Abstract

The need to create better implantation biomechanical systems is established. With more cases of bone dyscrasias emerging, the need is to create biomechanical materials that are less invasive, more biologically friendly and having superior mechanical strength. The need to create less invasive procedures and targeting only the affected areas of bone can help reduce morbidity and surgery time, and improve the time duration of healing. This study explores the use of injectable materials for similar purposes using sustained release systems.

I.

Introduction

The bones of the human body represent the structural component around which other organs of the body are arranged. This skeletal structure is an endoskeleton, which is present inside the body. Bone is a living connective tissue, arranged in the appendicular fashion. Human skeleton is the only skeleton that is of an upright nature. The similar skeletal structure is found among apes. Other skeletons are four legged in nature (Jae, 1998). Apart from the structural support role, bones also hold bone marrow which is responsible for production of blood cells. The shape and structure of the bones in any animal clearly indicate the kind of stress bearing positions the animal holds, its locomotion style and the evolution of the components to create features where maximum benefit is attained. Bones are comprised of organic and inorganic elements. Organic elements include sodium, calcium phosphate and magnesium. The inorganic elements include collagen, lipids, glycoproteins and peptides respectively (Cameron, 1972).

(a) cortical and cancellous bone; (b) osteons with Haversian systems; (c) lamellae; (d) collagen fibre assemblies of collagen fibrils; (e) bone mineral crystals, collagen molecules, and non-collagenous proteins.
Figure I.1 Hierarchical Structure of Bone.

Silicon is a trace element present in the human body which helps carry out many important functions. It helps in improving flexibility of the joints, makes bones stronger and keeps skin healthier. It helps in improving the function of other elements in the body such as sodium, glucosamine, calcium and vitamin D. Silicon is found in the cartilage, as orthosilicic acid in the body fluids or among different smaller compounds. It plays a critical role in the formation of bones, deposition of calcium and calcium matrix, production of collagen and in the formation of connective tissue fibers (Seaborn et al., 2002; Schwartz et al., 1972; Edith, 1972; Seaborn et al., 1994).

The importance of silicon in the body was first established through the silicon deprivation experiment carried out by Edith Carlisle and Schwartz in the 1970s. These experiments used rats and chickens, with controls fed with normal diet while the experimental animals were given a silicon deficient diet. Those animals that were given silicon deficient diets displayed abnormal skull and bone formation. There was also a deficiency of cartilage tissues among the experimental animals. These experiments proved the importance of silicon in the structural formation and maintenance of bone and cartilage in a body (Seaborn 1994; Seaborn, 2002). Silicon creates important links between complexes of proteoglycans. These proteoglycans interlace with collagens and help create a structural integrity of the skeletal system (Nielsen, 1996; Groff, 1995). Silicon sources in diet include calcium silicate, metasilicate and ground horsetail grass (John, and Alexander, 2000).

(a) Silicon diet fed chicken

(b) Silicon deficient diet fed chicken

Figure I.2 Importance of Silicon

Silicon in free form is not found. It is found in various silicates and dioxide forms, of which the soluble form, orthosilicic acid is found in foods such as whole grain. Orthosilicic acid can also be found in drinking water (Sangester et al, 2000). Orthosilicic acid is essential to transport calcium into the bones. Calcium is needed for maintain bones and cartilages, and silicon ensures its supply (Reffitt et al, 2003; Eisinger and Clariet, 1993). Presence of orthosilicic acid helps in the creation of type I collagen in the body (Reffitt et al, 2002). Bone tissue formation through artificial means, also known as bone tissue engineering, has many prerequisites. These materials must be both osteo-conductive and osteogenic in nature. The materials must also be biocompatible to other tissues in the body. Since bone tissue alternatives replace bone, they must display mechanical strength. Alongside, bioactivity and biodegradability are needed. The group of such materials are known as bioactive materials (Hideki and Akira, 2005). ORTHOSILICIC ACID AND THE BONE The role of orthosilicic acid must be understood in order to identify its importance for bone health. OSA or orthosilicic acid helps in the formation of type I collagen in the body. It also has been found in

researches to stimulate osteoblastic differentiation in humans. Topical applications of OSA lead to increased collagen type I concentrations in the skin. New therapies and researches show promise of using OSA as a preventive and therapeutic agent in bone pathologies (Spector et al, 2008) USE OF POLYMERS IN THE BIOMEDICAL FIELD Polymers have found a variety of uses in medical industry. These include delivery of drugs, implants, and sustained release combinations for drugs within body systems. These polymeric materials are appreciated for their biocompatibility, biodegradability and non-toxic nature. Now polymers and their derivatives are used to make artificial organs and grafts, scaffolds and bone plates. Other uses include artificial arteries, screws, pacemakers, stitching threads and replacement organs respectively (Ramakrishna et al, 2001; Joseph, 1999).

Scaffolds

Implant screws

Stitching Thread
Figure I.3 Biomedical Polymers (Ian Lee, 2012)

Arteries

Of the many types of polymers used in the industry today, Poly Lactic-co-glycolic acid or PLGA is considered the best. It has been approved by the Food and Drug Administration as having both biodegradable and biocompatible nature. Mostly it is used as scaffold to bear high mechanical strength. It has also found use as a medium for sustained drug delivery systems (Hirenkumar and Steven, 2011). The drawback of PLGA is its lack of bioactivity and the creation of inflammatory response (Lang et al, 2011). Aim of the research Bone regeneration is a constantly evolving field demanding creation of new systems that are more similar to bone structures. Bone surgeries are highly complicated, with high changes of

complications and side effects. Therefore, grafts that are applied should be selected carefully. The most readily available and commonly used techniques for grafts are allografts and autografts. Autografts contain a very high risk of morbidity. The grafting material is mostly obtained from bone. However, there is still a chance that immune-rejection or infection will take place (Susan et al, 1997). This research aims to use the favourable qualities of sodium metasilicate and PLGA, in order to create a sustained release system of orthosilicic acid at defected sites. Sodium metasilicate can help in creation of new bone due to its superior in vivo qualities of being biocompatible and biodegradable. In this research, orthosilicic acid was released in vitro. These levels were then determined and assessed. Alongside, distribution of the particle sizes and images were also used to trace the morphology of the release system micro particles. Many polymers and ceramics have been used in the past. Although structures such as hydroxyapatite have been applied, which are bioactive in nature, they lack in biodegradability (Jie et al, 2009). In surgeries such as implants where biocomposite scaffolds were used, failure to occupy the defect area led to failures (Pereira et al, 2005; Wenyuan and Jiang, 2005; Hideki and Akira, 2005). In this research, the aim is to encapsulate different pH values of metasilicate in to the PLGA microsphere. By making it an injectable system, the deposition can take place directly at the site of defect, thereby avoiding major surgery. This system can also help in improving the recovery rate, and will help in sustained release of orthosilicic acid, which will aid in bone mineralization. It is expected that the different properties of the two materials will help in creating an improved and controlled delivery system both in vivo and in vitro. Materials and Methods All the materials were purchased from Sigma-Aldrich UK unless stated otherwise. Calcium Citrate tetra-hydrate, Ca(NO3)2.4H2O; Ethylene Glycol, C2H6O2; 1-Amino-2-naphthol-4-sulfonic acid, C10H9NO4S; Sodium Metasilicate, Na2O3Si.9H2O; Hydrochloric Ace, HCl; Oxalic Acid, H2C2O4; Citric Acid, C6H8O7; Colloidal Silica of 40% weight suspended in water, SiO2 and Polyvinyl Alcohol, PVA. Ammonium Molybdate, (NH4)6Mo7O24.4H2O, was purchased from Riedel-de Haen, Germany. Dichloromethane (HPLC grade), DCM; Acetic Acid, C2H4O2; and Sodium Hydroxide, NaOH were supplied by Fisher chemical, UK. Sodium Bisulfite and Poly Lactic-co-Glycolic acid were supplied by Acros Organics and Lakeshore Biomaterial respectively from USA. Solutions of sodium metasilicate were prepared on different pH value. 13.4mg/ml solution of sodium Metasilicate was prepared and pH was adjusted by HCl for the value of 1.80 and 7, and pH 13 was adjusted by NaOH. For pH measurement, pH meter manufactured by Metller Toledo (UK) was used.

A.

Synthesis of Microspheres

The solution of PVA 0.5% (w/v) was prepared in the distilled water. Hot plate magnetic stirrer (BibbyB212, UK) used for 3 hours of stirring at temperature 200oC and then left it to cool down over night. PLGA 2% (w/v) was prepared in Dichloromethane, and the solution was placed in a sonicator (Clifton, UK) for one hour. Water in oil in water (w/o/w) emulsion technique is used to synthesise the microspheres. PLGA with Sodium metasilicate in proportion of 4:1 was homogenized for 20 seconds. Two different solutions

were prepared to be used as an emulsifier containing PVA only and 90 ml of PVA with 10 ml of Colloidal silica. The solution of PLGA and Sodium metasilicate was then mixed into several solutions of emulsifier and stirred for 15 minutes at 750 rpm and the stirring speed was decreased to 500rpm for 3hours. Vacuum filter was used to filter the microspheres and left to dry overnight. Every batch was replicated.

Figure I.4 Formation of Microspheres

B.

Characterisation of Microspheres

During the formation of microspheres, the morphology of microspheres was determined by light microscopy supplied by Carl Zeiss-Axiolab, UK. Several images were taken with a Canon PC1049 (manufactured in Japan). After the complete formation of the microspheres, they were examined under Scanning Electron Microscopy (SEM - Jeol 6060LV, Japan). Mastersizer 2000 SM (UK) was used to determine the particle size distribution.

C.

Encapsulation Efficiency

Encapsulation efficiency of sodium metasilicate was determined by mixing the microspheres in a tube with 5ml DCM and 5ml distilled water. The sample was vortexed (Genie2, UK) and left for 90 minutes. PLGA was degraded because of DCM and silicic acid was released in water phase. 3ml from the water phase was collected to determine the encapsulation efficiencies, by using following formula: ( )

Water + Silicic Acid Layer of Polymer DCM

Figure I.5 Encapsulation Efficiency

D.

Drug Loading
( )

E.

Yield
( )

F. 1. a)

Colorimetric reagents Preparation of Yellow assay Ammonium Molybdate reagent

100 mg/ml solution of Ammonium Molybdate was prepared and pH was adjusted by 1 M solution of sodium hydroxide at 7.

b)

Oxalic Acid

100 mg/ml solution of Oxalic acid was prepared. Mixture was stirred for about 2-3 hours and filtered to remove any solid particles.

c) 2.

Hydrochloric Acid Preparation of Blue assay reducing reagent

37% of Hydrochloric acid was mixed with distilled water of same proportion i.e. 1:1.

In 500 ml of distilled water, 500 mg of 1-amino-2-naphthol-4-sulfonic acid and 1 g of Sodium bisulfite was mixed by continuous stirring. It was then filtered out using vacuum filter when cloudy solution was obtained. This solution was kept in dark because it has great affinity for the absorbance of light. So, it was covered with aluminium foil and kept in refrigerator.

G.

Detection of Silicic acid

For the detection of silicic acid in the solution, 3 ml from each sample were examined with the help of reagents used to determine the concentration of the silicic acid. Following are the reagents: 60 l of Hydrochloric Acid 120 l of Ammonium Molybdate 60 l of Oxalic Acid.

Using these reagents produces yellow colour. Each sample was measured 3 times. UV spectrometer (Cecil 7500, Aquarius, UK) was used to determine the absorbance at the wavelength of 410 nm. As the concentration of silicic acid was low, therefore, 60 l of Blue assay reducing reagent was also used with other reagents. The colour of the sample solution changes to blue and from the UV spectrometer absorbance was determined at the wavelength of 610 nm.

H.

Release Studies

In vitro release studies were carried out on the basis of encapsulation efficiencies of solutions having different pH values of sodium metasilicate and the conditions they were prepared. Three batches of each solution containing sodium metasilicate of pH 1.80 and 7, and one batch of control were studied. In each batch, equal weight of microspheres were used and dissolved in 100 ml of distilled water. These batches were then placed in a shaker (Gallenkamp, UK) at the speed of 1000 rpm and at the temperature of 37C. The absorbance of these batches were measured after a fixed time intervals by adding colorimetric assay and orthosilicic acid (OSA) was determined in terms of cumulative release.

II.
A. Morphology of Microspheres

Results

Morphology of microspheres was studied by using the light microscopy and scanning electron microscopy (SEM). Several images were taken at different magnification from SEM which shows that the porosity and shape of the microspheres depends on the speed of stirrer and the molecular weight of PLGA. Fig. III.1 shows the microspheres of the sample having pH 1.80.

(A) Microspheres with PLGA 2.0A MW

(B) Microspheres with PLGA 2.5A MW

(C) Microspheres with PLGA 3.5A MW

Figure II.1 SEM for Microspheres of Sample having pH 1.80

We can clearly see that the porosity of these microsphere decreases as the molecular weight of PLGA increases. The shape and the surface also affected by the molecular weight of PLGA, in (A) and (B) particles have smooth surfaces and spherical in comparison with microspheres in (C). Fig. III.2 shows the microspheres of the sample having pH 7. These microspheres showed quite similar trend to the sample having pH 1.80.

(A) Microspheres with PLGA 2.0A MW

(B) Microspheres with PLGA 2.5A MW

(C) Microspheres with PLGA 3.5A MW

Figure II.2 SEM for Microspheres of Sample having pH 7

Microspheres of water control, Fig III.3, shows high porosity with each molecular weight of PLGA but they adopted disc like shape which might be cause of vacuum took place during SEM.

(A) Microspheres with PLGA 2.0A MW

(B) Microspheres with PLGA 2.5A MW

(C) Microspheres with PLGA 3.5A MW

Figure II.3 SEM for Microspheres of Water Control

B.

Particle Size Distribution of Microspheres

Particle size distribution of microspheres from each samples were determined several times, different lines having different colour shows one measurement at a time. The curves of these shapes are called bell curve. Fig. III.4 shows the particle size distribution of a water control. These curves indicate that 10% particles at the lower end have mean size of 52.57 m. 80% microspheres of the this sample have a mean size of 169.75 m. At the higher end, rest of the 10% particles have mean diameter of 423.29 m.

Figure II.4

The encapsulated microspheres of sodium metasilicate having pH 1.80 showed that 80% of the particles have mean size of 95.02 m represented by Fig. III.5. The 10% particles at the lower end have the mean size of 45.52 m and the other 10% of the particles at the higher end have mean size of 219.3 m.

Figure II.5

Particle size distribution of the microspheres having pH 7 is shown in Fig. III.6. 10% of the particles at the lower end have mean particle size of 66.42 m. And the other 10%, at higher end, have mean particle size of 333.80 m. Rest of the 80% of particles have mean particle size of 153.20 m.

Figure II.6

C.

Calibration Curves

Calibration curves of Ammonium Molybdate assays are shown in Fig. III.7. Yellow assay was used to determine higher concentrations of orthosilicic acid (OSA) and blue assay was used for the higher

concentrations of orthosilicic acid (OSA), these assays were also used for the determination of encapsulation efficiencies of the microspheres. The equations generated from these curves were used to calculate the concentration of orthosilicic acid during the release studies.

(A) Yellow Assay

Figure II.7

(B) Blue Assay

D.

Release Studies

Release studies were conducted for 21 days in dissolution media which was distilled water. Colorimetric assays were used to determine the release of OSA from microspheres. Fig III.8 shows the release study of OSA of two different, pH 1.80 and pH 7, from the microspheres. Burst release of OSA was observed during the first 6 hours, and then the concentration remained constant. There were different stages in which release studies carried out for both microspheres. Firstly, initial burst was observed then plateau was observed followed by released and again plateau. After initial burst the release rate was low in comparison with first 6 hours. After 180 hours, steady increase in the cumulative release of OSA was seen. The small error bars on the curves tells us that there is no significant differences in release of OSA from both the microspheres. Total release of OSA from pH 7 and pH 1.80 was 0.52 g/ml and 0.70 g/ml respectively, after 480 hours and the release became constant.
0.8

Cumulative release of OSA (g/ml)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 100 200 300 400 Time (hours)
Figure II.8

Control pH = 7 pH 1.80

500

600

Percentage cumulative release of OSA, shown in Fig. III.9. This percentage is a comparison between both the microspheres. The release of OSA from microspheres was in stages i.e. early burst, plateau, and release and then again plateaus. The release of OSA of the sample having pH 1.80 was higher than that of the sample having pH 7. After 21 days, 480 hours, the percentage cumulative release of the sample having pH 1.80 was 62.68% much higher than that of the sample having pH 7 which was 16.73%. 70 60 50 40 30 20 10 0 0 100 200 300 400 500 600 pH = 7 pH = 1.80

Cumulative Release %

Time (Hours)
Figure II.9

III.
A.

Discussion

Selection of pH values of sodium metasilicate

There were several method used to achieve higher encapsulation efficiency. Firstly, sodium metasilicate having pH 13 was entrapped in PVA which gives us very low encapsulation efficiency. Then 10 ml of colloidal silica was saturated with 90ml of PVA was used to encapsulate same sodium metasilicate which produces gel like solution and no microspheres. Following the flowchart, Fig. IV.1, several experiment were performed and at the end sodium metasilicate of pH 7 and sodium metasilicate of pH 1.80 were used separately with the emulsifier saturated with colloidal silica, the encapsulation efficiency from these microspheres was 1.02% and 0.23% respectively. From these experiments we also came to know that sodium metasilicate on basic pH value have poor encapsulation efficiency and also produce less number of microspheres.

Figure III.1

B.

Mixing Strategy

High encapsulation efficiency was achieved by making the homogenised solution of sodium metasilicate and PLGA. Homogeniser, vortex and sonicator were used for mixing purposes. Among these three, homogeniser showed the appropriate mixing.

C.

Molecular weight of PLGA

Molecular weight of PLGA affects the morphology of microspheres in terms of shape and porosity. Microspheres containing sodium metasilicate of two different pH values were synthesised from three different molecular weight of PLGA, 2.0A, 2.5A and 3.5A. These microspheres were analysed under scanning electron microscope, as shown in Fig. IV.2, and was observed to be more porous and their surface seems quite smooth when made with lower molecular weight of PLGA than those made with higher molecular weight of PLGA, they were very less porous and have rough surfaces. But the microspheres of water control (g,h,i) have the same level of porosity which tells us that they were not affected by the molecular weight of PLGA and adopted disc like shape.

(A) pH 1.80, PLGA 2.0A MW

(B) pH 1.80, PLGA 2.5A MW

(C) pH 1.80, PLGA 3.5A MW

(D) pH 7, PLGA 2.0A MW

(E) pH 7, PLGA 2.5A MW

(F) pH 7, PLGA 3.5A MW

(G) WC, PLGA 2.0A MW

(H) WC, PLGA 2.5A MW

Figure III.2

(I) WC, PLGA 3.5A MW

D.

Speed of stirrer

Stirring is usually required for the formation of microsphere and to evaporate DCM or excess solvent from the solution. The speed of stirring was of great importance during the formation of microspheres. From the images of light microscopy, Fig IV.3, it was seen that the microspheres when led to form for 3 hours at the speed of 750 rpm were mostly homogenous in shape and shape after first hour. But after 3 hours when they are formed and dried they were clumped with each other. This is because high stirring speed is good for making microspheres but once they are synthesised, the same high speed will led them to break caused by the collisions with the walls and other

microspheres. Once the microspheres are broken the polymer from these microspheres adhere on other microspheres and joined them with others and formed cluster like structure after drying.

(a) pH 1.80, After 1st Hour

(b) pH 1.80, After formation

(c) pH 7, After 1st Hour

Figure III.3

(d) pH 7, After formation

To avoid getting the clusters and breakage of microspheres the speed of stirring was adjusted. Initially the stirrers were operated at 750 rpm for 15 minutes and then the speed was decreased to 400 rpm. For first 15 minutes, the formation was carried out at high speed because it is beneficial for the formation of microspheres and cause of decreasing the speed of stirrer after first 15 minutes was to maintain the structure of the microspheres and it also facilitates in the evaporation of excess solvent. Fig IV.4 shows the images of first hour during formation and after the formation, these images confirm that speed is a critical parameter for the formation of these microspheres.

(a) pH 1.80, After 1st hour

(b) pH1.80, After formation

(c) pH 7, After 1st Hour

Figure III.4

(d) pH 7, After formation

IV.

Conclusion

The study was able to show a positive response in the test samples. This improved rate of delivery, sustained release and rapid response suggest that the methods can be refined and improved. Not only will this system help in reducing morbidity, but also prevent extensive surgery, thus improving prognosis and healing rates. Further researches to replicate results and comment on new areas of concern and development should be made. Ageing bones have more susceptibility to infection and delayed healing. This conservative method will allow better implantation of the material, sustained result, localized effect, and more efficient healing.

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