Bone Allografts Dario Bertossi, MD; Massimo Albanese, MD; Pier F. Nocini, MD, DDS; Antonio DAgostino, MD; Lorenzo Trevisiol, MD; Pasquale Procacci, MD Objective: To present our experience in the use of fresh- frozen human bone allograft as an interpositional graft- ing material for sliding genioplasty to correct chin deformities. Methods: Ten patients underwent sliding genioplasty using morcellized and corticospongious fresh-frozen human bone. Four patients underwent orthognathic surgery associated with genioplasty. Six patient under- went genioplasty associated with rhinoplasty. Panorex, lateral, and frontal cephalogram and computed tomo- graphic scans have been performed for each case pre- operatively and 12 months after surgery. One patient subsequently asked for plate removal, and with his consent, a bone biopsy specimen was obtained during the operation. Results: Stable aesthetic and functional results were ob- served in all cases. No infections occurred, and no bone resorption has been clinically or radiologically observed. Conclusion: The use of fresh-frozen bone allograft re- duces patient morbidity and operative time, providing a stable and excellent aesthetic result. JAMA Facial Plast Surg. 2013;15(1):51-57. Published online November 12, 2012. doi:10.1001/jamafacial.2013.224 T HE CHINIS ONE OF THE MOST prominent facial struc- tures, and maxillofacial de- formities are commonly as- sociated with the chin area. Appropriate size, shape, andpositionof the chin can enhance the global facial har- mony and symmetry. Thus, surgical cor- rection of chin deformities is often re- quired to improve harmony of the face. 1 Two principal methods, alloplastic im- plantation and sliding genioplasty, are available for chinaugmentation. Bothtech- niques achieved excellent results. Sliding genioplasty is more commonly used by maxillofacial surgeons because it allows to correct bone abnormalities in all 3 dimen- sions, therefore representing a more flex- ible technique. This procedure is gener- ally preferred in malocclusion associated with chin retrusion but is also indicated in association with rhinoplasty. 2,3 The use of interpositional bone grafts proved to offer several advantages owing to their mechanical and biological behav- ior. Interpositional bone grafts provide a stable mechanical structure that de- creases vertical height relapse, acts like a matrix for ossification, 4-7 andprevents soft- tissue herniation into the osteotomy site. Together, these factors help to avoid the formation of a deep angular labiomental fold and to improve osseointegration and stabilization of the sliding segment. 8 Various autologous, heterologous, and alloplastic interpositional grafts have been described as grafting interpositional ma- terials in cases of mandibular advance- ment, maxillary Le Fort I osteotomy, and chin osteotomies. 9 The main types of graft materials are autogenous bone grafts, 4 freeze-dried bone, 10 alloplastic material (Proplast; Vitek Inc) blocks, 11 solid block of hydroxyapatite, and porous block of hydroxyapatite. 12 The autogenous bone grafts represent the gold standard because of their os- teogenic potential, osteoconductivity, and osteoinduction, 13 but the use of autoge- nous grafts implies the availability of a do- nor site, thus increasing morbidity and op- erative time. 14,15 Alloplastic materials require a long healing periodandthenhave high rates of infections and relapse. 16 Fresh-frozen homologous bone, be- cause of its osteoinductivity and osteo- conductivity, has been widely used in different operative theaters, such as or- thopedic surgery and spine surgery. 17,18 Recently, several authors described the use Author Aff Oral and M Departmen University Italy. Author Affiliations: Section of Oral and Maxillofacial Surgery, Department of Surgery, University of Verona, Verona, Italy. JAMA FACIAL PLAST SURG/ VOL 15 (NO. 1), JAN/FEB 2013 WWW. JAMAFACIAL.COM 51 2013 American Medical Association. All rights reserved. Downloaded From: http://archfaci.jamanetwork.com/ by a Wegner Health Science Info Ctr & USD User on 08/29/2013 of fresh-frozen bone (FFB) allografts as a reliable surgi- cal alternative in maxillofacial and oral surgery. 19-21 Although many different grafting materials have been described, there is still no consensus in the literature. We present herein our clinical experience with 10 cases of sliding genioplasty using FFB allografts as a new graft- ing material. METHODS The present study includes 10 patients who underwent verti- cal height augmentation genioplasty by means of a horizontal sliding osteotomy of the symphysis with interpositional ho- mologous FFB allografts at the Department of Oral and Max- illofacial Surgery, Policlinico G. B. Rossi, University of Ve- rona, Verona, Italy, from2008 to 2009. All cases were performed by 1 surgeon (D.B.). Informed consent about the surgical pro- cedure, the grafting material, and the use of personal data for research purposes, approved by the local research ethics com- mittee and written by the local tissue bank, was obtained from patients. All of these patients, 3 men and 7 women, were white. Genioplasty was associated with bimaxillary surgery in 4 cases and with rhinoplasty in 6 cases. Initial consultation included a complete medical history and physical examination. Dental history was investigated by means of an occlusal evaluation, standard facial photographs, lateral cephalogram, anterior-posterior skull radiography, and pan- orex. In addition, in cases of dentoskeletal deformities, dental models were developed. Functional and cosmetic goals were determined according to the patients expectations and to the facial analysis. All surgical procedures were performed with the patient un- der general anesthesia. After local infiltration with anesthetic and vasoconstrictor, a vestibular incision of the mucosa was made only superficially, 6- to 8-mm labial to the depth of the oral vestibulum, and then directed horizontally to the alveolar process fromone cuspid to the other. After an accurate dissec- tion of the branches of the mental nerves, the chin promi- nence was gently degloved, providing a direct access to the an- terior surface of the symphysis. The osteotomy line was marked. Osteotomy of the chin was then performed by means of a piezo- electric scalpel (Piezosurgery Medical II; Mectron) using the Piezosurgery MT1-10 insert (Mectron), and when the oste- otomy was completed, the sliding segment was sectioned. After fixation with 3 titanium bone plates (2.0 Orthog- nathic system; Osteomed), fresh-frozen corticospongious bone blocks were shaped and positioned throughthe bone gap. Fresh- frozen morcellized bone chips were then used to fill in conti- nuity solutions between the bone grafts and the native bone to make the bone surface regular. Bioengineered membranes (Geistlich Biogide; Geistlich Pharma AG) were positioned as a barrier on the anterior surface to prevent migration of FFB chips and to reduce the ingress of saliva and oral bacterial flora. Clo- sure was then obtained with resorbable sutures (Polysorb 4.0; Covidien). Lengthening of the chin was obtained in each case, with a mean value of 3.5 mm, while the mean value of chin advancement was 3.7 mm (Table). Fresh-frozen bone was prepared in chips or blocks in a ster- ile environment by the Regional Tissue Bank of Treviso. The iliac crests were removed in a sterile setting. Asample was taken fromthe surface of the bone for bacterial studies, together with a bone biopsy specimen, and stored in 4 vials. The epiphysis or the iliac crest were then maintained in a refrigerator at 80C. The FFB was a mineralized, nonirradiated, and only disin- fected frozen homologous bone. Lateral cephalograms of hard and soft tissues were taken pre- operatively, immediately, and1 month, 6 months, and/or a mini- mum of 1 year postoperatively. An orthognathic patient un- derwent a computed tomographic (CT) cone-beamexamination preoperatively and 6 and 12 months after surgery. A compari- son study of hard- and soft-tissue changes after genioplasty in all 10 patients was performed with immediately postoperative and 1-year postoperative CT scans. The stability of the grafted FFB was assessed by measuring vertical resorption through the comparison of hard- and soft-tissue changes. The superimpo- sition of preoperative and postoperative lateral cephalogramal- lowed for checking any eventual height resorption of the skel- etal chin. One patient underwent the removal of osteosynthetic plates, and a bone biopsy was taken from the anterior surface of the interpositional graft by means of a trephine burr (diameter, 3 mm). Bone cores were immediately fixed in 10% neutral buff- ered formalin for 24 to 36 hours, decalcified in EDTA diso- dium salt acid buffer (Osteodec; Bio-optica) for 3 hours, and conventionally embedded in paraffin. Sections were immuno- stained with the following antibodies: CD56/NCAM (Clone 123C3.D5; NeoMarkers), cathepsin-K (Clone CK4; Novocas- tra), tenascin (Clone 49; Novocastra), and CD34 (Clone QBEND/10; Novocastra). Heat-induced antigen retrieval was used when requested by the standardized protocol. All samples were processed using a sensitive Bond Polymer Refine de- tection system in an automated Bond immunostainer (Vision- Biosystem; Menarini). Sections incubated without the pri- mary antibody served as a negative control. The bone cores were histologically analyzed to investigate the nature of the bone, Table. Surgical Treatment and Related Amount of Bone Shift Patient No./ Sex/Age, y Type of Surgery Advancement, mm Vertical Height Augmentation, mm 1/M/22 Genioplasty, rhinoplasty, and mandibular angles remodeling 4 4 2/F/24 Genioplasty and rhinoplasty 5 3 3/F/20 Bimaxillary orthognathic surgery and genioplasty 3 3 4/F/26 Genioplasty and rhinoplasty 4 3 5/F/21 Bimaxillary orthognathic surgery and genioplasty 3 3 6/M/29 Genioplasty and rhinoplasty 4 4 7/F/22 Genioplasty and rhinoplasty 3 4 8/F/23 Bimaxillary orthognathic surgery and genioplasty 4 4 9/F/26 Genioplasty and rhinoplasty 3 3 10/M/19 Bimaxillary orthognathic surgery and genioplasty 4 4 Mean value 3.7 3.5 JAMA FACIAL PLAST SURG/ VOL 15 (NO. 1), JAN/FEB 2013 WWW. JAMAFACIAL.COM 52 2013 American Medical Association. All rights reserved. Downloaded From: http://archfaci.jamanetwork.com/ by a Wegner Health Science Info Ctr & USD User on 08/29/2013 the presence of inflammatory cells throughout the grafting ma- terial, and the entity of osseointegration and healing. RESULTS REPORT OF A CASE A 22-year-old woman was referred to the Department of Oral and Maxillofacial Surgery, Policlinico G. B. Rossi, University of Verona, because of a severe hypoplasia of the chin and laterodeviation of the nose (Figure 1). The patient did not allow any orthodontic and orthognathic treatment and just asked for an aesthetical correction of the facial balance with minimally invasive surgery. Ac- cording to the patients expectations, a sliding genio- plasty and a simultaneous lipofilling of the upper lip as- sociated with a rhinoplasty were planned. Fresh-frozen corticocancellous and morcellized bone was used to fill the vertical gap between the sliding bone segment and the mandible (Figure 2). Open rhinoplasty was then performed. After a follow-up of 12 months, no adverse reaction occurred (Figure 3). Lateral cephalogram and frontal cranial radiography at 12 months after surgery showed a complete healing of the bone gap of the chin(Figure4). Nevertheless, the patient needed to extract all third mo- lars, and she asked to undergo plate removal at the same time. Twelve months after surgery, extractionof thirdmo- lars and plate removal were performed and, with the pa- tient consent, a bone biopsy specimen was harvested (Figure 5). CLINICAL OUTCOMES AND HISTOLOGICAL RESULTS The median follow-up time was 14 months. Immediate healing was achieved without any complications or ad- verse reactions. All patients healed without any major postoperative problems. No paresthesia of the mental nerves occurred. The clinical outcome was stable in every case, with- out any aesthetic adverse effects or vertical height modi- fication. In lateral cephalograms and panorex, bone heal- ing and integration to upper and lower chin bone was completed within 8 months after surgery in all cases. No changes of hard tissue were observed within 14 months after surgery. The histological analysis at the time of implant fix- ture placement revealed newbone formation and simul- taneous grafted bone chip resorption (Figure 6A). Ho- mologous bone chips were embedded in newly formed bone. Direct connection between homologous bone chips and newly formed bone trabeculae was also clearly dis- played in the sections. Newly formed bone was charac- terized by lacunae containing osteocytes (Figure 6B). Bone-lining cells and osteoblasts expressing CD56/ NCAM were observed on the boundary with the newly formed bone (Figure 6C). The newly formed bone had an inner space filled with a well-vascularized connec- tive tissue withevidence of ongoing fibrogenesis (as shown by the expression of the extracellular matrix protein tena- scin) (Figure 6D), effective angiogenesis, and no sign of inflammation or foreign body reaction (as demon- A B Figure 1. Preoperative patient front view (A) and profile (B). Figure 2. Intraoperative view of the final result of a sliding genioplasty and interpositional bone grafting using corticospongious fresh-frozen allografts. JAMA FACIAL PLAST SURG/ VOL 15 (NO. 1), JAN/FEB 2013 WWW. JAMAFACIAL.COM 53 2013 American Medical Association. All rights reserved. Downloaded From: http://archfaci.jamanetwork.com/ by a Wegner Health Science Info Ctr & USD User on 08/29/2013 strated by CD34 immunostaining) (Figure 6E). Osteo- clast activity was also revealed in sections immunos- tained for cathepsin-K. The osteoclast activity seemed to be effective in both homologous bone trabeculae and newly formed bone trabeculae. Remains of homologous bone chips, characterized by empty lacunae, seemed to be well integrated within the bone tissue and showed an intense osteoclastic resorption. Each patient underwent a radiological follow-up that provided a lateral cephalogram 7 days after surgery and 1 year after surgery. The cephalometric data confirmed that no alterations of bone height or bone width of the chin occurred. COMMENT The vertically shortened chin represents an anatomical facial deficiency commonly managed by maxillofacial and plastic surgeons because of its fundamental importance for global harmony of the face. Although vertical defi- ciency of the facial lower third is a proper character of class II dentofacial deformity and, for this reason, the orth- odontic and orthognathic surgical treatment is com- monly associated with sliding genioplasty for chin cor- rection, several patients are not interested in such treatment option. Moreover, patients affected by facial lower-third hypoplasia and associated nasal deformities often complain only about the aesthetic appearance of their nose. The importance of the chin projection on the global aesthetic facial impression is often unknown. Re- gardless, the vertically shortened chin represents an ana- tomical defect of the mandibular bone profile with a di- rect influence in the aesthetic appearance. A complete knowledge of facial analysis is then required to obtain a precise evaluation of each pathological scenario. Amax- illofacial surgeon should be able to propose the best sur- A B Figure 3. Postoperative patient front view (A) and profile (B). A B Figure 4. Preoperative (A) and postoperative (B) cephalograms showing the good result of the genioplasty without any radiological sign of bone resorption. JAMA FACIAL PLAST SURG/ VOL 15 (NO. 1), JAN/FEB 2013 WWW. JAMAFACIAL.COM 54 2013 American Medical Association. All rights reserved. Downloaded From: http://archfaci.jamanetwork.com/ by a Wegner Health Science Info Ctr & USD User on 08/29/2013 gical option to correct all the possible deformities of the facial lower third, such as class II dentofacial deformi- ties and isolated chin retrusion. 10 The use of interpositional grafts in mandibular oste- otomies for sliding genioplasty is not widely reported in the literature. There are limited studies that describe the use of various material in chin osteotomies. The use of interpositional grafts facilitates bone healing because it provides a matrix and a layer for secondary ossification and, furthermore, represents a static stop to relapse and to an eventual soft-tissue herniation. Actually, interpo- sitional grafts provide an acceleration of bony union and reduce relapse. It has been reported that interpositional grafts for slid- ing chin osteotomies could be performed with alloplas- tic material or autogenous bone. The advantages of the use of alloplastic material are simplicity, easy shaping, absence of a donor site morbidity, and absence of grafts reabsorption. The disadvantages are a higher rate of in- fections, graft failure, and reabsorption of the bone re- cipient site. The autogenous bone graft is currently more common than the alloplastic one and, moreover, repre- sents the gold standard in oral and maxillofacial sur- gery. Autogenous corticocancellous bone graft har- vested fromthe iliac crest is characterized by a lower rate of infection and no failure. The autogenous bone graft provides a predictable result because of its osteogenesis, osteoinductivity, and osteoconductivity. Nevertheless, it has been reported that corticocancellous bone from the iliac crest may have a high reabsorption rate and may re- sult in higher morbidity because of donor site harvest- ing, as well as a higher cost due to a longer operative time. Fresh-frozen corticocancellous and morcellized bone grafts can represent an attractive alternative because of their own biological and mechanical behavior. Primar- ily, allografts are osteoconductive and osteoinductive. Fresh-frozen allografts are not subjected to any chemi- cal or mechanical treatment that may damage their own natural mechanical and protein structure. Therefore, FFB allografts provide a physical structure and a matrix for secondary ossification without the need for any cell trans- plant. The advantages of using FFB include decreased op- erative trauma and time for the patient, decreased bleed- ing, no donor site morbidity, and a virtually unlimited amount of grafting material. 22 Furthermore, the use of FFB reduces surgical costs because FFB represents a less expensive grafting material compared with the costs of autologous bone grafts harvesting and with the costs of all the other bone substitutes. One of the main concerns about using FFB is the trans- mission of infectious diseases such as AIDS or hepatitis. In a recent article by Costain and Crawford, 23 a careful review of the literature on allograft bone processing re- ported no new cases of human immunodeficiency virus (HIV) transmission since 1985. The estimated risk of HIV transmission is 1 of 1.6 million procedures. Strict alle- giance to tissue bank guidelines on donor recruitment, bone harvesting and storage, and adequate record- keeping procedures make the use of safe allogeneic bone trustworthy. Actually, the risk of viral diseases transmis- sion is less than that calculated after a blood transfu- sion. Although irradiation is widely used to sterilize al- logenic bone, the usual upper limit of gamma irradiation exposure to bone allograft in the United States is 25 kGy, and it has been reported that up to 50 kGy may be nec- essary to inactivate HIV. However, there is no evidence supporting the need to irradiate to improve immunotol- erance and incorporation. Moreover, various studies have found that irradiation causes a clear weakening of the bio- mechanical and biological properties of the graft. 24 The major objection to allografting is the potential risk of host immune response. Unlike other tissues used in transplantation surgery, there is no evidence to intro- duce the use of haplotype matching with allogeneic bone. No increase of anti-HLAantibodies has been detected af- ter the use of frozen allogeneic bone. Although the in- A B C Figure 5. Front views 12 months after surgery and bone biopsy site. A, Postoperative front view of the vestibular aspect of the chin 12 months after surgery. Complete osteointegration of the interpositional allograft is evident. B, After plates removal, the allograft was undetectable, as shown in this postoperative front view. C, Bone biopsy was performed to examine the quality of bone through the entire width of the allograft. JAMA FACIAL PLAST SURG/ VOL 15 (NO. 1), JAN/FEB 2013 WWW. JAMAFACIAL.COM 55 2013 American Medical Association. All rights reserved. Downloaded From: http://archfaci.jamanetwork.com/ by a Wegner Health Science Info Ctr & USD User on 08/29/2013 fluence of freezing on the prevention of immune-based response has not been completely clarified, it has been demonstrated that FFB allografts present better results in terms of osteointegration and biomechanical proper- ties, compared withfreshor freeze-dried bone allografts. 23 Interpositional bone allografts have been covered in all cases by resorbable barrier membranes, according to the guided bone regeneration technique. Several studies dem- onstratedthat the use of resorbable membranes, whichdis- appear 1monthafter surgery, favorites bone allografts heal- ing during the first phase of the osseointegration process. Indeed, the use of resorbable membranes makes it pos- sible to maintain a free space and prevents the ingrowth of surrounding soft tissue. Many studies have demon- strated the predictability of guided bone regeneration in improving bone augmentation and reducing bone resorp- A B C D E Figure 6. Histological analysis. A, Histological section of entire bone biopsy specimen with evidence of homogeneous bone regeneration, graft reabsorption, and normal vascularization (hematoxylin-eosin, original magnification 5). B, Magnified view of a grafted bone chip invaginated by a continued layer of newly formed bone. Osteocytes are observed inside the vital bone, while grafted bone is characterized by empty lacunae (hematoxylin-eosin, original magnification 30). C, Histological findings 7 months after surgery. A continued front of bone apposition is evident all around the newly formed bone boundaries (CD 56/NCAMclone 123c3.D5 [NeoMarkers, immunostaining], original magnification 10). D, Histological section showing the mature aspect of the bone architecture and a normal well-vascularized inner space (Clone 49 [Novocastra], original magnification 50). E, CD34 immunostaining demonstrated effective angiogenesis and no sign of inflammation or foreign body reaction (Clone QBEND/10 [Novocastra], original magnification 50). JAMA FACIAL PLAST SURG/ VOL 15 (NO. 1), JAN/FEB 2013 WWW. JAMAFACIAL.COM 56 2013 American Medical Association. All rights reserved. Downloaded From: http://archfaci.jamanetwork.com/ by a Wegner Health Science Info Ctr & USD User on 08/29/2013 tion after autologous or heterologous bone grafts, which coulddisturbbone healing, inorder toregenerate newbone tissue in the previously free space. 25 The use of bone allografts decreases costs by reduc- ing operative time, morbidity, and hospitalization com- paredwithautogenous bone andalloplastic materials. Slid- ing genioplasty using FFB allografts showed satisfactory and stable results in all patients, as demonstrated by the cephalometric analysis in all the study patients. In conclusion, there are currently no studies in the literature, to our knowledge, that examine the use of FFB allograft in chin osteotomies. Our clinical experi- ence indicates that the use of this material, already applied in many fields of medicine and in oral and max- illofacial surgery, has proved itself to be a reliable alter- native for interpositional bone grafting in sliding oste- otomies. Therefore, assuming that further extensive follow-up studies will be necessary, FFB allograft can be considered an encouraging material in the treatment of chin deformities. Accepted for Publication: November 3, 2011. Published Online: November 12, 2012. doi:10.1001 /jamafacial.2013.224 Correspondence: Dario Bertossi, MD, Section of Oral and Maxillofacial Surgery, Department of Surgery, Univer- sity of Verona, Policlinico Giovanni Battista Rossi, Piazzale Ludovico Antonio Scuro, 10, 37134 Verona, Italy (dario.bertossi@univr.it). Author Contributions: Dr Bertossi had full access to all of the data in the study and takes full responsibility for the integrity of the data and accuracy of the data analy- sis. Study concept and design: Bertossi, Albanese, Nocini, Trevisiol, and Procacci. Acquisition of data: Bertossi, Nocini, Trevisiol, and Procacci. Analysis and interpreta- tion of data: Albanese and DAgostino. Drafting of the manuscript: Bertossi, Albanese, Nocini, DAgostino, Tre- visiol, and Procacci. Statistical analysis: DAgostino. Study supervision: Bertossi, Albanese, Nocini, and Procacci. Conflict of Interest Disclosures: None reported. REFERENCES 1. 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De Santis D, Trevisiol L, DAgostino A, Cucchi A, De Gemmis A, Nocini PF. Guided bone regeneration with autogenous block grafts applied to Le Fort I os- teotomy for treatment of severely resorbed maxillae: a 4- to 6-year prospective study. Clin Oral Implants Res. 2012;23(1):60-69. JAMA FACIAL PLAST SURG/ VOL 15 (NO. 1), JAN/FEB 2013 WWW. JAMAFACIAL.COM 57 2013 American Medical Association. All rights reserved. Downloaded From: http://archfaci.jamanetwork.com/ by a Wegner Health Science Info Ctr & USD User on 08/29/2013