified as either CONDEX or PASTEX depending on whether they had received conditioning exercise or not. A colt is a young male whereas a filly means a young female. The complete list can be found on page 232.

Horse ID G1 G3 G13 G16 G17 G19 G20 G24 G29 G30 G31 G33

Euthanized @ 18-months

Exercised

Group CONDEX PASTEX PASTEX

Sex (C=Colt, F=Filly) C C F F C C F C C F F F

Birth (Y/M/D) 2000/9/16 2000/9/28 2000/10/15 2000/10/24 2000/10/27 2000/11/7 2000/11/9 2000/11/21 2000/11/29 2000/12/14 2000/12/28 2000/11/2

CONDEX PASTEX CONDEX CONDEX PASTEX CONDEX PASTEX CONDEX PASTEX

1964 1965 1966 1967 1968 1969 1970 1971 From birth to approximately 18 months of age (the typical age at which race training is initiated), both groups were housed in pastures with full freedom of movement. All horses were observed daily for clinical abnormalities, and they were weighed and condition scored every 2 weeks, and underwent clinical examination by a veterinarian every month. On top of the autonomous pasture exercise, the CONDEX group received additional exercise on a turf-based oval track five days a week with the running direction alternated between clockwise and counter-clockwise.

Global Equine Research Alliance

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1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993

The exercise program for the CONDEX horses commenced at the age of 21 19 days (mean SD) and ended when the horses were approximately 18 months old. The CONDEX horses were exercised such that mean target speeds were regulated by two all-terrain vehicles (ATV) in unison, and they were constrained by an 8-m horizontal pole attached to the rear of each vehicle, which formed a barrier across the track. There were 3 phases to the exercise program. The first phase of the exercise program, from birth to weaning, lasted approximately 120 days at a mean target speed of 5.4 m/s. During the second phase, from weaning to the first sprint, the duration was approximately 100 days and the mean target speed was 7.5 m/s. The final phase lasted approximately 300 days to the end of the 18-month exercise program, and the mean target speed was 9.6 m/s with a brief sprint at a rate of 12.5 m/s for the remaining distance of 129 m. During the 18-month exercise period, none of the horses had any obvious lameness although several in both groups did show brief periods of mild effusion [500]. The intensity of the exercise regime was judged to be well within the safe margins for non-injurious loading in the young animals both in an immediate sense and in the longer term. At approximately 18 months (535 30 days, mean SD), the 12 CONDEX and PASETX horses were euthanized by free bullet at a commercial slaughterhouse approved under New Zealand slaughter regulations and the middle carpal joints were removed from both fore limbs. The joints were then plastic wrapped and frozen then transported to the University of Auckland and kept at -20 C condition prior to thawing for subsequent analysis.

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4.1 ANALYSIS 1 - CARTILAGE LESION MAPPING ON THE MIDCARPAL JOINT

4.1.1 METHODS
The mid-carpal joints were dissected and examined for evidence of general and focal lesions on the cartilage surface and then classified as normal (smooth surface without any abnormal undulation or superficial disruption), mild (some abnormal undulation but no disruption) or severe (disruption and/or cartilage lose). These sites were photographed, with special attention given to the dorsal surfaces of the third and radial carpal bones where carpal fractures are common (see section Intraarticular fractures of carpus on page 25). The gross appearances of the lesions were recorded, such as surface smoothness, disruption, lesion depth, fibrillation, markings, undulation, fissure, soft/firmness and overall dimensions to make comparative assessments over the entire cohort of samples at a later stage. The lesions sites were then cut into smaller osteochondral blocks, fixed using 10% CPC formaldehyde for approximately 12 hours, then gently decalcified using 10% formic acid buffered with sodium formate for three to five days [501, 502]. The samples were then rinsed in cold running water for 1 hour to remove residual chemicals and then using a sledging microtome2 and maintaining the tissue in a semi-frozen state, 30m thick cross-sectional slices were obtained. These slices were washed gently to remove any extraneous debris and excess fat, wet-mounted on a glass slide under a cover slip and digitally photographed using bright field optical microscopy3 (x2 x2 setting with 0.5-1x mode with an wide aperture setting) for in-depth analysis of the lesion previously identified.

2 3

Leica Instruments GmbH, Nussloch, Germany, Model SM 2000R Nikon Instruments Inc, Japan, Model AZ100

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4.1.2 RESULTS
Both the exercised (CONDEX) and control (PASTEX) groups had a wide range of gross lesions in the articular cartilage of the bones of the proximal and distal rows of the midcarpal joint, except for 2 CONDEX horses which were entirely free of any surface lesions (see Figure 63 for individual lesion mapping of each horse). The lesions ranged from slight discoloration of the cartilage to full-thickness cartilage loss, but without any evidence of severe fracture or osteoarthritis. The lesions were distributed across the joint surfaces at specific sites toward the dorsal or palmar aspects of the second (C2), third (C3), and radial (Cr) carpal bones. By carefully observing all twelve subjects the recurring lesion sites were identified, colour coded and labelled as five different lesion classes as shown in Figure 63 on page 119 .

2031

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G1 CONDEX

2033

Cr

Ci Cu Cu

Ci

Cr

G1
C4 C3 C2 C2 C3 C4

2034 2035 2036 2037 2038

Figure 28 No visible cartilage surface damage was detected in G1. Unfortunately no photographic records exist for this particular joint.

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2039

G3 CONDEX

2040

Cr

Ci Cu Cu

Ci

Cr

G3
C4 C3 C2 C2 C3 C4

2041

2042 2043 2044 2045 2046 2047 2048

Figure 29 G3: The joints were overall very clean, pinkish in colour and had healthy appearances with an exception of a small blister (GREEN) which is magnified in Figure 30 (arrow). Two minor rubbing type lesions were presented on both ridges of the intermediate facet of the palmar aspect in C3 in left and right limb (YELLOW). Y

[90]

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Cr

Ci Cu Cu

Ci

Cr

G3
C4 C3 C2 C2 C3 C4

2053

2054 2055 2056 2057 2058 2059

Figure 30 - (A) Two minor rubbing type lesions were presented on both ridges of the intermediate facet of the palmar aspect in C3 in left and right limb (YELLOW). They were glossy with mild disruptions on the cartilage surface. (B) A minor Y blister type feature was found (GREEN) which was less than 2mm in diameter. A similar blister feature was also presented in G19 (see Figure 52).

2060 [91]

2061

G13 PASTEX

2062

Cr

Ci Cu Cu

Ci

Cr

G13
C4 C3 C2 C2 C3 C4

2063

2064 2065 2066 2067

Figure 31 - G13 was present with various types of lesions from superficial (CYAN, YELLOW) to severe cartilage-loss (BLUE). The most severe type of lesions was found at the dorsal aspect of C3 and Cr in both left and right legs (RED). This type of lesion was later identified as osteochondrosis.

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C
B

2069 2070 2071 2072 2073 2074 2075 2076 2077

Figure 32 - (A) The RED regions in the previous figure do not show any obvious signs of cartilage damages. (B, C) However, when the RED regions were examined under the microscope, it revealed the calcified region was enlarged at the expense of the subchondral bone below (blue arrow in C). The cartilage surface has no superficial disruption (i.e. no fibrillation) or mass necrotic activities but present with multiple cracks in the calcified cartilage layer (red arrow in B). Whether these are cutting artefacts or genuine cracks resulted from impact are uncertain. However, since the cracks are shown to be consistent in terms of their morphology and orientation to the collagen matrix in the calcified cartilage layer, the cracks are likely to be microcracks created in vivo.

2078 2079 2080 2081 2082

Figure 33 - The lesion shown in (A) occurred in both left and right limb of C3 (BLUE). There were localised softening and cartilage losses at these sites. The calcified cartilage and (B) the subchondral fronts are severely disrupted (red arrow in B) and mass necrosis is evident together with (C) the prominent chondrocyte clone formation in the middle, deep zone and in the calcified cartilage layer (red arrow in C). Due to possible necrosis, overall cellular density is low in the region.

[93]

2083

2084 2085 2086 2087 2088 2089 2090

Figure 34 - (A) The ridge located in the palmar aspect of C3 (YELLOW) was one of the most frequent sites of rubbing type Y lesions. Here is one of the most severe cases where there was actually partial cartilage loss (black arrow). (B, C) The lesion site is filled with multicellular hypertrophic chondrons (blue arrow in B and C) unevenly distributed. The site contains a large crevice in the cartilage layer continuing beyond the calcified cartilage layer, and the disruption continues to the subchondral front (red arrow in B and D).

2091

D
B

2092 2093 2094 2095 2096 2097

Figure 35 - (A) The edges on C2 (CYAN) showed frequent rubbing lesions in nearly every horse, (B, C) including loss of cartilage surface (blue arrow in B) and numerous fissures (red arrow in B and C). The cartilage under the microscope shows no articular surface, while there are several hypertrophic chondrons and no normal chondrocytes. The calcified cartilage layer is disrupted compared to the adjacent unaffected regions. (D) Also, there is a cyst in the subchondral bone (blue arrow in D).

[94]

G13 Left

Cr

Palmar Dorsal

2098 2099 2100 2101

C3
Figure 36 - G13 left limb showing osteochondrosis in both C3 and Cr in the dorsal region of radial facet (blue arrows). The affected regions showed the enlarged calcified cartilage, disrupted cement line interface (blue arrows) and collapsed subchondral bone possibly due to the high porosity observed in the trabecular bone architecture (red arrows). These weakening features were consistently seen in all osteochondrosis affected sites.

G13 Right

Cr

Dorsal

Palmar

2102 2103 2104 2105

C3
Figure 37 - G13 right limb showing osteochondrosis in both C3 and Cr in the dorsal region of radial facet (blue arrows). The affected regions showed enlarged calcified cartilage, disrupted cement line interface (blue arrows) and collapsed subchondral bone possibly due to the high porosity observed in the trabecular bone (red arrows). These high porosities in the subchondral bone were consistently seen in all osteochondrosis affected sites.

2106

G16 CONDEX

2107

Cr

Ci Cu Cu

Ci

Cr

G16
C4 C3
2108

C2

C2 C3

C4

2109 2110 2111 2112 2113 2114

Figure 38 - The G16 exhibited a set of very healthy joints with only minor rubbing type lesions in the ridge of intermediate facet at the palmar aspect (YELLOW) of C3. Y

[97]

2115

Cr

Ci Cu Cu

Ci

Cr

G16
C4 C3
2116
A
B

C2

C2 C3

C4

2117

2118 2119 2120 2121 2122

Figure 39 - (A) The ridges on the intermediate facets of C3 (YELLOW) showed (B) minor local necrosis (red arrow in B) and Y (C) a fissure (red arrow in C) at the site of the lesion. (D) At the lesion site, there is a clear disruption in the subchondral plate contour (red arrow in D) possibly due to the same load that was responsible for inducing the lesion.

[98]

2123

G17 PASTEX

2124

Cr

Ci Cu Cu

Ci

Cr

G17
C4 C3 C2 C2 C3 C4

2125

2126 2127 2128 2129 2130 2131 2132 [99]

Figure 40 - G17 was healthy-pink in appearance without any disruptions on the cartilage surface. However the left dorsal aspect, near the edge, showed a small white region (RED) which was later found to be an osteochondrosis (see Figure 41). The joint surfaces were presented with a minor amount of rubbing lesions in the C2 ridges, a common type of lesion often found in the other horses (CYAN).

2133

Damaged

2134

Undamaged

2135

D
Dam

B
aged

C
Unda

ma g

ed

2136 2137 2138 2139 2140 2141 2142 2143 2144 2145 2146
Figure 41 - (A) The white region on the surface of C3 (RED region in the previous figure) was an enlarged calcified cartilage (i.e. osteochondrosis) hidden underneath the undisrupted cartilage surface. The two microphotographs on the top row (B, C) show a direct comparison between the injured and the uninjured region in the same osteochondral slice in (D). The thickness of calcified cartilage and interface to the subchondral bone (red arrow) are very different between these two sites, although the overall thickness of hyaline cartilage is similar. The (damaged) site has a very fragmented interface, whereas the (undamaged) site has an intact division between the calcified cartilage layer to the subchondral bone front with morphologically normal vascular channels. There are multiple cracks in the calcified cartilage layer in the (damaged) site (blue arrow).

[100]

2147 2148 2149 2150

A

2151

Cr Dorsal C3
C

Palmar

2152 2153 2154 2155 2156 2157 2158 2159 2160 2161 2162
Figure 42 - (A) Superficially, the dorsal region of the Cr showed no damages on the cartilage with only a faint whitening feature oppositional to the site of osteochondrosis in C3 as shown in the previous figure. (B) Both C3 and Cr cross sectional view showed nearly identical damages in the same contacting region (red and blue arrows in B). (C) Histologically, the Cr showed a crushed subchondral interface with an enlarged calcified cartilage layer (red arrow). (D) The two histological images from C3 and Cr were combined to provide the overall C3-Cr lesion morphology (red and blue arrows). The size of the subchondral lesion in C3 was about 6mm long, 2mm deep, and it was slightly smaller in Cr. Both calcified cartilage layers were seen with multiple parallel cracks. Whether these features are cutting artefacts or genuine microcracks from the subsequent traumatic impacts is uncertain. Additional staining methods developed for detecting microcracks in the subchondral bone [125] may have to be used to identify the origin of these cracks.

[101]

G17 Left

Cr

Dorsal

Palmar

2163 2164 2165

C3
Figure 43 - G17 left limb showing osteochondrosis in both C3 and Cr in the dorsal region of radial facet (blue arrows). The affected regions showed the enlarged calcified cartilage, disrupted cement line interface (blue arrows) and collapsed subchondral bone possibly due to the high porosity observed in the trabecular bone architecture (red arrows).

G17 Right

Cr

Dorsal

Palmar

C3
2166 2167 2168
Figure 44 - C3 and Cr of G17 right limb showed no signs of damage in the dorsal region of radial facet unlike the left limb. The calcified cartilage thickness is even throughout the dorsal to the palmar aspects. The trabecular bones were appeared to be much more densely packed with little porosities, strikingly different from the C3 and Cr of left limb in the previous figure.

2169

G19 CONDEX

2170

Cr

Ci Cu Cu

Ci

Cr

G19
C4 C3 C2 C2 C3 C4

2171

2172 2173 2174 2175 2176 2177 2178

Figure 45 - Superficially, G19 appeared to be normal but the dorsal aspects of radial facets in the left C3 and Cr (RED) were affected with osteochondrosis. At the same time, both the left and right C3s suffered multiple cartilage cracks at the ridge between the radial and intermediate facets in C3 (MAGENTA). There was a circular mark (GREEN) which was probably not a lesion, but it was likely to be a synovial fossa [503]. Other abrasive lesions (YELLOW and CYAN) were also Y present on both joints, however they were very minor.

[104]

2179 2180

2181

2182 2183 2184 2185 2186

Figure 46 - (A, B) Similar to G17, osteochondrosis was present underneath the intact cartilage. However, the osteochondrosis was not as severe as G17s. (C) The slice shown above has a crevice in the centre (blue arrow) however it is thought to be a cutting artefact only.

2187 2188 2189 2190 2191

Figure 47 - (A) Osteochondrosis in Cr was presented with distinct multi-cellular hypertrophic chondrons in the zone of calcified cartilage (red arrow in B, C). The degree of lesion severity in Cr was more prominent than the C3s.

[105]

2192 2193
G19 Left Cr

2194 2195
G19 Right Cr

2196 2197 2198 2199 2200 2201

Figure 48 - The entire dorsal sections highlighting osteochondrosis from left (TOP) and right (BOTTOM) Cr bones. The approximate dimensions of the osteochondrosis lesions were about 3mm long, 0.6mm deep each. The affected sites also showed abnormally large porosities (red circles) in the trabecular bone underneath the osteochondrosis.

[106]

Dorsal

Palmar

2202 2203 2204 2205 2206 2207

Figure 49 - The same osteochondral slice of Cr from the G19 right limb demonstrates how the trabecular bone architecture is significantly different from the dorsal region to the palmar region. The photograph also shows the subchondral collapse (red arrow) might be due to the large porosity (red circle) in the subchondral bone architecture in the dorsal region.

Hyaline cartilage

Normal CC

Damaged CC

Subchondral bone
2208 2209 2210 2211 2212 2213 2214 2215 2216
Figure 50 - A close-up view (G19 Right Cr) of the region marked by a red arrow in the previous figure showing two different of calcified cartilage (CC) matrices; damaged calcified cartilage in the left and normal calcified cartilage in the right divided by a red line. The difference between the damaged and normal calcified cartilage matrices is substantial. The normal matrix is homogeneous and has an amorphous appearance, whereas the damaged matrix is very inhomogeneous as if two different types of matrices are fused together. The green line indicates the cement line. There is also a substantial difference in terms of the morphology in the normal cement line compared to the damaged cement line as already demonstrated in Figure 41.

[107]

2217 2218

2219 2220 2221 2222 2223 2224 2225

A
B
Figure 51 - (A) Abrasive lesion on the intermediate facet ridges (YELLOW) on both right and left C3s are shown. (B) Y Histologically, underneath the lesions (red arrow) show deformed subchondral plates (red line). There was a minor loss of cartilage surface at the site but overall, the cartilage underneath was not degenerative. The cause of subchondral plate deformation is likely to be the load through the oppositional joint surface.

2226 2227 2228 2229 2230 2231 2232 2233 2234

Figure 52 - (A) A small blister type lesion (MAGENTA, black arrow in A, red arrow in B and C) at the ridge between the intermediate and radial facets only occurred in the right. Also near this site in both left and right C3 bones, there were multiple parallel cracks on the ridges that were about 3 to 5mm long, 2 to 3 cracks per site. (B) The blister lesion had an intact articular cartilage surface and (C) its core matrix was made up of denatured collagen fibrils with high amount of visible aggregations in the matrix (red arrow in C). In this zone, the matrix was completely cell-less, neither normal chondrocytes nor hypertrophic chondrons. The calcified cartilage layer in this region was also observed with a much aggregated collagen fibrillar formation in the calcified matrix, distinctly different from the normal tissue, suggesting the formation of the denatured collagen was not recent.

[108]

G19 Left

Cr

Dorsal Palmar

2235 2236 2237

C3
Figure 53 - G19 left limb showing osteochondrosis in both C3 and Cr in the dorsal region of radial facet (blue arrows). The affected regions showed the enlarged calcified cartilage, disrupted cement line interface (blue arrows) and collapsed subchondral bone possibly due to the high porosity observed in the trabecular bone architecture (red arrows).

G19 Right Cr

Dorsal

Palmar

C3
2238 2239 2240
Figure 54 - G19 right limb showing osteochondrosis in Cr in the dorsal region of radial facet (blue arrow). The affected region showed the enlarged calcified cartilage, disrupted cement line interface (blue arrow) and collapsed subchondral bone possibly due to the high porosity observed in the trabecular bone architecture (red arrows).

2241

G20 CONDEX

2242

Cr

Ci Cu Cu

Ci

Cr

G20
C4 C3 C2 C2 C3 C4

2243

2244 2245 2246 2247

Figure 55 - The cartilage surfaces of G20 was excellent with no visible lesions except a minor abrasive type lesion at the CYAN region indicated above on the C2 ridge.

[111]

2248

G24 PASTEX

2249

Cr

Ci Cu Cu

Ci

Cr

G24
C4 C3 C2 C2 C3 C4

2250

2251 2252 2253 2254 2255 2256

Figure 56 - The large reddish marks (GREEN) shown on the surfaces of the left and right C3s are freezing artefacts from the uncompleted thawing process. The joints were relatively healthy without any severe form of lesions. Minor abrasive type lesions were visible on the ridges of the intermediate facets (YELLOW). Also there was a minor cartilage disruption Y at radial facet ridge in the right Cr (ORANGE).

[112]

2257 2258 2259 2260 2261

A

2262

D
C B

2263 2264 2265 2266 2267 2268 2269

Figure 57 - (A) The ORANGE region in Cr ridge was slightly disrupted with minor surface damage and unevenness. (B, D) Under the microscope, the region was affected with necrosis and multicellular chondron formation (red arrows) (C, D) as well as disrupted calcified cartilage layer and the subchondral bone (blue arrows). It is interesting to note that the sharp crest between two facets of Cr shown in (C) and (D), underneath this crest, there is a collapse in the calcified cartilage front with a rip (blue arrows).

[113]

2270

G29 CONDEX

2271

Cr

Ci Cu Cu

Ci

Cr

G29
C4 C3 C2 C2 C3 C4

2272

2273 2274 2275 2276 2277

Figure 58 - G29 appeared to in an excellent condition with only minor abrasive type lesions on the ridges of the intermediate facets in C3 (YELLOW) and C2 (CYAN). A dark focal region on the edge of the radial facet (red arrow, Y GREEN) in the left C3 was found to be a non-lesion feature.

2278 [114]

2279

G30 PASTEX

2280

Cr

Ci Cu Cu

Ci

Cr

G30
C4 C3 C2 C2 C3 C4

2281

2282 2283 2284

Figure 59 - G30 was completely free from any damages in the cartilage and underlying bone.

2285

[115]

2286

G31 CONDEX

2287

Cr

Ci Cu Cu

Ci

Cr

G31
C4 C3 C2 C2 C3 C4

2288

2289 2290 2291 2292

Figure 60 - G31 was free from any form of cartilage damage. The reddish marks on the radial facet of the left C3 (red arrow, GREEN) are thought to be non-pathological cartilage thinning from under-development process in the joint.

[116]

2293

G33 PASTEX

2294

Cr

Ci Cu Cu

Ci

Cr

G33
C4 C3 C2 C2 C3 C4

2295

2296 2297 2298 2299 2300 2301 [117]

Figure 61 - G33 was in an excellent condition with a very mild form of an abrasive type lesion on the ridges of the C3 intermediate facets (YELLOW). Y

2302

S UMMARY OF THE LESION FOUND

Trauma

Ci

Cu

1 2 3 4

a
10mm

b
10mm

c
10mm

Cr

C2 C3
Unique
4 3 2 1

C4

Ci

Cu

1 2 3 4

10mm

10mm

10mm

Cr

C2 C3
C3 ridge
4 3 2 1

C4

Ci

Cu

1 2 3 4

10mm

10mm

10mm

Cr

C2 C3
Cr ridge
4 3 2 1

C4

Mild a b

Moderate c

Severe

Ci

Cu

1 2 3 4

10mm

10mm

10mm

Cr

C2 C3
C2 ridge
4 3 2 1

C4

Ci

Cu

1 2 3 4

5mm

5mm

5mm

Cr

C2 C3
4 3 2 1

C4

Mild

Severe

Severe

2303 2304 2305

Figure 62 - A summary of each lesion type found on the surfaces of the midcarpal joint.

[118]

CONDEX/Exercised
Left
Cr Ci Cu Cu

PASTEX/Control
Left
Cr Cr Ci Cu Cu

Right
Ci

Right
Ci Cr

G1
C4 C3 C2 C2 C3 C4 C4 C3 C2

G3
C2 C3 C4

Cr

Ci Cu Cu

Ci

Cr

Cr

Ci Cu Cu

Ci

Cr

G16
C4 C3 C2 C2 C3 C4 C4 C3 C2

G13
C2 C3 C4

Cr

Ci Cu Cu

Ci

Cr

Cr

Ci Cu Cu

Ci

Cr

G19
C4 C3 C2 C2 C3 C4 C4 C3 C2

G17
C2 C3 C4

Cr

Ci Cu Cu

Ci

Cr

Cr

Ci Cu Cu

Ci

Cr

G20
C4 C3 C2 C2 C3 C4 C4 C3 C2

G24
C2 C3 C4

Cr

Ci Cu Cu

Ci

Cr

Cr

Ci Cu Cu

Ci

Cr

G29
C4 C3 C2 C2 C3 C4 C4 C3 C2

G30
C2 C3 C4

Cr

Ci Cu Cu

Ci

Cr

Cr

Ci Cu Cu

Ci

Cr

G31
C4 C3 C2 C2 C3 C4 C4 C3 C2

G33
C2 C3 C4

2306 2307 2308

Traumatic

Unique

C3 ridge

Cr ridge

C2 ridge

Figure 63 - A summary of lesions (colour coded as shown above) found in each horse in the midcarpal joint surfaces.

[119]

2309

4.1.3 DISCUSSION

2310 2311 2312 2313 2314 2315 2316 2317 2318 2319 2320 2321 2322 2323 2324 2325 2326 2327 2328

T YPES OF LESIONS
The lesions observed in the midcarpal joints of the CONDEX and PASTEX horses are summarised in a graphical form in Figure 62. The lesions were manifested either as subtle focal blisters (Figure 30B, Figure 52), undulations (Figure 31), fissures (Figure 39), mild fibrillation (Figure 35), superficial to partial cartilage loss (Figure 34) or osteochondrosis (Figure 41, Figure 42 etc) together with several sightings of cellular necrosis and hypertrophic multicellular chondron formation (Figure 34, Figure 35, Figure 57). At the dorsal edges in the radial facets of the C3 and Cr from G13, G17 and G19 (Figure 64, Figure 65 and Figure 66), the calcified cartilage and subchondral bone were affected with localised damages without any obvious degenerations in the cartilage. This type of lesion is thought to be a mild form of traumatic osteochondrosis [504, 505], a common joint lesion in the Thoroughbreds [506]. Since there was no sustained inflammatory response or lameness in the horses during the entire 18months [500], it is speculated that these subchondral defects may have not yet transformed into full-blown clinical lesions [337, 507-510]. Repeated cyclic fatigues or acute traumatic loading is known to induce osteoarthritic degeneration in cartilage [511] and the lesions identified in this study could well be precursors to a secondary osteoarthrosis in the joint [512, 513]. If it is the case that such subchondral lesion are the result of trauma, it is of obvious concern that they would go undetected (Figure 67) when using common cartilage examination methods such as arthroscopy.
A
B

2329 2330 2331

Figure 64 - C3 (A) and Cr (B) microphotographs from G17 showing the concurrent calcified cartilage enlargements (C) and disrupted subchondral bone fronts from the osteochondrosis [504, 505].

[120]

G13 L

G13 R

G17 L

G17 R

G19 L

G19 R

2332 2333 2334 2335 2336 2337 [121]

Figure 65 - Most of the G13, G17 and G19 mid-carpal joints were found with traumatic osteochondrosis [514] (red arrows). The shape and size of lesions were nearly oppositionally symmetrical between C3 and Cr (G13, G17) suggesting the traumatic loads were the cause of these lesions. The trabecular bones beneath the lesion sites were shown to be more porous than the sites without the lesions (compare between G17L and G17R).

A - G13 L C3

B - G13 L Cr

C - G13 R C3

D - G13 R Cr

E - G17 L C3

F - G17 L Cr

G - G19 L C3

H - G19 L Cr

I - G19 R Cr

2338 2339 2340 2341 2342 2343 2344 2345 2346 2347 2348 2349 2350 2351
Figure 66 - Microphotographs of the selected regions in the calcified cartilage that were affected from the osteochondrosis in G13, G17 and G19. The calcified cartilage matrices were either containing hypertrophic chondrocytes (blue arrows) or normal chondrocyte with the columnar structure duplicating the deep zone of hyaline cartilage. Sometimes the calcified cartilage layer contained multiple microcracks (A, E, F; red arrows). (C) shows a distinctively large crack that completely splitting the calcified cartilage in the middle and extends to the hyaline cartilage. Most of the cracks were aligned with the orientation of the collagen fibrils. All of the observations have shown the crushed appearance in the subchondral fronts suggesting each and every osteochondrosis affected site was induced by traumatic loads.

The cartilage matrices at the osteochondrosis affected sites, although appearing normal superficially and histologically, produced a higher level of average swelling strain of +4.2% (P = 0.04) compared to the non-affected normal sites (see page 145) suggesting that subtle cartilage matrix changes had taken place.

[122]

2352 2353 2354

The cause of the osteochondrosis is likely to be traumatic forces acting on the dorsal regions in the C3 and Cr as the locations, sizes and shapes of the lesions detected were similar, oppositionally matching as shown in Figure 65 and Figure 67.

Cr Dorsal C3 Palmar

2355 2356 2357 2358 2359 2360 2361 2362 2363 2364 2365 2366 2367 2368 2369 2370 2371 2372 2373 2374
Figure 67 - The two histological images from C3 and Cr were combined to provide the overall C3-Cr lesion morphology. C3 and Cr of G17 showed no damages on the cartilage, however under this undamaged cartilage layer, there were severe disruptions in the calcified cartilage as well as subchondral bone. The size of the subchondral lesion in C3 was about 6mm long, 2mm deep while it was slightly smaller in Cr. Both calcified cartilage layers were seen with multiple parallel cracks. Whether these features are cutting artefacts or genuine microcracks from the subsequent traumatic impacts is uncertain. Additional staining methods developed for detecting microcracks in the subchondral bone [125] may have to be used to identify the origin of these cracks.

N EO - CALCIFIED CARTILAGE IN OSTEOCHONDROSIS

Microscopically, the osteochondrosis affected C3 and Cr of G13, G17 and G19 exhibited crushed subchondral bone fronts with the enlarged calcified cartilage occupying the region of original subchondral bone space (Figure 64, to Figure 68). It is likely that under the action of the traumatic force(s) the subchondral bone is pressed down creating a void space between the hyaline cartilage and the new subchondral bone front resulting in the crushed appearance. The traumatic damage would have triggered a repair response from the nearby chondrocytes and stem cells from the vascular haemorrhage, initiating the synthesis of new matrix which fills the region. This newly synthesized matrix may have converted into calcified cartilage. This neo-calcified region contained to varying degrees both hypertrophic chondrons as well as normal looking columnar chondrons (Figure 66). Whether this enlarged calcified cartilage region would be remodelled into replacement subchondral bone is uncertain, but it does seem probable as

[123]

2375 2376 2377 2378 2379 2380 2381 2382 2383 2384

there is evidence that some of the vascular channels had begun to resorb the calcified cartilage and lay down new layers of lamellar bone. However, this was not always the case, as some of the vascular channels were still missing their lamellar caps (Figure 66 and Figure 70). The calcified cartilage matrix texture in this region also exhibited an unusual and uneven appearance compared to the adjacent normal calcified cartilage, which was smooth and amorphous in appearance (Figure 68, Figure 69). During traumatic loading, the original calcified cartilage is likely to have been fractured into smaller pieces. It is possible that these calcified cartilage fragments with their embedded chondrocytes were subsequently fused with the neo-calcified cartilage matrix as shown in Figure 69 and thus giving rise to its unusual appearance.

Hyaline cartilage

Normal CC

Damaged CC

Subchondral bone
2385 2386 2387 2388 2389 2390 2391 2392 2393 2394 [124]
Figure 68 - A close-up of the calcified cartilage region (G19 Right Cr) affected by osteochondrosis showing two different of calcified cartilage (CC) matrices; damaged calcified cartilage in the left and normal calcified cartilage in the right divided by a red line. The difference between the damaged and normal calcified cartilage matrices is substantial. The normal matrix is homogeneous and has an amorphous appearance, whereas the damaged matrix is very blotchy as if two different types of matrices fused together. The green line indicates the cement line. There is also a substantial difference in terms of the morphology in the normal cement line in comparison to the damaged cement line as already demonstrated in Figure 41.

2395

OLD Matrix NEW Matrix OLD Matrix

NEW Matrix OLD Matrix

Subchondral bone

2396 2397 2398 2399 [125]

Figure 69 - Magnified view of the region (from G19 Right Cr) indicated with a red arrow in Figure 68, showing an uneven mixture of old and new calcified matrices.

Damaged

2400

Undamaged

2401 2402 2403 2404 2405 2406 2407 2408 2409 2410 2411
Figure 70 - A comparison of a damaged and a normal calcified cartilage-subchondral bone interface (red arrows). The damaged calcified cartilage layer also shows a numerous oblique angled microcracks (blue arrows).

The calcified cartilage layers contained multiple parallel cracks arranged at an oblique angle approximately 45 to the articular surface (Figure 70) and it is possible that these were induced as a result of repeated concussive loading of the calcified cartilage repair tissue after the neo-matrix formation. These microcracks were observed at four sites in the calcified cartilage layer (Figure 66) and the consistency of their morphology suggests that they are a genuine trauma-induced feature rather than cutting artefacts arising from sectioning. It is recommended that further analysis be carried out using additional staining methods developed by Mori et al. [125] to verify their origin.

2412 2413 2414 2415 2416 2417 2418 2419

T HE WEAKENING OF TRABECULAR BONE

The osteochondrosis resulted from the collapse of the subchondral plate by traumatic force, as demonstrated in Figure 71, is, in part, a consequence of inadequate support from the trabecular bone architecture from the below. The dorsal regions of both C3 and Cr exhibited obvious sclerosis (green zone in Figure 71) with large hollow spaces consistently observed in all 9 traumatic osteochondrosis sites (see red arrows in Figure 71 and Figure 72). It would be interesting to explore whether if there is a significant correlation between the degree of porosity in the trabecular bone and the frequency of subchondral collapse (i.e. traumatic osteochondrosis).

[126]

G19 Cr Right

G19 Cr Right

G19 Cr Left

2420 2421 2422 2423 2424

Figure 71 - The entire dorsal sections of the osteochondral slices with calcified cartilage lesion from right and left G19 Cr bones. The microphotographs show that the bone underneath the lesion affected areas exhibit large porosities in both examples (red arrows). The approximate dimensions of the lesions are about 3mm long, 0.6mm deep.

[127]

G13 L

G13 R

G17 L

G17 R

G19 L

G19 R

2425 2426 2427 2428 2429

Figure 72 - G13, G17 and G19 were found with osteochondrosis [514]. The shape and size of lesions were nearly oppositionally symmetrical in G13 and G17 suggesting the traumatic load was the cause of these lesions. The trabecular bone regions beneath the lesions were shown to be more porous (red arrows) than sites without the lesions (i.e. G17R).

[128]

2430 2431 2432 2433 2434 2435 2436 2437 2438 2439 2440 2441 2442 2443 2444 2445 2446 2447 2448 2449 2450 2451 2452 2453 2454 2455 2456 2457 2458 2459 2460 2461

Regardless of the exercise program, osteochondrosis was present in both the CONDEX and PASTEX (G13, G17 and G19), and with each observation of osteochondrosis, the subchondral bone architecture beneath the lesion site was porous. If such weakening in the subchondral bone is a normal developmental response in the Thoroughbred then perhaps it should be investigated whether restricting the intensity of their autonomous activities can reduce the high level of porosity observed in the trabecular bone. It might be that the animals have a natural tendency to selfexercise with greater intensity than what is physiologically safe. Possibly restricting the level of overall autonomous exercise of the animals should be investigated in order to protect them from accidental self-harm and at the same time review how Thoroughbreds should be raised and trained. Whether the problem of subchondral bone porosity can be alleviated using dietary changes that encourage the development of less porous bone architecture, might well be a fruitful line of investigation. Further, the level and frequency of exercise could be re-assessed to determine whether it is possible to achieve skeletal remodelling that does not result in this weakening of the trabecular bone architecture in these important weight bearing regions. It should be noted that these weakened regions have a pore feature dimension of about 2~4mm so if a series of high resolution radiographic examinations could be performed at regular intervals over the developmental and career life of the horse, such developmental weakness might well able to be tracked and ultimately avoided. Considering all of the above it is hypothesized that the cartilage calcification layer functions as an attenuating stress barrier that provides the subchondral bone with a degree of protection from traumatic impact. The calcified cartilage is less brittle than subchondral bone [515], and it may act in part as a sacrificial layer under harsh loading conditions. This layer absorbs the initial high impact energy by shattering, and thus reducing the mechanical energy actually transmitted into the subchondral bone. It should also be noted that the subchondral bone contains many cells and is richly vascularised. It seems reasonable to conclude that the joint incorporates structural features that are able to protect this region from the potentially damaging consequences of repeated impact loading. Based on an analysis of nine osteochondrosis affected sites, it is proposed that a repair mechanism operates that involves the calcified cartilage functioning as a temporary scar tissue for the subchondral bone following traumatic impact. The damage triggers the resident chondrocytes and stem cells to initiate the repair response by synthesizing new matrix to effectively patch fill the damaged site which is then hardened via calcification. The crevices are filled quickly and then [129]

2462 2463 2464 2465 2466 2467 2468 2469 2470 2471 2472 2473 2474 2475 2476 2477 2478 2479 2480 2481 2482 2483 2484 2485 2486 2487 2488 2489 2490

changed into a hard substrate. In this way the joint is able to support the load without damaging the articular cartilage even if the sunken underlying subchondral bone is still recovering. Furthermore, the calcified cartilage is readily resorbed again by the vascular invasions and thus the subchondral bone front is able to recover eventually to the previous level. This is considered a plausible interpretation because of the microstructural evidence presented in Figure 64 to Figure 71. These evidences are summarised as follows; 1. The subchondral bone front of the osteochondrosis always appeared to be crushed downwards, thus indicating that mechanical forces were involved in the region (Figure 66). 2. The lesions were present in both matched opposing joint surfaces of C3 and Cr and they were nearly identical in terms of shape and size suggesting impact forces being transmitted through the midcarpal joint axis (Figure 64, Figure 65 and Figure 67). 3. The damaged region is always completely filled with the calcified cartilage without a deformed cartilage surface profile or superficial damage. This suggests that the hyaline cartilage was well supported by the calcified cartilage even after the trauma and the loss of subchondral front. However, in order to accomplish that the new calcified cartilage layer must have been quickly synthesized to compensate the loss of subchondral bone (Figure 66). 4. The new calcified cartilage had a different texture (and possibly composition) compared to the undamaged sites. The occasional presence of what was thought to be fragments of old calcified cartilage within the new matrix suggests that the new calcified cartilage is a mixture of old and new matrices, akin to scar tissue formation (Figure 68 and Figure 69). 5. The new matrix contained hypertrophic multi-cellular chondrons indicating new cartilage matrix synthesis and this adds supports to the scar calcified cartilage hypothesis (Figure 68 and Figure 69). 6. The tips of the vascular channels at the damaged subchondral front were also shown to be damaged from the traumatic impact as the surrounding lamellar bone continuity is disrupted along the vascular channels. This indicates that the trauma must have resulted in a localised haemorrhage from the vascular channels in the region, and this may have triggered the repair response and provided the stem cells, growth factors and nutritions for new cartilage matrix synthesis to take place (Figure 66).

[130]

2491 2492 2493 2494 2495

7. Some of the vascular channels at the damaged subchondral front had just begun to start the resorption process and lay down new lamellar bone around them. This suggests that the damaged subchondral bone has the capacity to recover from the osteochondrosis (Figure 66).

2496 2497 2498 2499 2500

E FFECT OF EXERCISE ON LESION DEVELOPMENT

A comparison of lesion formation in the CONDEX and PASTEX groups revealed no significant differences between the groups (Figure 73 and Table 7) except for the number of osteochondrosis in the dorsal region of C3 and Cr. In this region the PASTEX was higher by 3 observations than the CONDEX (i.e. 6 vs. 3).
CONDEX/Exercised
Ci Cu Cu

Left
Cr Ci

Right
Cr Cr

Left
Ci

PASTEX/Control

Right
Ci Cr

Cu

Cu

C4 C3

C2

C2 C3

C4

C4 C3

C2

C2 C3

C4

Traumatic

Unique

C3 ridge

Cr ridge

C2 ridge

2501 2502 2503 2504 2505

Figure 73 - Superimposed lesions from the lesion affected sites in the CONDEX and PASTEX groups. Table 7 - A summary of the number lesions found in the CONDEX and PASTEX. Except the traumatic osteochondrosis lesions found at the dorsal radial facet of C3/Cr, overall there is no clear difference between the groups.

Osteochondrosis CONDEX PASTEX 2506 2507 2508 2509 2510 2511 2512 3 6

Unique 2 2

C3 ridge 6 8

Cr ridge 2 2

C2 ridge 4 4

The application of an imposed exercise in the first 18-month postnatally is an unorthodox method but the evidence shown here suggests that early exercise could have benefited the CONDEX group by reducing the prevalence of osteochondrosis in the dorsal site of C3 and Cr. It is possible that the early exercise may have increased the resistance in the CONDEX against the lesion formation through stronger and improved joint conformation thus allowing the horse to more effectively respond to an accidental traumatic loading. This hypothesis also is supported by Smith et al. [516] [131]

2513 2514 2515 2516 2517 2518 2519 2520

where they suggested an appropriate level of exercise in young horses may lead to a lower incidence of injury. However, when the total number of horses with such lesions is taken into account (3 horses out of 12), the higher lesion observations in the PASTEX could also be a random effect from the small sample size used in this study. It would be interesting to see results from a larger scale experiment using two different stables, where one would apply the exercise in the pre-18-month phase, while the other would delay training until the post-18-month phase. A comparison of the incidence of traumatic osteochondrosis in the C3 and Cr could then be made between the two stables.

2521 2522 2523 2524 2525 2526 2527 2528 2529 2530 2531 2532 2533 2534 2535 2536 2537 2538 2539 2540 2541

L ESION SYMMETRY
All the lesions with their approximate sizes and locations were combined into two lesion maps as shown in Figure 73. Most lesions were found to be symmetrically arranged when comparing left and right limb and/or on oppositional sites between proximal and distal rows of the midcarpal joint. Most traumatic lesions had an intra-joint oppositional symmetry such that a lesion located in the dorsal aspect of the proximal surface of the C3, was matched by a lesion of similar nature and size on the corresponding distal surface of the Cr (red regions in the Figure 73). Also nearly all other types of lesion (25/30 incidences) had a bilateral symmetry such that their locations on the surfaces of the C2 or C3 were mirrored on the same sites on the contra-lateral limb. The symmetric distribution of lesions at particular sites could be related to joint morphology and the loading environment of the midcarpal joint. For example, at high speeds, the forelimbs are likely to undergo hyperextension of the carpal joint which is attributable to muscle fatigue [517-521]. This configuration results in transmission of the axial dynamic loads through a restricted area located towards the dorsal aspect of the opposing surfaces of the C3 and Cr (i.e. the sites with traumatic osteochondrosis in Figure 73) [520, 522, 523]. Over time, a gradual degeneration of the cartilage and related bone sclerosis develops in this region [507, 520, 524]. A significant level of calcified cartilage, subchondral bone damage and sclerosis was observed in the dorsal regions of C3 and Cr, although the cartilage layers above were found to be relatively free from lesions (Figure 31, Figure 32 and Figure 40 to Figure 50). This damage was probably a result of the high intermittent stresses induced in the dorsal region and caused localised damage in the calcified cartilage and subchondral bone as demonstrated in Figure 42; and therefore developed at cartilage

[132]

2542 2543 2544 2545 2546 2547 2548 2549 2550 2551 2552

contact sites in the C3 and Cr surfaces (i.e. oppositionally) as well as bilaterally (i.e. left and right limbs). By contrast, the lesions observed at the sites in C2, C3 and Cr (Figure 62 and Figure 73) developed only bilaterally and were possibly caused by abrasion induced by the intercarpal expansion resulting from high dynamic loads [520, 525], Such loading would produce a distinct rubbing effect on the opposing surfaces in the joint as shown in Figure 30A, Figure 34 and Figure 39. However, this proposed mechanism is insufficient to explain the origin of the unique lesions also observed at the joint ridge of the radial and intermediate facets of C3. Without being able to propose a cause for this type of lesion, it is suggested that the relationship between lesion formation, load environment and midcarpal joint congruency is an area of potentially important research.

[133]

2553 2554 2555 2556 2557 2558 2559 2560

4.2 ANALYSIS 2 INFLUENCE OF EXERCISE ON EXTRACELLULAR MATRIX

SWELLING BEHAVIOUR

4.2.1 METHODS
Four adjacent sites on each of the midpoints of the opposing surfaces of the third and radial carpal bones were marked with a temporary fuchsin stain (Figure 74). These marks were used to identify a consistent sampling region that would yield contiguous osteochondral blocks free of obvious disruption.

2561 2562 2563 2564 2565 2566 2567 2568 2569 2570
Figure 74 - Temporary red fuchsin stains the site of swelling sampling region on C3 and Cr.

The intent was to provide serial slices of the cartilage matrix through the entire tissue depth. Sites 1 and 2 were toward the dorsal aspects of the third and radial carpal bones and corresponded to locations where it is known that there are high contact stresses and where regional adaptive bone changes and frequent osteochondral fractures have been reported [507, 522, 526](Figure 75). The sites toward the palmar aspects (S3 and S4) were chosen as locations with comparatively lower loads. The sites 1 to 4 on the radial carpal bone matched exactly with the sites 1 to 4 on the opposing surface of the corresponding third carpal bone (Figure 75).

[134]

A
Ci Cu

Dorsal
1 2 3 4

B
Cr

Cr

Cr

|1| | 2| |3| |4|

C2

Palmar C4
4 3 2 1
Defect

1 2 3 4
C3 C3
Medial

C3

Latera l

Swelling

Histomorphometric

Dorsal

2571 2572 2573 2574 2575 2576 2577 2578 2579 2580 2581
Figure 75 - [A] Schematic of the procedure used to obtain serial slices of cartilage to evaluate swelling behaviour of the ECM from the surfaces of the third and radial carpal bones. Sampling sites (1 through 4) of cartilage from the opposing surfaces of the third and radial carpal bones are shown above. The midcarpal joint was incised and reflected along its palmar aspect such that proximal surfaces of the second (C2), third (C3), and fourth (C4) and distal surfaces of the ulnar (Cu), intermediate (Ci), and radial (Cr) carpal bones were exposed. Immediately adjacent to the swelling sampling site, a histomorphometric sample was also obtained in the medial aspect, where both samples sharing same lesion; [B] Crosssectional view of both C3 and Cr showing how sampling sites 1 to 4 from both bones are well matched each other. [C] Thinly cut out slice of C3 and Cr (stained with toluidine blue and back-lit micro-photographed) where corresponding region to sites 1 to 4 who localised bone sclerosis (arrow) in sites 1 and 2.

Ci Cu

1 2 3 4

Cr

4
3

1
C2
4 3 2 1

C4

C3

2. Two sample cutouts from C3 & Cr

1. Carpus joint

3. Swelling blocks

4 mm
m 4m
2.2 mm

2.2 mm

2.2 mm

4. Single swelling block

2582 2583 2584 2585 2586

5. Double-cruciform vertical cuts of 2.2mm made

Figure 76 - Schematic of the procedure used to obtain serial slices of cartilage to evaluate swelling behaviour of the ECM from the surfaces of the third and radial carpal bones. The articular surface of each of the sampling sites was subjected to double-cruciform vertical cuts, which resulted in a remaining area of 2.2 X 2.2 mm, which was then sectioned to provide 30-m-thick serial sections.

1
[135]

2.2

mm

6. Redundant pieces are discarded

7. 30 -thick swelling m slices

2587 2588 2589 2590 2591 2592

Osteochondral blocks with en face dimensions of approximately 4 X 4 mm were sawn from the 4 sites on each of the opposing surfaces of the third and radial carpal bones (Figure 76); all osteochondral blocks were maintained in a fully hydrated condition. The blocks were blotted dry and further processed by use of a custom-built cutting system to make a double-cruciform vertical cut of defined dimensions (2.2 X 2.2 mm) through the full depth of the articular cartilage (Figure 77).

2593 2594 2595 2596

Figure 77 - [a] a custom-built cutting system to make a double-cruciform vertical cut of defined dimensions (2.2 X 2.2 mm). [b] Birds eye view of the device showing a cruciform slot where the cutter is inserted with precision. [c,d] An osteochondral block glued on top of the sample plate with the cut.

Normal

27%

Degenerate

115%
1mm

2597 2598 2599 2600 2601 2602 2603 2604 2605 2606 2607 2608 2609 2610
Figure 78 - Examples of low and high swelling strains in photomicrographs of representative cartilage slices obtained from the proximal surface of the third carpal bone. Swelling increased 27% and 115% depending on the tissue health, respectively, following immersion in 0.15M saline (NaCl) solution for 24 hours at 4C. Bar = 1 mm.

Using a sledge microtome (a generic type), 30-mthick serial sections of initial en face dimensions (2.2 x 2.2 mm) were obtained where each osteochondral block yielded between 5 and 20 transverse serial slices depending on the cartilage thickness, which varied across the joint surface. The original thickness of cartilage determined the total number of 30-m slices (n) that could be obtained and used to develop the swelling profile along the cartilage depth. All cartilage thicknesses were therefore normalized to a value of 1 and each slice in the sequential series numbered with slice #1 being uppermost and slice #n closest to the subchondral bone. The slices were immersed in 0.15M saline (NaCl) solution and allowed to swell freely for a minimum of 24 hours at 4C. All of the slices were then digitally photographed under identical magnifying conditions (x2.5) to determine their in-

[136]

2611 2612 2613 2614 2615 2616 2617 2618 2619 2620 2621 2622 2623 2624 2625 2626 2627 2628 2629 2630 2631 2632 2633 2634 2635 2636 2637 2638

plane swollen dimensions relative to their standard as-cut dimensions (Figure 78). Swelling strains were calculated as the percentage increase in the area (i.e., [(Areapost-swelling Areapre-swelling) / Areapreswelling]

x 100%) by use of an image analysis software4.

4.2.2 STATISTICAL ANALYSIS AND RESULTS

The swelling strains were analysed by use of linear mixed modelling5 to determine whether there was significant early exercise effect on the swelling behaviour of the cartilage extracellular matrix (ECM). Variables analysed included treatment (PASTEX or CONDEX group), limb (left or right), bone of origin (third or radial carpal bone), sampling site (osteochondral sites 1, 2, 3, or 4), and depth of the cartilage slice beneath the articular surface (normalized as 0 to 1). Because there were 6 horses nested within each treatment, horse was set as a random effect (i.e. the horses were considered to be a random sample from a population, and the conclusions were generalized to this population). A large degree of complexity was encountered in the variance structure so the analysis was adjusted to anticipate 2 additional effects, (i) the horse-specific degenerative changes among the horses (likelihood ratio test; 2 = 389.2; 4 df; P < 0.001) and (ii) biological swelling variance with depth of the cartilage slice among horses (likelihood ratio test; 2 = 74.9; 4 df; P < 0.001). These additional effects were set as random variables within the model. The fixed effects to be estimated included a full expansion that included all the interaction terms of treatment, limb, bone of origin, sampling site, and depth of cartilage slices. The depth was included as linear and quadratic terms. For the SAS related data and raw output for the swelling analysis please refer to page 233 in the Appendices. A total of 1,521 swelling measurements were made where the CONDEX and PASTEX swelling samples produced 755 and 766 observations respectively. A left midcarpal of PASTEX G3 was not available for the swelling analysis and therefore it was not included in the data set. The number of swellings slices produced from each osteochondral block (from sites 1 to 4 and from C3 and Cr) varied from as little as 4 to up to 20 due to changing cartilage thickness along the sampling axis. Thus it was more sensible to use relative depth (0 to 1) as an x-axis rather than absolute nth slice in the calculation and plotting. The C3 and Cr CONDEX and PASTEX swelling strains were plotted in the figures below (Figure 79 and Figure 80).

4 5

Image J version 1.39d, National Institutes of Health, Bethesda, Md. USA PROC MIXED, SAS, version 9.1, SAS Institute Inc, Cary, NC. USA

[137]

2639 2640 2641 2642 2643 2644 2645 2646 2647

Direct averaging of the absolute swelling strain (%) was avoided. Rather, the entire data set was first processed with the SAS statistical package to detect the signal and noise patterns using linear mixed modelling and then produced the mean swelling data, which was used to plot the relevant swelling strain curves for each osteochondral sampling sites as shown in Figure 81 on page 140 (for the raw data, see page 268 in the Appendices). The curves were fitted to the plots of mean swelling strain of cartilage ECM versus cartilage depth for each combination of the sampling site (1, 2, 3, or 4), the bone of origin (third or radial carpal bone), and the limb (left or right). Overall, the swelling strain increased gradually as the depth of the slice relative to its thickness increased, peaking around at the middle/deep zone of the cartilage matrix then falling without ever reaching for zero.

C3-1

160

C3-2

140 120 100

140 120
Swelling strain [%]

Swelling strain [%]

100

80 60 40 20 0

80 60
40

20 0
-20 70 60 50

0.0

0.5
Depth

1.0

0.0 -20 80

0.5

1.0

Depth

C3-3

C3-4

70
60

Swelling strain [%]

40 30 20 10 0 0.0 -10 0.5 1.0

Swelling strain [%]

50

40
30 20

10
0

Depth

-10

0.0

0.5 Depth

1.0

2648 2649 2650 2651 2652 2653 2654 [138]

CONDEX (exercised)

PASTEX (control)

Figure 79 - Scatter plots of swelling strains in the C3 from the CONDEX and PASTEX. Each plot represents each sampling site, from 1 to 4. All thickness value of the strain data points were normalized to 1.

2655 2656 2657 2658 2659 2660

Cr-1

100

Cr-2

120
100 80

80

Swelling strain [%]

Swelling strain [%]

60

60

40

40 20

20

0 0.0 0.5
Depth

1.0
-20 120 100

0.0

0.5
Depth

1.0

-20 70 60 50
Swelling strain [%]

Cr-3

Cr-4

80
Swelling strain [%]

40 30 20 10 0 0.0 -10 0.5 1.0

60 40
20 0 0.0 -20 0.5 1.0

Depth

Depth

2661 2662 2663 2664 2665 [139]

CONDEX (exercised)

PASTEX (control)

Figure 80 - Scatter plots of swelling strains in the Cr from the CONDEX and PASTEX. Each plot represents each sampling site, from 1 to 4. All thickness value of the strain data points were normalized to 1.

2666
Left C3 Site 1 Right C3 Site 1 Left Cr Site 1 Right Cr Site 1

80 70

80 70

80 70

80 70

Swelling strain [%]

Swelling strain [%]

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Left C3 Site 2
80 70

Right C3 Site 2

Left Cr Site 2

Right Cr Site 2

80

80 70

80 70

Swelling strain [%]

Swelling strain [%]

70

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Left C3 Site 3
80 70

Right C3 Site 3
80 70

Left Cr Site 3

Right Cr Site 3

80

80 70

Swelling strain [%]

Swelling strain [%]

70

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Left C3 Site 4

Right C3 Site 4

Left Cr Site 4

Right Cr Site 4

80 70

80 70

80 70

80 70

Swelling strain [%]

Swelling strain [%]

Swelling strain [%]

Swelling strain [%]

0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

2667 2668 2669 2670 2671 2672 2673 2674

PASTEX

CONDEX

Figure 81 - Cohort mean ECM swelling strain versus cartilage depth plots for each combination of the sampling sites (1, 2, 3, or 4), bone of origin (third [C3] or radial carpal [Cr]), and limb (left or right) of the CONDEX (x) and PASTEX (o) groups. Statistically fitted curves for the CONDEX (----) and PASTEX (- - -) are indicated. Plots shown are for site 2 on the C3 bone of the left limb (A) and site 2 in the radial carpal bone of the left limb (B). Only site 2 on the radial carpal bone of the left limb (B) in the CONDEX horses was marginally higher than in the PASTEX horses (orange box). *All cartilage thickness values were normalized to 1.

[140]

2675 2676 2677 2678 2679 2680 2681 2682

Analysis of the fitted curves from the CONDEX and PASTEX groups indicated that overall there was little difference between the two. There was only 1 of 16 sites (sampling site 2 in the left radial carpal bone shown in orange box in Figure 81) where the swelling strain of the cartilage ECM from the CONDEX group was higher than that of the PASTEX group.

Table 8 - A summary of excerpted data from the linear mixed modelling analysis performed. Overall, there was no evidence of a detectable treatment effect (F1,9 = 0.07; P = 0.795). But some variables were shown to be moderately significant, such as effect of site, bone and limb.

Null hypothesis Treatment Limb Site Site*Limb Bone*Site distance*Site dist2*Site

Num df 1 1 3 3 3 3 3

Den df 9 9 30 90 90 30 30

F Value 0.07 0.9 6.54 4.43 2.86 30.42 24.26

Pr > F 0.7951 0.3674 0.0016 0.006 0.0411 <.0001 <.0001

2683 2684 2685 2686 2687 2688 2689 2690 2691 2692 2693 2694 2695 2696 2697 An overall effect of early exercise on the mean swelling strain of the cartilage ECM was not detected (F1,9 = 0.07; P = 0.7951, see Table 8). The mean SE6 was 16.93 3.227% for the CONDEX group and 15.71 3.248% for the PASTEX group. The difference between the mean values of swelling strain had wide variability (mean, 1.23%; 95% C.I.7, 9.132 to 11.580) showing that the power to detect a difference between the groups was low (6 animals per treatment, 12 in total). Thus the mean swelling strain of cartilage ECM from the CONDEX group could be as much as 12% greater or 9% less compared with the mean swelling strain from the PASTEX group (Please refer to page 234 for the entire SAS output). Although it was shown that early conditioning not have a significant overall effect on swelling behaviour of the cartilage ECM, there was an influence of the biological variables on swelling behaviour (i.e. within the joint irrespective of whether CONDEX or PASTEX) . Also, there was a significant difference in the pattern of swelling behaviour between sampling sites (F3,30 = 6.54, P = 0.0016) (Figure 82).

6 7

Standard Error Confidence Interval

[141]

(a) Cohort mean swelling strain

30 25 35 30

(b) Mean swelling strain C3 & Cr

C3 Cr

(c) Mean swelling strain

35 30

Right & Left limbs

Right Left

Swelling strain [%]

20

18
15 10 5 0

Swelling strain [%]

16 10

20
15 10 5 0

Swelling strain [%]

22

25

25 20 15

10
5 0

2698 2699 2700 2701 2702 2703 2704 2705 2706 2707 2708 2709 2710 2711 2712 2713 2714 2715 2716 2717 2718 2719 2720 2721 2722 2723 2724

S1

S2

Site

S3

S4

S1

S2

Site

S3

S4

S1

S2

Site

S3

S4

Figure 82 - Plots of the estimated mean values of cartilage swelling strain for each sampling site (1 through 4) as follows: (A) both treatment groups plus both left and right plus C3 and Cr bones combined; (B) both treatment groups plus both left and right combined, but with C3 and Cr bones separated; (C) both treatment groups plus C3 and Cr bones combined, but with left and right separated. The error bars indicated 95% confidence intervals.

A higher swelling strain of 7.4% (P = 0.0016) was detected in the high-stress sites in the dorsal aspect of the third and radial carpal bones relative to the low-stress sites in the palmar aspect of these surfaces (Figure 82a). And the swelling strain of the third carpal bone was higher than that of the radial carpal bone in the dorsal sites (Figure 82b: site*bone interaction; F3,90 = 2.86; P = 0.041); there was also a limb interaction effect on the pattern of swelling behaviour detected among the sampling sites (Figure 12C: site*limb interaction; F3,90 = 4.43; P = 0.006). Interestingly, the cartilage lesions at or close to a sampling site significantly influenced swelling behaviour of the cartilage ECM, increasing the mean swelling strain by 4.2% (F1,994 = 4.23, P = 0.04). Of the many interaction terms involving the treatment, only those involving the shape of the curve that related mean swelling strain to cartilage depth were significant. The linear part of the quadratic expression depended on the treatment, limb, bone of origin, and sampling site within the bone (i.e., the depth* treatment*site*bone*limb interaction was significant; F6,85 = 2.36; P = 0.037). And the quadratic term depended on the same factors through the 2 simpler interactions, thus depth2*treatment *site*limb (F3,79 = 3.43; P = 0.021) and depth2 *treatment*bone*site (F3,79 = 3.16; P = 0.029) were also significant. In summary, the effect of exercise in the CONDEX was not detectable in the cartilage matrix swelling analysis (P = 0.7951). However, as for the sampling sites where mild osteochondrosis was observed, a modest increase in swelling of +4.2% (P = 0.04) was detected. As for the high-loaded dorsal sites (sites 1 and 2) of C3 and Cr, there was a significant increase of +7.4% in the swelling strain compared with that in the lower-load sites 3 and 4 in the palmar aspect (P = 0.002).

[142]

2725

4.2.3 DISCUSSION

2726 2727 2728 2729 2730 2731 2732 2733 2734 2735 2736 2737 2738 2739 2740 2741 2742 2743 2744 2745 2746 2747 2748 2749 2750 2751 2752 2753

I NFLUENCE OF E XERCISE ON MATRIX SWELLING BEHAVIOUR

The effect of early exercise in the extracellular matrix swelling behaviour was not detectable in the CONDEX (F1,9 = 0.07; P = 0.795, see Figure 81). The swelling behaviour of the cartilage ECM is highly dependent on the integrity of the collagen fibrillar network [527-530] and its glycosaminoglycan (GAG) content [528, 529, 531]. However as the tissue degenerates (e.g. as in osteoarthritis), the interconnectivity within the collagen network is reduced, which induces the network to destructure into more aggregated fibrillar bundles (Figure 52) with less effective entrapment of the hydrated GAGs [527, 532, 533]. Therefore, often an increased degree of swelling in cartilage is associated with osteoarthritis [528-530, 534-536] with a drastic reduction of its ability to resist the tensile, compressive, and shear stresses [83, 327]. To make matters more complicated, a moderately high level of swelling is not always a pathologic sign as the concentrations of collagen and GAG in cartilage can vary substantially depending on the joint type [537], site within the joint [536, 538-542], and cartilage depth [538, 543] plus the age and exercise regimen (type, timing and intensity) [477, 544, 545]. Since the technique used in the study was somewhat novel it was impossible to link the data with those of previous studies. For this reason, determining to what extent the swelling behaviour observed in this study is of an adaptive or pathologic response is difficult. However, the swelling analysis was able to indicate whether or not there were matrix changes due to the exercise treatment by directly comparing the overall swelling data from each group. The horses used in this study were also investigated by other groups but using different regions of the limbs as part of the GEXA global collaboration [1-3, 471, 546]. Of these, three have published their cartilage composition results [1-3, 466]. Nugent et al. [1] examined the cartilage at the lateral and medial condyles of the metacarpal bone, while both Van Weeren et al. [2] and Brama et al. [3] examined the proximal phalanx. Studies [1-3] revealed significant topographical variations in the ECM structural, biochemical and biomechanical properties; however, only Van Weeren et al. and Brama et al. [2, 3] reported any significant exercise effect on the collagen-related changes and GAG concentrations between the CONDEX and PASTEX, while Nugent et al. [1] reported negative results.

[143]

2754 2755 2756 2757 2758 2759 2760 2761 2762 2763 2764 2765

In the studies by Van Weeren et al. and Brama et al., the imposed exercise [500] in the CONDEX increased the rate of remodelling in the GAGs and collagen type II [2] resulting in a lower collagen content in the high intermittently loaded sites with a higher degree of cross-linking in the matrix in the CONDEX [2, 3]. In the present study, if similar maturation-related changes had developed in the comparatively high-load region [2, 3] of sampling sites 1 and 2, we would have expected a lower swelling strain in the CONDEX compared with that of the PASTEX; however, such effect was not detected, as also reported by Nugent et al. [1]. It seems the overall amount exercise was either too mild to trigger any significant effect to the cartilage matrix in the CONDEX or the swelling measurement technique used in this study may have not been sufficiently sensitive. However, the technique was successful in detecting minute swelling variations from site-to-site, from left-to-right limb and in the sites where there was obvious degeneration-related focal changes in the articular cartilage.

2766 2767 2768 2769 2770 2771 2772 2773 2774 2775 2776 2777 2778 2779 2780 2781 2782 2783

D EPTH AND SITE -SPECIFIC VARIATIONS IN MATRIX SWELLING

This study found that overall both the CONDEX and PASTEX cartilage matrices exhibited significant depth and site-dependent swelling characteristics (Psite = 0.0016, Pdepth = 0.0002). The swelling strain versus depth plots (Figure 81) indicated that the cartilage displayed classical swelling behaviour [531, 535, 547] (i.e. lowest in the surface layer, increasing to a maximum in the middle zone, and diminishing in the deep zone). This characteristic trend in swelling behaviour reflected the natural zonal differentiation of the cartilage matrix [548], behaviour that was highlighted in the much earlier cartilage swelling experiments conducted by Maroudas [549]. The dorsal aspect of the radial facet in C3 represented by sites 1 and 2 is also a well known region for cartilage loss, osteochondral fracture, ischemic sclerosis and osteochondrosis [505, 506, 523], presumably due to its exposure to the high intermittent loading. There were significant topographical variations in the swelling behaviour of the cartilage extracellular matrix (ECM) among the 4 sampling sites (Figure 82a, Psite = 0.0016). The dorsal sites (1 and 2) displayed significantly higher swelling strains than the palmar sites (3 and 4), which indicated that a positive correlation exists between the degree of swelling and the intensity of loading in the different areas of the joint [523, 526, 550]. It has been reported that a high intensity exercise can decrease HP cross-links (hydroxylysylpyridinoline, see page 73) in the collagen matrix resulting in a loose collagen network and increased

[144]

2784 2785 2786 2787 2788 2789 2790 2791 2792 2793 2794

swelling behaviour [285, 286, 293, 482]. But a matrix which received moderate levels of cyclic loads may also exhibit high swelling because such loading is known to encourage proteoglycan and protein synthesis [291, 292, 294]. Thus, both high and moderate intensity loads can result in elevated matrix swelling. Although it was stated earlier that the exercise treatment may have been too weak to trigger any significant changes in the extracellular matrix of the CONDEX animals, there are clear indications that of distinct site-specific changes in the matrix swelling behaviour that differentiate between sites 1 and 2, and sites 3 and 4. A possible reason for this difference is that the autonomous pasture exercise (which both the CONDEX and PASTEX equally received) was more influential in bringing about an adaptive response across the joint than any change arising from imposed versus nonimposed exercise.

2795 2796 2797 2798 2799 2800 2801 2802 2803 2804 2805 2806 2807 2808 2809 2810 2811 2812 2813

T RAUMA EFFECT IN MATRIX SWELLING

It is not possible to determine the mode, magnitude and duration of the stress experienced at the dorsal sites of C3 (and Cr) during the 18-months period in vivo. Further, there no literature available to indicate the level of stress the carpal joint experiences during various gait modes (stance, walking, trotting and galloping). The only available mechanically related data comes from a static compression study [53] but this study falls short of indicating the actual stress experienced by the joint and the mode of loading was far removed from loading experienced in vivo. However, it is well known that the dorsal region of the third carpal bone is exposed to a much higher level of stress than other regions of the joint, thus making this region a common site of various joint diseases. The region is often acutely overloaded because of the brief hyperextension induced during a fast gallop. All of the initial impact force from the weight and moment is focused into this region as the forelimb lands. Trauma from high intensity exercise is a well known cause of matrix degeneration. It can decrease the collagen content [551], loosen the fibrillar network [482], cause rupture of the fibrils [552], while decreasing the rate of proteoglycan biosynthesis [551] and cause cell death [552]. It is thought that the nine observations of osteochondrosis reported in this study were caused by such acute levels of stress. These acute stress may have induced localised degenerative matrix changes in the dorsal sites reflected by an increase in matrix swelling in these regions (+4.2%, P = 0.04).

[145]

2814 2815 2816 2817 2818 2819 2820 2821 2822

B ONE EFFECT IN C3 AND C R

Referring to the cohort mean swelling strains (both the CONDEX+PASTEX combined, Figure 82b), the dorsal sites (1, 2) from the C3 exhibited swelling strains that were consistently higher (approximately +5%, P < 0.001) than the opposing site on the Cr. It is difficult to postulate exactly what caused this difference in the level of swelling between the C3 and Cr in these regions. It is known that the subchondral plate in the distal row of the mid carpal joint is thicker than that of the proximal row. Similarly, the different swelling patterns may be due to the bone-specific (C3 vs. Cr) compositions of the extracellular matrix affirming that the two opposing layers of cartilage are different, even if they are proximal to each other and subjected to the same level of loading [64].

2823 2824 2825 2826 2827 2828 2829 2830 2831 2832 2833 2834 2835 2836 2837 2838 2839 2840 2841 2842 2843 2844

L ATERALITY IN SWELLING BEHAVIOUR

The cohort mean swelling strain of both the left and right limbs showed that there is clear laterality (handedness) in the horses. At site 1, the swelling strains in the left limb were significantly higher than those in the right (+9.4%, P = 0.006) and this distinction was reduced in site 2 and 3, and then reversed at site 4 though only slightly (Figure 82c). It is well known that horses have some degree of handedness [553, 554], being naturally right brain dominant; this allows the horse to support its weight most efficiently when turning to the left [555]. Also the Thoroughbreds have a greater reliance for support on the right forelimb than on the left forelimb [554] with a greater contact duration on the leading (right) forelimb than on the trailing (left) forelimb [556]; thus the right limb is usually the stronger of the two limbs [557]. Concerning the potential role of laterality in injuries, many Thoroughbred horses in the U.S. experience a greater number of carpal slab fractures [58] and proximal sesamoid fractures in the right forelimb. However, this may well be because in the U.S. the horses race counter-clockwise whereas in Europe and Australia they run clockwise. In this study, the CONDEX horses were trained bidirectionally and alternated daily in counter/clockwise directions [500] so that lateral development of the joint could be minimised. The laterality in the swelling strain, therefore, can be only explained by either some biologically random variable or by some inherent overall laterality. It is interesting to note that the bidirectional training did not eliminate the laterality in the CONDEX; it would thus seem that all horses developed their laterality through their free-pasture exercise. Thus, it could be speculated that the free-pasture exercise had a greater influence than the imposed exercise. [146]

2845 2846 2847 2848 2849 2850 2851 2852 2853 2854

4.3 ANALYSIS 3 - HISTOMORPHOMETRIC ANALYSIS

4.3.1 METHODS
In order to investigate how exercise might have influenced the histomorphometric features of the matrix in relation to matrix swelling behaviour and lesion development, the histomorphometric sampling was conducted in those regions immediately adjacent to the swelling regions shown in Figure 83. Using a fine toothed saw osteochondral slabs of approximately 5mm x 30mm x 10mm (width x length x height) were obtained from the medial aspect immediately adjacent to the swelling sampling region. These slabs were then wet-ground on a flat surface using fine sand paper8 under running water.

C2

Palmar C4
4 3 2 1

Lateral

Medial

C3

Swelling sample

HistomorphoDefect metric sample

Dorsal

2855 2856 2857 2858 2859 2860 2861 2862 2863 2864 2865 2866 2867
Figure 83 - (Left) Sample schema showing the approximate location where a histomorphometric sample was obtained in relation to the swelling sample, a medial side of the C3. The both samples were immediately adjacent to each other and if possible, were sawn out so that they both share the site of a lesion as shown in the figure above. (a, b) Photographs of a histomorphometric osteochondral slab photographed on an actual hand drawn schema where the red box indicates the approximate size and location of the actual cutting site.

Each slab was then fixed using 10% CPC formaldehyde for approximately 12 hours then gently decalcified using 10% formic acid buffered with sodium formate for three to five days [501, 502]. The samples were then rinsed in cold running water for 1 hour to cleanse the residual chemicals and then mounted on a sledging microtome9 in a semi-frozen state where up to 12 full depth crosssectional slices (thickness = 30m) were obtained. The slices were then gently washed to remove any extraneous debris and excess fat, then wet-mounted on a glass slide under a cover slip and

8 9

Silicon carbide Leica Instruments GmbH, Nussloch, Germany, Model SM 2000R

[147]

2868 2869 2870 2871 2872 2873 2874 2875 2876 2877 2878 2879 2880 2881 2882 2883 2884

digitally photographed using bright field optical microscopy10 (x2 x2 x 0.5-1 mode with wide aperture setting) without any histo-staining. Typically, 12 slices were produced and the best five slices with the least amounts of cutting damage were used for the analysis. Since the microscopes field of view was limited, 10 to 15 5-megapixel digital images were taken for each slice and then digitally stitched together using a digital image stitching programme11 so as to produce high resolution images of the full cartilage-bone cross-section, approximately 350MB in file size each (Figure 84). Each image was then overlaid with four 3000 x 3000 m matrix grid squares (which was pre-scaled to the microscope magnification using a micrometer) corresponding to the four sampling sites used in the swelling analysis. Within each square, using a pen-tablet digitiser12 with a digital imaging software13, all quantifiable histomorphometric features were traced by hand with three colours; blue (hyaline cartilage), green (calcified cartilage) and red (vascular channel). These were then readily transformed into binary (black & white) colours for efficient image analysis which included measurement of area, average hyaline and calcified cartilage thickness, vascularity of the ZCC, cement line rugosity and preferential angles of the collagen matrix and vascular branching. The analysis was performed using an image analysis software14 (Figure 84 to Figure 86, Table 9).

10 11

Nikon Instruments Inc., Japan, Model AZ100 PTGui Pro version 8.3.6, New House Internet Services BV, Rotterdam, The Netherlands 12 Wacom Co., Ltd., Saitama, Japan, Model Graphire CTE-630 13 Adobe Photoshop version CS, Adobe Systems Inc., San Jose, CA, USA 14 Image J, version 1.39d, National Institutes of Health, Bethesda, MD, USA

[148]

Dorsal

Site 1
S2

S4 S3

Palmar

A B Hyaline cartilage C

E
1 2

D Calcified cartilage
3

J
7 4 5 6 8 9 10 11

F J I

G,H
3

Subchondral bone

2885 2886 2887 2888 2889 2890

Figure 84 - (Top) Microphotograph of a slice after completing the image stitching process (which typically resulted in an 18,000 x 4000 pixel image of 350MB file size). (Bottom left) Site 1 is shown as an example of how outlines were first drawn using a digitizer and overlaid with fill-in of blue (for hyaline cartilage), green (for calcified cartilage) and red (for vascular channels) in order to quantify their respective areas (refer the table below). (Bottom right) The same image was also used to measure the preferred orientation of the collagen matrix, calcified cartilage and vascular channels. Furthermore, the rugosity of the cement line (green line) was calculated given that the width of the grid overlay was constant (3000m). The alphabetical letters (A to I) shown above are explained in Table 9.

2891 2892 2893

S1 S2 S3

S4

Hyaline cartilage (HCt) Calcified cartilage (ZCCt) Cement line (rug.) Vascular channel (Vca, VCn)

2894 2895 2896

S1

S2

S3

S4

Figure 85 - An example of how each osteochondral slice was divided into 4 sites (S1, S2, S3 and S4) and then coloured using red, blue and green for each histomorphometric feature to quantify the area, thickness, rugosity of each region. From this process, the site specific differences in the hyaline cartilage, calcified cartilage, vascularity and cement line were analysed.

2897 2898 2899 2900 2901 2902

Figure 86 - An example of a completed histomorphometric index of an osteochondral slice G30 R 4 (i.e. 4 slice of the C3 extracted from the right limb of G30). The index graphically summarises histomorphometric results of the hyaline cartilage (hc), zone of calcified cartilage (zcc), cement line (bl) and vascular channels (vc). A complete collection of the indices is available in the appendices on page 297.

th

[151]

2903
Tissue region

Table 9 - A summary of descriptions and symbols used in the histomorphometric analysis.

Label A B C

Data output type Total area Average thickness Preferential matrix angle Total area Average thickness Preferential matrix angle Total number Total area occupied by Average branching angle Rugosity (unevenness)

Acronyms HCa HCt HCang ZCCa ZCCt ZCCang VCn VCa VCang Rug

Unit m
2

Sample size 10 10 5~20 -

Hyaline cartilage

m deg m
2

Calcified cartilage

D E F

m deg m 2

Vascular channel

G H I

deg

2904 2905 2906 2907 2908 2909 2910 2911 2912 2913 2914 2915 2916 2917 2918 2919 2920

Cement line

The decision as to which vascular channels were selected for quantification was based on two conditions; whether the vascular channels (1) protruded into or touched the calcified cartilage and (2) had a length greater than 200m. The aim was to try and measure only those vascular channels believed to play a significant role in the remodelling of the cement line via their advancement into the ZCC, a process known to be assisted by angiogenesis factors (VEGF) [187, 558-560]. In the angular measurements of the VCang, HCang as well as ZCCang, the articular cartilage surface was used as the reference as it was consistently flat within the measurement area, whereas the calcification and subchondral bone fronts were poor candidates for such a reference line due to the relative irregularity of their contours. The rugosity of the cement line (Rug) was obtained by measuring the full coastal length and dividing it by the horizontal dimension of the grid (i.e. LengthCement line /3000m) so that if the cement line was very flat, the Rug would be close to 1, whereas if the line was very irregular due to high vascularity, the value would be higher due to longer parameter length.

4.3.2 STATISTICAL ANALYSIS AND RESULTS

2921 2922 2923

O VERALL DISTRIBUTION AND MEAN OF HISTOMORPHOMETRIC DATA

Each osteochondral slice produced 10 different types of data; with a total of 360 osteochondral slices available, 360 sets of 10 histomorphometric measurements were made. These results are [152]

2924 2925 2926 2927 2928 2929 2930 2931 2932 2933 2934 2935 2936 2937 2938 2939 2940 2941 2942

graphically summarised in an index form as shown in Figure 86 and the complete set can be found in the appendices section on page 297. The entire data sets were also scatter-plotted in Figure 87 in page 154 (see page 286 in the appendices section for the entire data set). The histomorphometric data contains the sample data from 9 horses (5 CONDEX, 4 PASTEX) out of 12, where the three other horses were excluded due to unavailability of the sampling site. As with the swelling analysis, the histomorphometric data sets were analysed by use of a statistical software package15 to determine whether there were significant influences of early exercise on the quantified histomorphometric features of the four sites in the C3. Initially, a multivariate analysis of variance (MANOVA) was conducted (using the same error terms as a mixed model formulation would require) to determine the effect of early exercise in the CONDEX (exercised) in comparison to the PASTEX (control) using 10 response variables (HCa, HCt, HCang, ZCCa, ZCCt, HCang, VCn, VCa, VCang, Rug, see Table 9). In order to minimise the effects of noise, all data showing evidence of trauma (the 20 data points affected by osteochondrosis from the 2 PASTEX horses G13 & G17) were removed (compare Figure 87 and Figure 89). Excluding these data did not affect the overall significance of the test results on the treatment effect. This was verified by an exact chi-square significance test, which showed no detectable difference in the overall difference by removing these trauma-affected data (2 = 3.2143, P = 0.1699).

15

SAS, version 9.2, SAS Institute Inc, Cary, NC. USA

[153]

900

1200000

A 800
700

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1000000

40
35

30 800000
25

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600000

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400 300

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15

400000 10
200000 5 0 0 500 1000 1500 0 200 400 600 800 1000 1200 1400

200
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2.8 900

HCt
900

HCt

2.6 2.4 2.2 2

E 800
700 600 500

800 700 600 500

Rug
1.8 1.6 1.4 1.2 1

ZCCt
400 300 200 100 0

ZCCt
400 300 200 100 0

500

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PASTEX (Control)
S1 S2 S3 S4

VCa
CONDEX (Exercised)
S1 S2 S3 S4

VCn
Trauma
S1 S2

2943 2944 2945 2946 2947

Figure 87 - Matrix scatter plots of histomorphometric data where each plot represents two of five histomorphometric variables (HCt, ZCCt, VCa, VCn, Rug, ang(HC, CC, VC)); see Table 9 for the explanation of the symbols) were drawn in order to detect possible site-specific relationships and treatment effects. The orange toned colours (see the legend) represent four sampling sites (site 1, 2, 3 & 4) from the control (PASTEX) and the blue toned colours represent four sampling sites from the exercised animals (CONDEX), while the red-magenta colours (X, +) represent the sites with traumatic damages in the calcified cartilage. Only sites 1 and 2 of the trauma-animals (G13, G17, and G19) were affected with the damages. (continued on to the next page)

900

40

2.8

2.8

G 800
700
600

35
30 25

I2.6
2.4
2.2 2

J 2.6
2.4
2.2 2

500

ZCCt
400
300

20 VCn

Rug
1.8
1.6

Rug
1.8
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15 10
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-60 -40 -20

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VCang

-10 -20 -30

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-20
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HCang
PASTEX (Control)
S1 S2 S3 S4

-60

HCang
CONDEX (Exercised)
S1 S2 S3 S4

-60

VCang
Trauma
S1 S2

-60

2948 2949 2950 2951 2952 2953 2954

The plots A to L are described as follows; (A) Hyaline cartilage thickness [m] against calcified cartilage thickness [m], (B) Hyaline cartilage thickness [m] against vascular channel area [m ], (C) 2 Hyaline cartilage thickness [m] against vascular channel number, (D) Hyaline cartilage thickness [m] against rugosity of cement line, (E) Vascular channel area [m ] against calcified cartilage thickness, (F) Vascular channel number against calcified cartilage thickness, (G) Rugosity of cement line against calcified cartilage thickness, (H) Vascular channel area against calcified vascular 2 channel number, (I) Vascular channel area [m ] against rugosity of cement line, (J) Vascular channel number against rugosity of cement line, (K) Vascular channel preferential angle [deg]against calcified cartilage collagen matrix preferential angle [deg], (L) Hyaline cartilage collagen matrix preferential angle [deg] against vascular channel preferential angle [deg] and (M) Vascular channel preferential angle [deg] against calcified cartilage collagen matrix preferential angle [deg]. The data used to create these plots can be found in the appendices section on page 286.

2955 2956 2957 2958 2959 2960 2961 2962 2963 2964 2965

From the results of the statistical analysis, overall it was not possible to detect evidence of an early exercise treatment effect in the variables (Pillai's Trace: 0.6930, F5, 3 = 1.35, P = 0.4271). The only significant term was site (Pillai's Trace: 2.0550, F15, 54 = 7.83, P =< 0.0001 as in Table 10). After the MANOVA had failed to show any treatment effect, the corresponding univariate mixed model analyses (ANOVAs using SAS PROC MIXED) were also performed on each of the response variables to further investigate any potential treatment effects. The SAS code and the full output can be found in the appendices section on page 278. The summary of the results and estimated mean with the 95% confidence intervals are presented in Table 11 and Figure 88;

Table 10 - A summary of excerpted data from the multivariate ANOVA (MANOVA) results. Only the site variable was statistically significant (P = <0.0001) whereas the treatment variable was not (P = 0.4271).

Null hypothesis Treatment Site Leg Treatment*Leg Leg*Site Treatment*Site Treatment*Leg*Site Horse(Treatment) Horse*Site(Treatment) Horse*Leg(Treatment) Horse*Leg*Site(Treatment)

Pillai's trace 0.693 2.055 0.481 0.324 0.892 0.418 0.688 2.244 1.980 1.419 1.281

Num df 5 15 5 5 15 15 15 35 100 35 90

Den df 3 54 3 3 48 54 48 1360 1360 1360 1360

F Value 1.35 7.83 0.56 0.29 1.36 0.58 0.95 31.64 8.91 15.40 5.21

Pr > F 0.4271 <.0001 0.7355 0.8934 0.2084 0.8741 0.5168 <.0001 <.0001 <.0001 <.0001

2966 2967 2968 2969 2970

Table 11 - A summary of excerpted data from the univariate ANOVA. In terms of the exercise treatment, only the VCa (vascular channel area) was shown to be significant (P = 0.0461) among the variables tested. However, overall, the site was again found to be a highly significant factor in all variables.

Variable HCt ZCCt VCa VCn Rug

Null hypothesis Treatment Treatment Treatment Treatment Treatment

Num df 1 1 1 3 3

Den df 7 7 7 18 18

F Value 0.12 1.04 5.86 0.05 0.33

Pr > F 0.7385 0.3413 0.0461 0.8234 0.5848

2971

[156]

Exercise effect likely?

E - Calcified cartilage thickness (ZCCt)
350 300
250

Exercise effect not likely?

B - Hyaline cartilage thickness (HCt)
1200 1000 800 600 400 200 0

CONDEX PASTEX

CONDEX PASTEX

Thickness [m]

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H - Vascular channel area (VCa)

800000

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Number of vascular channels found
CONDEX PASTEX

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J - Cement line rugosity (Rug)

2.2
2.0 CONDEX PASTEX

Hyaline cartilage
Rugosity

1.8

1.6
1.4

E
1 2

Calcified cartilage
3

G,H
3

7 4 5 6 8

9 10

J
11

1.2 1.0

Subchondral bone

S1

S2

Site

S3

S4

2972 2973 2974 2975 2976 2977

Figure 88 - The HCt, ZCCt, VCn, VCa and Rug histomorphometric results. The capital letters in the title in each plot correspond to the region in the histological diagram above. While (E) the calcified cartilage thickness (ZCCt) and (H) the vascular channel area (VCa) showed some positive responses to the exercise treatment, other variables such as (B) the hyaline cartilage thickness (HCt), (G) the vascular channel number (VCn) and (J) the cement line rugosity (Rug) have shown to be a little or no effect. The error bars indicate 95% confidence intervals.

[157]

2978 2979 2980 2981 2982 2983 2984 2985 2986 2987

The statistical significance of early exercise was not detectable in 9 of the 10 variables analysed, the exception being the vascular channel area (VCa, see Figure 88H, F1,7 = 5.86, P = 0.0461). The estimated mean of VCa in the CONDEX was shown to be consistently higher than the PASTEX by 35% to 57% in all four sampling sites (S1-S4) with a confidence interval (C.I. = 95%) of approximately 26% (Table 12).

Table 12 - The mean of the VCa and ZCCt in the CONDEX and PASTEX, and their percentile differences. The results are also shown graphically in Figure 88. The VCa shows that the CONDEX values are relatively higher throughout the sites. However for the ZCCt, it is the opposite, the PASTEX values are higher than of the CONDEX albeit the differences are now much smaller.

Vascular channel area 2 [VCa/m ] Site CONDEX PASTEX (CONDEX-PASTEX) /PASTEX % S1 533212 340173 +57% S2 318713 234747 +36% S3 254859 181301 +41% S4 206252 133814 +54% S1 221 260 -15%

Calcified cartilage thickness [ZCCt/m] S2 256 271 -5% S3 201 221 -9% S4 148 160 -7%

2988 2989 2990 2991 2992 2993 2994 2995 2996 2997 2998 2999 3000 3001 3002

Also, although the effect of early exercise on the thickness of the calcified cartilage (ZCCt) in the CONDEX was not very significant (F1,7 = 1.04, P = 0.3413), the calculated means plotted in Figure 88E suggest that there might be a small treatment effect with a mean % increase of 5% to 15% in the PASTEX (Table 12). As in the swelling analysis, the site effect was found to be consistently significant (F15,54 = 7.83, P < 0.0001). And of the other terms (including 1st and 2nd order interaction terms) only the leg*site interaction for ZCCt was highly significant (F3,18 = 3.96, P = 0.0248). In conclusion, out of 35 significance tests performed, 9 out of 10 variables did not show any evidence of early exercise effect in the CONDEX relative to the PASTEX. Only the VCa (vascular channel area) variable was found to be significant (P = 0.0461) for the early exercise treatment in the CONDEX.

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3003 3004 3005 3006 3007 3008 3009

I NTER - VARIABLE CORRELATION OF HISTOMORPHOMETRIC VARIABLES

In order to investigate the possible relationships between any of two variables among the histomorphometric variables (i.e. HCt, HCang, ZCCt, HCang, VCn, VCa, VCang, Rug) 12 two-variable scatter plots were drawn in Figure 89 and simple linear regression analyses were performed and summarised below in Table 13.

Table 13 - The CONDEX and PASTEX correlation results where each row corresponds to the scatter plot in Figure 89.

CONDEX
A B C D E F G H I J K L M HCt ZCCt HCt VCa HCt VCn HCt Rug VCa ZCCt VCn ZCCt Rug ZCCt VCa VCn VCa Rug VCn Rug HCang ZCCang HCang VCang VCang ZCCang

Multiple R Correlation 0.5877 0.1716 0.3385 0.2016 0.3592 0.4160 0.2958 0.0636 0.0381 0.4214 0.9299 0.6705 0.6359

Adjusted R Square 0.3420 0.0245 0.1101 0.0358 0.1246 0.1689 0.0829 -0.0010 -0.0036 0.1734 0.8639 0.4467 0.4014

df 198 198 198 198 198 198 198 198 198 198 198 198 198

Coefficients 0.1857 199.7693 0.0087 0.0002 0.0001 5.1364 75.4374 0.0000 0.0000 0.0204 1.1793 0.5667 0.9542

t Stat. 10.2200 2.4512 5.0627 2.8968 5.4154 6.4376 4.3569 -0.8973 0.5359 6.5378 35.5611 12.7153 11.5945

P-value < 0.0001 0.0151 < 0.0001 0.0042 < 0.0001 < 0.0001 < 0.0001 0.3707 0.5926 < 0.0001 < 0.0001 < 0.0001 < 0.0001

3010

PASTEX
A B C D E F G H I J K L M HCt ZCCt HCt VCa HCt VCn HCt Rug VCa ZCCt VCn ZCCt Rug ZCCt VCa VCn VCa Rug VCn Rug HCang ZCCang HCang VCang VCang ZCCang

Multiple R Correlation 0.0836 0.0700 0.2279 0.3841 0.2674 0.3265 0.2491 0.0178 0.0875 0.3461 0.9280 0.4876 0.5178

Adjusted R Square -0.0002 -0.0023 0.0451 0.1414 0.0648 0.1001 0.0553 -0.0069 0.0005 0.1134 0.8601 0.2322 0.2628

df 138 138 138 138 138 138 138 138 138 138 138 138 138

Coefficients 0.0411 70.9725 0.0075 0.0006 0.0001 4.8760 79.4214 0.0000 0.0000 0.0162 1.2319 0.3977 0.8427

t Stat. 0.9860 0.8246 2.7497 4.8874 3.2602 4.0572 3.0220 0.2091 -1.0315 4.3341 29.2491 6.5607 7.1099

P-value 0.3258 0.4110 0.0068 < 0.0001 0.0014 < 0.0001 0.0030 0.8346 0.3041 < 0.0001 < 0.0001 < 0.0001 < 0.0001

[159]

500

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VCa

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3011 3012 3013 3014 3015

CONDEX (exercised)

PASTEX (control)

Figure 89 - The same matrix scatter plots shown as Figure 87 was redrawn without the bias osteochondrosis data. The plots are now coloured according to the treatment group (CONDEX and PASTEX). The plots A to L are described as follows; (A) Hyaline cartilage thickness [m] against calcified cartilage thickness [m], (B) Hyaline cartilage thickness [m] against vascular channel area 2 2 [m ], (C) Hyaline cartilage thickness [m] against vascular channel number, (D) Hyaline cartilage thickness [m] against rugosity of cement line, (E) Vascular channel area [m ] against calcified cartilage thickness, (F) Vascular channel number against calcified cartilage thickness, (G) Rugosity of cement line against calcified cartilage thickness, (continued on to the next page)

500

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2.6 2.4
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1.8 1.6 1.4 1.2

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-20 -30 -40 -50 -60

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3016 3017 3018 3019 3020

CONDEX (exercised)

PASTEX (control)

(H) Vascular channel area against calcified vascular channel number, (I) Vascular channel area [m2] against rugosity of cement line, (J) Vascular channel number against rugosity of cement line, (K) Vascular channel preferential angle [deg]against calcified cartilage collagen matrix preferential angle [deg], (L) Hyaline cartilage collagen matrix preferential angle [deg] against vascular channel preferential angle [deg] and (M) Vascular channel preferential angle [deg] against calcified cartilage collagen matrix preferential angle [deg]. The data used to create these plots can be found in page 286 in the appendices.

3021 3022 3023

Table 14 - Excerpts from the regression analysis, where each row shows correlation value of each histomorphological variable combination in the CONDEX and PASTEX, and also their difference. The alphabetical letters correspond to the letters in Figure 89 in the previous pages.

Treatment X-Y

CONDEX
Rp 0.59 0.17 0.34 0.20 0.36 0.42 0.30 0.06 0.04 0.42 0.93 0.67 0.64 P-value < 0.0001 0.0151 < 0.0001 0.0042 < 0.0001 < 0.0001 < 0.0001 0.3707 0.5926 < 0.0001 < 0.0001 < 0.0001 < 0.0001

PASTEX
Rp 0.08 0.07 0.23 0.38 0.27 0.33 0.25 0.01 0.09 0.34 0.93 0.49 0.52 P-value 0.3258 0.4110 0.0068 < 0.0001 < 0.0001 < 0.0001 0.0030 0.8346 0.3041 < 0.0001 < 0.0001 < 0.0001 < 0.0001

CONDEXPASTEX Rp +0.50 +0.1 +0.11 -0.18 +0.09 +0.09 +0.05 +0.05 -0.05 +0.08 0.00 +0.18 +0.12

A B C D E F G H I J K L M
3024 3025 3026 3027 3028 3029 3030 3031 3032 3033 3034 3035 3036 3037 3038 3039

HCt ZCCt HCt VCa HCt VCn HCt Rug VCa ZCCt VCn ZCCt Rug ZCCt VCa VCn VCa Rug VCn Rug HCang ZCCang HCang VCang VCang ZCCang

Overall, the regression analyses showed that out of 12 two-variable combinations, up to 9 were found with moderate to high correlation values (i.e. 0.93 Rp 0.3) with high statistical significance in the CONDEX (i.e. P < 0.05, see Table 14). These combinations included most of the non-angular variables (HCt, ZCCt, VCa, VCn) and all angular variables (HCang, VCang and ZCCang). Between the CONDEX and PASTEX, the Rp values of the CONDEX were usually higher than the PASTEX by up to +0.5 where the single biggest CONDEX/PASTEX difference was in the HCt-ZCCt pair. This was due to a very scattered distribution of the PASTEX rather than a high correlation in the CONDEX (see Figure 27-A). Overall, the HCt, ZCCt and VCn were shown to be moderately well correlated among each other suggesting that if a tissue exhibited a higher hyaline cartilage thickness (HCt), it would be also likely to have a thicker calcified cartilage (ZCCt) and a relatively high level of vascularity in the calcified zone (VCn). Also very strong positive correlations were detected among the angular variables HCang, ZCCang and VCang, where HCang-ZCCang with Rp = 0.93 (P 0.0001) while HCang-VCang and VCang-ZCCang registered more moderate levels of Rp = 0.60 and Rp = 0.59 respectably. [162]

3040 3041 3042 3043 3044 3045 3046 3047 3048 3049 3050

In conclusion, in both the CONDEX and PASTEX, there was strong evidence (P < 0.0001) that up to 7 of 12 combinations displayed moderate to strong correlations (Rp = 0.33~0.93) between two variables involving the hyaline cartilage (HCt), calcified cartilage (ZCCt) and vascular channels (VCa, VCn). However, the rugosity was not strongly correlated (Rp = 0.25~0.30). Overall the CONDEX correlation values were higher than the PASTEX by 0.09 to 0.5 in nearly all variables. The angular variables (i.e. HCang, VCang and ZCCang) displayed the highest correlations among the measured values, one of which, the HCang-ZCCang pairs from both CONDEX and PASTEX scored near-perfect correlation values of Rp = 0.93. The non-angular variables, the HCt, ZCCt and VCn, showed moderate correlations of approximately 0.3 to 0.4. The rugosity established weak correlations with either the HCt of PASTEX or the ZCCt of CONDEX but not at the same time in both groups.

3051 3052 3053 3054 3055 3056 3057 3058 3059 3060 3061 3062 3063 3064 3065 3066

H ISTOMORPHOMETRIC CHANGES IN THE TRAUMA AFFECTED REGIONS

Potential histomorphometric changes (HCt, ZCCt, VCa and VCn) due to the presence of traumatic osteochondrosis in the site 1 dorsal region in G13, G17 and G19 (as shown in page 120) were also investigated. In the analysis, only site 1 of the normal and osteochondrosis affected regions was used as there were not enough observations in site 2 to make the confident comparisons. The total numbers of observations in the normal site 1 vs. traumatic site 1 were 65 vs. 25 respectably. The students t-test was performed to test HCt, ZCCt, VCa and VCn distributions but not the Rug as the lesion affected sites did not have intact cement lines.

[163]

3067 3068

H YALINE CARTILAGE THICKNESS (HC T )

Hyaline cartilage thickness (HCt) histogram

30 25

Normal
Traumatic

20
Frequency
15 10 5 0 400
3069 HCt Normal Traumatic 3070 3071 3072 3073 3074 3075 3076 3077 3078 The hyaline cartilage thickness distributions in the normal and traumatic regions (i.e. the osteochondrosis sites) were shown to be different with a high level of significance (P = 0.0137) (Figure 90). The mean HCt is higher in the trauma region by +79 m while the HCt distribution in the normal region was more variable (i.e. high variance) and skewed towards the lower values peaking at 600 m. Mean (m) Variance (m) 603 681 24738 19684 t-statistics -2.2728 P-value 0.0137

500

600

700

800

900

1000 More

HCt - Thickness (m)

Figure 90 - Histograms of hyaline cartilage thickness (HCt) in the normal and traumatic osteochondrosis sites.

[164]

3079 3080

C ALCIFIED CARTILAGE THICKNESS (ZCC T )

Caclfied cartilage thickness (ZCCt) histogram

30
Normal 25 Traumatic

Frequency

20 15

10
5 0

ZZCt - Thickness (m)

3081 ZCCt Normal Traumatic 3082 3083 3084 3085 3086 3087 3088 3089 3090 3091 The calcified cartilage distribution (ZCCt) in the traumatic tissue was found to be different from the normal tissue (Figure 91), as already demonstrated in the histological images in the section of the thesis describing the lesion analysis (i.e. Analysis1, P = 0. 000003). The ZCCt in the non-osteochondrosis affected sites exhibited a normal distribution with a mean thickness of 228 m and a relatively tight variance of 2099 m, whereas the traumatic tissue displayed a near random distribution ranging from 250 m to over 700 m (figure 91). Mean (m) Variance (m) 228 452 2099 38072 t-statistics 1.7081 P-value 0.000003

Figure 91 -Histograms of calcified cartilage thickness (ZCCt) in the normal and traumatic osteochondrosis sites.

[165]

3092 3093

V ASCULAR CHANNEL NUMBER (VC N )

Vascular channel number (VCn) histogram

25
Normal 20 Traumatic

Frequency

15 10 5 0 8 11 14 17 20 23 26 29 VCn - Frequnecy

3094 VCn Normal Traumatic 3095 3096 3097 3098 3099 3100 3101 3102 With respect to the vascular channel number VCn, although the mean values of both distributions were close to each other, their differences in variance, skewness and overall patterns indicated that the VCn of the traumatic tissue was significantly different from the normal tissue (P = 0.0107). Mean (m) Variance (m) 17.98 15.92 16.58 12.49 t-statistics 1.6759 P-value 0.0107

Figure 92 -Histograms of vascular channel number (VCn) in the normal and traumatic osteochondrosis sites.

[166]

3103 3104

V ASCULAR CHANNEL AREA (VCA)

Vascular channel area (VCa) histogram

16 14 12 Normal Traumatic

Frequency

10
8 6 4 2 0

3105 VCa Normal Traumatic 3106 3107 3108 3109 3110 3111 3112

VCa - Area ( m2 )
Mean (m2) Variance (m2) 490320 624893 4.31E+10 5.36E+10 t-statistics 1.6839 P-value 0.0076

Figure 93 -Histograms of vascular channel area (VCa) in the normal and traumatic osteochondrosis sites.

The vascular channel area (VCa) in the traumatic tissue was distinctively different from the normal tissue (Figure 93); the mean of the traumatic tissue is higher than the normal (624893 vs. 490320 m2) with a high degree of significance (P = 0.0076).

[167]

3113

4.3.3 DISCUSSION

3114

U NDERSTANDING THE EFFECT OF EARLY EXERCISE USING

1. N ON - ANGULAR VARIABLES (HC T , ZCC T , VC N , VC A & R UG ) While cyclic loading induced by moderate exercise is crucial to the healthy joint development, in the current investigation, the effect of early exercise in the CONDEX was not so clear. Initial multivariate analysis of variance (MANOVA) indicated that there was no detectable evidence of any positive exercise effect in the CONDEX (P = 0.4271); and the univariate analysis of variance (ANOVA) similarly showed that only one significant response was detected (i.e. the vascular channel area (VCa)) out of the 10 variables tested (P = 0.0461, Figure 88H); however, the calcified cartilage thickness (ZCCt) did suggest a weak but positive exercise effect (P = 0.3413, Figure 88E). The area and frequency of primary vascular channels (i.e. VCa, VCn: vascular channels that are near the cement line) were used as indirect indicators of the possible tissue response to exercise and trauma. Vascular channels are thought to initiate the mineralization of the overlying hyaline cartilage [91, 94], which is a necessary process in the advancing of the tidemark and remodelling of the subchondral bone. This vascularity may be considered as a stress indicator in the joint since the number of vascular channels has been positively correlated with the stress concentration in the tibial plateau and femoral head in humans [111, 165]. However, the relationship between vasculature and exercise is not so clear; one study [167] reported that fewer vascular invasions occurred with mild exercise, whereas another study [154] reported that there was no significant difference. With the above in mind, using the vascularity of 5 exercised (CONDEX) and 4 control (PASTEX) horses, a strong positive correlation was detected in the primary vasculature space (VCa) in the CONDEX with exercise treatment (+36% to +57% in sites 1 to 4). However, with respect to the frequency of the vasculature (VCn), there was no evidence of an exercise effect (Figure 88).

3115 3116 3117 3118 3119 3120 3121 3122 3123 3124 3125 3126 3127 3128 3129 3130 3131 3132 3133 3134 3135 3136 3137

[168]

Exercise effect likely?

E - Calcified cartilage thickness (ZCCt)
350 300
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Exercise effect not likely?

B - Hyaline cartilage thickness (HCt)
1200 1000 800 600 400 200 0

CONDEX PASTEX

CONDEX PASTEX

Thickness [m]

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S3

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Number of vascular channels found
CONDEX PASTEX

25

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2.2
2.0 CONDEX PASTEX

Hyaline cartilage
Rugosity

1.8

1.6
1.4

E
1 2

Calcified cartilage
3

G,H
3

7 4 5 6 8

9 10

J
11

1.2 1.0

Subchondral bone

S1

S2

Site

S3

S4

3138 3139 3140 3141 3142 3143

Figure 88 - The HCt, ZCCt, VCn, VCa and Rug histomorphometric results. The capital letters in the title in each plot correspond to the region in the histological diagram above. While (E) the calcified cartilage thickness (ZCCt) and (H) the vascular channel area (VCa) showed some positive responses to the exercise treatment, other variables such as (B) the hyaline cartilage thickness (HCt), (G) the vascular channel number (VCn) and (J) the cement line rugosity (Rug) have shown to be a little or no effect. The error bars indicate 95% confidence intervals.

[169]

3144 3145 3146 3147 3148 3149 3150 3151 3152 3153 3154 3155 3156 3157 3158 3159 3160 3161 3162 3163 3164 3165 3166 3167 3168 3169 3170 3171 3172 3173 3174

The role of this vasculature is not very well understood, but possible roles either separately or in combination have been suggested: 1) they provide nutritional channels into the deeper layers of the hyaline cartilage [152, 153], 2) provide signalling pathways between bone and cartilage [154] and/or 3) initiate mineralization of the hyaline cartilage [91, 94]; where all of these are driven by age [165, 166], stress [111, 154, 165], exercise [154, 167], trauma [168, 176, 177] and pathologic processes [158, 163, 175]. Since the horses in this study were of the same breed and age, and exhibited no signs of sustained effusion [500], the only reasonable factor that might have caused the observed difference in vascularity between CONDEX and PASTEX reduces to one factor, namely exercise. It has been reported that the vasculature and calcified cartilage thickness are positively correlated to each other [91, 94]. However, this study found otherwise. While the CONDEX VCa was significantly higher than the PASTEX (i.e. +36% to +57%, P = 0.0461) this increase in the VCa was not matched with similar levels of increase in the ZCCt in the CONDEX. Conversely, the ZCCt in the PASTEX was higher than the CONDEX by +5% to +15% in sites 1 through 4, albeit at a low level of significance (P = 0.3413, see Table 12, Figure 88E). The thickness of calcified cartilage is determined by the activity of chondrocytes in the zone of calcified cartilage and chondroclasts at the cement line [94-96]. Both cells readily respond to mechanical loading and it has been reported that high compressive loads induce an increase in volume fraction and a reduction in mineralization of both the subchondral bone and calcified cartilage [92, 93]. Moreover, it has been suggested that advancement of the subchondral plate is inhibited by intermittent hydrostatic pressure generated in the deep layers of cartilage during cyclic loadings from weight bearing and muscular contraction [141-143]. Several authors have speculated that tidemark and chondro-osseous interfaces are coupled since they are both affected by common factors (such as a lack of intermittent and static pressure on the joint) in promoting their advancements [94, 120, 132, 144, 145]. Thus it may be possible that in the present study the imposed exercise may have provided the extra levels of cyclic loading in the CONDEX that caused the inhibition of calcified cartilage advancement and therefore, resulting in a thinner calcified layer in the CONDEX relative to the PASTEX. However, with the low statistical significance of the ZCCt (P = 0.3413), the above interpretation must be offered with some degree of caution. Thickening of calcified cartilage has been reported to [170]

3175 3176 3177 3178 3179 3180 3181 3182 3183 3184 3185 3186 3187 3188 3189 3190 3191 3192 3193 3194 3195 3196 3197 3198 3199 3200 3201 3202 3203

contribute to the progressive erosion and ulceration of the cartilage in horses [97], the thinner calcified cartilage in the CONDEX may be a welcoming consequence of the early exercise in the CONDEX. Nevertheless, it is difficult to postulate what actually caused the differences in the VCa and ZCCt levels between the CONDEX and PASTEX groups. The overall effect of the imposed exercise in the CONDEX was not generally significant (P = 0.4271) but the VCa and ZCCt have indicated that there might be some effect of exercise (PVCa = 0.0461, PZCCt = 0.3413). The results overall are in close agreement with the swelling analysis presented in Section 2 (see page 143), where it was reported that there was no significant effect of exercise on matrix swelling behaviour. Another parallel histomorphometric study [92] of the same horse but using different bones (third metacarpal distal condyle) also concluded that the imposed exercise induced little effect. So did the early exercise work? Not as much as was originally intuitively anticipated. However, it may have affected some aspects of the cartilage-bone of C3, for example, the vascular channel area (VCa) near the cement line. Although, we did not see any difference between the number of vascular channels (VCn), the area occupied by these channels was significantly greater in the CONDEX (P = 0.0461) with the greatest difference occurring at site 1 and then slightly lower but still consistently different throughout sites 2, 3 and 4 (Figure 88). The lack of a detectable exercise effect in the other measured variables (i.e. HCt, VCn, and Rug) suggests that the imposed exercise program might have not been intense enough to cause any distinct changes in the third carpal bones. Furthermore, in the HCt, VCn and Rug, the free-pasture activities in the CONDEX may have been greater than the effect of the imposed exercise, masking any possible treatment effect. The detection of any further differences is clearly rendered more difficult by the limitations of sample size imposed on this study.

2. I NTER - VARIABLE CORRELATIONS OF NON - ANGULAR VARIABLES (HC T , ZCC T , VC N , VC A , R UG ) In order to investigate any pattern of correlations among the five histomorphometric variables (HCt, ZCCt, VCa, VCn and Rug), the Pearsons correlation (Rp) was calculated for each combination of the five variables using simple linear regression analysis.

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3204 3205 3206 3207 3208 3209 3210 3211

Overall, in the CONDEX 6 out of 10 relationships (A, C, E, F, G and J in Table 14 and see Figure 94) were shown to exhibit moderate to strong correlations between the variable pairs (0.3 Rp 0.59), 2 out of 10 relationships (B and D) showed a weak (0.1 Rp < 0.3) but significant correlation (P 0.005), and the remaining 2 relationships (H and I) formed null correlations (Rp < 0.1). In terms of the overall magnitude of these correlation values, the CONDEX was higher than the PASTEX.

Table 14 Excerpts from the regression analysis showing only above the moderate levels of correlation values (i.e. Rp > 0.3). The alphabetical letters correspond to the letters in Figure 94.

Treatment X-Y

CONDEX Rp 0.59 0.17 0.34 0.20 0.36 0.42 0.30 0.06 0.04 0.42 P-value < 0.0001 0.0151 < 0.0001 0.0042 < 0.0001 < 0.0001 < 0.0001 0.3707 0.5926 < 0.0001

PASTEX Rp 0.08 0.07 0.23 0.38 0.27 0.33 0.25 0.01 0.09 0.34 P-value 0.3258 0.4110 0.0068 < 0.0001 < 0.0001 < 0.0001 0.0030 0.8346 0.3041 < 0.0001

CONDEXPASTEX Rp +0.50 +0.1 +0.11 -0.18 +0.09 +0.09 +0.05 +0.05 -0.05 +0.08

A B C D E F G H I J
3212 3213 3214 3215 3216 3217 3218 3219 3220 3221 3222

HCt ZCCt HCt VCa HCt VCn HCt Rug VCa ZCCt VCn ZCCt Rug ZCCt VCa VCn VCa Rug VCn Rug

The complex inter-variable relationships were explained with a structural equality diagram (see Figure 95). This is a common statistical method where each pair of variables is linked with a line of different thickness to indicate the relative strength of the correlation relationship (Rp). The diagram aids in the clarification of the overall inter-correlational relationships among the variables and also allows detection of a possible exercise effect in the CONDEX relative to the PASTEX.

[172]

3223 3224
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2.6

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3225 3226 3227

CONDEX (exercised)

PASTEX (control)

Figure 94 - Matrix scatter plots of HCt, ZCCt, VCa, VCn, Rug of the CONDEX and PASTEX. Please refer Table 14 for the calculated correlation values.

[173]

3228
0.42 0.35

Rug.
30 0.

0.20

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59 0.
0.42

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0. 34

0.38

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08 0.
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Exercise effect?

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0.07

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CONDEX
3229 3230 3231 3232 3233 3234 3235 3236 3237 3238 3239 3240 3241 3242 3243 3244 3245 3246 3247 3248 3249 3250 3251

Figure 95 - Structural equality diagrams of the histomorphometric correlation factors. Histomorphometric correlation values between each of two variables were used as the link-strength to represent their relative correlation strengths to each other. From the diagrams, it is clear to see whether there is an overall structural difference between the CONDEX and the PASTEX. In the CONDEX, the overall correlation relationship is stronger than the PASTEX. At the centre the CONDEX diagram, a triangular relationship is presented, which is consisted of HCt, ZCCt and VCn (red). Whereas in the PASTEX, the diagram is made up of two sub groups, HCt-Rug and ZCCt-VCn (red).

For example, as can be seen from the CONDEX portion of the diagram in Figure 95, HCt and ZCCt have a strong correlation of Rp = 0.59. If the CONDEX HCt experience an increase, the CONDEX ZCCt would also respond by increasing its value with a relatively high probability since the correlation is high between these two variables. Also since the HCt is correlated to the VCn with a lesser correlation factor of Rp = 0.34, the VCn would also experience an increase, but with a weaker probability. Furthermore, there could be a secondary effect from the increase in the HCt. Since the ZCCt increases as a consequence of the HCts increase, both the VCa and Rug would also increase but to lesser degrees. And because the Rug is linked to both the ZCCt and the VCn, the Rug will have a higher probability of increasing than other variables for any primal increase in HCt. The VCa, on the other hand, might not experience any significant increase since its correlation value with the HCt via the ZCCt route is relatively low (0.59 x 0.36). Further, the direct effect from the HCt has a low probability since the HCt-ZCCt linkage is only Rp = 0.17. The CONDEX and PASTEX groups were tested in order to determine which demonstrated the stronger inter-variable correlation. This was done using the structural equality of permutation test

0.17

VCa

PASTEX

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3252 3253 3254 3255 3256 3257 3258 3259 3260 3261 3262 3263 3264 3265 3266 3267 3268 3269 3270 3271 3272 3273 3274 3275 3276 3277 3278 3279

incorporating Fishers Z transformation with 100,000 permutations16 (see page 284 for the R source code). It was found that the CONDEX was significantly stronger than the PASTEX (P = 0.0520). The biggest difference between the groups was the HCt-ZCCt pair, where the CONDEX was considerably higher (Rp = +0.5, P = 0.00127). This may have been caused by the exercise treatment. As graphically shown in Figure 95, the HCt, ZCCt and VCn of the CONDEX form a triangular relationship whereas the PASTEX exhibits two subgroups; Rug-HCt and ZCCt-VCn, these two being linked by the VCn-Rug pair. From the diagrams, it can be inferred that a similar increase in the HCt in the CONDEX and PASTEX would induce different tissue responses, especially in the ZCCt values. The histomorphometric variables in the CONDEX would be likely to experience a higher likelihood of a coordinated increase; however, the variables in the PASTEX would have a lower probability of a coordinated increase, simply because of the weaker inter-variable correlations. The use of this method does make it easier to appreciate the tissue behaviour in the two groups from a more integrated perspective. The method might be able to provide a better prediction of tissue responses, and thus allow for more efficient design of a treatment experiment aimed at inducing a particular tissue reaction. From the correlation diagram and the permutation test, it is obvious the CONDEX and PASTEX behave differently. Whether this is due to the effect of early exercise is uncertain. However, the analysis does suggest that the imposed exercise on the CONDEX may have enhanced in the intervariable tissue responsiveness of the CONDEX relative to the PASTEX, although the ANOVA and MANOVA tests did not detect this with any certainty. So what might this mean in practical terms? If an exercise or other given treatment has induced a change in one aspect of the cartilage properties in the CONDEX and PASTEX, the CONDEX has higher probability than the PASTEX of exhibiting coordinated positive responses in its other related properties. It should be emphasized that this analysis, by virtue of the limited sample population, is limited to probabilities. The method presented here does not report that the CONDEX is likely to have a higher increase than PASTEX, but it states that if both groups experience an identical level of change (either

16

R, version 2.10.1, R Development Core Team, 2009: Vienna, Austria

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3280 3281 3282 3283 3284 3285 3286 3287 3288 3289 3290 3291 3292 3293 3294 3295 3296 3297 3298 3299 3300 3301 3302 3303 3304 3305 3306 3307 3308

positive and negative), the CONDEX has a higher probability of responding with positive changes in other aspects of its tissue properties (i.e. ZCCt, VCn, Rug) than the PASTEX. The important principle here is that this method does not deal with how much a certain variable increases or decreases. Rather, it takes account of inter-variable correlations that express the tendency of one variable following anothers increase or decrease. In an investigation of the treatment-effect type described in this thesis in which there is a very limited population, the primary concern is not so much how much? but whether there is a null, positive or negative effect. This study detected a significant shift in the inter-variable relationships in the CONDEX relative to the PASTEX (P = 0.052) possibly due to the exercise treatment. However, many of the previous exerciseinduced tissue response studies have failed to show a consistent response trend, and some of these results also conflicted to each other [3, 466, 470, 471, 546, 561]. Perhaps this is because the statistical analysis methods these investigators used considered only the absolute magnitudes of the data (i.e. mean, variance and error) rather than the overall complexity of tissue property relationships described in detail above. The CONDEX and PASTEX correlation diagrams presented above suggest that the imposed exercise may have induced a more integrated set of tissue responses in the CONDEX in comparison to the PASTEX, i.e. if the trainer wanted to induce changes in the cartilage, the CONDEX would be likely to respond more readily than the PASTEX. However, whether this change is always beneficial is another question entirely. 3. D ISTRIBUTION OF VASCULAR CHANNELS (VC ANG ) Students t-tests were performed in order to calculate whether there was a difference in the pattern of orientation distribution of the primary vascular channels (VCang) in the calcified cartilage and subchondral bone from the exercise treatment in the CONDEX in comparison to the PASTEX. The VCang distributions (Figure 96) indicated that both groups show very similar distributions and an exercise effect was not found (t-stat = 0.45, P = 0.3263). The mean values of VCang were very similar between the CONDEX and PASTEX with only a negligible difference (VCangCONDEX = -3.2 2.1, VCangPASTEX = -2.5 2.3). However, the CONDEX variance was higher than the PASTEX (VARCONDEX = 237 vs. VARPASTEX = 186). Regardless of the exercise treatment, the directions of vascular channel

[176]

3309 3310 3311

growth were found to be mostly perpendicular to the hyaline cartilage surface, although they ranged from approximately -40 to +40.

VCang CO NDEX v s PASTEX

t-Test: Two-Sample Assuming Unequal Variances CONDEX Mean Variance Observations df t Stat P(T<=t) one-tail t Critical one-tail P(T<=t) two-tail t Critical two-tail -3.17 237 200 320 0.4507 0.3263 1.6496 0.6525 1.9674 -60 -40 PASTEX -2.46 186 140

Histogram of VCang
F-Test Two-Sample for Variances
40
PASTEX 35 PASTEX

Number of vascular channels

CONDEX

CONDEX

Mean -2.45574 -3.16987 30 Variance 185.7877 236.7657 25 Observations 140 200 20 df 139 199 F 0.78469 15 P(F<=f) one-tail 0.063428 10 F Critical one-tail 0.77003
5 0 -20 0 VCang [de g ] 20 40 60

3312 3313 3314 3315 3316 3317 3318 3319 3320 3321 3322 3323 3324 3325 3326 3327 3328 3329

Figure 96 - Histogram of the vascular channel orientation (VCang) in the CONDEX and PASTEX of sites 1 to 4. Due to different observation numbers (CONDEX = 200, PASTEX = 140), the frequency of the CONDEX is overall higher. There is a very little difference in terms of the mean VCang values and variances. Both distributions are very similar to each other indicating that there is probably no overall exercise effect in the VCang.

The VCang distribution mean values of both the CONDEX and PASTEX were close to 0 (i.e. VCangCONDEX = -3.2 2.1, VCangPASTEX = -2.5 2.3, mean 95% C.I.) and clearly indicating that the majority of the vascular channels had the tendency to grow towards the superficial layer of the articular cartilage with little lateral bias regardless of the exercise treatment or site. It is possible that the branching orientation of the vascular channels is dependent on the cartilage surface geometry which is defined by the loading direction during early growth. This quantification of vascular channel orientation in a series of four sites in the radial facet of the third carpal bone is probably the first study of its kind. The study has found no evidence an exercise effect on the vascular orientation (P = 0.3263). However, the results suggest that vascular channel growth is likely to be dependent on loading direction. Further tests will be required to establish firmer evidence of this positive relationship.

[177]

Swelling Analysis
(a) Cohort mean swelling strain
30
25 1200 1000

Histomorphometric Analysis
B - Hyaline cartilage thickness (HCt)
CONDEX PASTEX

350

E - Calcified cartilage thickness (ZCCt)

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G - Vascular channel number (VCn)

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J - Cement line rugosity (Rug)

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C3

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Rugosity

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3330 3331 3332

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Figure 97 - Figure 81(a) and Figure 88(B, E, G & J) were exhibited on the same page above to illustrate how there is a common recurring site-specific behaviour in the tissue between the matrix swelling behaviour (a) to the histomorphometric results (B, E, G & J). The error bars indicate 95% confidence intervals.

3333 3334 3335 3336 3337 3338 3339 3340 3341 3342 3343 3344 3345 3346 3347

S ITE SPECIFICITY IN
From Figure 97, it is interesting to note that both the matrix swelling results and four of the five histomorphometric measurements (i.e. HCt, ZCCt, VCn and Rug but not VCa) share similar patterns of site-dependency and yet they differ in terms of the underlying mechanisms involved (i.e. osmotic swelling vs. resorptive remodelling). This suggests that the direction and magnitude of loading experienced by the tissue could be one of the most influential factors controlling site-specific responses in the joint.

1. H YALINE CARTILAGE THICKNESS (HC T ) Hyaline cartilage (HCt) is known to vary in thickness from area to area within the joint [64]. This variation is thought to be mostly affected by the resultant contact stresses generated by weight bearing and movement as well as from variations in joint congruency [562-564]. The magnitude of loading assumed to be positively correlated with thicker hyaline cartilage [111, 165, 562, 564] whereas, a reduction in loading results in cartilage thinning [130].

B - Hyaline cartilage thickness (HCt)

1200 1000 CONDEX PASTEX

Thickness [m]

800 600 400 200 0

S1
3348 3349 3350

S2

Site

S3

S4

Figure 98 - Hyaline cartilage thickness responses of the CONDEX and PASTEX. The plot suggests that HCt does not respond to the exercise. However, both the CONDEX and PASTEX display strong site-specific behaviours.

[179]

3351 3352 3353 3354 3355 3356 3357 3358 3359 3360 3361 3362 3363 3364 3365 3366 3367 3368 3369 3370 3371 3372 3373 3374 3375 3376 3377 3378 3379 3380 3381

However, the effect of exercise is not always positively correlated with the cartilage thickness. A mild to high intensity level of exercise was found to increase cartilage thickness in the tarsi [103] and carpal bones in both mature and immature horses [32, 565]. In contrast, a similar study under a high-intensity treadmill exercise failed to induce the same cartilage thickening effect in the equine carpal bones, but increased the calcified cartilage thickness in the region [566]. In the current study, there was no detectable exercise treatment effect on the cartilage thickness (HCt, P = 0.7385) whereas both the CONDEX and PASTEX exhibited thickest cartilage at sites 2 and 3 (HCt = 720~790m) versus thinner cartilage at sites at 1 and 4 (520~600 m, Figure 98). This finding was also demonstrated by Firth [565]. Thus, the results suggest that the strong site-specific variations in the HCt may be induced by an adaptational response in the cartilage arising from the loading in this region [562-565]. Cartilage thickening is generally considered to come from three mains sources: genetical factors [567] persistent localized patterns of loading across the joint [111, 165, 562, 564] and, in certain circumstances, exercise [32, 103, 565-568]. However, the HCt results of this study and the above noted literature suggest that the influence of sustained application of load (e.g. normal weight-bearing) may play a greater role in influencing the final cartilage thickness than intermittent high loading (e.g. intense exercise). Therefore, the thicker cartilage at sites 2 and 3 in relation to sites 1 and 4 may be induced by higher weight bearing loads presented at sites 2 and 3. Whereas, thinner cartilage thickness at site 1 and site 4 was probably caused by an infrequent loading (albeit high in magnitude) at site 1 and lower levels of loading at site 4 [53].

2. C ALCIFIED CARTILAGE THICKNESS (ZCC T ) Calcified cartilage is an intermediate layer between the hyaline cartilage and the subchondral bone; its thickness being determined by the calcification and resorption rates at each junction [94], and these in turn influenced by mechanical factors [100-103] including mode of loading [97, 107], immobilisation of the joint [108] and age [104-106]. However, in this study, the most influential factor is likely from joint loading resulting from both the autonomous and imposed exercise. Removing the influence of body weight on rats limbs by casting [569], and imposing high-intensity exercises on horses [103, 480], although representing opposite extremes on the load scale, both produced similar thickening effects on the calcified cartilage. On the other hand, mild to moderate amounts of early exercise in carpal bones failed to influence the calcified cartilage thickness [32]. It, [180]

3382 3383 3384 3385 3386 3387 3388 3389 3390 3391 3392 3393 3394

therefore, seems that both a lack of loading or excessive loading have the potential to induce thicker calcified cartilage, whereas a moderate amount of intermittent cyclic loading may inhibit calcified cartilage thickness increase. In this current study, the imposed level of exercise was believed to be well within the physiologic level (i.e. mild to moderate intensity). Therefore, if the exercise had any effect on the calcified cartilage thickness (ZCCt), it is likely to have been inhibitory, and therefore the calcified cartilage would be thinner in the CONDEX than in the PASTEX. This is demonstrated in Figure 97 albeit with a marginally low statistical significance (P = 0.3413). However, this does not explain the site-specific thickness variations along the sampling sites 1 to 4 in both the CONDEX and PASTEX. And since both share similar site-specific patterns (Figure 97E), the tissue response must have been induced by a common factor(s) that was influential in both groups. It is suggested that this is most likely to be the loading generated during normal weight bearing as well as the free-pasture exercise.

Calcified cartilage % portion in total cartilage thickness (ZCCt/(HCt+ZCCt))

35 CONDEX

31
27 27

PASTEX

ZCCt % in total cartilage thickness

30 25 20 15 10 5 0

26

22 22

21 22

S1
3395 3396 3397 3398 3399 3400 3401

S2
Site

S3

S4

Figure 99 - The ratio of the calcified cartilage thickness (ZCCt) over the total cartilage thickness (HCt+ZCCt) in each site. The plot shows that much higher proportions of calcified cartilage exist in sites 1 and 2 when compared to site 3 and 4. The differences are believed to be due to the topographical variation in loading magnitude (i.e. sites 1 and 2 experience higher intermittent stress). Both the CONDEX and PASTEX have shown sites 1 and 2 exhibit higher values of ZCCt ratio, whereas than sites 3 and 4.

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3402 3403 3404 3405 3406 3407 3408 3409 3410 3411 3412 3413 3414 3415 3416 3417 3418 3419 3420 3421 3422 3423 3424 3425 3426 3427 3428 3429

There is a distinct change in the percentage ratio ZCCt/total cartilage thickness (see Figure 99) between sites 1 and 2 (dorsal) to sites 3 and 4 (palmar), where this ratio in the dorsal regions ranged from 26-31%, whereas in the palmar sites it was around 22%. Norrdin et al. [514] have reported that at the dorsal region of C3, which is known to experience high intermittent loads, the calcified cartilage was thinner than at the lower loaded palmar region. Conversely, Tranquille et al. [103] and Murray et al. [566] observed the opposite - they reported that the high loaded regions exhibited thicker calcified cartilage in the distal tarsal and midcarpal joints respectably. The results in the current study also agree with Tranquille et al. and Murray et al., i.e. sites 1 and 2 (high loaded region) exhibited distinctively thicker calcified cartilage than sites 3 and 4 in both the CONDEX and PASTEX (Figure 99). Sites 1 and 2 are regions of frequent degenerative change in cartilage and bone, and often present with localised cartilage fibrillation and erosion, ischemic sclerosis and osteochondral fracture [505, 523]. The calcified cartilage thickness is thought to be positively correlated with the acceleration in the degenerative process in the hyaline cartilage [123]. The site-specific difference in the calcified thickness could arise from localised adaptations to increase the overall congruency in the joint [92, 570] and a similar adaptive process in hyaline cartilage has been proposed [567]. Increasing congruency may lead to a better conformation in the joint and thus redirect some of the high intermittent stress towards the centre of the concave joint surface. This would reduce the traumatic stress at the dorsal region. It would be interesting to investigate possible relations between the calcified cartilage morphology in concave-convex type joints in the pasture-free versus mild, moderate and intensively exercised horses.

3. V ASCULAR CHANNEL (VC N , VC A ) There are few studies available to provide a definitive relationship between the vascularity and sitespecific stress. In high loaded regions of the human tibial plateau, the femoral head [111, 165] and the equine third carpal bones [514], the vascular channels (VCn) were more frequent. However, there is also counter evidence that in the low-loaded region of the equine third metacarpal bone exhibiting a higher number of vascular channels [154].

[182]

G - Vascular channel number (VCn)

25

H - Vascular channel area (VCa)

800000 700000 600000 CONDEX PASTEX

Number of vascular channels found

CONDEX PASTEX

20

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Area [ 2 m]

500000 400000 300000 200000 100000

10

3430 3431 3432 3433 3434 3435 3436 3437 3438 3439 3440 3441 3442 3443 3444 3445 3446 3447 3448 3449 3450

S1

S2

Site

S3

S4

S1

S2

Site

S3

S4

Figure 100 - (G) The number of vascular channels (VCn) and (H) the total vascular channel areas (VCa) in sites 1 to 4 in the CONDEX and PASTEX. In the VCn, there is virtually no difference between the CONDEX and PASTEX whereas the CONDEX VCa is higher than the PASTEX by a significant amount. The error bars indicate 95% confidence intervals.

The VCn levels in both the CONDEX and PASTEX resembled the pattern of other variables (Figure 100G) such as the thickness of both the hyaline cartilage (HCt) and calcified cartilage (ZCCt), and rugosity (Rug) as discussed above. Thus, as with the HCt, ZCCt and Rug, the frequency of vascular channels (VCn) is likely to correlate with the variation in load caused by joint congruency and thus higher in the regions of high weight bearing (i.e. sites 2 and 3). Curiously, although the VCn of both the CONDEX and PASTEX were nearly identical, there was a significant difference in the VCa (PVCa = 0.0461), the CONDEX exceeding the PASTEX by 36% to 57%. Furthermore, only VCa showed a site-specific profile that was distinctively different from HCt, ZCCt, VCn and Rug (Figure 100H). The average area of a single vascular channel (VCa/n) in each site was calculated by dividing the mean vascular channel area (VCa) by the number of vascular channels (VCn) as shown in Figure 101 in the next page. From the plot, the CONDEX and PASTEX VCa/n in site 1 were significantly higher than sites 2, 3 and 4. This demonstrates that there is a strong site-specific distribution of VCa/n. Moreover, since the VCa response profile is radically different from the HCt, ZCCt, VCn and Rug profiles, and this suggests that the VCa causal mechanism may be different from the others.

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3451 3452 3453 3454 3455 3456 3457

Two points should be noted. Firstly, site 1, which exhibits the highest VCa, is also the region that is known to experience a higher level of stress intermittently applied during normal activities that would include galloping. Secondly, in contrast to the profiles of the other variables (the HCt, ZCCt, VCn and Rug), in the CONDEX the VCa profile is consistently elevated above that of the PASTEX (figure 101, P = 0.0461). This suggests that VCa is more sensitive to these intermittently applied loads rather than to the more sustained applied loads from weight bearing.

Vascular channel area per channel (VCa/VCn)

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30437

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19733

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3458 3459 3460 3461 3462 3463 3464 3465 3466 3467 3468 3469 3470 3471 3472 4. R UGOSITY OF CEMENT LINE (R UG )

Site

S3

S4

Figure 101 - Estimated mean vascular channel area (VCa) is divided by the mean number of channel (VCn) at each site to produce average area of single vascular channel per site (VCa/n). The plot describes how at site 1, the average area occupied by a single vascular channel is much larger than any other sites 2, 3 and 4 by more than 2 folds.

The significance of this finding may be that the existence of the larger vascular channels induced by the imposed exercise may becomes the sites of potential weakness within the calcified cartilage layer and the subchondral bone, which may then progress to become the fracture initiation sites under a traumatic load.

The degree of inter-digitation at the cement line measured by the rugosity is thought to be a strength indicator in the anchorage between the calcified cartilage and the subchondral bone [86, 87], and this hypothesis was successfully demonstrated correct by an in vitro shear osteochondral fracture study [571]. [184]

3473 3474 3475 3476 3477 3478 3479 3480 3481 3482 3483

In terms of site-specific rugosity (Rug), there is only one known study by Norrdin et al. [514] which has methodically measured the irregularity (similar to the rugosity, Rug) of the cement line in the third carpal bone in horses. It was reported that the rugosity tended to be greater at sites of high stress and that this higher rugosity correlated with a thinner calcified cartilage (ZCCt). [514]. In our study, we found however that the ZCCt-Rug relationship was not as clear as in the Norrdin et al. study (Figure 88E & J). There was only a weak positive correlation between calcified cartilage thickness (ZCCt) and the rugosity (Rug) in both the CONDEX and PASTEX (Rp,CONDEX = 0.2958, Rp,PASTEX = 0.3265, P > 0.0001). Surprisingly however, there was a strong site-specific dependence. This suggests that the rugosity changes may be linked to the site-specific adaptive changes in the joint as with the HCt and VCn (Figure 102).

350 300

E - Calcified cartilage thickness (ZCCt)

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3484 3485 3486 3487

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Figure 88E & J - The cement line rugosity in sites 1 to 4 from the CONDEX and PASTEX. There is virtually no difference between the groups with a minor exception in the site 1. The error bars indicate 95% confidence intervals.

J - Cement line rugosity (Rug)

2.2
2.0 1.8 CONDEX PASTEX

1200
1000 800 600 400

B - Hyaline cartilage thickness (HCt)

Number of vascular channels found
CONDEX PASTEX

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200
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3488 3489 3490 3491

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Figure 102 - Rugosity (J) has site-specific behaviour similar to HCt (B) and VCn (G). The error bars = 95% confidence intervals.

[185]

3492 3493 3494 3495 3496 3497 3498 3499 3500 3501 3502 3503 3504 3505 3506 3507 3508 3509 3510 3511 3512

5. C OLLAGEN FIBRILLAR ORIENTATION (HC ANG , ZCC ANG ) HCang and ZCCang are variables which define the dominant angle of collagen fibrillar orientation in the hyaline cartilage (HC) and zone of calcified cartilage (ZCC). Since most of the literature assumes collagen fibrils are aligned radially in the deep zone along with the chondrocyte alignment [84], in reality not all collagen fibrils are oriented this way but have distinct site-specific collagen orientations. The angular deviations were measured from the reference line that is perpendicular to the articular cartilage surface as shown in Figure 103 and Figure 104; and the flat surface of the articular cartilage provided consistency for these angular measurements. When there was no difference between the collagen orientation to the reference line (i.e. 90 degrees to the articular surface), the HCang or ZCCang registered zero. But as the tilting (or deviation) increased, the value became negative for the left side tilting (dorsal) and positive for the right side tilting (palmar). 200 CONDEX and 140 PASTEX angular measurements were collected and scatter-plotted, and their relative correlation values were calculated in Figure 103. The correlations for the HCang and ZCCang relationship were extremely strong, producing near perfect Rp = 0.93 in both groups thus indicating that the angular association between the overall collagen orientations in the hyaline cartilage and the calcified cartilage were nearly identical. There was a distinct site-specific distribution in the HCang and ZCCang, where dorsal sites (sites 1 & 2) occupied the third quadrant (both X & Y are negatives), whereas the palmar sites (sites 3 & 4) occupied the first quadrant (both X & Y are positives). It is interesting to note that collagen orientations at different sites have unique angular deviations as demonstrated in Figure 104.

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S1

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3513 3514 Treatment X-Y Hcang ZCCang 3515 3516 3517 3518 3519 CONDEX Rp 0.93 P-value < 0.0001

S1

S2

S3

PASTEX Rp 0.93 P-value < 0.0001

CONDEX-PASTEX Rp 0

Figure 103 - ZCCang and HCang (X-Y) scatter plots of the CONDEX and PASTEX. Each distribution contains data from sites 1, 2, 3 and 4, marked by similar but different tone of colours, where the rd CONDEX is in maroon and PASTEX is in blue. Both plots show strong linear relationships between HCang and ZCCang where dorsal sites (site 1 and 2) occupy 3 quadrant (-ve, -ve), and palmar st sites (sites 3 and 4) occupy 1 quadrant (+ve, +ve). These plots demonstrate that regardless of treatment, 1) both the ZCCang and HCang behave in near-unison and 2) the overall deviation behaviour is extremely site-specific.

S1

S2

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28 37

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32 17

10 6

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28 10 7 32 27 12 3 16

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37 6 15 17 37 11 12 21

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3520 3521 3522 3523 3524 3525 3526 3527 3528 3529 3530 3531 3532 3533 3534 3535 3536 3537

CONDEX

PASTEX

Figure 104 - Collagen and chondrocyte preferential orientation angles in hyaline cartilage (HCang) and calcified cartilage (ZCCang) in sites 1, 2, 3 and 4 from the CONDEX and PASTEX. The black arrows indicate the direction of deviation (left = negative = dorsal, right = positive = palmar) and the values at the bottom of each cone is mean deviatoric angles [] in each site. There is a little difference between the CONDEX and the PASTEX, but both showing strong and consistent sitespecific angular behaviours.

The orientations of the sites 1 and 2 deviated to the dorsal aspects, whereas those at the sites 3 and 4 deviated to the palmar aspects. The degree of deviation was largest at site 1 and site 4, decreasing at site 2 and site 3 and reducing further to a non-deviatoric collagen angle orientation somewhere between sites 2 and 3 (i.e. HCang = ZCCang = 0). The obvious question is why C3 displays such a distinct site-specific pattern of collagen fibrillar orientation. It is probably related to the direction of stress applied to the cartilage surface during the maturation period. The centre of applied stress must be somewhere between sites 2 and 3 so that the collagen fibrils directly under the load are loaded in pure axial compression. Conversely, the adjacent fibrils that are not directly under the load axis are sheared and compressed at the same time.

[188]

S1

S2

S3

S4

(a) Neonatal blank cartilage

(b) Joint loading starts

(c) Collagen realigns to the loading

3538 3539 3540 3541 3542 3543 3544 3545 3546 3547 3548 3549 3550 3551 3552

(d) Deep zone collagen deviation calcified

(e) Continual calcification of collagen deviation

(f) Removal of load & relaxation

Figure 105 - A proposed model of how the site-specific collagen orientations might arise in the hyaline and calcified cartilage. (a) Initially the neonatal cartilage collagen fibrils (light blue) have relatively straight and parallel alignments that are perpendicular to the tangential line of the cartilage surface (navy blue) in both the hyaline cartilage and calcified cartilage (green). (b) As the animal grows, the heavier load is applied axially to the joint surface (c) which causes the realignment of the collagen fibrils under the surface. The collagen fibrils in the centre of the joint surface (i.e. S2 & S3) are axially compressed, while the collagen fibrils at the outer rims (i.e. S1 & S4) are both compressed and sheared. (d) While the collagen fibrils are realigned to accommodate the oppositional joint surface, the collagen fibrils in the deep zone are continuously calcified making a series of snapshots of whatever the site-specific alignment deviations of the fibrils at that moment (red). (e) This calcification process continues over the years. (f) Once the load is removed and the section is incised from the animal, the site-specific collagen angles are prominent in the calcified cartilage as well as in hyaline cartilage in a lesser degree. The microphotographs of site 0 and 4 demonstrate that each site has a distinct collagen orientation; (S0) has no direct load, thus the collagen orientation in this region is neutral, the (S1, S4) demonstrate extreme deviations due to the shear stress, while the (S2-S3) exhibit neutral without deviation.

S1

S2-S3

S4

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C3
3553

Dorsal

Palmar

[189]

3554 3555 3556 3557 3558 3559 3560 3561 3562 3563 3564 3565 3566 3567 3568 3569 3570 3571 3572 3573 3574 3575 3576 3577 3578

A model proposing how collagen fibrils in the hyaline and calcified cartilage at the dorsal and palmar regions in C3 form such distinct deviatoric orientation patterns is shown in Figure 105. Initially, collagen fibrils in neonatal cartilage are relatively straight and parallel to each other in the middle and deep zone. But once the joint is compressed during standing still position or exercise, the opposing surfaces are compressed together. The collagen fibrils are then re-aligned to accommodate the opposing surface. The centre of the joint surface is axially compressed while the dorsal and palmar aspects of the joint are pushed aside as a combination of axial and shear deformations. However, because the ends of the collagen fibrils are already anchored in the calcified cartilage, there is an overall deviation of fibrils from the radial axis. As the joint develops, this deviation is calcified at the deep zone and becomes a permanent feature in the calcified cartilage like a snapshot in time. The ongoing calcification process further embeds the deviated fibrils. As the load is removed, the collagen fibrils in the hyaline cartilage relax but to a limited amount. This could explain why the deviatoric angles of the collagen fibrils in the hyaline cartilage are slightly less steep than the collagen in the calcified cartilage. As shown in Figure 105, both sites 1 and 4 exhibits collagen fibrils orienting outwards towards the dorsal and palmar aspect of the C3 respectably, while site 2-3 have virtually no deviations. The site 0, which is at the very edge of C3, is free from any joint contact (i.e. zero stress) and thus no deviation is present. However, this proposed model, without investigating the same region (C3) in more horses of in different age groups, cannot be fully verified, on the basis of the available data. The implication of such findings is not yet clear. However, multiple microcracks were consistently observed following the lines of collagen orientations at the site of traumatic osteochondrosis affected calcified cartilage layers as shown in Figure 106. Perhaps a more detailed understanding of the influence of the collagen fibrillar arrangement in the calcified cartilage region may provide significant advances in our understanding of microcrack formation and fracture in the calcified cartilage.

Damaged

3579 3580 3581 3582

Figure 106 - A cross-sectional slice from C3 dorsal aspect from G17 showing potential microcracks following the direction of collagen orientation (blue arrow) at the site with osteochondrosis (red arrow).

[190]

3583 3584 3585 3586 3587 3588 3589 3590 3591 3592 3593 3594 3595

O STEOCHONDROSIS EFFECT ON DORSAL REGION

The cause of traumatic osteochondrosis is thought be joint overloading [504, 572] which results in the sclerotic thickening of subchondral bone, vascular disturbances, and subsequent subchondral bone necrosis due to ischemia and hypoxia [573, 574]. Eventually, the devitalised bone is resistant to remodelling and this is thought to allow accumulation of micro-damage [575, 576] leading to further destruction to the joint. Vascularity has recently been studied in relation to age, stress distribution, trauma, exercise and osteoarthritis [111, 154, 165-167]. However, there is still little understanding of the relationship between vascularity and functional adaptation in the joint, as well as its role in the pathogenesis of joint diseases such as osteochondrosis. In an attempt to explore the relationship between vascularity and osteochondrosis in the dorsal region of C3, the hyaline cartilage thickness (HCt), calcified cartilage thickness (ZCCt), vascular channel area (VCa) and its frequency (VCn) was histomorphometrically analysed.
Normal
G24 Left S1 G13 Left S1

Traumatic osteochondrosis
G13 Right S1

G20 Left S1

G19 Left S1

G19 Right S1

3596 3597 3598 3599 3600 [191]

Figure 107 - Histomorphometric segment images highlighting the vast difference in the vascular channel area (VCa) morphology between the "normal" and "traumatic" osteochondrosis tissues acquired from site 1. (Blue = hyaline cartilage, Green = calcified cartilage & Red = vascular channels)

3601
(A) Hyaline cartilage thickness (HCt) histogram
30
Normal 25
Frequency

(B) Caclfied cartilage thickness (ZCCt) histogram

30
Normal

Traumatic
Frequency

25
20

Traumatic

20 15
10

15 10
5

5 0 400 500 600 700 800 900 1000 More

HCt - Thickness (m)

ZZCt - Thickness (m)

(C) Vascular channel area (VCa) histogram

16
14

(D) Vascular channel number (VCn) histogram

25 Normal 20
Frequency

Normal
Traumatic

12
Frequency

Traumatic

10
8

15 10 5 0
8 11 14 17 20 23 26 29

6
4

2 0

3602 3603 3604 3605 3606 3607

VCa - Area ( m2)

VCn - Frequnecy

Figure 108 - 4 comparative histograms for the histomorphometric distributions (HCt, ZCCt, VCa, VCn) of the dorsal region of C3 from the normal and osteochondrosis affected regions.

Table 15 - A summary of the students t-test results from the four histomorphometric variables (HCt, ZCCt, VCa, VCn) comparing the normal to the osteochondrosis affected regions in site 1.

HCt (m) Normal

( SD)

ZCCt (m) 228

46

VCa (m2) 490320

207596

VCn 17.98
4.1

603
157

Trauma
( SD)

681
140

452
195

624893
231605

15.92
3.5

P-value 3608 3609

0.0137

<0.0001

0.0076

0.0107

[192]

3610 3611 3612 3613 3614 3615 3616 3617 3618 3619 3620 3621 3622 3623 3624 3625 3626 3627 3628 3629 3630 3631 3632 3633 3634 3635 3636 3637 3638 3639 3640

The overall results indicated that there is a relatively strong evidence that trauma affected all aspects of the histomorphometric features (i.e. HCt, ZCCt, VCa and VCn; see Figure 107). The comparative histomorphometric results from the normal and the osteochondrosis affected tissues are illustrated in Figure 108 and their t-test summary results in Table 15. Regarding the overall morphology, the traumatic tissues with obviously enlarged calcified cartilage regions (Figure 107) exhibited more prominent vasculature with thicker channels, developed directly towards the calcified cartilage layer, and forming approximately 90 without much deviation towards left or right. However, in the normal tissue the vascular morphology was vastly different from the trauma tissue; the channels were shorter and less prominent, more or less randomly oriented where they were present in a smaller volume fraction in the subchondral bone (Figure 107). As for the thickness measurements, the HCt and ZCCt were both higher in the trauma tissue by approximately +13% (HCt = 79 m) and 99% (ZCCt = 224 m) with high levels of significance (PHCt = 0.0137, PZCCt 0.0001). In terms of the vascularity, VCa was again higher in the trauma tissue by 27% ( = 134573 m2, P = 0.0076). Conversely, the VCn was higher in the normal tissue by a marginal value of +2.06; this difference being statistically significant (P = 0.0107). From the above, we can conclude that although trauma did not affect the hyaline cartilage thickness, it caused collapse of the subchondral bone and enlargement of the calcified cartilage. It should be noted that nearly all of the osteochondrosis affected sites displayed normal healthy articular surfaces without any obvious superficial damage. These findings suggest that the collapsing of the subchondral bone was either gradual, so that the cartilage was not exposed to the sudden loss of underlying support from the subchondral bone, or the collapse may have been quickly rectified by the wound healing process, with the chondrocytes and blood-borne stem cells filling the region with the newly synthesized collagen matrix that quickly calcified into a new supporting tissue in a relatively short time. This latter hypothesis would help explain why the scarred calcified cartilage matrix morphology is uneven, consistent with it being a mixture of old and new calcified matrix (see Figure 68 and Figure 69 on page 124). In terms of matrix quality, the hyaline cartilage in the trauma sites, although superficially normal in appearance, may be more degenerate as it was demonstrated earlier that a +4.2% increase in the matrix swelling strain was detected compared to the normal tissue (P = 0.04, see page 145).

[193]

3641 3642 3643 3644 3645 3646 3647 3648 3649 3650 3651 3652 3653 3654 3655 3656 3657 3658 3659 3660 3661 3662 3663 3664 3665 3666 3667 3668 3669 3670 3671 3672

The VCa level of the traumatic tissue was also 27% higher than the normal sites (P = 0.0076). This higher level of vascularity in these trauma regions may reflect the need to supply higher amount of oxygen and nutrients to support the remodelling activities in the bone. Alternatively, it might reflect support of the initial resorption process before laying down new bone tissue of a higher stiffness and mineralization. It is even possible that the increased vascularity could a result of the osteochondrosis, induced by the subsequent repair process in the subchondral bone. It seems counter-intuitive to imagine that the region which is subjected to high intermittent load is then destabilised via the excessive enlargement of the vascularity area (or marrow space) in the supporting architecture as discussed up to this point. However, this is what is observed in the regions adjacent to the osteochondrosis affected sites, where nearly all exhibited high vascular channel areas (VCa) in the subchondral bone regions proximal to the lesion locations. Furthermore, these tissues also presented with a large amount of porosity in the trabecular regions as shown in Figure 109. From these observations, a number of important questions arise: Is the large increase in the VCa a response to the high intermittent load, a natural remodelling process or a pathological process? Can it be prevented or controlled? How feasible is it to detect these signs early to prevent osteochondrosis? Answers to such questions are clearly required but they lie beyond the scope of the current study. VCn on the other hand was found to be higher in the normal tissue than in the trauma tissue (i.e. the osteochondrosis). This was somewhat unusual as the frequency of VCn is thought to be correlated positively to the highly loaded regions of the joint [111, 165, 514]. But perhaps the loading pattern was not sufficiently different between the normal and trauma tissues to induce changes in the VCn, while the damaging force that induced the osteochondrosis was abrupt and infrequent. However, this is really only speculation. The current study has demonstrated that VCa is highly dependent on the stress that is produced from mild to moderate levels of exercise, and the VCa enlargement is further magnified by the high intermittent load that is experienced in the dorsal regions of C3. It is therefore suggested that the high intermittent load induces high levels of vascular area (VCa) in the dorsal region of C3, which may have provided initial sites of destabilisation in the region susceptible to traumatic loading, resulting in the collapse of the subchondral bone and, in turn, increasing the probability of traumatic osteochondrosis in the dorsal regions of both C3 and Cr as shown in Figure 109.

[194]

G13 L

G13 R

G17 L

G17 R

G19 L

G19 R

3673 3674 3675 3676 3677 3678

Figure 109 - The dorsal regions of C3 and Cr of G13, G17 and G19 were found with osteochondrosis [514]. The shapes and sizes of the lesion were nearly oppositionally identical in G13 and G17 suggesting that traumatic loads were the cause of these lesions. The trabecular bone regions beneath the lesion sites were shown to be unusually porous (red arrows) than sites without the lesions when non-osteochondrosis G17R is examined in conjunction with the rest.

[195]

3679 3680 3681 3682 3683 3684 3685 3686 3687 3688 3689 3690 3691 3692 3693

E XERCISE TREATMENT AND SITE - SPECIFICITY

All histomorphometric variables measured in this investigation displayed site-specific responses, whereas, in terms of the effects of exercise, only the VCa (P = 0.0461) and ZCCt (P = 0.3413) displayed non-zero responses. With respect to site-specific behaviour, there were two somewhat different categories of behaviour observed; one in which the variables displayed a parabolic trend ( ) along the sites 1 to 4, where

the maximums occurred at site 2 or 3 (Figure 110), and the other displayed the highest values in the dorsal sites 1 and 2 and decreasing towards palmar sites 3 and 4 (
, Figure

111).
,,

Interestingly, those tissue responses with high values in the dorsal sites ( exercise treatment whereas those in the first category (

also responded to the

) did not. The hyaline cartilage thickness

(HCt), vascular channel number (VCn) and cement line rugosity exhibited behaviour in the first category (
, Figure

110). Conversely the vascular channel area (VCa) and calcified cartilage thickness ), and also exhibited a mild to moderate exercise

(ZCCt) exhibited second category behaviour ( effect (Figure 111).

B - Hyaline cartilage thickness (HCt)

1200

G - Vascular channel number (VCn)

25

J - Cement line rugosity (Rug)

2.2

1000

Number of vascular channels found

CONDEX PASTEX

CONDEX PASTEX

20

2.0
1.8

CONDEX PASTEX

Thickness [m]

800 600 400

Rugosity

15

1.6 1.4

10

200
0

1.2

1.0

3694 3695 3696 3697 3698 3699 3700 3701 3702

S1

S2

Site

S3

S4

S1

S2

Site

S3

S4

S1

S2

Site

S3

S4

Figure 110 - The hyaline cartilage thickness (HCt), vascular channel number (VCn) and cement line rugosity exhibited a non-response to the exercise treatment with a parabolic site-specific pattern.

Regardless of the exercise treatment, the HCt, VCn and Rug from both groups showed near-identical tissue responses (Figure 110) indicating that the imposed exercise had no effect on them. However, some stimulus must have triggered the parabolic site-specific behaviours ( ) seen in both the

CONDEX and PASTEX. The common stimulus for the HCt, VCn and Rug was clearly equally active in both groups and thus unlikely to have arisen from the imposed exercise (i.e. not CONDEX exclusive).

[196]

3703 3704 3705 3706 3707 3708 3709 3710 3711 3712 3713 3714 3715 3716 3717 3718

Rather, it probably arose either from the free-pasture exercise (which was available for both groups), or the weight bearing load, or a combination of these two. Also, another clue of the stimulus identity may exist in the parabolic shapes of these tissue responses. Since the shape of the site-specific response was parabola ( ), the magnitude of the

stimulus is likely to have followed a comparable parabolic pattern along the sites 1 to 4, and this could only have been achieved by a matching distribution of weight bearing stress across the physiological contour of the joint. On the other hand, if the free-pasture exercise was the stimulus for the HCt, VCn and Rug, being cyclic in nature, would have induced high stresses in the dorsal sites which decrease towards the palmar regions (i.e. second category response i.e. Rug did not exhibit the second category ( ). However, as demonstrated, the HCt, VCn and ); and therefore the ).

), but the first category response (

free-exercise is not likely to have affected the HCt, VCn and Rug variables (i.e.

It is therefore suggested that the HCt, VCn and Rug all responded to loads that were applied continuously, i.e. stresses generated from normal weight bearing. It is also possible that while these same stresses were still the major stimulant to induce site-specific changes in these variables, the exercise treatment was not significant enough to trigger any additive effects.

[197]

ZCCt % in total cartilage thickness

Calcified cartilage % portion in total cartilage thickness (ZCCt/(HCt+ZCCt)) 33 31 CONDEX

31
29 27

E - Calcified cartilage thickness (ZCCt)

350
CONDEX PASTEX

PASTEX

27
Thickness [m]

300

25 23
21

27

26

250

22
22

21

200

22

19 17
15

150

100

S1

S2
Site

S3

S4

S1

S2

Site

S3

S4

Vascular channel area per channel (VCa/VCn)

35000 30000 25000

H - Vascular channel area (VCa)

800000 700000 600000

30437

CONDEX PASTEX

CONDEX PASTEX

Area [ 2 m]

20000 15000 10000 5000 0

19733

15212

Area [ 2m]

500000

13068

13071

400000 300000 200000

11361

9502

8513

100000 0

S1

S2
Site

S3

S4

S1

S2

Site

S3

S4

3719 3720 3721 3722 3723 3724 3725 3726 3727 3728 3729 3730 3731

Figure 111 - The vascular channel area (VCa, VCa/n) and calcified cartilage thickness (ZCCt and its ratio), have exhibited a non-zero response to the exercise treatment with a high sensitivity in dorsal sites 1 and 2.

By contrast, the VCa and ZCCt may be sensitive to intermittent/cyclic loads, which only occur during the exercise activities such as in the imposed exercise and the free-pasture exercise. Evidence to support this proposition is obtained from the fact that both the VCa and ZCCt responded to the exercise treatment - the CONDEX VCa level increased and the CONDEX ZCCt decreased (Figure 111). This clearly indicates that the VCa and ZCCt are sensitive to the intermittent/cyclic type loads. Furthermore, the VCa and ZCCt responses were highest at the dorsal regions and drastically reduced in the palmar sites ( ), thus indicating the magnitude of the stimulus was also greatest in the dorsal ).

region [53] while decreased significantly at the palmar region (

These responses are consistent with the high dorsal stresses that are generated from the gallop exercise during the free-pasture and the imposed exercise treatment. Furthermore, since the VCa [198]

3732 3733 3734 3735 3736

and ZCCt were sensitive to the intermittent/cyclic type loads, the additionally imposed exercise in the CONDEX may have further accentuated the tissue response in the CONDEX VCa while inhibiting the CONDEX ZCCt levels; and such changes are demonstrated in Figure 112 thus strengthening the credibility of this hypothesis.

Tissue response

(4) High intermittent load = imposed exercise

(3) Intermittent load = pasture exercise

C ON DEX PASTE X
(2) Constant load = weight bearing (1) Neonatal = blank

VCa and ZCCt HCt, VCn and Rug CONDEX+PASTEX

S1

S2

S3

S4

Dorsal

3737 3738 3739 3740 3741 3742 3743 3744 3745 3746 3747 3748 3749 3750 3751 3752

Site

Palmar

Figure 112 -Proposed mechanism of how cartilage (HCt), calcified cartilage (ZCCt), vascular channel (VCa, VCn) and subchondral bone cement line (Rug) tissue develop their site-specific postnatal tissue response from moderate but regularly applied load (weight bearing) and high but intermittently applied load induced during galloping.

In brief, here is what is proposed to explain how the cartilage and bone of C3 might develop its sitespecific characteristics during the development and under the imposed exercise program based on what has been reported so far. Regardless of the exercise, the HCt, VCn and Rug respond to the constantly applied weight bearing loads during the stance position, causing localised adaptation of the tissues from their more homogeneous postnatal state [473, 577] to the parabolic profile that reflects the joint contour ((1) (2) in Figure 112). However, with respect to the VCa and ZCCt, their sensitivity to the intermittent/cyclic loads of the free-pasture activities transform their weight bearing response pattern (i.e. decay type response (
, (2)

) to a logarithmic

(3) in Figure 112). Because the VCa and ZCCt are sensitive to the

intermittent/cyclic loads, the extra amount of loading available in the CONDEX through the exercise treatment causes an overall increase in the VCa, while suppressing the ZCCt ((3) (4) in Figure 112).

[199]

3753 3754 3755 3756 3757 3758

The implications of the above findings, if they are correct, are considerable. Under a physiologic level of early exercise, the trainer has little or no control over the hyaline cartilage thickness (HCt), vascular channel number (VCn) or cement line rugosity (Rug). However, through moderate levels of early exercise, it might be possible to increase the vascular channel area (VCa) especially in the high intermittent loaded sites whilst simultaneously suppressing the thickness of the calcified cartilage (ZCCt).

3759

[200]

3760

[201]

3761 3762 3763 3764 3765

5. SUMMARY OF EACH FINDING

[202]

3766

3767

[203]

3768

OVERALL EXERCISE EFFECTS

Even at the relatively early age of 18 months, lesions were found at five distinct locations throughout the joint surface of both the CONDEX and PASTEX (Figure 113), where 10 out of 12 horses had some lesions, 3 out of 12 horses had signs of traumatic osteochondrosis, of which 2 were from the PASTEX, the control group (Table 17).
CONDEX/Exercised
Ci Cu Cu

3769 3770 3771 3772

Left
Cr Ci

Right
Cr Cr

Left
Ci

PASTEX/Control

Right
Ci Cr

Cu

Cu

C4 C3

C2

C2 C3

C4

C4 C3

C2

C2 C3

C4

Traumatic

Unique

C3 ridge

Cr ridge

C2 ridge

3773 3774 3775 3776 3777

Figure 113 - Superimposed lesions from the lesion affected sites in the CONDEX and PASTEX groups. Table 16 - A summary of the number lesions found in the CONDEX and PASTEX. Except the traumatic osteochondrosis lesions found at the dorsal radial facet of C3/Cr, overall there is no clear difference between the groups.

Osteochondrosis CONDEX PASTEX 3778 3779 3780 3781 3782 3783 3784 3785 3786 3787 3788 3 6

Unique 2 2

C3 ridge 6 8

Cr ridge 2 2

C2 ridge 4 4

The prevalence of osteochondrosis-type lesions, which is likely to have serious consequences during the race career of the horse, was surprisingly high even for pasture raised animals. This raises the question as to whether some of the tested horses were destined for lesions during their early growth phase even in conditions where they were not subjected to any kind of imposed exercise (i.e. PASTEX). The lower prevalence of traumatic osteochondrosis in the CONDEX is an encouraging finding but it must be remembered that the study was restricted to a very small sample size. The exercise program may have made the CONDEX develop stronger muscles and tendons around the joint, overall making the joint more resistant to low level trauma resulting from the rough and tumble of normal pasture life for the developing animal.

[204]

3789 3790 3791 3792 3793 3794 3795 3796 3797 3798 3799

Common sense tells us that adopting early exercise in children would make them more agile and stronger than inactive children. Once they experience the freedom and potential risks of the playground, the early exercised children would have the enhanced ability/agility to avoid injury. This may well have happened to the CONDEX horses. Concerning matrix swelling, no significant effect of exercise was detected (P = 0.7951, Table 17). However, there were significant differences in terms of matrix swelling between the sampling sites 1-4 (P = 0.0016), C3 and Cr (P = 0.041), as well as left to right (P = 0.3674, see page 137).

Table 17 - A summary of excerpted data from the univariate ANOVA. In terms of the exercise treatment, only the VCa (vascular channel area) was shown to be significant (P = 0.0461) among the variables tested. However, overall, the site was again found to be a highly significant factor in all variables.

3800 3801 3802 3803 3804 3805 3806 3807 3808 3809 3810 3811 3812 3813 3814 3815

Variable HCt ZCCt VCa VCn Rug

Null hypothesis Treatment Treatment Treatment Treatment Treatment

Num df 1 1 1 3 3

Den df 7 7 7 18 18

F Value 0.12 1.04 5.86 0.05 0.33

Pr > F 0.7385 0.3413 0.0461 0.8234 0.5848

Histomorphometric quantification of the hyaline cartilage, calcified cartilage, cement line and vasculariture, allowed a number of important conclusions to be drawn. The area occupied by the vascular channels (VCa) just beneath the calcified cartilage in C3 was significantly higher in the CONDEX by a considerable margin of +36 to +57% (P = 0.0461, see Figure 114). The CONDEX registered a thinner calcified cartilage layer by -5% to -15% in sites 1 through 4 albeit with a low statistical significance (P = 0.3413). It seems that early exercise may have caused more activity in the subchondral region of the C3 by increasing its vascularity (VCa) presence and therefore inducing more active advancement of the subchondral bone, and consequently reducing the overall thickness of the calcified cartilage layer (ZCCt) without sacrificing the hyaline cartilage thickness (HCt). Calculation of the inter-variable correlations of all the variables measured, revealed a positive effect of exercise in the CONDEX (P = 0.052, Figure 115) thus suggesting that these animals are more likely to undergo coherent adaptive changes in the hyaline cartilage (HCt), calcified cartilage (ZCCt), cement line (Rug) and vascularity (VCa, VCn) in response to the external stimuli of the exercise treatment than the PASTEX.

[205]

Exercise effect likely?

E - Calcified cartilage thickness (ZCCt)
350 300
250

Exercise effect not likely?

B - Hyaline cartilage thickness (HCt)
1200 1000 800 600 400 200 0

CONDEX PASTEX

CONDEX PASTEX

Thickness [m]

200 150 100 50

S1

S2

Site

S3

S4

Thickness [m]

S1

S2

Site

S3

S4

H - Vascular channel area (VCa)

800000

G - Vascular channel number (VCn)

Number of vascular channels found
CONDEX PASTEX

25

700000
600000

CONDEX PASTEX

20

Area [ 2 m]

500000

15

400000 300000
200000 100000

10

S1

S2

Site

S3

S4

S1

S2

Site

S3

S4

J - Cement line rugosity (Rug)

2.2
2.0 CONDEX PASTEX

Hyaline cartilage
Rugosity

1.8

1.6
1.4

E
1 2

Calcified cartilage
3

G,H
3

7 4 5 6 8

9 10

J
11

1.2 1.0

Subchondral bone

S1

S2

Site

S3

S4

3816

[206]

3817 3818 3819 3820 3821

Figure 114 - The HCt, ZCCt, VCn, VCa and Rug histomorphometric results. The capital letters in the title in each plot correspond to the region in the histological diagram above. While (E) the calcified cartilage thickness (ZCCt) and (H) the vascular channel area (VCa) showed some positive responses to the exercise treatment, other variables such as (B) the hyaline cartilage thickness (HCt), (G) the vascular channel number (VCn) and (J) the cement line rugosity (Rug) have shown to be a little or no effect. The error bars indicate 95% confidence intervals.
0.42 0.35

Rug.
30 0.

0.20

HCt
59 0.
0.42

Rug.
0. 34

0.38

HCt
08 0.
0.33

0. 23

25 0.

ZCCt
36 0.

VCn

Exercise effect?

ZCCt
27 0.

VCn

0.07

VCa

CONDEX
3822 3823 3824 3825 3826 3827 3828

Figure 115 - Structural equality diagrams of the histomorphometric correlation factors. In the CONDEX, the overall correlation relationship is stronger than the PASTEX. At the centre the CONDEX diagram, a triangular relationship is presented which is consisted of HCt, ZCCt and VCn (red). Whereas in the PASTEX, the diagram is made up of two sub groups, HCt-Rug and ZCCt-VCn (red).

The table below summarises, the above: Exercise effect? I. Lesion analysis No But...
Osteochondrosis observations were higher in the PASTEX VCa was significantly higher in the CONDEX (P = 0.0461)

II. Matrix swelling analysis

III. Histomorphometric analysis

IV. Structural equality test 3829 3830 3831 3832 3833 3834 3835 3836

The above findings indicate that the imposed early exercise may have reduced the incidence of traumatic osteochondrosis in the dorsal regions of C3 and Cr without triggering any significant changes in the matrix as monitored by the degree of swelling. The exercise in the CONDEX significantly increased the vascular channel area (VCa) in the subchondral plate and may have enhanced a more coherent tissue response in the cartilage and subchondral bone in comparison to the PASTEX. [207]

0.17

VCa

PASTEX

No (P = 0.795)

No (P = 0.4271)

Yes (P = 0.052)

3837

SITE SPECIFIC RESPONSE AND EXERCISE

Two different patterns of site-specific behaviour were observed; one that gave a parabolic trend ( ) along the sites 1 to 4 (Figure 116), and another pattern that exhibited high values in the dorsal sites 1 and 2 and then lower values at palmar sites 3 and 4, similar to a logarithmic decay response (
,

3838 3839 3840 3841 3842 3843

Figure 117). The parabolic response group (HCt, VCn and Rug) were insensitive to the exercise treatment whereas those exhibiting the logarithmic response (VCa and ZCCt) were sensitive to the exercise treatment.
B - Hyaline cartilage thickness (HCt)
1200

G - Vascular channel number (VCn)

25

J - Cement line rugosity (Rug)

2.2

1000

Number of vascular channels found

CONDEX PASTEX

CONDEX PASTEX

20

2.0
1.8

CONDEX PASTEX

Thickness [m]

800 600 400

Rugosity

15

1.6 1.4

10

200
0

1.2

1.0

3844 3845 3846 3847

S1

S2

Site

S3

S4

S1

S2

Site

S3

S4

S1

S2

Site

S3

S4

Figure 116 - The hyaline cartilage thickness (HCt), vascular channel number (VCn) and cement line rugosity exhibited a non-response to the exercise treatment in a parabolic site-specific pattern.

33

ZCCt % in total cartilage thickness

31
29 27

Calcified cartilage % portion in total cartilage thickness (ZCCt/(HCt+ZCCt)) 31 CONDEX

PASTEX

E - Calcified cartilage thickness (ZCCt)

350
CONDEX PASTEX

27
Thickness [m]

300

25 23
21

27

26

250

22
22

21

200

22

19 17
15

150

100

S1

S2
Site

S3

S4

S1

S2

Site

S3

S4

Vascular channel area per channel (VCa/VCn)

35000 30000 25000

H - Vascular channel area (VCa)

800000 700000 600000

30437

CONDEX PASTEX

CONDEX PASTEX

Area [ 2 m]

20000 15000 10000 5000 0

19733

15212

Area [ 2m]

500000

13068

13071

400000 300000 200000

11361

9502

8513

100000 0

S1

S2
Site

S3

S4

S1

S2

3848 3849 3850

Site

S3

S4

Figure 117 - The vascular channel area (VCa, VCa/n) and calcified cartilage thickness (ZCCt and its ratio), have exhibited a non-zero response to the exercise treatment with a high sensitivity in dorsal sites 1 and 2.

[208]

Tissue response

(4) High intermittent load = imposed exercise

(3) Intermittent load = pasture exercise

C ON DEX PASTE X
(2) Constant load = weight bearing (1) Neonatal = blank

VCa and ZCCt HCt, VCn and Rug CONDEX+PASTEX

S1

S2

S3

S4

Dorsal
3851 3852 3853 3854 3855 3856 3857 3858 3859 3860 3861 3862 3863 3864 3865 3866 3867 3868 3869

Site

Palmar

Figure 118 - Proposed mechanism of how cartilage (HCt), calcified cartilage (ZCCt), vascular channel (VCa, VCn) and subchondral bone cement line (Rug) tissue develop their site-specific postnatal tissue response from moderate but regularly applied load (weight bearing) and high but intermittently applied load induced during galloping.

It is hypothesised that the two categories of site-specific tissue responses demonstrated in the study arise in the following manner. The logarithmic response group responded to the intermittent/cyclic loads generated during the imposed and free-pasture exercise. By contrast, the parabolic response group may have responded only to the constantly applied weight bearing loads as shown in Figure 118. The logarithmic response group would have been further elevated with the additional imposed exercise. Implicit in the hypothesis proposed above is the idea that a physiologic level of early exercise can induce changes in the vascular channel area (VCa) and the calcified cartilage thickness (ZCCt) while the hyaline cartilage thickness (HCt), vascular channel number (VCn) and cement line rugosity (Rug) may remain unaffected. The consequences of there being such a drastic increase in the vascularity (VCa) may well be that there are now easy local sites for microfracture initiation; which might progress to catastrophic fracture upon continued application of traumatic loads. Please refer to page 179 for further details for this topic.

3870 [209]

3871

TRAUMATIC OSTEOCHONDROSIS EFFECTS

The influence of traumatic osteochondrosis in the dorsal region of C3, was investigated with respect to its hyaline cartilage thickness (HCt), calcified cartilage thickness (ZCCt), vascular channel area (VCa) and vascular channel frequency (VCn, see Figure 119).
Normal
G24 Left S1 G13 Left S1

3872 3873 3874

Traumatic osteochondrosis
G13 Right S1

G20 Left S1

G19 Left S1

G19 Right S1

3875 3876 3877 3878 3879 3880 3881

Figure 119 - Histomorphometric segment images highlighting the vast difference in the vascular channel area (VCa) morphology between the "normal" and "traumatic" osteochondrosis tissues acquired from site 1. (Blue = hyaline cartilage, Green = calcified cartilage & Red = vascular channels) Table 18 - A summary of the students t-test results from the four histomorphometric variables (HCt, ZCCt, VCa, VCn) comparing the normal to the osteochondrosis affected regions in site 1.

HCt (m) Normal

( SD)

ZCCt (m) 228

46

VCa (m2) 490320

207596

VCn 17.98
4.1

603
157

Trauma
( SD)

681
140

452
195

624893
231605

15.92
3.5

P-value 3882 3883

0.0137

<0.0001

0.0076

0.0107

[210]

3884 3885

The effects of trauma were detected in all four categories of measurements; ZCCt, VCa, VCn and HCt in the order of high to low significance (Table 18).
(A) Hyaline cartilage thickness (HCt) histogram
30
Normal 25

(B) Caclfied cartilage thickness (ZCCt) histogram

30
Normal

Traumatic

25

Traumatic

Frequency

Frequency
400 500 600 700 800 900 1000 More
HCt - Thickness (m)

20 15
10

20

15 10
5

5 0

ZZCt - Thickness (m)

(C) Vascular channel area (VCa) histogram

16
14

(D) Vascular channel number (VCn) histogram

25 Normal 20
Traumatic

Normal
Traumatic

12

Frequency

Frequency

10
8

15 10 5 0
8 11 14 17 20 23 26 29

6
4

2 0

3886 3887 3888 3889 3890 3891 3892 3893 3894 3895 3896 3897 3898 3899 3900 3901 3902 3903

VCa - Area ( m2)

VCn - Frequnecy

Figure 120 - 4 comparative histograms for the histomorphometric distributions (HCt, ZCCt, VCa, VCn) in the dorsal region of C3 from the normal and osteochondrosis affected tissue.

The results demonstrated that osteochondrosis dramatically enlarged both the ZCCt as well as VCa with very high significances (PZCCt 0.0001, PHCt = 0.0137), while inducing a minor decrease for the VCn and an increase in HCt. VCa in the trauma tissue was more prominent with thicker vascular channels (+27%) which developed radially towards the calcified cartilage layer compared to the less active vasculature of the normal tissue (Figure 119). However this increase in VCa was accompanied by a decrease in the VCn, the latter being slightly higher in the normal tissue ( = 2.06). As for the HCt, a minor increase in the trauma tissue was detected (+13%) which indicated that the trauma may have induced a thickening effect in the cartilage without damaging it superficially (see Figure 64 on page 120). This suggests that the subchondral bone collapse was either gradual, or the collapse may have been quickly rectified by the adaptive healing process (see page 123). We also have addressed the possibility that an excessive VCa enlargement may have provided initial sites of destabilising faults within the supporting architecture of the subchondral plate. These faults might then cause the subchondral bone to collapse resulting in osteochondrosis upon traumatic loading. Please refer to page 191 for further details on this topic.

[211]

3904

COLLAGEN FIBRILS NOT ALWAYS 90

On the radial facet of C3, the collagen alignment in the hyaline and calcified cartilage was not always perpendicular to the articular surface as is portrayed in Benninghoff's classic collagen arcade model [578]. Rather, the orientation was strongly site-dependent (Figure 121). It is suggested that this site-dependent deviation of the collagen orientation in the hyaline and calcified cartilage away from the perpendicular direction is linked to the sustained mode of loading in the joint. As the calcification front advances, this deviation in the collagen is petrified into the calcified cartilage as shown in Figure 121. Please refer to page 186 for further details on this topic.

3905 3906 3907 3908 3909 3910 3911

S1

S2

S3

S4

(a) Neonatal blank cartilage

(b) Joint loading starts

(c) Collagen realigns to the loading

3912 3913

(d) Deep zone collagen deviation calcified

(e) Continual calcification of collagen deviation

(f) Removal of load & relaxation

S1

S2-S3

S4

S1 S0 S0

S2

S3

S4

C3
3914 3915 3916 3917 3918
Dorsal Palmar Figure 121 - A proposed model of how site-specific collagen orientation might form in the hyaline and calcified cartilage. The microphotographs of site 0 and 4 demonstrate that each site has a distinct collagen orientation; (S0) has no direct load, thus the collagen orientation in this region is neutral, the (S1, S4) demonstrate extreme deviations due to the shear stress, while the (S2-S3) exhibit neutral without deviation.

[212]

3919

VASCULAR CHANNELS BRANCH 90

The effect of exercised was not detected in the growth direction of vascular channels (VCang) underneath the calcified cartilage layer of C3 in the CONDEX relative to the PASTEX (P = 0.3263, Figure 122). However the variances in their distributions were different, the CONDEX being higher than the PASTEX (VARCONDEX = 237, VARPASTEX = 186). The VCang distribution mean values of both the CONDEX and PASTEX were near 0 (i.e. CONDEX = 3.2 2.1, PASTEX = -2.5 2.3, mean 95% C.I) implying that the majority of the vascular channels have a tendency to grow radially towards the articular cartilage surface with little overall bias towards either left or right (their distributions ranged from approximately -40 to +40). These results suggest that the growth direction is likely to be dependent on the loading direction as reflected in the contour of the articular cartilage surface. Please refer to page 176 for further details on this topic.

3920 3921 3922 3923 3924 3925 3926 3927 3928 3929 3930 3931

VCang CO NDEX v s PASTEX

t-Test: Two-Sample Assuming Unequal Variances CONDEX Mean Variance Observations df t Stat P(T<=t) one-tail t Critical one-tail P(T<=t) two-tail t Critical two-tail -3.17 237 200 320 0.4507 0.3263 1.6496 0.6525 1.9674 -60 -40 PASTEX -2.46 186 140

Histogram of VCang
F-Test Two-Sample for Variances
40
PASTEX 35 PASTEX

Number of vascular channels

CONDEX

CONDEX

Mean -2.45574 -3.16987 30 Variance 185.7877 236.7657 25 Observations 140 200 20 df 139 199 F 0.78469 15 P(F<=f) one-tail 0.063428 10 F Critical one-tail 0.77003
5 0 -20 0 VCang [de g ] 20 40 60

3932 3933 3934 3935 3936 3937

Figure 122 - Histogram of the vascular channel orientation (VCang) in the CONDEX and PASTEX of sites 1 to 4. Due to different observation numbers (CONDEX = 200, PASTEX = 140), the frequency of the CONDEX is overall higher. There is a very little difference in terms of the mean VCang values and variances. Both distributions are very similar to each other indicating that there is probably no overall exercise effect in the VCang.

3938

[213]

3939

DEPTH EFFECT ON SWELLING

The matrix swelling strain behaviour (expansion ratio) of cartilage slices was found to be highly depth dependent (P = 0.0002) thus exhibiting classical swelling behaviour as reported in earlier studies [531, 535, 547]. The values were lowest in the surface layer, increasing to a maximum in the middle zone, and diminishing in the deep zone (Figure 123). This characteristic trend in swelling reflects the natural zonal differentiation of the cartilage matrix [548] and is consistent with the depth-dependent heterogeneity of the principal biochemical components of articular cartilage (e.g. collagen matrix architecture and proteoglycan concentration). Please refer to page 144 for further details on this topic.
Left C3 Site 1 Right C3 Site 1 Left Cr Site 1 Right Cr Site 1

3940 3941 3942 3943 3944 3945 3946 3947

80 70

80 70

80 70

80 70

Swelling strain [%]

Swelling strain [%]

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Left C3 Site 2
80 70

Right C3 Site 2

Left Cr Site 2

Right Cr Site 2

80

80 70

80 70

Swelling strain [%]

Swelling strain [%]

70

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Left C3 Site 3
80 70

Right C3 Site 3
80 70

Left Cr Site 3

Right Cr Site 3

80

80 70

Swelling strain [%]

Swelling strain [%]

70

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Swelling strain [%]

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

Left C3 Site 4

Right C3 Site 4

Left Cr Site 4

Right Cr Site 4

80 70

80 70

80 70

80 70

Swelling strain [%]

Swelling strain [%]

Swelling strain [%]

Swelling strain [%]

0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

60 50 40 30 20 10 0 0.00

60 50 40 30 20 10 0 0.00 0.50 Depth 1.00

3948 3949 3950 3951

PASTEX

CONDEX

Figure 123 - Cohort mean ECM swelling strain versus cartilage depth plots for each combination of samplings site (1, 2, 3, or 4), bone of origin (third [C3] or radial carpal [Cr]), and limb (left or right) of the CONDEX (x) and PASTEX (o) groups. Statistically fitted curves for the CONDEX (----) and PASTEX (- - -) are indicated

[214]

3952

BONE EFFECT IN THE DORSAL REGION

The study found that between the high loaded sites of the C3 and Cr, there was a significant bone effect in the matrix swelling behaviour, i.e. C3 was higher than Cr in the margin of +5% with a strong statistical significance (P = 0.0411, Figure 124). This suggests that there might be a difference in the biochemical/molecular composition of C3 and Cr even though, as paired opposing bones, they will experience the same magnitude of loading. Please refer to page 144 for further details for this topic.

3953 3954 3955 3956 3957 3958

Mean swelling strain C3 & Cr

35 30

C3 Cr

Swelling strain [%]

25
20

15
10 5 0

S1
3959 3960 3961 3962 3963 3964

S2

Site

S3

S4

Figure 124 - Plots of the estimated mean values of cartilage swelling strain for each sampling site (1 through 4) in C3 and Cr with combined results of both treatment groups and the left and right limbs. The plot shows significant difference between the C3 and Cr at site 1 and 2 (and perhaps site 4) with high significance of P = 0.0411. The error bars indicated 95% confidence intervals.

[215]

3965

LATERALITY EFFECT IN DORSAL REGION

The effect of laterality in the dorsal region was found to be significantly biased towards the left limb, regardless of treatment, by +9.4% (P = 0.006, Figure 125) even though the CONDEX were trained bidirectionally with daily alternation aimed at minimising any laterality development [500] . The combined laterality observed in the present study would appear to be explained either by some biologically random effect arising from the limited numbers of horses or, alternatively, there is an inherent laterality in the horse. It is possible that the bidirectional training failed to eliminate the laterality and that the free-pasture exercise in both the CONDEX and PASTEX may have had a greater influence on the animals. Please refer to page 146 for further details for this topic.

3966 3967 3968 3969 3970 3971 3972 3973

Mean swelling strain Right & Left limbs

35

30

Right Left

Swelling strain [%]

25 20 15 10 5 0

S1
3974 3975 3976 3977 3978 3979

S2

Site

S3

S4

Figure 125 - Plots of the estimated mean values of cartilage swelling strain for each sampling site (1 through 4) in left and right limbs with combined results of both treatment groups and C3 and Cr. The error bars indicated 95% confidence intervals. The plot shows significant difference between the left and right limbs at site 1 (and perhaps site 4) with high significance of P = 0.006.

[216]

3980

SYMMETRY ARRANGEMENT OF LESIONS

Nearly all lesions were arranged either bilaterally symmetrical (mirrored between left and right) and/or oppositionally symmetrical (between C3 and Cr, Figure 126).
CONDEX/Exercised
Ci Cu Cu

3981 3982

Left
Cr Ci

Right
Cr Cr

Left
Ci

PASTEX/Control

Right
Ci Cr

Cu

Cu

C4 C3

C2

C2 C3

C4

C4 C3

C2

C2 C3

C4

Traumatic

Unique

C3 ridge

Cr ridge

C2 ridge

3983 3984 3985 3986 3987 3988 3989 3990 3991 3992 3993 3994 3995 3996 3997
Figure 126 - Superimposed lesions from the lesion affected sites in the CONDEX and PASTEX groups.

8 of 9 traumatic osteochondrosis type lesions on the dorsal radial facet of the C3 and Cr had bilateral and oppositional symmetry, while 25 of 30 minor lesions on various sites on the C2, C3 and Cr had bilateral symmetry but not oppositional symmetry. It is suspected that the distribution of these lesions is probably related to joint morphology and the loading environment of the midcarpal joint. It is suggested that the traumatic lesions resulted from direct concussions from high impact loads between C3 and Cr, while the other lesions on the ridges of C3, C2 and Cr are probably linked to friction effects between the two opposing surfaces during intercarpal ligament expansion. As for the unique lesions (see blue regions in Figure 126), we do not have an explanation what might have caused these lesion development. Please refer to page 132 for further details for this topic.

3998

[217]

3999

CALCIFIED CARTILAGE , PROTECT AND REPAIR ROLE?

Close examination of the 9 sites of traumatic osteochondrosis from 3 horses (1 CONDEX, 2 PASTEX) led to the suggestion that the calcified cartilage layer acts as both a protective layer and scar tissue for the subchondral bone (Figure 127).

4000 4001 4002 4003

A - G13 L C3

B - G13 L Cr

C - G13 R C3

D - G13 R Cr

E - G17 L C3

F - G17 L Cr

G - G19 L C3

H - G19 L Cr

I - G19 R Cr

4004 4005 4006 4007 4008 4009 4010 4011 4012

Figure 127 - Microphotographs of the selected regions in the calcified cartilage that were affected from the osteochondrosis in G13, G17 and G19. The calcified cartilage matrices were either containing hypertrophic chondrocytes (blue arrows) or normal chondrocyte with the columnar structure duplicating the deep zone of hyaline cartilage. Sometimes the calcified cartilage layer contained multiple microcracks (A, E, F; red arrows). (C) shows a distinctively large crack that completely splits the calcified cartilage in the middle and extends to the hyaline cartilage. Most of the cracks were aligned with the angular orientation of the collagen fibrils. All of the observations have shown the crushed appearance in the subchondral fronts suggesting each and every osteochondrosis affected site was induced by traumatic loads.

[218]

4013 4014 4015 4016 4017 4018 4019 4020 4021 4022 4023

It is suggested that with traumatic impact, the calcified cartilage partially absorbs the impact energy by undergoing a fragmenting mode of fracture, absorbing energy and thus reducing the overall damage to the subchondral bone. As a part of the wound healing response which is aimed at regaining near-normal joint function in a situation where the subchondral bone is damaged, nearby stem cells from the haemorrhage and chondrocytes quickly patch up the region with newly synthesized collagen matrix, thus allowing the joint to regain some support. The newly formed calcified cartilage can now fully support the load as well as re-establish the protective plate for the trabecular bone from further impact damage. The calcified cartilage is then resorbed by the invading vascular channels, eventually leading to the recovery of the full subchondral front as before. Please refer to page 123 for further details for this topic.

Hyaline cartilage

Normal CC

Damaged CC

Subchondral bone
4024 4025 4026 4027 4028 4029 4030 4031 4032
Figure 128 - A close-up of the calcified cartilage region in G17 affected by osteochondrosis showing two different of calcified cartilage (CC) matrices; (damaged) calcified cartilage in the left and (normal) calcified cartilage in the right divided by a red line. The difference between the (damaged) and (normal) calcified cartilage matrices is substantial. The normal matrix is homogeneous and has an amorphous appearance whereas the damaged matrix is very blotchy as if two types of matrices attempted to fuse together. The green line indicates the cement line. There is also a substantial difference in terms of the morphology in the normal cement line compared to the damaged cement line as already demonstrated in Figure 41.

[219]

4033

POROSITY OF SUBCHONDRAL BONE ?

In all mild traumatic osteochondrosis affected sites (i.e. 9), a high degree of porosity was observed in the trabecular bone regions beneath these lesions (Figure 128, Figure 130). It is possible that the cause of subchondral collapse is due to this local weakness in the trabecular bone arising from this increased porosity. However it may also be possible that these local weaknesses are a consequence of destructive bone resorption processes triggered by the development of the traumatic lesion. Please refer to page 126 for further details for this topic.

4034 4035 4036 4037 4038 4039 4040

G19 Cr Right

G19 Cr Right

G19 Cr Left

4041 4042 4043 4044 4045 4046 [220]

Figure 129 - The entire dorsal section of the Cr osteochondral slices with calcified cartilage lesion from right (TOP, MIDDLE) and left (BOTTOM) legs from G19 which suffered the osteochondrosis. The microphotographs show that the bone underneath the lesion affected areas has relatively large marrow porosity in both examples (red arrows). The approximate dimensions of the lesions are about 3mm long, 0.6mm deep.

G13 L

G13 R

G17 L

G17 R

G19 L

G19 R

4047 4048 4049 4050 4051 [221]

Figure 130 - G13, G17 and G19 were found with osteochondrosis [514]. The shape and size of lesions were nearly oppositionally symmetrical in G13 and G17 suggesting the traumatic load was the cause of these lesions. The trabecular bone regions beneath the lesions were shown to be more porous (red arrows) than sites without the lesions (i.e. G17R).

4052 4053 4054 4055 4056 4057 4058 4059 4060 4061 4062 4063 4064 4065 4066 4067 4068 4069 4070 4071 4072 4073 4074 4075 4076 4077 4078 4079

6. SELF-ASSESSMENT

This study has attempted to push the boundaries by every possible means to investigate the effects of early exercise using mostly microscopy, image processing and photography techniques. Some of the techniques were developed in-house, such as those required for the multiple-layer matrix swelling strain measurements (see analysis 2), while the histomorphometry procedures gave what is believed to be a unique view of changes in the joint microanatomy in terms of structural resolution and differentiation and dimensional accuracy. Also the structural equality diagram used in analysis 3, inter-variable correlation, was a unique and yet simple and intuitive visual tool not often utilised in this kind of multivariate study. It is hoped that other researchers will be inspired to adopt this tool in the experimental design of their own studies. This tool proved extremely useful in detecting changes that the widely used MANOVA and ANOVA tools could not detect. Although not all aspects of the findings reported in the study (e.g. the site-specificity in the joint) are new, much of the investigations value resides in the quality of the multi-dimensionally collected data dealt with by the three separate analyses. These provided a glimpse of underlying changes in the cartilage and subchondral bone when considered within a programme of properly documented and controlled exercise. A major limitation of the present study was the low statistical power attributable to the small number of horses in each treatment (i.e. only six horses in each group). This limited the amount of tissue that could be allocated to the lesion mapping, swelling strain and histomorphometric analyses. However the procedures that were used sought to optimize the data that could be gathered from the limited area of cartilage available in the dorsal radial facet of C3 (and Cr). The sample size for the swelling and histomorphometric analysis were, therefore, critical factors in achieving this optimization.

[222]

4080 4081 4082 4083 4084 4085 4086 4087 4088 4089 4090 4091 4092 4093 4094 4095 4096 4097 4098 4099 4100 4101 4102 4103 4104 4105 4106 4107 4108 4109 4110

With regard to the size chosen for the swelling sample, it was important that the samples were sufficiently small to permit site-specific detection of differences and also permit the other types of analysis to be performed on the remaining tissue. Another constraint was that the swelling samples were required to be of a size under the microscope such that the post-swelling image was still within the field of view of a single frame of the digital photograph. Approximately 1,500 swelling slice samples were required to be photographed microscopically. If the swollen dimensions of the sample were larger than the field of view, more than one photograph of each slice would have been needed, and thus greatly increasing the amount of time spent on multiple-stitched images. In the end, the sample size became 2.2mm x 2.2mm with a 30m thickness. 30m was the minimum thickness that the sledging microtome could reproduce consistently without any significant thickness variation, while at the same time ensuring that at least five serial slices could be obtained at the site of thinnest cartilage. In the authors opinion, the 2.2mm x 2.3 mm x 30m sample dimensions for the swelling analysis was just right. The choice of a 30 m thickness for the sections could be criticized as it might not fairly represent the in vivo behaviour of extracellular matrix (ECM). However, when compared with the nano-level scale of the fibrillar network, a 30-mthick section would contain a substantial depth of ECM structure and thus was considered sufficient to detect meaningful differences in swelling behaviour. It is also suggested that these slices could be used to measure proteoglycan and collagen contents and thus be correlated with the swelling data. The matrix swelling technique is able to detect subtle changes in the articular cartilage without having to employ complex chemical analyses, even without the cartilage exhibiting overt disruption. The technique is simple and inexpensive, requiring only a relatively simple set of laboratory facilities such as a microtome, a microscope and a computer. Reflecting now on how the histomorphometric analysis was designed, various factors had to be considered; What could realistically be measured? How long would it take to hand trace each histomorphometric slice? How many slices should be processed? Would the computer be powerful enough to handle the data? What is the best balance between file size, image capture resolution and optical setting of the microscope? What level of accuracy in parameter measurement would be acceptable? In the end, it was decided that by digitally stitching 10 to 15 five-megapixel images this captured the important data in each osteochondral slice measuring about 2cm long (where there were 360 slices). [223]

4111 4112 4113 4114 4115 4116 4117 4118 4119 4120 4121 4122 4123 4124

This enabled an ultra high resolution image database to be built up from the entire C3 sample collection; this was achieved utilising the best optical and digital resolution the Nikon microscope system could offer. In the end, the accuracy of the histomorphometric analysis was within 5m producing nearly 122 GB of imaging data. Although the hand tracing of histomorphometric features was carried out as consistently as possible, the tracing was still based on the investigators subjective judgement, especially for measuring collagen orientation (based on the chondrocyte orientation) as well as choosing which vascular channel to be counted. But in the end, it was judged that the errors in these measurements would have been equally distributed in both groups and thus would not have significantly affected the overall variance of the data. Finally, it was a little disappointing that the horses were not able to be examined by the author while they were alive and undergoing their exercise treatment. The opportunity to interview those who personally conducted the exercise programme and tracked clinically the animals wellbeing could have provided additional insights.

4125

[224]

4126

[225]

4127 4128 4129 4130 4131 4132 4133 4134 4135 4136 4137 4138 4139 4140 4141 4142 4143 4144 4145 4146 4147 4148 4149 4150 4151 4152 4153 C AUSE OF OSTEOCHONDROSIS , IS IT TRABECULAR BONE S FAULT ?

7. FURTHER WORK

9 sites of mild osteochondrosis were detected in the dorsal regions of C3 and Cr. These sites were also seen with localised sclerosis, but at the centre of these features, there seems to be a high level of porosity in the trabecular bone associated with them. Is this a natural tendency of normal bone remodelling? Is there a good correlation between the osteochondrosis and the trabecular bone porosity? Which comes first, the osteochondrosis or the porosity? Can this be prevented? What measures might be employed to detect this feature early? Such a study could fruitfully develop a systemic method of detection, management and prevention of porosity in the trabecular bone and mild osteochondrosis in the C3 and Cr, and thus the possibility of reducing the level of joint disease in Thoroughbreds.

SEM CARTILAGE STUDY AND QUANTIFICATION OF MATRIX COLLAGEN AND PROTEOGLYCAN CONTENT In this study the matrix swelling data provides only an indirect indication of the collagen matrix integrity and proteoglycan content. However further examination is required to affirm the credibility of this correlation. A high resolution scanning electron microscopy (SEM) and collagen and proteoglycan content assay studies could be performed in conjunction with the swelling study in the future.

M ICROCRACKS IN THE OSTEOCHONDROSIS , ARE THESE REALLY MICROCRACKS ? We have observed microcrack-like features in the calcified cartilage lesions in the sites that were affected with osteochondrosis (Figure 66 on page 122). Are these really genuine microcracks? What is their distribution patterns like? Are they clinically significant? Do they ever recover and sealed up? [226]

4154 4155 4156 4157 4158 4159 4160 4161 4162 4163 4164 4165 4166 4167 4168 4169 4170 4171 4172 4173 4174 4175 4176 4177 4178

Are they potential initiation sites of more damaging progressive fractures? A structural study should be carried out in order to answer the questions above in the horses with various ages and histories.

E ARLY EXERCISE IN LARGER SCALE USING TWO STABLES A larger scale trial (n = 50 or more) would hugely improve the power of the study.

L ESION DISTRIBUTION PATTERN STUDY A range of lesions were present in the CONDEX as well as the PASTEX. Most of these lesions were location-specific. However, for one type labelled unique, it was not possible to find a plausible cause even though it was located bilaterally. However, it is suggested that the lesion formation was related somehow to the load environment and midcarpal joint congruency. This is an area of potential research which might help explain the pattern of traumatic stress applied on the joint.

D EVELOPMENT OF LEG EXERCISE MACHINE While it is common for athletes to tailor their exercise regime in the gym to target the areas of most use and high susceptibility to injuries, Perhaps horses should also work out as well. Is there a gym machine that allows weight training for the racing horses? Using what is known already about the role of specific muscles, tendons and ligaments involved in the high speed gallop, a gym machine for Thoroughbred might be developed to prevent the overextension of the forelimbs.

C OLLAGEN ORIENTATION - HOW THEY DEVELOP IN VARIOUS AGE GROUPS There was a consistent pattern of site-specific collagen orientation in C3 in the hyaline and calcified cartilage (see page 212). A larger scale investigation should be carried out to confirm whether this feature is Thoroughbred specific and how it might vary according to age.

4179

[227]

4180 4181 4182 4183 4184 4185 4186 4187 4188 4189 4190 4191 4192 4193 4194 4195 4196 4197 4198 4199 4200 4201 4202 4203 4204 4205 4206

8. CONCLUSIONS

The key question this thesis sought to address was whether there was an early exercise effect in the midcarpal joint of the CONDEX when compared to the PASTEX. To this end, three types of analysis were employed; (a) lesion mapping of the midcarpal cartilage surface with microscopic verification, (b) measurement of cartilage matrix swelling behaviour of C3 and Cr and (c) histomorphometric analysis of the osteochondral features in C3. In terms of number and pattern of lesions found on the midcarpal joint surfaces, there were no significant differences between the CONDEX and the PASTEX. However a higher frequency (n = +3) of mild traumatic osteochondrosis was detected in the PASTEX than in the CONDEX. In terms of extracellular matrix swelling behaviour, the influence of early exercise was not detectable in the CONDEX (P = 0.795). In terms of overall histomorphometric analysis, MANOVA could not detect any evidence of early exercise effect in any of the variables (P = 0.4271). However by analysing each histomorphometric variable separately using ANOVA, the vascular channel area (VCa, out of 10 variables tested) was shown to be significantly affected by the exercise treatment (P = 0.0461). Using the structural equality of permutation test, it was judged that the exercise may have caused the CONDEX to develop a more coherent tissue response than the PASTEX (P = 0.052). The most significantly affected variables were the hyaline cartilage thickness (HCt) and the calcified cartilage thickness (ZCCt, P = 0.00127). The above findings indicate that the imposed early exercise may have (1) reduced the number of traumatic osteochondrosis in the dorsal region of C3, (2) significantly increased the vascular channel area beneath the calcified cartilage layer, and (3) enhanced the coherence of the inter-tissue response between the hyaline cartilage, calcified cartilage and vascular channels.

[228]

4207 4208 4209 4210 4211 4212 4213 4214 4215

In conclusion, the findings of the study reported here together with others [1-3] have revealed that early exercise treatment may have induced a positive response in the CONDEX without causing a deleterious effect in the midcarpal joint relative to changes that result naturally from typical exercise in the pasture. The practical implications of the studys findings are of major potential significance for the equine industry. The results have provided a global assessment of the overall effectiveness of the early exercise trials at both the gross and histological levels. Furthermore the study points to the possibility of designing early exercise programmes that target specific aspects of tissue development without causing adverse effects on the midcarpal joints of young Thoroughbreds.

4216

[229]

4217 4218 4219

APPENDICES

[230]

4220

[231]

4221 4222

8.1 GEXA TEST SUBJECTS (18-MONTHS AND 36-MONTHS)

Horse ID G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20 G21 G22 G23 G24 G25 G26 G27 G28 G29 G30 G31 G32 G33 4223 4224 4225 4226 4227

Euthanized @ 18-mths

Exercised

Treatment CONDEX PASTEX

Sex (Colt/Filly) C F C C F F F F F F C F F F C F C F C F F C F C F F F F C F F F F

Birth (Y/M/D) 2000/9/16 2000/9/24 2000/9/28 2000/9/29 2000/9/30 2000/10/4 2000/10/5 2000/10/6 2000/10/6 2000/10/7 2000/10/10 2000/10/12 2000/10/15 2000/10/20 2000/10/22 2000/10/24 2000/10/27 2000/10/27 2000/11/7 2000/11/9 2000/11/18 2000/11/20 2000/11/21 2000/11/21 2000/11/12 2000/11/25 2000/11/25 2000/11/28 2000/11/29 2000/12/14 2000/12/28 2000/9/10 2000/11/2
th

PASTEX PASTEX

CONDEX PASTEX CONDEX CONDEX

CONDEX PASTEX CONDEX PASTEX

A complete list of Thoroughbreds which participated in the GEXA project. Only those euthanized at 18 month, were used in this project. The 18-month horses were classified as either CONDEX or PASTEX depending on whether they had received conditioning exercise or not. A colt is a young male whereas a filly means a young female.

[232]

4228 4229 4230 4231 4232 4233 4234 4235 4236 4237 4238 4239 4240 4241 4242 4243 4244 4245 4246 4247 4248 4249 4250 4251 4252 4253 4254 4255 4256 4257 4258 4259 4260 4261 4262 4263 4264 4265 4266 4267 4268 4269 4270 4271 4272 4273 4274 4275 4276

8.2 ANALYSIS 2 - SAS SOURCE CODES AND OUTPUT

8.2.1 LINEAR MIXED MODELLING CODE
options ps=10000; data dat1;set active.kims_data2; *SET MIN VALUE TO ZERO AND THEN DO LOG(X+1); lswelling=log(swelling+6.2); dist2=distance**2; dist3=distance**3; run; proc standard data=dat1 out=data2 mean=0;var dist3;run;

distance

dist2

proc mixed data=data2 covtest; *where site>1; where age=18; class treatment horse bone site limb; model swelling=treatment|limb| bone| site distance dist2 treatment*distance treatment*dist2 bone*distance bone*dist2 limb*distance limb*dist2 site*distance site*dist2 limb*site*distance limb*site*dist2 bone*site*distance bone*site*dist2 limb*bone*distance limb*bone*dist2 treatment*limb*distance treatment*limb*dist2 treatment*bone*distance treatment*bone*dist2 treatment*site*distance treatment*site*dist2 treatment*site*bone*distance treatment*site*bone*dist2 treatment*limb*site*distance treatment*limb*site*dist2 treatment*limb*bone*distance treatment*limb*bone*dist2 treatment*limb*bone*site*distance treatment*limb*bone*site*dist2/outpm=out ; */solution; random horse(treatment) limb*horse(treatment) bone*horse(treatment) site*horse(treatment) limb*bone*site*horse(treatment) distance*horse(treatment) dist2*horse(treatment) limb*distance*horse(treatment) limb*dist2*horse(treatment) bone*distance*horse(treatment) bone*dist2*horse(treatment) site*distance*horse(treatment) site*dist2*horse(treatment) site*distance*horse(treatment) site*dist2*horse(treatment) limb*bone*site*distance*horse(treatment) limb*bone*site*dist2*horse(treatment) ; lsmeans treatment/cl diff; run;

[233]

4277 4278 4279 4280 4281 4282 4283 4284 4285 4286 4287 4288 4289 4290 4291 4292 4293 4294 4295 4296 4297 4298 4299 4300 4301 4302 4303 4304 4305 4306 4307 4308 4309 4310 4311 4312 4313 4314 4315 4316 4317 4318 4319 4320 4321 4322 4323 4324 4325 4326 4327 4328 4329 4330 4331 4332 4333 4334 4335 4336 4337 4338 4339 4340 4341 4342 4343 4344 4345 4346 4347 4348

8.2.2 OUTPUT
Covariance Parameter Estimates Cov Parm Horse(Treatment) Horse*Limb(Treatmen) Horse*Bone(Treatmen) Horse*Site(Treatmen) Hor*Bon*Sit*Lim(Tre) distan*Horse(Treatm) dist2*Horse(Treatme) dist*Hors*Limb(Trea) dist*Hors*Limb(Trea) dist*Hors*Bone(Trea) dist*Hors*Bone(Trea) dist*Hors*Site(Trea) dist*Hors*Site(Trea) di*Ho*Bo*Si*Lim(Tre) di*Ho*Bo*Si*Lim(Tre) Residual Standard Estimate 45.4497 0.8527 8.3572 27.9879 72.8070 2293.61 1374.18 7.27E-17 0 18.5906 0 57.9412 62.0351 0 105.10 56.4971 Z Error 28.8855 5.5682 8.6659 13.1446 12.6128 1099.62 670.33 . . 21.0068 . 52.6020 43.5296 . 25.8733 2.4766 Value 1.57 0.15 0.96 2.13 5.77 2.09 2.05 . 0.88 . 1.10 1.43 . 4.06 22.81 Pr Z 0.0578 0.4391 0.1674 0.0166 <.0001 0.0185 0.0202 . . 0.1881 . 0.1353 0.0771 . <.0001 <.0001

Fit Statistics -2 Res Log Likelihood AIC (smaller is better) AICC (smaller is better) BIC (smaller is better) 10565.7 10589.7 10589.9 10595.5

Type 3 Tests of Fixed Effects Effect Treatment Limb Treatment*Limb Bone Treatment*Bone Bone*Limb Treatment*Bone*Limb Site Treatment*Site Site*Limb Treatment*Site*Limb Bone*Site Treatment*Bone*Site Bone*Site*Limb Treat*Bone*Site*Limb distance dist2 distance*Treatment dist2*Treatment distance*Bone dist2*Bone distance*Limb dist2*Limb distance*Site dist2*Site distance*Site*Limb dist2*Site*Limb distance*Bone*Site dist2*Bone*Site distance*Bone*Limb dist2*Bone*Limb distanc*Treatme*Limb dist2*Treatment*Limb distanc*Treatme*Bone Num DF 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 1 3 3 3 3 3 3 1 1 1 1 1 Den DF 9 9 9 10 10 90 90 30 30 90 90 90 90 90 90 9 9 9 9 10 10 9 9 30 30 85 79 85 79 85 79 9 9 10 F Value 0.07 0.90 0.55 1.24 3.07 0.27 0.33 6.54 0.34 4.43 0.92 2.86 0.83 1.44 0.20 35.16 26.03 0.08 0.16 2.02 0.24 0.79 0.62 30.42 24.26 2.58 3.35 10.61 8.59 2.06 2.97 13.99 18.46 14.41 Pr > F 0.7951 0.3674 0.4787 0.2910 0.1104 0.6032 0.5697 0.0016 0.7946 0.0060 0.4368 0.0411 0.4807 0.2376 0.8928 0.0002 0.0006 0.7820 0.6966 0.1856 0.6338 0.3963 0.4507 <.0001 <.0001 0.0588 0.0233 <.0001 <.0001 0.1553 0.0886 0.0046 0.0020 0.0035

[234]

4349 4350 4351 4352 4353 4354 4355 4356 4357 4358 4359 4360 4361 4362 4363 4364 4365 4366 4367 4368 4369 4370 4371

dist2*Treatment*Bone distanc*Treatme*Site dist2*Treatment*Site dist*Treat*Bone*Site dist*Treat*Bone*Site dist*Treat*Site*Limb dist*Treat*Site*Limb dist*Treat*Bone*Limb dist*Treat*Bone*Limb dis*Tre*Bon*Sit*Limb dis*Tre*Bon*Sit*Limb

1 3 3 3 3 3 3 1 1 6 6

10 30 30 85 79 85 79 85 79 85 79

14.99 1.82 1.94 2.30 3.16 2.36 3.43 0.04 0.10 2.36 2.07

0.0031 0.1643 0.1449 0.0833 0.0290 0.0771 0.0210 0.8502 0.7493 0.0370 0.0660

Least Squares Means Effect Treatment Treatment Treatment Control Exercis Estimate 15.7156 16.9427 Error 3.2483 3.2277 Standard DF t Value Upper 9 9 4.84 5.25 Pr > |t| 0.0009 0.0005 Alpha 0.05 0.05 Lower 8.3674 9.6411 CI 23.0638 24.2443

[235]

4372

8.3 ANALYSIS 2 - MATRIX SLICE SWELLING STRAIN DATA

Horse
G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 4 4 4 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2

Slice
1 2 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 1 2 2 3 3 4 5 1 2 3 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11

Total Slice
3 3 3 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 3 5 3 5 3 5 5 5 3 3 3 4 4 4 4 14 14 14 14 14 14 14 14 14 14 14

Relative depth
0.3 0.7 1.0 0.1 0.1 0.2 0.3 0.3 0.4 0.5 0.5 0.6 0.7 0.7 0.8 0.9 0.9 1.0 0.3 0.2 0.7 0.4 1.0 0.6 0.8 1.0 0.3 0.7 1.0 0.3 0.5 0.8 1.0 0.1 0.1 0.2 0.3 0.4 0.4 0.5 0.6 0.6 0.7 0.8

Swelling strain [%]

21.4 42.5 39.2 -1.4 2.3 4.3 8.6 13.2 9.8 33.2 33.2 49.0 27.6 69.7 73.5 45.1 53.7 42.6 3.3 11.6 5.0 14.6 8.0 20.4 28.3 26.6 0.6 3.2 8.0 0.3 9.0 15.7 20.4 4.0 37.9 52.2 32.7 48.2 58.8 51.2 57.3 50.0 42.4 34.0

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[236]

Horse
G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
L L L L L L L L L L L L L L L L L R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
2 2 2 3 3 3 3 3 4 4 4 4 4 4 4 4 4 1 1 1 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 1 1 1 1 1 1 1 1 1 2

Slice
12 13 14 1 2 3 4 5 1 2 3 4 5 6 7 8 9 1 2 3 1 2 3 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 1

Total Slice
14 14 14 5 5 5 5 5 9 9 9 9 9 9 9 9 9 1 1 1 3 3 3 9 9 9 9 9 9 9 9 9 6 6 6 6 6 6 9 9 9 9 9 9 9 9 9 13

Relative depth
0.9 0.9 1.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 1.0 2.0 3.0 0.3 0.7 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.2 0.3 0.5 0.7 0.8 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1

Swelling strain [%]

46.2 20.6 14.1 -2.4 0.7 7.2 16.6 20.1 0.5 3.2 5.4 18.8 20.5 32.3 40.5 30.2 35.5 -4.8 11.8 31.0 9.8 39.9 46.6 -2.6 -1.4 2.9 4.0 8.7 9.4 12.8 13.9 15.5 1.5 3.7 5.2 5.5 6.9 6.5 0.8 4.5 6.1 8.9 13.6 19.2 17.1 26.0 32.2 9.2

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[237]

Horse
G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R L L L L L L L L L L L L L L L L

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2

Slice
2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9

Total Slice
13 13 13 13 13 13 13 13 13 13 13 13 9 9 9 9 9 9 9 9 9 11 11 11 11 11 11 11 11 11 11 11 7 7 7 7 7 7 7 10 10 10 10 10 10 10 10 10

Relative depth
0.2 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Swelling strain [%]

10.1 17.0 22.0 25.2 35.4 39.6 36.0 34.8 34.4 20.0 6.9 4.7 -0.3 6.4 11.8 15.9 15.7 20.7 18.7 13.8 10.3 -1.3 -0.5 2.6 4.3 6.8 9.6 12.5 15.7 14.9 12.5 10.6 2.3 7.2 9.8 14.9 21.6 20.0 18.5 3.4 6.4 16.0 22.6 23.4 21.1 19.4 17.8 14.3

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[238]

Horse
G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3

Site
2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 1 1 1 1 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 1

Slice
10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 1 2 3 4 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 1

Total Slice
10 10 10 10 10 10 10 10 10 10 10 8 8 8 8 8 8 8 8 4 4 4 4 9 9 9 9 9 9 9 9 9 11 11 11 11 11 11 11 11 11 11 11 4 4 4 4 5

Relative depth
1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.3 0.5 0.8 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.3 0.5 0.8 1.0 0.2

Swelling strain [%]

11.5 1.7 3.6 6.5 9.2 10.7 12.5 12.7 10.5 8.8 7.4 -0.1 0.5 1.9 2.6 5.6 5.6 6.5 9.0 1.6 5.4 9.7 20.7 4.9 22.9 33.1 40.0 31.0 30.4 25.9 22.8 17.0 1.8 3.9 8.0 14.5 22.5 27.6 40.1 44.3 38.0 33.2 36.9 2.3 3.5 2.8 3.8 3.8

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[239]

Horse
G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2

Slice
2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 1 2 3 1 2 3 4 5 6 7 8 9 10 11 12 13

Total Slice
5 5 5 5 13 13 13 13 13 13 13 13 13 13 13 13 13 9 9 9 9 9 9 9 9 9 6 6 6 6 6 6 3 3 3 13 13 13 13 13 13 13 13 13 13 13 13 13

Relative depth
0.4 0.6 0.8 1.0 0.1 0.2 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.2 0.3 0.5 0.7 0.8 1.0 0.3 0.7 1.0 0.1 0.2 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.8 0.9 1.0

Swelling strain [%]

7.7 9.5 20.5 35.6 -0.7 3.2 7.4 11.0 12.5 18.9 24.3 26.6 27.5 24.5 19.6 13.2 12.9 1.6 3.8 12.6 15.1 17.0 16.3 17.3 16.3 15.5 7.7 27.8 24.8 19.1 48.5 70.6 -0.1 5.3 10.7 1.0 3.7 11.0 12.3 16.8 18.6 19.6 17.1 13.0 11.1 9.7 7.0 4.0

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[240]

Horse
G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
R R R R R R R R R R R R R R R R R R L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr

Site
3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 1 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 1 1 2

Slice
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 1 2 3 4 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 1 2 1

Total Slice
8 8 8 8 8 8 8 8 10 10 10 10 10 10 10 10 10 10 8 8 8 8 8 8 8 8 4 4 4 4 10 10 10 10 10 10 10 10 10 10 5 5 5 5 5 13 13 13

Relative depth
0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.3 0.5 0.8 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.1

Swelling strain [%]

2.4 5.0 6.6 9.2 8.1 7.7 7.6 5.5 1.7 5.0 6.3 9.3 15.6 25.1 28.3 33.3 35.3 59.1 3.2 6.4 30.2 66.8 35.7 28.9 37.3 37.9 5.1 4.5 28.7 37.2 12.2 21.9 23.2 24.2 26.2 32.1 24.5 22.5 21.3 15.0 8.2 9.0 10.3 8.1 7.2 6.4 91.9 1.1

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N Y Y Y

[241]

Horse
G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L R R R R R R R R R R R R R R R R R R R R R R

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4 4 4 4 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3

Slice
2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 1 2 3 4 5 6 7 8 1 2 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7

Total Slice
13 13 13 13 13 13 13 13 13 13 13 13 6 6 6 6 6 6 8 8 8 8 8 8 8 8 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 8 8 8 8 8 8 8

Relative depth
0.2 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.2 0.3 0.5 0.7 0.8 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.1 0.2 0.1 0.2 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9

Swelling strain [%]

3.9 8.9 14.3 16.4 21.2 37.0 40.9 51.7 49.6 73.8 55.6 48.3 12.4 17.7 29.5 38.6 34.8 30.5 5.8 10.2 13.6 18.4 20.0 18.2 15.7 13.6 0.4 4.2 -2.2 -1.5 1.0 3.1 9.3 18.7 20.8 27.3 37.8 42.1 41.5 34.5 31.2 7.3 14.3 27.4 41.5 36.1 34.7 31.0

Defect
Y Y Y Y Y Y Y Y Y Y Y Y N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[242]

Horse
G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
3 4 4 4 4 4 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4

Slice
8 1 2 3 4 5 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6

Total Slice
8 5 5 5 5 5 8 8 8 8 8 8 8 8 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13

Relative depth
1.0 0.2 0.4 0.6 0.8 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.1 0.1 0.2 0.3 0.3 0.4 0.5 0.5 0.6 0.7 0.7 0.8 0.9 0.9 1.0 0.1 0.2 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.2 0.2 0.3 0.4 0.5

Swelling strain [%]

17.3 5.2 8.6 18.6 16.5 15.6 -1.8 1.9 7.6 19.8 21.5 31.5 19.3 71.7 2.3 6.1 8.4 13.9 21.7 26.0 39.9 37.7 50.6 67.0 97.3 60.5 57.9 74.2 48.4 2.7 5.0 7.4 10.4 23.5 32.8 29.3 32.8 36.9 47.7 44.8 22.3 16.7 7.4 19.3 36.0 72.9 113.1 89.1

Defect
N N N N N N Y Y Y Y Y Y Y Y N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[243]

Horse
G19 G19 G19 G19 G19 G19 G19 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
R R R R R R R L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L R R R R R R

Bone
Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3

Site
4 4 4 4 4 4 4 1 1 1 2 2 2 2 2 2 2 2 2 3 3 4 4 4 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 4 4 1 1 1 1 1 1

Slice
7 8 9 10 11 12 13 1 2 3 1 2 3 4 5 6 7 8 9 1 2 1 2 3 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 1 2 1 2 3 4 5 6

Total Slice
13 13 13 13 13 13 13 3 3 3 9 9 9 9 9 9 9 9 9 2 2 3 3 3 5 5 5 5 5 11 11 11 11 11 11 11 11 11 11 11 2 2 6 6 6 6 6 6

Relative depth
0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.3 0.7 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.5 1.0 0.3 0.7 1.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.5 1.0 0.2 0.3 0.5 0.7 0.8 1.0

Swelling strain [%]

69.5 69.8 59.3 50.7 40.2 32.5 24.5 5.4 10.5 10.8 1.1 3.8 10.0 14.5 22.4 21.9 25.3 20.8 20.0 5.4 4.3 -0.5 2.3 5.4 0.6 4.8 8.1 10.3 9.0 0.9 8.9 15.4 27.2 27.5 32.6 42.6 20.1 19.2 9.4 8.3 -0.1 2.9 -0.3 1.9 5.7 7.4 8.6 9.1

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[244]

Horse
G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G29 G29

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R L L

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3

Site
2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 1 1

Slice
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 6 1 2 3 4 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 1 2 3 4 5 1 2

Total Slice
9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 1 6 4 4 4 4 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 6 6 6 6 6 9 9

Relative depth
0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 1.0 1.0 0.3 0.5 0.8 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.2 0.3 0.5 0.7 0.8 0.1 0.2

Swelling strain [%]

-2.2 1.5 2.7 11.4 18.7 25.1 26.7 25.0 19.7 3.3 7.8 12.9 18.2 18.4 25.1 15.1 14.0 12.1 5.1 8.7 5.7 8.7 12.2 8.5 5.6 7.3 15.2 20.0 19.7 17.3 13.1 12.1 3.9 5.5 7.1 9.7 10.7 14.1 12.2 10.0 8.7 0.1 -0.1 2.1 6.2 7.9 26.8 37.1

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[245]

Horse
G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 1 1 1 2 2

Slice
3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 1 2

Total Slice
9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 12 12 12 12 12 12 12 12 12 12 12 12 8 8 8 8 8 8 8 8 10 10 10 10 10 10 10 10 10 10 21 21

Relative depth
0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.3 0.4 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.1

Swelling strain [%]

64.0 64.3 138.6 84.9 75.0 59.8 45.8 6.2 58.6 50.2 61.4 76.7 114.9 89.7 74.0 68.7 -2.3 1.1 5.4 9.6 14.5 25.1 25.2 26.7 26.7 23.9 28.9 25.2 2.6 4.3 6.2 9.4 8.4 10.9 12.1 11.6 -0.8 6.1 18.0 29.1 32.8 38.3 48.6 41.2 35.6 43.2 0.1 3.6

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[246]

Horse
G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L R R R R R R R R R

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 1 1 2 2 2 2 2 2 2

Slice
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 1 2 1 2 3 4 5 6 7

Total Slice
21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 12 12 12 12 12 12 12 12 12 12 12 12 8 8 8 8 8 8 8 8 2 2 18 18 18 18 18 18 18

Relative depth
0.1 0.2 0.2 0.3 0.3 0.4 0.4 0.5 0.5 0.6 0.6 0.7 0.7 0.8 0.8 0.9 0.9 1.0 1.0 0.1 0.2 0.3 0.3 0.4 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.5 1.0 0.1 0.1 0.2 0.2 0.3 0.3 0.4

Swelling strain [%]

6.4 13.1 22.7 26.8 30.9 57.8 51.9 65.9 63.2 69.4 46.5 46.3 36.3 20.8 15.7 9.6 7.7 7.0 2.6 8.5 18.0 19.4 17.9 28.9 41.3 36.8 26.2 23.2 26.3 19.9 20.6 -0.2 5.3 8.8 11.4 19.6 21.7 17.5 16.1 13.6 53.4 -2.7 0.4 4.4 10.6 15.3 29.3 29.9

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N Y Y N N N N N N N

[247]

Horse
G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2

Slice
8 9 10 11 12 13 14 15 16 17 18 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 12

Total Slice
18 18 18 18 18 18 18 18 18 18 18 12 12 12 12 12 12 12 12 12 12 12 12 8 8 8 8 8 8 8 8 5 5 5 5 5 16 16 16 16 16 16 16 16 16 16 16 16

Relative depth
0.4 0.5 0.6 0.6 0.7 0.7 0.8 0.8 0.9 0.9 1.0 0.1 0.2 0.3 0.3 0.4 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.2 0.4 0.6 0.8 1.0 0.1 0.1 0.2 0.3 0.3 0.4 0.4 0.5 0.6 0.6 0.7 0.8

Swelling strain [%]

52.9 41.8 66.1 26.7 65.3 72.3 66.7 67.9 61.0 50.0 23.4 -2.4 -1.1 5.2 14.1 15.3 20.8 23.2 27.6 27.0 26.0 24.7 22.9 -2.3 0.8 3.9 7.2 8.8 11.9 14.3 10.9 16.9 27.8 57.6 53.7 50.6 -0.9 4.0 6.5 11.6 22.3 32.6 37.7 41.8 46.7 62.9 46.9 40.1

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[248]

Horse
G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R L L L L L L L L L L L L L L L

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2

Slice
13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8

Total Slice
16 16 16 16 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 11 11 11 11 11 11 11 11 11 11 11 7 7 7 7 7 7 7 9 9 9 9 9 9 9 9

Relative depth
0.8 0.9 0.9 1.0 0.1 0.1 0.2 0.2 0.3 0.3 0.4 0.4 0.5 0.6 0.6 0.7 0.7 0.8 0.8 0.9 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9

Swelling strain [%]

35.0 27.7 17.6 15.4 -3.2 0.1 2.2 6.2 10.2 12.0 16.9 16.9 22.2 25.9 26.2 23.7 27.3 26.4 18.3 12.9 7.2 7.1 1.4 4.8 4.6 6.2 8.4 21.5 23.0 15.0 17.8 15.3 17.7 0.2 0.7 3.1 7.3 9.8 12.5 13.6 -1.0 0.9 5.0 7.3 10.6 11.8 14.6 16.2

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[249]

Horse
G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L R R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 2 3 3 3 3 3 4 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 3

Slice
9 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 1 1 2 3 4 5 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 1

Total Slice
9 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1 5 5 5 5 5 9 9 9 9 9 9 9 9 9 10 10 10 10 10 10 10 10 10 10 11 11 11 11 11 11 11

Relative depth
1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 1.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.1

Swelling strain [%]

10.6 1.6 2.2 10.2 8.6 9.4 14.9 19.8 20.0 15.7 14.2 0.7 2.4 7.4 9.6 12.1 5.5 3.3 6.4 10.7 12.4 9.5 -0.1 2.4 4.5 6.0 8.6 9.3 10.4 9.7 9.9 -1.8 0.1 1.4 4.1 7.1 10.2 13.5 21.0 45.8 24.8 12.5 15.9 26.1 -1.3 3.6 13.8 3.8

Defect
N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[250]

Horse
G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
3 3 3 3 3 3 3 3 3 3 4 4 4 4 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4

Slice
2 3 4 5 6 7 8 9 10 11 1 2 3 4 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8

Total Slice
11 11 11 11 11 11 11 11 11 11 4 4 4 4 4 4 4 4 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 10 10 10 10 10 10 10 10

Relative depth
0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.3 0.5 0.8 1.0 0.3 0.5 0.8 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Swelling strain [%]

8.0 8.0 12.9 17.6 22.4 31.2 28.1 19.3 21.8 14.8 -0.8 -5.2 3.8 4.0 1.1 3.7 12.2 12.5 0.0 0.7 3.3 6.8 8.3 8.4 9.7 9.4 7.0 6.2 4.9 -1.3 0.1 2.3 4.2 6.4 11.8 12.9 14.4 12.7 13.2 11.8 -2.6 -1.2 1.3 3.1 5.8 9.5 9.8 11.2

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[251]

Horse
G31 G31 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13

Treatment
CONDEX CONDEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
R R L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L

Bone
Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
4 4 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3

Slice
9 10 1 2 3 4 5 6 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3

Total Slice
10 10 6 6 6 6 6 6 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Relative depth
0.9 1.0 0.2 0.3 0.5 0.7 0.8 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.2 0.3 0.5 0.7 0.8 1.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.1 0.3 0.4

Swelling strain [%]

10.4 9.6 1.2 7.8 12.3 23.0 26.6 20.5 3.2 7.4 10.2 11.3 13.8 17.0 16.1 15.8 3.5 5.3 8.9 11.9 14.9 17.6 16.7 17.3 17.4 -1.9 0.9 1.9 4.4 5.5 4.8 -1.6 -0.3 1.2 3.1 7.9 9.8 9.1 3.0 4.3 2.2 9.2 10.8 12.0 10.3 4.1 7.9 9.9

Defect
N N Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N N N N N N N N N N N N N N Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N N

[252]

Horse
G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
L L L L L L L L L L L R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
3 3 3 3 4 4 4 4 4 4 4 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4

Slice
4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1

Total Slice
7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 14 14 14 14 14 14 14 14 14 14 14 14 14 14 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 10

Relative depth
0.6 0.7 0.9 1.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.1 0.1 0.2 0.3 0.4 0.4 0.5 0.6 0.6 0.7 0.8 0.9 0.9 1.0 0.1 0.1 0.2 0.3 0.3 0.4 0.5 0.5 0.6 0.7 0.7 0.8 0.9 0.9 1.0 0.1

Swelling strain [%]

11.9 12.8 12.6 12.0 1.3 4.1 5.0 9.1 8.8 9.7 8.8 1.1 4.1 9.6 11.5 15.5 19.3 22.7 -0.1 3.0 8.6 15.7 18.9 23.6 39.5 35.5 38.2 38.6 34.8 33.6 29.9 23.9 0.9 3.5 6.5 7.3 10.8 12.5 16.7 21.0 23.7 23.9 32.4 29.5 21.2 20.9 14.9 -2.0

Defect
N N N N N N N N N N N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N N N N N N N N N N N N N N N

[253]

Horse
G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G17 G17

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R L L

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3

Site
4 4 4 4 4 4 4 4 4 1 1 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 1

Slice
2 3 4 5 6 7 8 9 10 1 2 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2

Total Slice
10 10 10 10 10 10 10 10 10 2 2 7 7 7 7 7 7 7 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 12 12

Relative depth
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.5 1.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.1 0.1 0.2 0.3 0.4 0.4 0.5 0.6 0.6 0.7 0.8 0.9 0.9 1.0 0.1 0.1 0.2 0.3 0.4 0.4 0.5 0.6 0.6 0.7 0.8 0.9 0.9 1.0 0.1 0.2

Swelling strain [%]

1.8 2.5 4.4 8.4 7.1 7.8 6.9 7.4 7.1 1.6 27.3 28.4 39.7 37.7 33.3 30.5 22.2 13.6 2.5 4.4 8.1 11.6 18.5 19.3 27.8 26.6 25.9 26.2 27.2 19.5 18.2 11.5 -0.1 1.2 3.2 4.6 6.5 9.3 10.9 15.6 14.6 14.3 11.8 11.6 10.6 9.4 12.6 25.4

Defect
N N N N N N N N N Y Y Y Y Y Y Y Y Y N N N N N N N N N N N N N N N N N N N N N N N N N N N N Y Y

[254]

Horse
G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4

Slice
3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6

Total Slice
12 12 12 12 12 12 12 12 12 12 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14

Relative depth
0.3 0.3 0.4 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.1 0.2 0.2 0.3 0.3 0.4 0.4 0.5 0.6 0.6 0.7 0.7 0.8 0.8 0.9 0.9 1.0 0.1 0.1 0.2 0.3 0.4 0.4 0.5 0.6 0.6 0.7 0.8 0.9 0.9 1.0 0.1 0.1 0.2 0.3 0.4 0.4

Swelling strain [%]

87.4 75.2 83.7 91.2 108.7 69.3 74.4 82.9 57.1 41.2 0.2 2.5 5.5 8.1 11.5 17.9 23.0 30.9 37.1 46.7 38.1 45.1 44.9 41.6 35.0 41.6 26.9 20.6 3.0 5.3 16.5 19.1 20.4 26.4 26.3 29.7 24.4 33.0 24.3 20.4 16.8 10.9 1.0 3.9 6.6 12.6 14.4 17.4

Defect
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N N N N N N N N N N N N N N N N N N N

[255]

Horse
G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
4 4 4 1 1 1 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2

Slice
7 8 9 1 2 3 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2

Total Slice
14 14 14 3 3 3 9 9 9 9 9 9 9 9 9 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 14 14 14 14 14 14 14 14 14 14 14 14 14 14 13 13

Relative depth
0.5 0.6 0.6 0.3 0.7 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.1 0.2 0.3 0.4 0.4 0.5 0.6 0.6 0.7 0.8 0.9 0.9 1.0 0.1 0.2

Swelling strain [%]

19.3 16.8 12.6 51.7 34.9 54.7 2.9 8.2 10.9 20.5 35.7 32.1 37.9 35.0 21.3 7.0 8.5 11.2 18.5 21.9 31.6 28.6 28.1 1.0 2.0 3.9 7.2 9.6 16.6 18.3 12.6 9.7 0.3 2.3 7.7 31.0 34.2 53.1 50.6 43.1 42.7 38.9 36.5 24.2 20.3 13.5 -2.1 -0.5

Defect
N N N Y Y Y Y Y Y Y Y Y Y Y Y N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[256]

Horse
G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 4 4 4 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Slice
3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Total Slice
13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17

Relative depth
0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.2 0.2 0.3 0.1 0.2 0.2 0.3 0.4 0.1 0.2 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.1 0.2 0.2 0.3 0.4 0.4 0.5 0.5 0.6 0.6 0.7 0.8 0.8 0.9

Swelling strain [%]

4.5 13.2 20.1 27.0 31.5 58.8 45.0 36.5 41.0 28.6 23.6 4.5 6.2 8.3 23.1 13.6 21.7 15.6 21.1 27.2 3.1 5.4 19.1 30.7 29.9 49.5 48.2 38.0 50.7 35.9 46.7 39.7 34.6 -0.8 1.5 1.6 7.4 11.2 17.1 19.1 23.1 29.5 28.4 26.2 21.2 19.5 16.1 13.1

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[257]

Horse
G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
R R R R R R R R R R R R R R R R R R R R L L L L L L L L L L L L L L L L L L L L L L L L L L L L

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3

Slice
16 17 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7

Total Slice
17 17 12 12 12 12 12 12 12 12 12 12 12 12 6 6 6 6 6 6 13 13 13 13 13 13 13 13 13 13 13 13 13 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Relative depth
0.9 1.0 0.1 0.2 0.3 0.3 0.4 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.2 0.3 0.5 0.7 0.8 1.0 0.1 0.2 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Swelling strain [%]

9.4 6.8 1.9 6.0 8.7 13.4 19.4 20.8 22.9 19.0 17.3 14.0 11.0 10.9 39.9 49.0 60.3 85.8 35.8 29.8 -0.5 3.5 8.0 17.0 22.9 33.0 35.2 43.8 37.7 46.9 41.5 37.6 30.1 2.0 9.2 27.0 49.5 63.7 70.6 66.5 49.0 10.0 13.9 16.0 19.2 26.2 38.6 28.5

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[258]

Horse
G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
3 3 3 4 4 4 4 4 4 1 1 1 1 1 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 1 2 2 2 2 2 2 2 2 2 2 2 2 3

Slice
8 9 10 1 2 3 4 5 6 1 2 3 4 5 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 1 1 2 3 4 5 6 7 8 9 10 11 12 1

Total Slice
10 10 10 6 6 6 6 6 6 5 5 5 5 5 4 4 4 4 12 12 12 12 12 12 12 12 12 12 12 12 4 4 4 4 1 12 12 12 12 12 12 12 12 12 12 12 12 11

Relative depth
0.8 0.9 1.0 0.2 0.3 0.5 0.7 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.3 0.5 0.8 1.0 0.1 0.2 0.3 0.3 0.4 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.3 0.5 0.8 1.0 1.0 0.1 0.2 0.3 0.3 0.4 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1

Swelling strain [%]

31.3 32.8 36.1 0.2 3.2 9.3 20.7 37.0 42.3 -0.5 17.3 29.1 40.9 57.0 5.7 15.8 28.5 18.6 0.6 3.7 11.1 36.9 26.1 35.0 28.9 30.3 32.6 25.7 18.1 9.4 1.5 11.8 19.4 30.5 -0.8 3.6 11.0 28.5 35.5 49.0 71.8 59.9 103.7 75.4 64.0 53.0 49.4 3.5

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[259]

Horse
G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 1 2 2 2 2 2 2 2 2 2 2 2 2 4 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3

Slice
2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 1 1 2 3 4 5 6 7 8 9 10 11 12 1 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3

Total Slice
11 11 11 11 11 11 11 11 11 11 5 5 5 5 5 1 12 12 12 12 12 12 12 12 12 12 12 12 1 4 4 4 4 12 12 12 12 12 12 12 12 12 12 12 12 11 11 11

Relative depth
0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.2 0.4 0.6 0.8 1.0 1.0 0.1 0.2 0.3 0.3 0.4 0.5 0.6 0.7 0.8 0.8 0.9 1.0 1.0 0.3 0.5 0.8 1.0 0.1 0.2 0.3 0.3 0.4 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.2 0.3

Swelling strain [%]

10.5 24.4 32.0 61.1 52.5 55.1 53.5 58.8 40.6 43.3 0.7 2.8 10.1 8.7 7.7 1.3 0.9 4.1 8.4 16.4 18.6 30.4 38.3 41.3 46.3 39.2 24.7 15.8 0.8 -1.1 3.6 8.4 11.1 -1.7 -0.3 4.6 6.0 15.5 13.8 23.1 23.5 21.6 19.0 17.0 15.1 -4.7 -2.1 2.6

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[260]

Horse
G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3 G3

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
3 3 3 3 3 3 3 3 4 4 4 4 4 4 1 1 1 1 1 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4

Slice
4 5 6 7 8 9 10 11 1 2 3 4 5 6 1 2 3 4 5 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

Total Slice
11 11 11 11 11 11 11 11 6 6 6 6 6 6 5 5 5 5 5 9 9 9 9 9 9 9 9 9 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Relative depth
0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.2 0.3 0.5 0.7 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Swelling strain [%]

10.8 22.0 21.8 27.1 34.7 27.6 28.5 26.6 1.8 4.2 9.7 6.8 10.6 8.9 3.2 4.6 7.0 10.9 13.3 -1.1 0.7 2.6 3.8 7.7 7.8 6.7 5.7 5.1 2.9 5.4 8.1 10.5 15.8 13.1 11.7 9.9 7.5 7.4 0.7 3.0 6.0 9.5 11.5 10.2 14.8 9.2 8.0 7.2

Defect
N N N N N N N N N N N N N N Y Y Y Y Y N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[261]

Horse
G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 1 1 1 1 2 2 2 2 2 2

Slice
1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 1 2 3 4 1 2 3 4 5 6

Total Slice
7 7 7 7 7 7 7 9 9 9 9 9 9 9 9 9 14 14 14 14 14 14 14 14 14 14 14 14 14 14 8 8 8 8 8 8 8 8 4 4 4 4 11 11 11 11 11 11

Relative depth
0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.1 0.2 0.3 0.4 0.4 0.5 0.6 0.6 0.7 0.8 0.9 0.9 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.3 0.5 0.8 1.0 0.1 0.2 0.3 0.4 0.5 0.5

Swelling strain [%]

0.2 3.8 6.6 16.0 17.6 22.9 24.8 6.5 18.8 34.0 41.7 47.4 64.2 61.5 57.5 55.2 -0.4 2.5 5.7 8.9 12.6 19.1 34.5 23.3 36.6 24.5 21.4 18.2 16.1 10.6 -1.6 0.1 1.5 4.2 4.3 12.1 6.4 22.0 5.1 9.3 15.6 19.3 0.0 3.5 8.0 14.2 17.7 16.3

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[262]

Horse
G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L R R R R R R R R R R R R R R R R R R

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2

Slice
7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7

Total Slice
11 11 11 11 11 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 10 10 10 10 10 10 10 10 10 10 11 11 11 11 11 11 11 11 11 11 11 9 9 9 9 9 9 9

Relative depth
0.6 0.7 0.8 0.9 1.0 0.1 0.1 0.2 0.3 0.3 0.4 0.5 0.5 0.6 0.7 0.7 0.8 0.9 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8

Swelling strain [%]

12.7 11.7 9.2 7.7 5.8 -1.5 0.4 4.2 10.3 21.5 17.7 38.3 27.8 42.9 30.9 61.3 17.7 21.0 -0.9 23.3 0.6 1.2 3.2 9.0 5.6 7.2 8.1 8.8 13.0 10.6 -1.5 1.3 5.0 15.2 23.1 28.5 35.2 34.0 33.9 24.9 24.0 4.5 11.8 24.6 55.0 74.8 35.0 75.9

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[263]

Horse
G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3

Slice
8 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 1 2 3 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11

Total Slice
9 9 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 7 7 7 7 7 7 7 3 3 3 10 10 10 10 10 10 10 10 10 10 11 11 11 11 11 11 11 11 11 11 11

Relative depth
0.9 1.0 0.1 0.1 0.2 0.3 0.3 0.4 0.5 0.5 0.6 0.7 0.7 0.8 0.9 0.9 1.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.3 0.7 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0

Swelling strain [%]

33.3 22.0 -0.5 3.2 5.1 9.8 13.0 17.8 23.8 25.0 26.3 25.1 29.2 26.0 19.8 20.6 14.3 -2.2 -0.1 1.0 2.0 3.1 3.3 5.1 3.2 10.1 13.9 2.8 5.2 11.1 17.8 27.4 30.3 38.2 30.2 27.2 22.4 -1.4 1.7 6.0 9.0 10.9 13.2 19.4 14.9 15.1 10.7 10.3

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[264]

Horse
G30 G30 G30 G30 G30 G30 G30 G30 G30 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
R R R R R R R R R L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr

Site
4 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 1 1 1

Slice
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 1 2 3

Total Slice
9 9 9 9 9 9 9 9 9 8 8 8 8 8 8 8 8 12 12 12 12 12 12 12 12 12 12 12 12 11 11 11 11 11 11 11 11 11 11 11 5 5 5 5 5 4 4 4

Relative depth
0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 1.0 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.1 0.2 0.3 0.3 0.4 0.5 0.6 0.7 0.8 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.2 0.4 0.6 0.8 1.0 0.3 0.5 0.8

Swelling strain [%]

0.3 2.6 4.8 6.3 11.0 7.5 8.1 8.3 8.2 -0.4 2.0 5.3 7.1 15.2 34.6 25.3 18.4 4.1 4.8 7.5 14.4 17.8 21.8 28.7 22.3 21.6 18.2 18.3 11.5 1.6 4.6 7.0 10.7 13.2 13.0 19.1 16.5 15.5 12.3 9.5 2.7 3.9 4.7 7.1 6.4 3.0 6.0 8.0

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[265]

Horse
G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
L L L L L L L L L L L L L L L L L L L L R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site
1 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3

Slice
4 1 2 3 4 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10

Total Slice
4 4 4 4 4 10 10 10 10 10 10 10 10 10 10 5 5 5 5 5 7 7 7 7 7 7 7 11 11 11 11 11 11 11 11 11 11 11 10 10 10 10 10 10 10 10 10 10

Relative depth
1.0 0.3 0.5 0.8 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.2 0.4 0.6 0.8 1.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Swelling strain [%]

9.6 5.3 9.6 13.9 12.7 0.8 3.2 5.5 8.0 9.9 14.6 13.9 14.1 13.4 15.6 1.3 3.8 6.9 5.8 4.6 0.4 1.4 4.3 8.9 6.9 9.6 2.8 1.1 2.2 3.9 7.2 9.7 14.1 20.7 23.0 22.2 24.2 14.3 1.4 4.6 8.1 12.6 15.2 20.0 21.2 22.3 22.1 20.4

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

[266]

Horse
G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33 G33

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Limb
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

Bone
C3 C3 C3 C3 C3 C3 C3 C3 Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site
4 4 4 4 4 4 4 4 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4

Slice
1 2 3 4 5 6 7 8 1 2 3 4 5 6 1 2 3 4 5 6 7 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10

Total Slice
8 8 8 8 8 8 8 8 6 6 6 6 6 6 7 7 7 7 7 7 7 6 6 6 6 6 6 10 10 10 10 10 10 10 10 10 10

Relative depth
0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 0.2 0.3 0.5 0.7 0.8 1.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 0.2 0.3 0.5 0.7 0.8 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Swelling strain [%]

0.1 1.2 3.7 5.7 5.6 7.5 6.9 7.9 3.9 4.7 6.8 9.1 11.1 11.0 2.8 7.1 11.9 14.3 22.1 13.9 6.7 1.3 3.9 7.6 11.6 11.4 10.7 0.4 2.7 4.8 4.7 5.9 7.1 7.5 8.3 7.6 6.7

Defect
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

4373 4374

[267]

4375

8.4 ANALYSIS 2 - MEAN SWELLING STRAIN DATA (SAS)

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX Horse 17 13 13 17 13 17 13 13 17 13 Limb Left Left Left Left Left Left Left Left Left Left Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Site 1 1 1 1 1 1 1 1 1 1 Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94 Mean 2.975 9.525 32.2 29.625 35.76 45.925 36.9 51.15 35.725 27

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 16 29 1 16 19 16 1 19 16 1

Limb Left Left Left Left Left Left Left Left Left Left

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 1 1 1 1 1 1 1 1 1 1

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 8.125 37.1 17.5166667 26.85 66.8 49.125 33.86 51.95 32.4 27.6333333

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 13 24 13 13 30 13 13 24 13 13

Limb Left Left Left Left Left Left Left Left Left Left

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 1 1 1 1 1 1 1 1 1 1

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean -1.6 -0.5 14.875 9.25 7.65 16.1 21.4 21.5 9.8 29.94

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 19 19 1 20 1

Limb Left Left Left Left Left

Bone Cr Cr Cr Cr Cr

Site 1 1 1 1 1

Depth 0.04 0.14 0.24 0.34 0.44

Mean 2.8 32.8666667 6.63333333 16.95 15.7333333

[268]

CONDEX CONDEX CONDEX CONDEX CONDEX

20 29 1 29 1

Left Left Left Left Left

Cr Cr Cr Cr Cr

1 1 1 1 1

0.54 0.64 0.74 0.84 0.94

23.2 48.6 19.225 35.6 23.325

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 13 17 13 13 13 13 17 13 13 13

Limb Left Left Left Left Left Left Left Left Left Left

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 2 2 2 2 2 2 2 2 2 2

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 3.08333333 9.28 17.1 28.85 33.475 40.8833333 48.6 34.8428571 32.08 25.775

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 1 1 1 1 1 1 1 1 1 1

Limb Left Left Left Left Left Left Left Left Left Left

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 2 2 2 2 2 2 2 2 2 2

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 1.76666667 14.8 15.4428571 23.12 23.575 35.96 44.2166667 41.6 37.35 31.7666667

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 13 17 13 13 24 13 13 17 13 13

Limb Left Left Left Left Left Left Left Left Left Left

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 2 2 2 2 2 2 2 2 2 2

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 1.96666667 5.85 6.84 12.3 14.85 19.2 18.2 22.375 18.2333333 13.74

Treatment CONDEX

Horse 29

Limb Left

Bone Cr

Site 2

Depth -0.06

Mean 0.1

[269]

CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

1 1 1 1 1 1 1 1 1 1

Left Left Left Left Left Left Left Left Left Left

Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

2 2 2 2 2 2 2 2 2 2

0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

8.4 18.9428571 25.5333333 42.9 42.6571429 48.2428571 37.8666667 34.1428571 24.5571429 14.6857143

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 13 13 13 13 17 13 13 13 13 13

Limb Left Left Left Left Left Left Left Left Left Left

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 3 3 3 3 3 3 3 3 3 3

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 3.64285714 9.2 11.98 17.1857143 22.64 26.6571429 24.02 21.84 19.1285714 16.9

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 16 1 1 1 16 1 1 1 16 1

Limb Left Left Left Left Left Left Left Left Left Left

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 3 3 3 3 3 3 3 3 3 3

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 3.3 8.08 9.7 14.22 15.36 21.02 17.74 21.9833333 18.675 14.3857143

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 13 24 13 13 17 13 13 17

Limb Left Left Left Left Left Left Left Left

Bone Cr Cr Cr Cr Cr Cr Cr Cr

Site 3 3 3 3 3 3 3 3

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74

Mean 1.9 3.7 14.5285714 14.58 25.9 24.04 29.84 24.34

[270]

PASTEX PASTEX

13 13

Left Left

Cr Cr

3 3

0.84 0.94

15.4666667 17.68

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 16 1 16 1 16 1 16 1 16 1

Limb Left Left Left Left Left Left Left Left Left Left

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 3 3 3 3 3 3 3 3 3 3

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 5.15 7.04 15.75 12.625 30.225 23.7 36.3666667 25.2166667 26.55 23.52

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 17 13 13 17 13 17 13 13 30 13

Limb Left Left Left Left Left Left Left Left Left Left

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 4 4 4 4 4 4 4 4 4 4

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 1.1 1.9 4.2 9.3 8.675 9.6 12.55 15.425 6.4 18.875

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 16 19 1 16 16 16 1 16 16 1

Limb Left Left Left Left Left Left Left Left Left Left

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 4 4 4 4 4 4 4 4 4 4

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 1.06666667 5.3 2.46 6.675 8.03333333 8.1 2.75 8.2 9.3 8.24

Treatment PASTEX PASTEX PASTEX PASTEX

Horse 13 17 13 13

Limb Left Left Left Left

Bone Cr Cr Cr Cr

Site 4 4 4 4

Depth 0.044210967 0.14 0.24 0.34

Mean 0.96666667 1.5 3.175 6.25

[271]

PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

24 13 13 17 13 13

Left Left Left Left Left Left

Cr Cr Cr Cr Cr Cr

4 4 4 4 4 4

0.44 0.54 0.64 0.74 0.84 0.94

8.7 8.2 11.1666667 15.5 11.7666667 12.84

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 1 1 1 1 16 1 1 1 1 1

Limb Left Left Left Left Left Left Left Left Left Left

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 4 4 4 4 4 4 4 4 4 4

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 1.5 2.8 5.54 11.8 8.3 17.175 20.8 18.72 18.275 13.6333333

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 13 17 3 13 3 13 13 3 13 3

Limb Right Right Right Right Right Right Right Right Right Right

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 1 1 1 1 1 1 1 1 1 1

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 0.52 4.5 8.08 23.28 26.45 28.28 23.825 26.2666667 19.66 12.2166667

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 19 16 20 1 16 20 16 1 20 16 31 1

Limb Right Right Right Right Right Right Right Right Right Right Right Right

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 1 1 1 1 1 1 1 1 1 1 1 1

Depth 0.04 0.14 0.24 0.27 0.34 0.44 0.54 0.60 0.64 0.74 0.84 0.94

Mean -0.7 1.95 1.65 -4.8 5.9 8.8 9.85 11.8 10.45 16.7 45.8 30.78

[272]

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 17 3 17 3 13 3 17 3 17 3

Limb Right Right Right Right Right Right Right Right Right Right

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 1 1 1 1 1 1 1 1 1 1

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 3.1 7.9 12.8666667 17.25 26.525 22.5 23.3 26.15 39.7 16.9

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 1 1 1 1 19 1 1 1 1 1

Limb Right Right Right Right Right Right Right Right Right Right

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 1 1 1 1 1 1 1 1 1 1

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean -0.5 10.7 2.94 14.7666667 10.7333333 30.9 12.25 25.34 22.65 31.0333333

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 3 3 3 3 3 3 3 3 3 3

Limb Right Right Right Right Right Right Right Right Right Right

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 2 2 2 2 2 2 2 2 2 2

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 1.18571429 5.32857143 16.5 27.0428571 29.6285714 44.4285714 44.8 43.3777778 31.3714286 24.7166667

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 16 16 16 1 16 16

Limb Right Right Right Right Right Right

Bone C3 C3 C3 C3 C3 C3

Site 2 2 2 2 2 2

Depth 0.04 0.14 0.24 0.27 0.34 0.44

Mean 0.85 5.3125 14.5833333 9.8 19.1166667 20.2714286

[273]

CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

16 1 16 16 16 1

Right Right Right Right Right Right

C3 C3 C3 C3 C3 C3

2 2 2 2 2 2

0.54 0.60 0.64 0.74 0.84 0.94

33.08 39.9 45.6 41.2857143 36.74 26.76

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 3 3 3 3 17 3 3 3 3 3

Limb Right Right Right Right Right Right Right Right Right Right

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 2 2 2 2 2 2 2 2 2 2

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 4.92857143 3.8 13.7857143 18 27.6 25.5 26.85 26.3333333 16.6 11.7333333

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 1 1 1 1 1 1 1 1 1 1

Limb Right Right Right Right Right Right Right Right Right Right

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 2 2 2 2 2 2 2 2 2 2

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 3.4125 8.2 14.3 22.9 26.93 34.6714286 44.7333333 26.1222222 26.325 14.9166667

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 3 3 3 3 3 3 3 3 3 3

Limb Right Right Right Right Right Right Right Right Right Right

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 3 3 3 3 3 3 3 3 3 3

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 1.475 5.58571429 12.3875 17.14 28.7888889 30.44 31.4285714 32.84 24.8142857 23.9

Treatment

Horse

Limb

Bone

Site

Depth

Mean

[274]

CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

1 1 1 1 19 1 1 1 1 1

Right Right Right Right Right Right Right Right Right Right

C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

3 3 3 3 3 3 3 3 3 3

0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

1.83333333 3.42 10 15.4833333 25.575 22.4333333 21.3 21.7428571 20.2833333 16.35

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 3 3 3 3 3 3 3 3 3 3

Limb Right Right Right Right Right Right Right Right Right Right

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 3 3 3 3 3 3 3 3 3 3

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 2.06 4.5 8.61666667 15.34 16.0166667 21.58 16.68 15.8166667 13.38 10.16

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 1 1 1 1 16 1 1 1 1 1

Limb Right Right Right Right Right Right Right Right Right Right

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 3 3 3 3 3 3 3 3 3 3

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 0.61428571 4.68571429 8.4 13.3857143 18.6166667 18.9 22.85 23.5625 12.4285714 10.0166667

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 13 3 3 13 3 13 3 3

Limb Right Right Right Right Right Right Right Right

Bone C3 C3 C3 C3 C3 C3 C3 C3

Site 4 4 4 4 4 4 4 4

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74

Mean 2.375 8.32 5.78 7.82 7.93333333 6.2 5.9 8.425

[275]

PASTEX PASTEX

13 3

Right Right

C3 C3

4 4

0.84 0.94

5.86666667 7.34

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 29 1 1 19 1 19 1 1 29 1

Limb Right Right Right Right Right Right Right Right Right Right

Bone C3 C3 C3 C3 C3 C3 C3 C3 C3 C3

Site 4 4 4 4 4 4 4 4 4 4

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean -2.3 4.8 7.875 6.25 8 13.7 12.3 17.52 14.3 18.7833333

Treatment PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Horse 3 3 3 3 3 3 3 3 3 3

Limb Right Right Right Right Right Right Right Right Right Right

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 4 4 4 4 4 4 4 4 4 4

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 0.5 10.28 13.84 7.26 22.15 11.7 25.98 14.64 9.22 10.35

Treatment CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Horse 1 1 1 1 1 1 1 1 1 1

Limb Right Right Right Right Right Right Right Right Right Right

Bone Cr Cr Cr Cr Cr Cr Cr Cr Cr Cr

Site 4 4 4 4 4 4 4 4 4 4

Depth 0.04 0.14 0.24 0.34 0.44 0.54 0.64 0.74 0.84 0.94

Mean 1.32 9.07142857 14.6 27.2 25.3777778 27.98 22.3833333 25.1428571 21.2 21.7

4376 4377

[276]

4378

8.5 ANALYSIS 2 - COHORT MEAN SWELLING STRAIN DATA (SAS)

Site (a) Cohort S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4 Estimate Mean Swelling [%] 17.8759 22.2065 15.5537 9.6806 19.8313 25.2847 16.7712 7.4536 15.9205 19.1282 14.3362 11.9076 22.5819 22.8105 15.4032 7.2867 13.1699 21.6025 15.7042 12.0745 S.E. 2.9244 2.8828 2.8961 2.9024 3.27 3.2225 3.2258 3.276 3.3301 3.2366 3.2804 3.2493 3.2985 3.2377 3.2581 3.2536 3.2303 3.1355 3.1703 3.187 C.I. CI Upper 11.9034 16.3189 9.639 3.753 13.3348 18.8826 10.3626 0.9452 9.3048 12.6982 7.819 5.4523 16.0289 16.3783 8.9305 0.8229 6.7523 15.3732 9.4059 5.7429 C.I. Lower 23.8484 28.094 21.4684 15.6081 26.3277 31.6869 23.1799 13.962 22.5362 25.5583 20.8533 18.3628 29.1349 29.2427 21.8759 13.7504 19.5875 27.8318 22.0026 18.4061 Pr > |t| <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 0.0253 <.0001 <.0001 <.0001 0.0004 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 0.0276 0.0003

(b)

C3

Cr

(c)

Left

Right

4379 4380

[277]

4381 4382 4383 4384 4385 4386 4387 4388 4389 4390 4391 4392 4393 4394 4395 4396 4397 4398 4399 4400 4401 4402 4403

8.6 ANALYSIS 3 - SAS SOURCE CODES AND OUTPUT

8.6.1 MULTIVARIATE ANOVA (MANOVA) CODE

proc glm data=dat; class horse treatment leg site; model logHCT--logBLF=treatment horse(treatment) site site*treatment site*horse(treatment) leg leg*treatment leg*horse(treatment) site*leg*horse(treatment) site*leg site*leg*treatment ; random horse(treatment) site*horse(treatment) leg*horse(treatment) site*leg*horse(treatment)/test; manova h=treatment e=horse(treatment) ; manova h=site e=site*horse(treatment) ; manova h=leg e=leg*horse(treatment) ; manova h=treatment*leg e=leg*horse(treatment) ; manova h=site*leg e=site*leg*horse(treatment) ; manova h=site*treatment e=site*horse(treatment) ; manova h=site*leg*treatment e=site*leg*horse(treatment) ; manova h=horse(treatment) ; manova h=site*horse(treatment) ; manova h=leg*horse(treatment) ; manova h=site*leg*horse(treatment) ; run;

[278]

4404 4405 4406 4407 4408 4409 4410 4411 4412 4413 4414 4415 4416 4417 4418 4419 4420 4421 4422 4423 4424 4425 4426 4427 4428 4429 4430 4431 4432 4433 4434 4435 4436 4437 4438 4439 4440 4441 4442 4443 4444 4445 4446 4447 4448 4449 4450 4451 4452 4453 4454 4455 4456 4457 4458 4459 4460 4461 4462 4463 4464 4465 4466 4467

8.6.2 OUTPUT

MANOVA Test Criteria and Exact F Statistics for the Hypothesis of No Overall Treatment Effect H = Type III SSCP Matrix for Treatment E = Type III SSCP Matrix for Horse(Treatment) S=1 Statistic Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root M=1.5 Value 0.30697365 0.69302635 2.25760861 2.25760861 N=0.5 F Value 1.35 1.35 1.35 1.35 Num DF 5 5 5 5 Den DF 3 3 3 3 Pr > F 0.4271 0.4271 0.4271 0.4271

MANOVA Test Criteria and F Approximations for the Hypothesis of No Overall Site Effect H = Type III SSCP Matrix for Site E = Type III SSCP Matrix for Horse*Site(Treatmen) S=3 Statistic Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root M=0.5 N=7 Num DF 15 15 15 5 Den DF 44.57 54 25.399 18 Pr > F <.0001 <.0001 <.0001 <.0001

Value 0.01105350 2.05500029 16.06051267 12.03364297

F Value 12.22 7.83 16.27 43.32

MANOVA Test Criteria and Exact F Statistics for the Hypothesis of No Overall Leg Effect H = Type III SSCP Matrix for Leg E = Type III SSCP Matrix for Horse*Leg(Treatment) S=1 Statistic Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root M=1.5 Value 0.51867333 0.48132667 0.92799581 0.92799581 N=0.5 F Value 0.56 0.56 0.56 0.56 Num DF 5 5 5 5 Den DF 3 3 3 3 Pr > F 0.7355 0.7355 0.7355 0.7355

MANOVA Test Criteria and Exact F Statistics for the Hypothesis of No Overall Treatment*Leg Effect H = Type III SSCP Matrix for Treatment*Leg E = Type III SSCP Matrix for Horse*Leg(Treatment) S=1 Statistic Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root M=1.5 Value 0.67599742 0.32400258 0.47929559 0.47929559 N=0.5 F Value 0.29 0.29 0.29 0.29 Num DF 5 5 5 5 Den DF 3 3 3 3 Pr > F 0.8934 0.8934 0.8934 0.8934

MANOVA Test Criteria and F Approximations for the Hypothesis of No Overall Leg*Site Effect

[279]

4468 4469 4470 4471 4472 4473 4474 4475 4476 4477 4478 4479 4480 4481 4482 4483 4484 4485 4486 4487 4488 4489 4490 4491 4492 4493 4494 4495 4496 4497 4498 4499 4500 4501 4502 4503 4504 4505 4506 4507 4508 4509 4510 4511 4512 4513 4514 4515 4516 4517 4518 4519 4520 4521 4522 4523 4524 4525 4526 4527 4528 4529 4530 4531 4532 4533 4534

H = Type III SSCP Matrix for Leg*Site E = Type III SSCP Matrix for Hors*Leg*Site(Treat) S=3 Statistic Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root M=0.5 N=6 Num DF 15 15 15 5 Den DF 39.049 48 21.68 16 Pr > F 0.1872 0.2084 0.1838 0.0122

Value 0.30109552 0.89253966 1.72021621 1.32138943

F Value 1.42 1.36 1.52 4.23

MANOVA Test Criteria and F Approximations for the Hypothesis of No Overall Treatment*Site Effect H = Type III SSCP Matrix for Treatment*Site E = Type III SSCP Matrix for Horse*Site(Treatmen) S=3 Statistic Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root M=0.5 N=7 Num DF 15 15 15 5 Den DF 44.57 54 25.399 18 Pr > F 0.8769 0.8741 0.8568 0.1799

Value 0.61274277 0.41896201 0.58098295 0.47912387

F Value 0.58 0.58 0.59 1.72

MANOVA Test Criteria and F Approximations for the Hypothesis of No Overall Treatment*Leg*Site Effect H = Type III SSCP Matrix for Treatment*Leg*Site E = Type III SSCP Matrix for Hors*Leg*Site(Treat) S=3 Statistic Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root M=0.5 N=6 Num DF 15 15 15 5 Den DF 39.049 48 21.68 16 Pr > F 0.2824 0.5168 0.1437 0.0030

Value 0.34010949 0.68795111 1.85836674 1.81378181

F Value 1.24 0.95 1.64 5.80

MANOVA Test Criteria and F Approximations for the Hypothesis of No Overall Horse(Treatment) Effect H = Type III SSCP Matrix for Horse(Treatment) E = Error SSCP Matrix S=5 Statistic Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root M=0.5 Value 0.00328481 2.24412093 40.41288107 36.05544235 N=133 F Value 93.41 31.64 307.96 1401.01 Num DF 35 35 35 7 Den DF 1129.8 1360 750.22 272 Pr > F <.0001 <.0001 <.0001 <.0001

MANOVA Test Criteria and F Approximations for the Hypothesis of No Overall Horse*Site(Treatmen) Effect H = Type III SSCP Matrix for Horse*Site(Treatmen) E = Error SSCP Matrix S=5 Statistic M=7 Value N=133 F Value Num DF Den DF Pr > F

[280]

4535 4536 4537 4538 4539 4540 4541 4542 4543 4544 4545 4546 4547 4548 4549 4550 4551 4552 4553 4554 4555 4556 4557 4558 4559 4560 4561 4562 4563 4564 4565 4566 4567 4568 4569 4570 4571 4572

Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root

0.03169555 1.97979116 7.49621863 4.44656628

13.50 8.91 19.98 60.47

100 100 100 20

1312.1 1360 1028.5 272

<.0001 <.0001 <.0001 <.0001

MANOVA Test Criteria and F Approximations for the Hypothesis of No Horse*Leg(Treatment) Effect H = Type III SSCP Matrix for Horse*Leg(Treatment) E = Error SSCP Matrix S=5 Statistic Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root M=0.5 Value 0.09891769 1.41933449 4.37137544 3.01265070 N=133 F Value 23.67 15.40 33.31 117.06 Num DF 35 35 35 7 Den DF 1129.8 1360 750.22 272 Pr > F <.0001 <.0001 <.0001 <.0001

MANOVA Test Criteria and F Approximations for the Hypothesis of No Overall Hors*Leg*Site(Treat) Effect H = Type III SSCP Matrix for Hors*Leg*Site(Treat) E = Error SSCP Matrix S=5 Statistic Wilks' Lambda Pillai's Trace Hotelling-Lawley Trace Roy's Greatest Root M=6 N=133 F Value 7.11 5.21 10.12 40.54 Num DF 90 90 90 18 Den DF 1304.7 1360 1005.4 272 Pr > F <.0001 <.0001 <.0001 <.0001

Value 0.14424133 1.28135872 3.41610974 2.68253586

[281]

4573 4574 4575 4576 4577 4578 4579 4580 4581 4582 4583 4584 4585 4586 4587 4588

8.6.3 UNIVARIATE MIXED MODEL ANALYSES (ANOVA)

proc glm data=dat plot=diagnostics; class horse treatment leg site; model logHCT--logBLF=treatment horse(treatment) site site*treatment site*horse(treatment) leg leg*treatment leg*horse(treatment) site*leg*horse(treatment) site*leg site*leg*treatment ; random horse(treatment) site*horse(treatment) leg*horse(treatment) site*leg*horse(treatment)/test; means treatment/cldiff t e=horse(treatment); run;

8.6.4 OUTPUT
log(HCT) Num DF 1 3 3 1 1 3 3 Den DF 7 18 18 7 7 18 18

Effect Treatment Site Treatment*Site Leg Treatment*Leg Leg*Site Treatment*Leg*Site 4589 4590 4591 4592 4593

F Value Pr > F 0.12 22.94 0.96 1.78 0.04 0.63 0.05 0.7385 <.0001 0.4316 0.2243 0.8538 0.6026 0.9859

log(ZCCT) Num DF 1 3 3 1 1 3 3 Den DF 7 18 18 7 7 18 18

Effect Treatment Site Treatment*Site Leg Treatment*Leg Leg*Site Treatment*Leg*Site 4594 4595 4596 4597 log(VCA)

F Value Pr > F 1.04 24.94 0.2 0.49 0 3.96 2.89 0.3413 <.0001 0.8979 0.5051 0.995 0.0248 0.064

[282]

Effect Treatment Site Treatment*Site Leg Treatment*Leg Leg*Site Treatment*Leg*Site 4598 4599 4600 log(VCN)

Num DF 1 3 3 1 1 3 3

Den DF 7 18 18 7 7 18 18

F Value Pr > F 5.86 43.19 0.35 0.1 0.01 1.6 0.18 0.0461 <.0001 0.7874 0.7613 0.9115 0.2252 0.9065

Effect Treatment Site Treatment*Site Leg Treatment*Leg Leg*Site Treatment*Leg*Site 4601 4602 4603 log(BLF)

Num DF 1 3 3 1 1 3 3

Den DF 7 18 18 7 7 18 18

F Value Pr > F 0.05 13.68 0.01 0.42 0.36 0.18 0.24 0.8234 <.0001 0.9978 0.5396 0.5653 0.9095 0.8682

Effect Treatment Site Treatment*Site Leg Treatment*Leg Leg*Site Treatment*Leg*Site 4604

Num DF 1 3 3 1 1 3 3

Den DF 7 18 18 7 7 18 18

F Value Pr > F 0.33 3.93 0.23 0.28 2.91 0.77 1.56 0.5848 0.0256 0.8729 0.6153 0.1318 0.5252 0.2327

[283]

4605

8.6.1 STRUCTURE EQUALITY MODELLING / PATH ANALYSIS SOURCE CODE (R)

4606 4607 4608 4609 4610 4611 4612 4613 4614 4615 4616 4617 4618 4619 4620 4621 4622 4623 4624 4625 4626 4627 4628 4629 4630 4631 4632 4633 4634 4635 4636 4637 4638 4639 4640 4641 4642 4643 4644 4645 4646

W ITH F ISHER . TRANSFORMATION

Fisher.trans=function(r) { if (length(r)>1) {z=0.5*log((1+r)/(1-r))} } compare2cormats=function(d,fac,nperm=1000) { nfac=unique(fac) m1=d[fac==nfac[1],] m2=d[fac==nfac[2],] c1=Fisher.trans(cor(m1)) c2= Fisher.trans(cor(m2)) diag(c1)=1 diag(c2)=1 dif=(c1-c2)^2 dif=sum((c1-c2)^2) pdif=NULL for (i in 1:nperm) { perm=sample(1:nrow(d)) pd=d[perm,] pm=pd[fac==nfac[1],] pw=pd[fac==nfac[2],] pc1=Fisher.trans(cor(pm)) pc2= Fisher.trans(cor(pw)) diag(pc1)=1 diag(pc2)=1 pdif=append(pdif,sum((pc1-pc2)^2)) } p.value=mean(pdif>dif) p.value } compare2cormats(d,as.factor(data[,2]))

[284]

4647 4648 4649 4650 4651 4652 4653 4654 4655 4656 4657 4658 4659 4660 4661 4662 4663 4664 4665 4666 4667 4668 4669 4670 4671 4672 4673 4674 4675 4676 4677 4678

W ITHOUT F ISHER .TRANSFORMATION

compare2cormats=function(d,fac) { nfac=unique(fac) m1=d[fac==nfac[1],] m2=d[fac==nfac[2],] c1= cor(m1) c2= cor(m2) dif=(c1-c2)^2 dif=sum((c1-c2)^2) pdif=NULL for (i in 1:10000) { perm=sample(1:nrow(d)) pd=d[perm,] pm=pd[fac==nfac[1],] pw=pd[fac==nfac[2],] pc1= cor(pm) pc2= cor(pw) pdif=append(pdif,sum((pc1-pc2)^2)) } p.value=mean(pdif>dif) p.value } compare2cormats(d,as.factor(data[,2]))

[285]

4679

8.7 ANALYSIS 3 - OSTEOCHONDRAL HISTOMORPHOMETRIC DATA

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Trauma

G17 G17 G17 G17 G17 G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G17 G17 G17 G17 G17

Horse

HCt [m]
646.37 643.56 641.33 642.35 643.70 593.08 607.17 605.81 597.51 763.53 669.95 662.26 671.47 668.02 672.38 560.41 530.24 505.86 518.90 590.85 580.99 579.41 566.37 588.39 584.03 611.09 598.54 589.80 592.20 601.10 671.79 678.80 678.54 679.15 676.12 724.05 716.05 722.86 722.67 715.80

HCang [deg]
-30 -29 -27 -29 -27 -27 -28 -28 -26 -28 -27 -31 -30 -28 -31 -28 -27 -30 -27 -24 -23 -23 -24 -26 -23 -7 -5 -12 -8 -3 -24 -28 -28 -28 -17 -14 -18 -15 -17 -14

ZCCa 2 [m ]
816158.4 913231.5 834042 832388.9 802247.6 726381.8 743806.1 775179.9 728775.1 917266.4 667614.6 580481.4 540194.8 609202.1 634963.6 598065.6 620297.4 653903.8 610904.1 634965.3 722084.2 770518.3 743225.3 718871.8 694135.3 1321115.1 1317966.1 1213614.1 1309414.5 1224699.4 885412.6 849142.6 904307.4 914834.1 885151.9 803482.1 733228.6 725079.4 774662.5 690209.4

ZCCt [m]
272.05 304.41 278.01 277.46 267.42 242.13 247.94 258.39 242.93 305.76 222.54 193.49 180.06 203.07 211.65 199.36 206.77 217.97 203.63 211.66 240.69 256.84 247.74 239.62 231.38 440.37 439.32 404.54 436.47 408.23 295.14 283.05 301.44 304.94 295.05 267.83 244.41 241.69 258.22 230.07

ZCCang [deg]
-38 -40 -41 -37 -40 -43 -43 -42 -43 -42 -41 -40 -41 -41 -39 -29 -31 -34 -29 -27 -32 -33 -34 -35 -31 -26 -18 -22 -22 -17 -8 0 2 -10 -10 -17 -20 -18 -15 -17

VCa 2 [m ]
176216 255877 243853 500978 550670 350510 301179 458201 530358 609117 548617 404907 515236 318725 496953 202575 313375 455751 354970 140905 259620 473401 379524 442522 493466 23023 332485 437471 184104 170804 286291 139481 244547 274139 245719 234276 282797 136612 316330 379322

VCn

VCang [deg]
-19 5 -10 -16 -7 -12 -33 -35 -19 -31 -26 -46 -12 -12 10 -7 -7 -11 -15 -11 -12 8 -10 -10 -8 -22 -13 -13 2 -13 -7 -22 -7 -7 -12 3 5 7 11 -19

Rug

Limb
R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R R R R R R

Slice
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

Site
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

23 22 16 20 20 18 19 18 14 12 16 18 14 18 22 16 17 12 16 22 14 18 18 13 16 23 21 21 21 16 23 19 14 17 21 19 24 21 25 22

2.3 2.5 1.8 2.3 1.9 1.6 1.7 1.7 1.6 1.7 1.7 1.7 1.6 2.0 1.5 1.8 1.6 1.5 1.5 1.9 1.7 1.8 1.7 1.4 1.5 1.9 1.8 1.7 1.6 1.9 1.8 2.0 1.9 2.1 1.8 1.8 2.2 2.0 2.0 2.0

[286]

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Trauma

G24 G24 G24 G24 G24 G24 G24 G24 G24 G24 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G24 G24 G24

Horse

HCt [m]
757.87 747.13 754.07 738.98 947.95 780.86 783.59 787.89 789.12 801.11 760.65 748.40 757.33 755.71 774.68 749.61 738.30 733.69 727.53 747.79 566.71 557.85 589.93 575.92 569.53 838.47 845.32 856.21 850.18 846.89 862.43 873.72 864.03 842.72 877.38 782.04 771.94 766.36 790.47 778.85 841.70 851.33 835.73

HCang [deg]
-5 -6 -4 -5 -2 -15 -20 -18 -18 -12 -9 -5 -5 -7 -7 -10 -4 -8 -6 -4 -13 -17 -11 -12 -10 5 10 10 10 9 1 -1 -1 -5 -5 -5 -4 -6 -5 -8 4 4 2

ZCCa 2 [m ]
670686.3 667533.2 639849.2 664198 889944.8 676246.4 618946.2 646750.4 637318.2 638382.2 772626.6 780519.4 790437.9 753738.2 750542.9 770524.1 761471.3 728484.3 753291.9 757041.1 947654.2 900310.4 820113.7 849255.2 920059.5 698698.5 684375.8 652166.2 656518.2 624503.6 687054.4 730459.2 723470.9 688004.1 704457.4 730023.2 750842.9 732592.9 762621.9 699724.9 467140.9 477245.5 516869.3

ZCCt [m]
223.56 222.51 213.28 221.40 296.65 225.42 206.32 215.58 212.44 212.79 257.54 260.17 263.48 251.25 250.18 256.84 253.82 242.83 251.10 252.35 315.88 300.10 273.37 283.09 306.69 232.90 228.13 217.39 218.84 208.17 229.02 243.49 241.16 229.33 234.82 243.34 250.28 244.20 254.21 233.24 155.71 159.08 172.29

ZCCang [deg]
7 -5 -1 2 -7 -14 -17 -18 -20 -21 -10 -10 -9 -13 -6 -10 -9 -11 -7 -4 0 -7 -8 -8 4 17 16 20 25 20 10 10 17 12 11 0 5 3 -2 1 15 7 13

VCa 2 [m ]
277316 270390 286154 159664 466197 344661 141026 323570 615074 280452 304961 231421 242817 72657 214414 368272 212954 254455 147291 290841 107874 243226 215807 167782 204043 72220 79335 77861 66199 160836 269970 185346 346080 349025 272301 76378 213437 114255 311727 374657 188152 133214 145294

VCn

VCang [deg]
-2 -21 -9 5 -7 -9 -14 3 3 -15 -1 -9 8 1 -6 -1 0 -8 -1 -4 -11 7 -12 -10 -1 5 7 -15 3 -7 2 -12 -11 27 -11 -12 -6 -2 2 -8 -1 -6 0

Rug

Limb
L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R L L L

Slice
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3

Site
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

20 21 21 18 16 26 22 18 12 19 27 22 20 20 21 25 31 21 25 18 26 26 14 14 26 21 20 20 19 20 22 16 17 21 22 15 13 16 20 18 18 21 22

1.9 2.1 2.1 1.8 2.2 2.0 2.0 2.0 1.8 1.9 2.0 1.7 2.0 1.9 1.8 1.6 1.7 1.8 1.8 1.9 2.1 1.9 2.1 1.9 2.3 1.7 2.2 2.1 1.9 2.3 1.9 1.7 1.7 1.9 1.8 1.9 2.0 1.9 1.9 1.8 1.7 1.9 1.8

[287]

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Trauma

G24 G24 G24 G24 G24 G24 G24 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G13 G13 G13 G13 G13 G13 G13 G13 G13 G13 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17 G24 G24 G24 G24 G24 G24

Horse

HCt [m]
843.94 1057.13 785.22 783.57 781.40 776.23 762.48 789.59 824.28 819.00 823.29 784.55 870.56 904.77 892.76 906.53 845.93 488.69 493.52 494.37 492.98 502.94 476.71 510.86 487.02 497.57 478.09 705.19 705.04 696.15 697.93 698.24 743.22 743.33 733.24 726.07 740.56 634.81 630.34 636.92 629.05 810.33 723.55

HCang [deg]
3 3 11 10 8 6 11 7 8 6 4 7 11 11 10 10 13 22 20 16 13 14 24 35 26 26 23 17 16 16 19 16 12 18 15 18 15 7 13 12 10 8 4

ZCCa 2 [m ]
534134.8 653066.2 643310.5 660707.7 632161.3 633938.4 611442.4 500089.5 561040.8 543687.4 532291.6 525783.9 612866.2 728341.9 711656.1 757868.2 651837.8 681091.9 684668.2 704894.7 685964.9 687690.8 503143.2 516873.1 533609.3 550660.2 533653.7 521477.8 487414 484985.8 449782.5 489348.2 603932.8 569756.6 497650.4 592514.2 571514.4 418371.8 360350.8 405181.9 448131.3 594082.1 511206.6

ZCCt [m]
178.04 217.69 214.44 220.24 210.72 211.31 203.81 166.70 187.01 181.23 177.43 175.26 204.29 242.78 237.22 252.62 217.28 227.03 228.22 234.96 228.65 229.23 167.71 172.29 177.87 183.55 177.88 173.83 162.47 161.66 149.93 163.12 201.31 189.92 165.88 197.50 190.50 139.46 120.12 135.06 149.38 198.03 170.40

ZCCang [deg]
12 9 22 21 18 14 16 18 16 14 12 17 18 20 19 18 19 22 22 24 26 24 28 29 23 21 27 28 24 27 29 26 22 24 23 25 21 13 17 16 15 20 9

VCa 2 [m ]
169077 203859 143563 255260 402539 227489 219567 173828 126046 116738 154316 189102 232862 207935 196489 338034 295981 174279 125725 137610 66352 145329 120115 44453 121834 50651 131167 84897 245477 157377 175214 254946 140743 207014 171344 141269 328989 142100 129647 142304 113637 109428 103232

VCn

VCang [deg]
5 0 4 7 -5 4 17 -13 -8 -27 7 -8 4 -15 9 3 -3 23 6 4 28 -19 -10 -36 16 -2 -12 21 22 19 21 25 17 13 21 20 19 3 -2 23 14 4 -6

Rug

Limb
L L R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R

Slice
4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1

Site
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

21 11 22 20 19 26 19 16 16 14 21 15 25 27 23 22 16 13 17 21 15 21 14 17 23 13 23 17 16 20 15 17 18 20 19 21 19 21 16 11 15 9 16

1.8 2.3 2.1 2.0 1.9 2.1 1.9 1.5 1.7 1.8 1.7 1.8 1.8 2.1 1.9 2.0 1.8 1.7 1.5 1.8 1.6 1.6 1.9 2.0 1.7 2.0 1.7 1.7 1.8 2.0 1.8 1.8 1.9 2.0 1.8 1.7 1.9 1.7 1.5 1.7 1.7 1.9 2.0

[288]

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Trauma

G24 G24 G24 G24 G30 G30 G30 G30 G30 G30 G30 G30 G30 G30 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G20 G20 G20 G20 G20 G20 G20 G20 G20

Horse

HCt [m]
716.82 701.04 712.97 664.90 451.21 429.52 436.67 434.88 435.22 535.23 564.01 541.31 542.57 536.95 518.22 534.11 564.48 571.25 564.58 554.19 545.42 570.16 559.17 544.92 823.83 750.43 834.18 825.77 828.59 752.91 738.71 758.84 747.81 715.87 424.35 433.33 419.85 404.27 416.17 448.83 469.82 464.33 462.59

HCang [deg]
7 1 4 8 16 15 9 14 13 23 20 21 19 21 -32 -29 -25 -24 -25 -30 -33 -30 -34 -31 -28 -29 -26 -25 -28 -26 -28 -26 -28 -27 -37 -42 -34 -42 -42 -38 -32 -31 -34

ZCCa 2 [m ]
485526.5 465935.5 465958.7 440350.3 283696.4 322971.8 284600.5 339530.8 283288.5 490088.3 523627.4 405075.8 455350.8 417418 517722.7 652664.2 670151.8 693637.5 679426.4 542578.7 514287.2 579906.2 566579.5 530630.5 816749.5 720629.8 809648.8 848020.6 934814.8 722180.2 702474.9 769119.9 673219.2 677684.3 790943.4 774398.9 777939.6 795263.1 768695.5 646422.9 613473.4 642479.1 640295

ZCCt [m]
161.84 155.31 155.32 146.78 94.57 107.66 94.87 113.18 94.43 163.36 174.54 135.03 151.78 139.14 172.57 217.55 223.38 231.21 226.48 180.86 171.43 193.30 188.86 176.88 272.25 240.21 269.88 282.67 311.60 240.73 234.16 256.37 224.41 225.89 263.65 258.13 259.31 265.09 256.23 215.47 204.49 214.16 213.43

ZCCang [deg]
9 2 7 13 21 15 12 16 15 27 19 28 22 28 -38 -32 -25 -30 -26 -40 -35 -42 -38 -39 -50 -52 -51 -50 -51 -41 -41 -40 -42 -41 -42 -40 -41 -39 -40 -37 -31 -35 -38

VCa 2 [m ]
237433 221200 196985 34748 105365 268188 117832 138694 158137 76441 141389 108187 183096 142067 382571 337429 262900 481317 169316 422582 904533 727098 283305 215206 663652 727159 1035669 374957 988340 832982 520720 475139 794302 525641 751587 380996 451395 725302 708306 610974 914784 1097820 906108

VCn

VCang [deg]
-13 9 -13 6 0 -3 17 0 -10 11 8 26 6 8 -34 -38 -19 -25 0 -46 -43 -36 -38 -51 -18 -6 -9 1 3 -20 -2 -33 -31 -11 1 -18 -1 -34 -6 -13 -30 -28 -30

Rug

Limb
R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R

Slice
2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4

Site
4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

15 19 22 5 14 18 11 18 15 12 13 13 13 16 20 19 28 19 17 22 15 16 18 18 19 8 21 25 14 17 17 18 12 18 16 16 22 14 21 21 12 14 8

1.9 1.9 1.7 1.7 1.8 1.7 1.5 1.7 1.8 1.7 1.7 1.6 1.8 1.6 1.7 2.0 1.9 2.2 2.0 2.1 1.8 1.8 2.0 1.8 1.6 1.7 1.6 1.7 1.6 1.8 1.7 1.8 2.0 2.0 1.6 1.6 1.8 1.7 1.8 1.7 1.8 1.6 2.0

[289]

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Trauma

G20 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G20 G20

Horse

HCt [m]
451.63 998.42 929.04 1032.53 974.43 985.86 833.85 846.94 832.08 822.61 810.47 396.31 393.51 402.68 399.11 401.53 584.88 568.42 569.43 582.39 581.37 572.55 596.19 604.77 593.35 594.54 608.92 592.19 610.05 602.98 597.26 979.76 867.34 978.74 994.09 997.25 782.75 782.60 788.24 785.03 748.88 552.06 544.16

HCang [deg]
-34 -22 -25 -25 -23 -26 -27 -27 -29 -27 -26 -21 -22 -19 -21 -21 -29 -25 -24 -21 -19 -21 -11 -8 -4 -5 2 0 1 -4 -4 -16 -22 -28 -18 -22 -21 -14 -14 -13 -14 -4 -7

ZCCa 2 [m ]
670562.3 813974.3 919900.1 918667.6 884214.2 814547.9 762513.9 843457.9 805921.4 860107.2 865560.8 349249.1 345090.1 439584.4 448633.3 430674 595235.9 571155.4 614909.2 549629.6 592767.8 632717.9 734812.8 732916.4 794499.2 786126.2 668066.2 660207.2 625780.1 693741.8 643163.1 885987.5 787063.6 895749.1 848082.6 851576.8 834241.6 826743.6 824474.2 787945.4 775346.6 639163.2 681750.2

ZCCt [m]
223.52 271.32 306.63 306.22 294.74 271.52 254.17 281.15 268.64 286.70 288.52 116.42 115.03 146.53 149.54 143.56 198.41 190.39 204.97 183.21 197.59 210.91 244.94 244.31 264.83 262.04 222.69 220.07 208.59 231.25 214.39 295.33 262.35 298.58 282.69 283.86 278.08 275.58 274.82 262.65 258.45 213.05 227.25

ZCCang [deg]
-39 -38 -41 -39 -42 -40 -42 -41 -38 -41 -31 -22 -21 -21 -20 -21 -30 -32 -26 -25 -26 -22 -18 -7 -6 -11 -1 -11 -1 -7 -13 0 -8 -16 -8 -15 -17 -15 -15 -12 -13 8 1

VCa 2 [m ]
435060 472699 614030 815903 590284 782763 585891 632703 712656 505156 681760 793044 291774 429583 489471 378654 719761 218642 342066 477109 390787 483886 659667 299416 257257 58295 184767 503706 546583 265425 525674 318305 223478 311925 202666 517079 663157 290673 390643 636491 328326 415969 305227

VCn

VCang [deg]
-32 -8 3 -6 -26 -8 1 3 -6 9 2 -20 -26 -23 -16 -23 -22 -26 -22 -19 -27 -20 -13 0 -6 1 -9 -14 -16 1 0 -6 -10 -16 -10 0 -6 -4 -23 -21 -10 -1 1

Rug

Limb
R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R L L

Slice
5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2

Site
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

11 22 25 16 20 19 19 21 19 29 28 16 14 16 17 18 14 21 24 13 19 18 17 26 29 17 18 24 24 24 28 16 16 19 14 18 21 16 21 16 24 19 19

1.5 2.0 2.0 2.0 2.2 1.7 2.1 2.3 2.3 2.4 2.4 1.9 1.6 1.8 1.6 2.0 1.6 2.0 1.7 1.6 2.0 2.0 2.0 2.3 2.2 2.2 2.1 2.0 2.2 2.0 2.3 1.6 1.5 1.5 1.5 1.7 1.9 2.0 2.1 1.7 1.8 1.8 1.9

[290]

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Trauma

G20 G20 G20 G20 G20 G20 G20 G20 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G16 G16 G16 G16 G16 G16 G16 G16 G16 G16 G19 G19 G19 G19 G19

Horse

HCt [m]
539.75 537.06 533.33 602.13 598.70 610.95 612.43 612.41 1068.40 1059.62 1157.21 1060.61 1060.71 1006.21 992.77 985.99 992.34 985.73 590.09 595.17 619.58 579.25 593.54 678.64 679.86 681.05 676.17 682.92 595.16 586.94 581.56 565.89 583.48 650.04 631.39 645.39 658.23 650.93 765.43 692.02 769.16 772.87 774.78

HCang [deg]
-6 -11 -20 1 -8 -1 -7 -11 -15 -12 -14 -17 -12 -20 -16 -14 -20 -10 -11 -11 -12 -11 -10 -3 -3 1 1 -2 0 7 9 19 14 7 5 6 6 7 11 13 15 16 14

ZCCa 2 [m ]
655678.6 716544.6 687839.3 592301.1 663678.6 684331.6 656203.8 662705.8 1143999.6 1023715.4 1239873.1 1050794.8 1128364.1 1024646 912888.8 1009916.5 937125 936415.2 646143.8 650887.5 624148.6 665549.7 673278.9 688373.9 686508 720385.7 737594.8 724762 764167.2 745522.6 761374.9 728378.2 786644.6 611084.5 535281.2 517876.9 518736.6 527294.5 577880.6 530258.7 566981.4 595506.5 579822.4

ZCCt [m]
218.56 238.85 229.28 197.43 221.23 228.11 218.73 220.90 381.33 341.24 413.29 350.26 376.12 341.55 304.30 336.64 312.38 312.14 215.38 216.96 208.05 221.85 224.43 229.46 228.84 240.13 245.86 241.59 254.72 248.51 253.79 242.79 262.21 203.69 178.43 172.63 172.91 175.76 192.63 176.75 188.99 198.50 193.27

ZCCang [deg]
3 2 4 7 9 12 14 15 -8 -9 -8 -14 -6 -10 -14 -14 -16 -10 -11 -13 -12 -7 -10 -1 -4 1 -1 -3 3 13 15 22 20 14 20 15 19 18 18 18 22 23 22

VCa 2 [m ]
635797 553513 550716 717209 292861 427849 224927 341454 302465 369140 554526 684737 263584 519188 397668 348911 394482 541171 269632 88046 263702 111621 106637 218346 127276 203299 191099 169315 452283 263560 287789 177855 157640 238384 402837 320670 232415 279919 234072 262146 280690 180670 235206

VCn

VCang [deg]
-2 -13 -6 -3 -5 3 -1 0 -9 -8 -8 2 -1 9 -8 11 -9 -1 -9 2 -4 -2 0 -3 -8 10 -5 7 2 14 -5 14 -1 0 -17 -7 20 -5 1 -7 -4 0 2

Rug

Limb
L L L R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L

Slice
3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

Site
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

19 21 20 20 17 17 17 20 26 26 23 28 35 25 26 28 20 22 23 26 20 19 20 20 25 28 17 17 18 20 20 22 24 26 18 28 32 27 14 19 13 14 18

2.2 2.1 2.1 1.8 1.7 1.6 1.8 1.7 2.1 1.8 2.1 2.4 1.7 2.0 1.7 2.1 1.9 1.7 2.1 2.1 1.9 1.9 1.8 2.0 2.0 1.8 2.0 2.3 2.1 1.8 1.9 1.7 2.4 2.3 1.9 1.8 2.1 2.0 1.8 1.8 1.6 1.7 1.7

[291]

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX

Trauma

G19 G19 G19 G19 G19 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G16 G16 G16 G16 G16 G16 G16 G16

Horse

HCt [m]
804.71 807.37 811.16 807.73 770.83 625.00 612.59 619.77 624.26 472.91 598.02 589.27 582.27 582.48 587.17 990.67 954.46 1037.52 988.69 981.58 972.81 983.64 967.43 977.48 971.44 619.01 650.52 650.77 634.98 673.12 801.17 819.92 801.12 781.68 796.34 469.68 458.13 451.38 448.09 457.39 459.68 477.71 481.10

HCang [deg]
-4 -1 0 2 0 2 0 -1 0 5 12 7 12 8 10 15 11 15 18 13 7 10 7 7 5 4 8 8 5 -1 11 6 11 6 6 32 32 33 29 29 7 15 16

ZCCa 2 [m ]
506776.3 519611.5 544072.6 510018.6 470299.8 694969.1 733195.7 769245.9 746600.6 511181.3 694713.3 693724.9 696688.1 724660.8 693387.8 607413.4 605541.3 635994.6 612049 563326.4 480470 520691.9 514495.8 545941.9 490138.3 590357.4 612294.9 566028.8 606427.8 662941.2 613225.9 684919.1 587777.8 579440.6 564501.5 573180.1 598667.2 499955.4 564214.3 533045.5 478706.1 507818.5 464363

ZCCt [m]
168.93 173.20 181.36 170.01 156.77 231.66 244.40 256.42 248.87 170.39 231.57 231.24 232.23 241.55 231.13 202.47 201.85 212.00 204.02 187.78 160.16 173.56 171.50 181.98 163.38 196.79 204.10 188.68 202.14 220.98 204.41 228.31 195.93 193.15 188.17 191.06 199.56 166.65 188.07 177.68 159.57 169.27 154.79

ZCCang [deg]
17 17 22 16 19 14 15 14 14 5 17 13 15 15 13 25 18 21 22 24 13 18 16 17 11 6 10 11 10 8 11 9 12 8 13 32 32 32 28 26 15 19 23

VCa 2 [m ]
193321 171272 157325 472036 259979 269086 511143 245877 180658 290471 264995 260801 406465 202309 263572 317762 466426 499716 332808 409219 349845 484225 187981 419257 407353 285641 116938 152739 104634 105099 218224 199852 269119 178310 134155 403119 137154 227127 143722 147303 502444 131997 391286

VCn

VCang [deg]
14 -2 19 1 6 7 8 -2 -10 -1 -1 17 4 9 -5 -10 2 -3 -2 -9 1 -6 -2 -7 -7 -4 -8 4 1 17 2 -2 6 -5 6 25 26 30 21 29 -31 7 -12

Rug

Limb
R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R

Slice
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3

Site
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4

N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

18 14 13 12 16 19 15 18 17 13 22 18 22 24 16 26 29 19 21 24 19 21 17 14 22 25 21 18 21 23 30 18 24 20 20 14 16 18 17 14 14 15 21

1.7 1.6 1.6 1.6 1.5 1.9 2.1 2.3 2.4 1.9 2.0 1.7 1.7 1.9 2.0 2.3 2.2 2.4 2.5 2.1 2.0 1.8 2.0 1.8 1.6 2.0 1.9 1.9 2.0 2.2 2.0 1.8 1.8 1.8 2.0 1.9 1.8 1.9 1.7 2.1 1.6 1.7 1.9

[292]

Treatment
CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX CONDEX PASTEX

Trauma

G16 G16 G19 G19 G19 G19 G19 G19 G19 G19 G19 G19 G20 G20 G20 G20 G20 G20 G20 G20 G20 G20 G29 G29 G29 G29 G29 G29 G29 G29 G29 G29 G31 G31 G31 G31 G31 G31 G31 G31 G31 G31 G13

Horse

HCt [m]
471.56 472.21 611.68 556.93 622.76 622.23 627.53 691.05 687.15 693.21 681.56 653.08 494.63 490.00 471.49 474.60 472.85 469.98 483.79 470.53 466.59 479.94 611.30 599.23 680.91 625.69 657.54 649.88 661.61 629.29 625.44 633.99 430.57 430.30 430.68 427.31 431.12 486.62 486.33 488.14 478.25 488.68 502.90

HCang [deg]
13 17 18 21 22 21 21 9 14 20 14 18 -4 9 2 2 7 27 23 27 25 29 15 15 19 17 19 6 9 -1 1 3 16 15 16 11 9 6 9 13 1 6 -32

ZCCa 2 [m ]
443425.1 443926.6 484255.2 443739.8 484400.5 507373.2 394618 538533.9 501059.3 520625.1 507179.4 505720.2 482133.1 522092.2 476276.5 515136.8 511063.1 465745.6 582768.2 529419.6 526869.2 473197.1 375507.7 466290.1 442077.1 423984.2 413216.8 428273.2 429779.5 472346.3 439973 470272.7 286643.4 294784.4 278946.8 303841 306813 420951.8 369561.1 346438.6 287668.7 344674.6 1438100.2

ZCCt [m]
147.81 147.98 161.42 147.91 161.47 169.12 131.54 179.51 167.02 173.54 169.06 168.57 160.71 174.03 158.76 171.71 170.35 155.25 194.26 176.47 175.62 157.73 125.17 155.43 147.36 141.33 137.74 142.76 143.26 157.45 146.66 156.76 95.55 98.26 92.98 101.28 102.27 140.32 123.19 115.48 95.89 114.89 479.37

ZCCang [deg]
17 18 22 25 23 19 22 23 24 23 25 25 12 7 7 5 9 29 24 26 29 28 6 -1 8 11 13 10 11 4 9 4 15 16 19 17 13 10 13 12 13 15 -50

VCa 2 [m ]
101516 362549 159400 205790 259460 345707 242375 220003 201385 137763 195342 164860 320132 174305 238528 149563 290429 162590 90689 197561 210776 207292 376291 235303 459313 235846 490286 268940 170019 281880 236565 177354 237436 123320 263836 102029 108175 168921 127329 160688 164314 162609 897550

VCn

VCang [deg]
-20 11 18 16 24 26 17 18 20 35 27 23 -1 6 -5 -9 0 20 6 11 23 21 -20 -4 13 17 -1 5 13 15 -6 -15 3 5 15 9 -1 3 16 1 14 10 18

Rug

Limb
R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R L L L L L R R R R R L

Slice
4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1

Site
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1

N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N Y

19 14 15 13 18 14 18 15 14 15 13 17 19 18 13 19 13 18 17 14 17 17 13 17 13 26 14 15 14 18 19 15 17 13 14 17 14 13 17 13 21 17 13

1.7 1.7 2.0 1.9 1.8 1.7 1.9 2.0 1.9 1.9 2.0 1.8 1.7 1.8 1.8 1.7 1.9 1.9 1.5 1.6 1.6 1.7 1.6 1.8 1.9 1.6 1.9 1.6 1.9 1.8 1.9 1.6 1.6 1.7 1.5 1.6 1.7 1.5 1.9 1.8 1.6 1.7 1.6

[293]

Treatment
PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX PASTEX

Trauma

G13 G13 G13 G13 G13 G13 G13 G13 G13 G17 G17 G17 G17 G17 G17 G17 G17 G17 G17

Horse

HCt [m]
494.94 486.27 487.31 488.35 515.52 555.64 544.49 549.35 554.93 806.88 804.77 829.56 809.94 811.59 870.14 852.35 864.27 851.33 858.33

HCang [deg]
-28 -27 -33 -30 -33 -32 -38 -32 -31 -30 -30 -31 -30 -29 -14 -22 -21 -24 -20

ZCCa 2 [m ]
1441543.6 1089294.5 1161886.4 1384080 1614714.2 1736580.6 1554944.5 1852100.4 1725482.4 2205641.2 2211116 2283051.8 2309187.2 2202625.8 1556373.5 1671279.8 1742593.4 1782478.8 1577799.6

ZCCt [m]
480.51 363.10 387.30 461.36 538.24 578.86 518.31 617.37 575.16 735.21 737.04 761.02 769.73 734.21 518.79 557.09 580.86 594.16 525.93

ZCCang [deg]
-45 -42 -43 -41 -42 -42 -37 -47 -43 -49 -48 -50 -47 -51 -23 -26 -22 -26 -19

VCa 2 [m ]
1056270 760132 650214 260712 605424 715767 499799 660905 631119 229555 374233 611264 313334 417500 192284 252361 727610 326628 446116

VCn

VCang [deg]
7 2 16 17 5 10 3 -2 1 1 9 9 -6 7 -9 -24 -8 -6 -12

Rug

Limb
L L L L R R R R R L L L L L L L L L L

Slice
2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

Site
1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2

Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

13 14 17 15 17 20 17 17 18 13 17 13 12 13 17 18 14 14 17

1.5 1.8 1.6 1.5 2.0 1.8 1.5 1.4 1.6 1.3 1.4 1.3 1.5 1.3 1.8 1.6 1.5 1.4 1.5

4680 4681 4682

[294]

4683 4684

8.8 ANALYSIS 3 - MEAN HISTOMORPHOMETRIC DATA (SAS)

Hyaline cartilage thickness [m]

HCt S1 S2 S3 S4

CONDEX 606 721 723 529

CI Lower 499 595 596 436

CI Upper 735 875 877 642

PASTEX 587 730 795 586

CI Lower 467 587 641 472

CI Upper 739 908 987 727

4685 4686

Calcified cartilage thickness [m]

ZCCt S1 S2 S3 S4

CONDEX 222 257 201 149

CI Lower 187 216 170 125

CI Upper 264 305 239 176

PASTEX 260 271 221 160

2

CI Lower 209 223 182 132

CI Upper 324 330 268 194

4687 4688

Vascular channel area [m ]

VCa S1 S2 S3 S4

CONDEX 533212 318713 254859 206253

CI Lower 412477 246547 197151 159551

CI Upper 689287 412003 329458 266624

PASTEX 340173 234747 181301 133814

CI Lower 246037 174663 136050 100415

CI Upper 470219 315500 241602 178320

4689 4690

Vascular channel number

VCn S1 S2 S3 S4

CONDEX 17.5 21.0 19.5 15.8

CI Lower 15.6 18.7 17.4 14.1

CI Upper 19.6 23.5 21.9 17.7

PASTEX 17.2 20.7 19.1 15.7

CI Lower 14.8 18.1 16.8 13.8

CI Upper 20.1 23.6 21.7 17.9

4691 4692

Rugosity of cement line

Rug S1 S2 S3 S4

CONDEX 1.9 1.9 1.9 1.8

CI Lower 1.7 1.8 1.8 1.6

CI Upper 2.0 2.1 2.1 1.9

PASTEX 1.8 1.9 1.9 1.8

CI Lower 1.6 1.8 1.8 1.6

CI Upper 1.9 2.1 2.1 1.9

4693 4694

[295]

4695

[296]

4696 4697 4698

8.9 ANALYSIS 3 - HISTOMORPHOMETRIC INDICES

[297]

4699

[298]

4700 [299]

4701 [300]

4702 [301]

4703 [302]

4704 [303]

4705 [304]

4706 [305]

4707 [306]

4708 [307]

4709 [308]

4710 [309]

4711 [310]

4712 [311]

4713 [312]

4714 [313]

4715 [314]

4716 [315]

4717 [316]

4718 [317]

4719 [318]

4720 [319]

4721 [320]

4722 [321]

4723 [322]

4724 [323]

4725 [324]

4726 [325]

4727 [326]

4728 [327]

4729 [328]

4730 [329]

4731 [330]

4732 [331]

4733 [332]

4734 [333]

4735 [334]

4736 [335]

4737 [336]

4738 [337]

4739 [338]

4740 [339]

4741 [340]

4742 [341]

4743 [342]

4744 [343]

4745 [344]

4746 [345]

4747 [346]

4748 [347]

4749 [348]

4750 [349]

4751 [350]

4752 [351]

4753 [352]

4754 [353]

4755 [354]

4756 [355]

4757 [356]

4758 [357]

4759 [358]

4760 [359]

4761 [360]

4762 [361]

4763 [362]

4764 [363]

4765 [364]

4766 [365]

4767 [366]

4768 [367]

4769 [368]

4770 [369]

4771 [370]

4772 [371]

4773 [372]

4774 [373]

4775 [374]

4776 [375]

4777 [376]

4778 [377]

4779 [378]

4780 [379]

4781 [380]

4782 [381]

4783

[382]

4784

[383]

4785

[384]

4786

[385]

4787

[386]

4788

[387]

4789

[388]

4790 4791 4792 4793 4794 4795 4796 4797 4798 4799 4800 4801 4802 4803 4804 4805 4806 4807 4808 4809 4810 4811 4812 4813 4814 4815 4816 4817 4818 4819 4820 4821 4822 4823 4824 4825 4826 4827 4828 4829 4830 4831 4832 1. 2.

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18.

Nurgent, G.E., et al., Site- and exercise-related variation in structure and function of cartilage from equine distal metacarpal condyle. Osteoarthritis and Cartilage, 2004. 12(10): p. 826-833. Van Weeren, P.R., et al., Early exercise advances the maturation of glycosaminoglycans and collagen in the extracellular matrix of articular cartilage in the horse. Equine Veterinary Journal, 2008. 40(2): p. 128-135. Brama, P.A., et al., Influence of exercise and joint topography on depth-related spatial distribution of proteoglycan and collagen content in immature equine articular cartilage. Equine Vet J, 2009. 41(6): p. 557-63. Milner, M., in The Godolphin Arabian: the story of the Matchem line. 1990, J. A. Allen. p. 36. Wall, J.F., in Famous running horses, their forebears and descendants. 1949, Infantry Journal Press: Washington. p. 8. Pickrell, J. 95% of thoroughbreds linked to one superstud New Scientist 2005 6th September 2005 [cited 2009 11th June]; Available from: http://www.newscientist.com/article/dn7941-95of-thoroughbreds-linked-to-one-superstud.html. Wikipedia. Thoroughbred. 2009 19 October 2009 [cited; Available from: http://en.wikipedia.org/wiki/Thoroughbred. Brown-douglas, C. and J. Pagan. Body Weight, Wither Height and Growth Rates in Thoroughbreds Raised in America, England, Australia, New Zealand and India. 2009 [cited 2009 11th June]; Available from: www.ker.com/library/Proceedings/06/2_BodyWitherGrowthRatesTB_p15.pdf Smith, G.R., Why racehorses are cracking up, in The Age. 2002. The Associated Press, Barbaro injury focuses industry on safety, in MSNBC.com. 2006. McIlwraith, C., Intraarticular Fractures of the Carpus, in Adams' lameness in horses, T. Stashak, Editor. 2002, Lippincott Williams & Wilkins Baltimore. p. 847-858. Beyer, A., For Run of Fatal Breakdowns, No Easy Place to Fix Blame, in Washington Post. 2006. Kluger, J., Bred for Speed ... Built for Trouble, in Time. 2006. The Jockey Club. Throughbred Racing and Breeding Worldwide. Online Fact Book n.d. [cited 2009 11th June]; Available from: http://www.jockeyclub.com/factbook.asp?section=17. Club, T.J., 2003 Fact Book: A Guide to the Thoroughbred Industry in North America. 2003 New York: The Jockey Club. 36. The Jockey Club. About the Registry. The Jockey Club Website n.d. [cited 2009 11th June]; Available from: http://www.jockeyclub.com/registry.asp. Australia Racing Board. International Racing. Australian Racing Fact Book 2007/08 2008 [cited 2009 11th June]; Available from: http://www.australianracingboard.com.au/factbook/07_08_factbook/arbFinalBook.PDF. British Horseracing Authority. British Breeding: Overview. British Horseracing Authority Website n.d. [cited 2009 11th June]; Available from: http://www.britishhorseracing.com/owning_breeding/breeding/3.2.3.asp. [389]

4833 4834 4835 4836 4837 4838 4839 4840 4841 4842 4843 4844 4845 4846 4847 4848 4849 4850 4851 4852 4853 4854 4855 4856 4857 4858 4859 4860 4861 4862 4863 4864 4865 4866 4867 4868 4869 4870 4871 4872 4873 4874 4875 4876 4877 4878 4879 4880 4881 4882 4883

19.

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23.

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