A Vascularized Elbow Allotransplantation Model in the Rat

J Shoulder Elbow Surg (2015) 24, 779-786

www.elsevier.com/locate/ymse

A vascularized elbow allotransplantation model in the rat

Juyu Tang, MDa, Hainan Zhu, MDb, Xusong Luo, MDb, Qinfeng Li, MDb, L. Scott Levin, MDc, LCDR Scott M. Tintle, MDd,* aDepartment of Hand and Microsurgery, Xiangya Hospital of Central South University, Changsha, China bDepartment of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University Medical College, Shanghai, China cDepartment of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA, USA dDepartment of Orthopaedic Surgery, Walter Reed National Military Medical Center, Bethesda, MD, USA

Background: The aim of this research was to develop a rat model for vascularized composite allotransplantation (VCA) of the elbow. Methods: We developed an animal model for VCA of the elbow in rats. Microvascular VCA was performed in 9 rats across a major histocompatibility barrier. Three different immunosuppressive regimens were provided. Joint mobility and weight-bearing capability were assessed throughout 90 days of life. Pedicle patency, bone blood ﬂow, and histologic analyses were performed. Results: In the cyclosporine group, forelimb activity was recovered during the postoperative 90 days. The extremity that was operated on was used in daily activities. There was minimal motion or use of the limb in the cyclosporine taper and control groups. The vascular pedicles were patent at the time of death in the cyclosporine-treated group but not in the remaining groups. Micro-computed tomography scan performed 3 months after transplantation revealed union at the bone junctions, and the elbow joint appeared grossly normal on death in the cyclosporine treatment group only. Incomplete healing was observed in the other 2 groups, and the elbow joints were grossly destroyed. Histologic examination revealed normal cartilage and bone cells in the cyclosporine-treated group, whereas the nontreated groups demonstrated lymphocytic inﬁltration and loss of normal histologic features. Flow cytometry of blood samples obtained on days 14, 30, 60, and 90 showed no recipient cell chimerism in any of the groups. Conclusions: We developed an animal model for elbow VCA. Immunosuppressed animals regained nearly normal function of forelimbs and maintained grossly normal elbow cartilage. Without cyclosporine treatment, the elbow transplants were rejected. Level of evidence: Basic Science, In Vivo Animal Study. Published by Elsevier Inc. on behalf of Journal of Shoulder and Elbow Surgery Board of Trustees. Keywords: Vascularized; elbow; allotransplant; transplant; arthritis; rat

This study was approved by the University of Pennsylvania Institutional Department of Defense, nor the U.S. Government. Nothing in the pre- Animal Care and Use Committee: No. 804260. This study took place at the sentation implies any Federal/DOD/DON endorsement. None of the au- University of Pennsylvania, Philadelphia, PA, USA. thors received financial support for this study. One of the authors is an employee of the US Government and this work *Reprint requests: LCDR Scott M. Tintle, MD, Walter Reed National was prepared as part of his official duties. The views expressed in this Military Medical Center, 8901 Wisconsin Avenue, Bethesda, MD 20889, article are those of the authors and do not necessarily reflect the official USA. policy or position of the Department of the Navy, Department of the Army, E-mail address: [email protected] (LCDR S.M. Tintle).

1058-2746/$ - see front matter Published by Elsevier Inc. on behalf of Journal of Shoulder and Elbow Surgery Board of Trustees. http://dx.doi.org/10.1016/j.jse.2015.01.006 780 J. Tang et al.

End-stage elbow arthritis in the young patient is a vexing The following day, the skin of the rat was removed. The whole problem. Potential surgical solutions include elbow joint arm was exposed, and the neurovascular structures were closely debridement, resurfacing arthroplasty, nonvascularized dissected and studied. elbow allografts, and total elbow replacement. None of these options, however, currently offers a realistic and Experimental design, part II long-term solution to alleviate pain and to maintain motion.1,3,6,7,9,10,15 Tissue-engineered joint replacements In the second part of the experiment, 9 vascularized elbow joints currently remain only a theoretical possibility.11 Whereas from brown Norway rats with residual biceps and triceps tendons vascularized bone and joint autografts have proved suc- and the median nerve were transplanted across a strong major histocompatibility complex barrier to Lewis rats. The animals cessful for small joint reconstruction, expendable autograft were then randomly divided into 3 groups with different immu- sources are limited and do not provide viable donors for nosuppressive regimens. elbow reconstruction.2,8,22,24 The only viable solution is often surgical arthrodesis or Animals and anesthesia resection arthroplasty, leaving a patient with a minimally useful extremity with minimal or no motion.10,16 The ideal Brown Norway rats were also used as the donors in the second replacement for a destroyed elbow joint would be a living part of the experiment, and male Lewis rats (genetic expression, joint allogeneic transplant that precisely matches the di- RT1l) weighing 250 to 275 g were used as the recipient animals. mensions and structural properties of the impaired joint. Both elbow joints of 1 donor rat were used for 2 transplantation Despite the potential for this technology, few living vas- procedures (N ¼ 5). Donor rats were anesthetized with isoflurane cularized transplantations of any large joint have been inhalation at a dose of 3% for induction and 2% for maintenance performed. There have been a few studies evaluating knee by inhalation. After graft harvest, they were euthanized with an joint allotransplantation in rats, and there have even been overdose of isoflurane (5%). Recipient rats were anesthetized with vascularized allotransplantations in human knees, with only isoflurane as described for the donors. The rats were divided into 3 short-term success and then failure.4,5,14 Given the current different immunosuppressive regimens. immunosuppressive regimens, elective joint allotransplantation and the inherent side effects may not be Group 1: cyclosporine treatment group widely accepted. The scale, however, could realistically be Three of the 9 recipients were given cyclosporine immunosuppression at a dose of 16 mg/kg/day subcutaneously for the first tipped in favor of performing elbow allotransplantation if week. The dose was then tapered down to a maintenance dose of the regimens could be improved by decreasing side effects 2 mg/kg/day thereafter. of the medications.12,14 An animal model is important to study this possibility Group 2: temporary cyclosporine treatment group further. The rat has been frequently used in vascularized Three recipients were given cyclosporine at the dose of 16 mg/kg composite allotransplantation (VCA) experimentation for 10 days, and then the immunosuppression was stopped. because of its easy handling, adequate size for microsurgery, and affordability. Multiple VCA models have previously Group 3: control group 13,14,21,25,26 been established in the rat. We are, however, Three recipients received no immunosuppression. unaware of a rat elbow joint allotransplantation model. Harvest of vascularized elbow joint allotransplant (donor) Materials and methods An anterior longitudinal incision was made over the entire arm All animal care complied with the guidelines of the Institutional extending to the chest wall. The axillary vessels were dissected all Animal Care and Use Committee, the National Institutes of the way to the entrance of the thorax by elevating the pectoralis Health, and all national laws on the use of laboratory animals. major. After ligation, all major branches from the pedicle to the axillary and brachial arteries were isolated and preserved. The Experimental design, part I median nerve was identified near the brachial artery. The ulnar nerve was identified at the midbrachial level and protected to In the first part of the experiment, inbred male brown Norway rats preserve the ulnar collateral artery. The ulnar collateral artery (genetic expression, RT1n) weighing 250 to 275 g were anes- traversing with the ulnar nerve was identified, and a muscle cuff thetized and eventually euthanized with carbon dioxide; they were was preserved to protect this vessel as it provides arterial supply to used to study the rat elbow and its vascular anatomy. Once it was the joint. The major anterior muscle groups of the arm were adequately anesthetized, the rat was perfused with heparinized identified. The biceps was transected at a distal level, leaving only saline, followed by 30 mL of red latex or Microfil (17.1 mL a small tendinous portion. The flexors were identified and trans- contrast agent, 0.9 mL catalyst; Flow Tech, Inc., Carver, MA, ected at the midforearm level. At this time, the median, radial, and USA). When the rat expired (termination of heartbeat and lung ulnar arteries along with muscle perforators were identified, movement), it was placed in 4C overnight to allow the latex to ligated, and cut. The ulnar recurrent vessel was identified, and a polymerize. muscle cuff was saved to protect this vessel to the joint. Vascularized elbow allotransplantation model in the rat 781

Figure 1 (A) Elbow allograft after brachial artery and median nerve anastomoses. (B) Elbow allograft after internal ﬁxation of elbow and coverage with recipient musculature.

from the middle arm to the middle forearm. The recipient elbow joint was removed by cutting the brachial bone at the deltoid tuberosity level and the radius and ulna 2 mm distal to the insertion of the biceps. The donor and recipient elbow joints were then held side by side, and any adjustments to the length of the bones was performed to create a proper fit of the allograft (Fig. 1). Intramedullary fixation and 90/90 wiring were used. Early in our preliminary study design, 90/90 wiring failed to produce reliable union, and intramedullary fixation was selected for stabilization with improved results (Fig. 2). A 20-gauge needle and two 27-gauge needles were used as intramedullary rods for the humerus and the ulna and Figure 2 Radiograph of rat after transplantation and fixation radius, respectively, after reaming with a slightly smaller needle. All with intramedullary needles. recipient muscle groups (intact) were then attached to their corre- sponding donor parts with nonabsorbable 7-0 monofilament suture. The mediannervewas coaptedtothe recipient mediannerve in anend- The elbow joint of the rat was flexed, and another 2.5-cm to-side pattern to improve proprioception to the joint. A 2-cm incision incision posteriorly over the brachium extending distally over the was then made at the cervical region of the recipient rat. The external subcutaneous border of the ulna was made. The triceps was jugular vein was exposed and prepared as the recipient vein. The in- transected, leaving a small tendinous portion inserting into the ternal carotid artery was found just medial to the sternocleidomastoid ulna. The extensors were transected, and the posterior interosseous muscle and prepared as the recipient artery. The recipient artery (in- artery was identified between the ulna and the radius. The recur- ternal carotid) and the recipient vein (external jugular) were ligated rent interosseous artery, a branch from the posterior interosseous and transected as distal as possible to leave long recipient vessels for artery, was identified and preserved as it went into the joint. The anastomosis to the donor vessels. The donor pedicle was then tunneled humerus, radius, and ulna were then further exposed in a sub- to the neck region, and the end-to-end anastomosis was performed periosteal fashion. The bones were then cut with a small power with 11-0 nylon sutures under a surgical microscope. Finally, the saw. The elbow allograft was taken just proximal to the deltoid wounds were closed with absorbable sutures. tuberosity and 2 mm distal to the origin of the recurrent interosseous artery. The pedicle of the graft was then transected as In vivo assessment proximal as possible at the level of the axilla. The arms that were operated on were assessed daily for signs of Preparation of recipient bed and transfer of rejection, such as color changes, edema, and decreased motion. vascularized elbow joint Other complications, such as bleeding, wound healing, and signs of infection, were also assessed daily for the first 10 days, then every other day. In the recipient Lewis rat, an anterior incision, from the distal third of the forelimb to the proximal third of the brachium, was made over the elbow joint. The brachial artery and the median and ulnar Assessment of pedicle patency, graft blood flow, nerves were identified. The ulnar nerve was isolated from the and joint architecture cubital tunnel and was transposed anteriorly. The radial nerve was identified, and special care was paid to isolate the radial nerve at After 90 days of survival, patency of the pedicle was evaluated the radiocapitellar joint where the nerve lies close and is easily in a second nonsurvival anesthetic procedure. The transplant was damaged during the removal of the recipient elbow. Four muscle exposed under the operating microscope, and the pedicles were groups, biceps, triceps, flexors, and extensors, were identified and carefully tested for patency by downstream occlusion and cut off at their insertions. Then, the humerus, radius, and ulna release. Blood flow was then measured by performing the pre- were exposed and isolated from the soft tissue subperiosteally viously discussed contraction agent perfusion (Microfil), 782 J. Tang et al.

Figure 3 Anatomic basis of vascularized elbow joint harvesting. The 4 arterial branches constitute the periarticular network (after latex perfusion). Also noted is the large nerve branch from the median nerve to the elbow joint. followed by gross inspection and micro-computed tomography In vivo assessment (micro-CT) scanning. Micro-CT scanning was performed both before and after the blood supply Microfil test using the in vivo Group 1: cyclosporine treatment group micro-CT 40 (Scanco Medical AG, Bruttisellen,€ Switzerland) at In the cyclosporine-treated group, postoperative edema was 10-mm resolution. Three-dimensional angiograms were then reconstructed. The bone morphologic changes were then observed in the first 5 days after the operation. No elbow compared with a normal rat elbow joint. joint rigidity was noted throughout the experiment, and the shoulder joint regained normal activity and range of motion within 10 days after operation. The elbow joint motion as Histologic assessment of viability and well as the motion of the wrist and fingers continuously inflammation improved in the 3 months after the operation. The rats used the forelimb that was operated on in daily life for holding The transplanted elbows were then sectioned for histologic food and scratching their faces. These animals also ambu- assessment of the elbow joint and the cortical bone. Assessments lated with normal quadrupedal ambulation. No signs of of cartilage structure were made by looking for any signs of chondrocyte and matrix degeneration. rejection were observed (Fig. 4). Group 2: short-term cyclosporine treatment group; and Recipient chimerism evaluation group 3: control group, no immunosuppression In the short-term cyclosporine-treated group and in the A chimerism analysis was performed on 1 104 cells using control group receiving no immunosuppression, similar FACScan (BD Biosciences, San Diego, CA, USA). postoperative edema was noted for about 5 days. No obvious signs of rejection were demonstrated after the operation; however, the grafted side showed less shoulder Results and elbow range of motion compared with the contralateral side and compared with the cyclosporine treatment group. Part I: vascular anatomy of the rat elbow Throughout the 3-month survival, these elbows became progressively stiffer until they were rigid by the time of In the anatomic study, we found that 4 arterial branches sacrifice. constitute the periarticular network. These branches were consistently identified in the 3 rats studied. We identified Assessment of pedicle patency, graft blood flow, (1) the recurrent interosseous artery, (2) the recurrent ulnar bone union, and joint architecture artery, (3) the ulnar collateral artery, and (4) the collateral radial artery (Fig. 3). The transplant was exposed under the operating microscope, and the pedicles were carefully tested for patency by Part II: rat elbow allotransplantation downstream occlusion and release. Blood flow was then measured by performing the previously discussed contrac- The mean surgical duration for the donor elbow harvest was tion agent perfusion (Microfil), followed by gross inspec- 2 hours, and the mean time for the recipient transplantation tion and micro-CT scanning. was 4.5 hours. Warm ischemia time was 30 minutes. Three rats failed to recover from anesthesia and subsequently Group 1: cyclosporine treatment group died. The remaining animals returned to their normal ac- The pedicles remained grossly open on visual inspection as tivities the second day after transplantation. well as with the patency test. Radiologic evaluations after Vascularized elbow allotransplantation model in the rat 783

Figure 5 Note the preserved main pedicle (brachial artery).

Figure 4 Note the active use of the transplanted left forelimb in the cyclosporine (CsA) group (top), whereas the control rat is not weight bearing and holding the elbow ﬂexed (bottom).

Microﬁl perfusion demonstrated the patency of the pedicle and the circulation of the graft (Fig. 5). In the cyclosporine- treated group, plain radiographs and CT scans of the graft showed good position and good ﬁxation of the transplanted elbow 1 week after the operation (Fig. 6). Bone union was demonstrated at 90 days postoperatively. Histologic samples revealed viable bone without signs of ischemic Figure 6 Healed osteotomies with preserved joint structure. necrosis or rejection in the cyclosporine-treated group at Note that the elbow is relatively extended. post-transplantation day 90. Viable bone marrow was also observed in the bone samples. Discussion

Groups 2 and 3: short-term cyclosporine group and Previous studies in a rat population have shown that control group surgical neoangiogenesis has allowed short-term bone The pedicles were grossly constricted and did not have flow allotransplants to survive without immunosuppression.14 with the patency test. Microfil perfusion showed vascular Whereas a great deal of research is currently under sprouting around the graft in a highly abnormal pattern with way in promoting tolerance of VCA, the allotransplant of no evidence of the main pedicle (Fig. 7). No bone healing an elbow joint may differ significantly. As in our model, was seen 3 months after the operation, and micro-CT the skin is not necessary to transplant the joint, and showed obvious degeneration, absorption, and fractures of thus the antigenicity of the transplant may differ signif- the joint (Fig. 8). Histologic sections revealed little or no icantly from a transplant that contains highly antigenic viable bone marrow and minimal viable cartilage at the skin. elbow joint. There was a large amount of lymphocyte During the past decades, osteoarticular elbow allograft infiltration visible in both of these groups (Fig. 9). replacement has become a potential option for elbow reconstruction in the severely damaged elbow.2,3,6-11,14-16,22,24 Recipient-donor chimerism evaluation The initial postsurgical results were often satisfactory, yet most cases inevitably resulted in severe and obvious degen- No obvious chimerism was evidenced on day 10, day 30, erative joint changes in the midterm follow-up.10 With the and day 60 after transplantation. rapid advances of VCA, vascularized joint allograft 784 J. Tang et al.

Figure 7 The main pedicle (brachial artery) is difficult to see, Figure 8 Note the dislocated, severely destroyed elbow joint in and note the abundant neovasculature present. this elbow without immunosuppression. transplantation might become the solution for end-stage transplant. The in vivo assessments demonstrated a large elbow arthritis if immunosuppression can be minimized.14 difference between group 1 and groups 2 and 3. We were Diefenbeck et al4 have published their results of knee allo- not able to distinguish groups 2 and 3 either clinically or in transplantation, which have unfortunately all been lost the laboratory, and therefore the results of both have been because of graft vasculopathy. presented together. The clinical differences between the In a beautifully performed study, Wavreille et al23 cyclosporine group 1 and the other 2 groups were obvious. published their anatomic research for a potential vascu- The immunosuppressed rats’ forelimb use increased over larized elbow joint transplant in humans in 2006. time, whereas the other 2 groups’ limbs became stiff and Although this would be the ultimate goal (vascularized were used minimally. elbow allotransplantation), more basic science research is The patency of the pedicle was verified surgically as clearly needed before this endeavor. An animal model is well as with angiography using Microfil and then by micro- needed to further progress this potential surgical treat- CT scanning of the elbow joints. The main pedicle of the ment. Our research aimed to develop a vascularized elbow brachial artery was seen in group 1, whereas in the other allograft model in the rat across a major histocompati- groups, the main pedicle was no longer visualized patent bility barrier. crossing the joint. In the other groups, we found an abun- dance of neovascular tissue surrounding the joint, which Part I: vascularity of the rat elbow appeared to be an attempt to revascularize the elbow joint unsuccessfully. The use of the micro-CT scanner allowed us to verify that there was indeed union at our osseous We determined, as previously noted, that 4 main branches junctions in the group 1 rats treated with cyclosporine, vascularize the elbow joint. In addition, we noted that there whereas nonunions were obvious in the other 2 groups. In was a large branch from the median nerve to the elbow addition, the joint destruction was seen when micro-CT joint. We thought that this was likely a major propriocep- images of the nonimmunosuppressed elbows were per- tive component to the elbow joint and thus performed an formed. The histologic slides of the joint graphically end-to-side coaptation to the recipient’s median nerve. It demonstrate the preservation of the joint structure in the was our intent that this coaptation will help the proprio- immunosuppressed group and progressive destruction in ceptive function of the joint and prevent neuropathic the other 2 groups. degeneration. Chimerism has been demonstrated after bone marrow transplantation in rat models17,19,20; however, no chimerism Part II: rat elbow allotransplantation has been reported in humans to date.14,18 We did not detect any donor-specific chimerism in our animal model, and this This study demonstrates that a vascularized elbow allograft result was not unexpected. transplantation can be performed in the rat across a major We did note some problems in our experimentation histocompatibility complex barrier. We have additionally worthy of note. Early on, we used 90/90 wiring and noted confirmed that short-term immunosuppression and no that the rats had a difficult time healing the transplant bone immunosuppression lead to rejection of the allograft junctions, so we switched to intramedullary fixation with Vascularized elbow allotransplantation model in the rat 785

Figure 9 The upper left image is a control elbow that did not have surgery. The upper right is a transplanted elbow that received cyclosporine; note the relatively preserved joint architecture. The lower left is a destroyed elbow joint in the short-term cyclosporine group. The lower right is the control group that was transplanted without immunosuppression. needles. During the postoperative 90 days, there were no References severe complications necessitating graft removal. There were superficial wound infections in 2 animals that healed 1. Athwal GS. Revision total elbow arthroplasty for prosthetic fractures. after debridement. J Bone Joint Surg Am 2006;88:2017. http://dx.doi.org/10.2106/JBJS. E.00878 2. Chen SH, Wei FC, Chen HC, Hentz VR, Chuang DC, Yeh MC. Conclusion Vascularized toe joint transfer to the hand. Plast Reconstr Surg 1996; 98:1275-84. Despite the low number of transplants performed, this 3. Delloye C, Cornu O, Dubuc JE, Vincent A, Barbier O. Recon- struction du coude par allogreffe massive osteo-articulaire totale: study confirms the surgical feasibility and survival of echec precoce par instabilite. Rev Chir Orthop Reparatrice Appar a vascularized elbow allograft transplant in a rat Mot 2004;90:360-4. model when it is properly immunosuppressed. Short- 4. Diefenbeck M, Nerlich A, Schneeberger S, Wagner F, Hofmann GO. term follow-up demonstrated no obvious degenerative Allograft vasculopathy after allogeneic vascularized knee trans- joint changes, and the animals appeared to be using plantation. Transpl Int 2010;24:e1-5. http://dx.doi.org/10.1111/j.1432- 2277.2010.01178.x their limbs normally. Despite the feasibility of this 5. Diefenbeck M, Wagner F, Kirschner MH, Nerlich A, Muckley€ T, procedure, more transplants need to be performed, Hofmann GO. Outcome of allogeneic vascularized knee transplants. and a more quantitative approach to results mea- Transpl Int 2007;20:410-8. http://dx.doi.org/10.1111/j.1432-2277. surements would improve the validity of this study in 2007.00453.x the future. 6. Ehsan A, Lee B, Itamura JM. Total elbow allografts with collateral ligament reconstruction for posttraumatic elbow injuries. J Orthop Sci 2010;15:795-803. http://dx.doi.org/10.1007/s00776-010-1540-7 7. Funovics PT, Schuh R, Adams SB, Sabeti-Aschraf M, Dominkus M, Kotz RI. Modular prosthetic reconstruction of major bone defects of Disclaimer the distal end of the humerus. J Bone Joint Surg Am 2011;93:1064-74. http://dx.doi.org/10.2106/JBJS.J.00239 The authors, their immediate families, and any research 8. Hierner R, Berger AK. Long-term results after vascularised joint transfer for finger joint reconstruction. J Plast Reconstr Aesthet Surg foundation with which they are affiliated have not 2008;61:1338-46. http://dx.doi.org/10.1016/j.bjps.2007.09.035 received any financial payments or other benefits from 9. Jaffe KA, Morris SG, Sorrell RG, Gebhardt MC, Mankin HJ. Massive bone any commercial entity related to the subject of this allografts for traumatic skeletal defects. Southampt Med J 1991;84:975. article. 10. Kharrazi FD, Busfield BT, Khorshad DS, Hornicek FJ, Mankin HJ. Osteoarticular and total elbow allograft reconstruction with severe 786 J. Tang et al.

bone loss. Clin Orthop Relat Res 2008;466:205-9. http://dx.doi.org/10. 19. Plissonnier D, Nochy D, Poncet P, Mandet C, Hinglais N, Bariety J, 1007/s11999-007-0011-8 et al. Sequential immunological targeting of chronic experimental 11. Kosuge D, Khan WS, Haddad B, Marsh D. Biomaterials and scaffolds arterial allograft. Transplantation 1995;60:414. in bone and musculoskeletal engineering. Curr Stem Cell Res Ther 20. Siemionow M, Klimczak A, Unal S, Agaoglu G, Carnevale K. He- 2013;8:185-91. http://dx.doi.org/10.2174/1574888X11308030002 matopoietic stem cell engraftment and seeding permits multi- 12. Kremer T, Giusti G, Friedrich PF, Willems W, Bishop AT, lymphoid chimerism in vascularized bone marrow transplants. Am J Giessler GA. Knee joint transplantation combined with surgical Transplant 2008;8:1163-76. http://dx.doi.org/10.1111/j.1600-6143. angiogenesis in rabbitsda new experimental model. Microsurgery 2008.02241.x 2012;32:118-27. http://dx.doi.org/10.1002/micr.20946 21. Siemionow M, Klimczak A. Advances in the development of experi- 13. Kulahci Y, Siemionow M. A new composite hemiface/mandible/ mental composite tissue transplantation models. Transpl Int 2010;23: tongue transplantation model in rats. Ann Plast Surg 2010;64:114-21. 2-13. http://dx.doi.org/10.1111/j.1432-2277.2009.00948.x http://dx.doi.org/10.1097/SAP.0b013e3181a20cca 22. Silberman J, Kanat IO. Total joint replacement in digits of the foot. J 14. Larsen M, Friedrich PF, Bishop AT. A modiﬁed vascularized whole Foot Surg 1984;23:207-12. knee joint allotransplantation model in the rat. Microsurgery 2010;30: 23. Wavreille G, Remedios Dos C, Chantelot C, Limousin M, Fontaine C. 557-64. http://dx.doi.org/10.1002/micr.20800 Anatomic bases of vascularized elbow joint harvesting to achieve 15. Larson AN, Adams RA, Morrey BF. Revision interposition arthro- vascularized allograft. Surg Radiol Anat 2006;28:498-510. http://dx. plasty of the elbow. J Bone Joint Surg Br 2010;92:1273-7. http://dx. doi.org/10.1007/s00276-006-0130-z doi.org/10.1302/0301-620X.92B9.24039 24. Wetke E, Zerahn B, Kofoed H. Prospective analysis of a ﬁrst MTP 16. Moghaddam-Alvandi A, Dremel E, Guven€ F, Heppert V, Wagner C, total joint replacement. Evaluation by bone mineral densitometry, Studier-Fischer S, et al. Arthrodesis of the elbow joint. Unfallchirurgie pedobarography, and visual analogue score for pain. Foot Ankle Surg 2010;113:300-7. http://dx.doi.org/10.1007/s00113-009-1722-y 2012;18:136-40. http://dx.doi.org/10.1016/j.fas.2011.07.002 17. Muramatsu K, Doi K, Kawai S. Limb allotransplantation in rats: 25. Yazici I, Carnevale K, Klimczak A, Siemionow M. A new rat model of combined immunosuppression by FK-506 and 15-deoxyspergualin. J maxilla allotransplantation. Ann Plast Surg 2007;58:338-44. http://dx. Hand Surg Am 1999;24:586-93. http://dx.doi.org/10.1053/jhsu.1999. doi.org/10.1097/01.sap.0000237683.72676.12 0586 26. Yazici I, Unal S, Siemionow M. Composite hemiface/calvaria trans- 18. Petruzzo P, Dubernard JM. The International Registry on Hand and plantation model in rats. Plast Reconstr Surg 2006;118:1321-7. http:// Composite Tissue allotransplantation. Clin Transplant 2011:247-53. dx.doi.org/10.1097/01.prs.0000239452.31605.b3