CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Number 425, pp. 237–243 © 2004 Lippincott Williams & Wilkins Feasibility of Percutaneous Gene Transfer to an Atrophic Nonunion in a Rabbit Christian Lattermann, MD*,†; Axel W. Baltzer, MD†,‡; Boris A. Zelle, MD*; Janey D. Whalen, PhD*; Christopher Niyibizi, PhD*; Paul D. Robbins, PhD†; Christopher H. Evans, PhD, DSc*†; and Gary S. Gruen, MD* Treatment of atrophic nonunions is a challenge to orthopae- which 5–10% percent result in a delayed union or non- dic surgeons. Growth factors potentially are valuable factors union.25 In addition, the costs for the treatment of a non- for improvement of tissue healing. The use of growth factors, union range approximately between $10,000 and $50,000 however, is limited by their short half-lives. Gene therapy per case and pose a substantial socioeconomic burden.3,13 has the potential to improve the treatment. This study aimed to establish and validate an atrophic nonunion model in a Although the introduction of intramedullary nailing re- rabbit for the use of a percutaneous in vivo gene therapy duced surgical invasiveness and reduced the risk for hy- protocol. An atrophic tibial nonunion was established in 24 pertrophic nonunions, this improvement still has not found New Zealand White rabbits. Radiologic and histologic fol- its match in atrophic nonunions. As a result, atrophic non- lowup was for 64 weeks. The rabbit tibias showed no radio- unions are challenging for the orthopaedic surgeon be- logic or histologic signs of healing. In addition, an adenoviral cause of their greatly impaired healing capacity and mar- vector carrying a marker gene was injected percutaneously ginal vascularity. To treat an atrophic nonunion, restora- into the nonunion site in 12 rabbits. Expression of the tion of vascularity to allow for a healing response to occur marker gene was assessed for as many as 4 weeks. The per- is necessary. Current treatment options therefore still in- cutaneous gene delivery resulted in transgene expression in the nonunion site for as many as 4 weeks. The described volve extensive surgical debridement of the nonunion site model reliably leads to an atrophic tibial nonunion in rab- to reestablish vascularity to the site. After this occurs, bits. Adenoviral percutaneous gene delivery into the non- growth factors, competent osteoprogenitor cells, and other union site is feasible and leads to transgene expression locally blood-borne components of the healing cascade can gain for at least 1 month. This study provides investigators with a access to the nonunion. However, extensive surgical ap- reliable and reproducible model of an atrophic nonunion. proaches additionally endanger an already impaired blood flow at the nonunion site. Additionally, there is a high risk of infection associated with these surgical procedures.28 An impaired healing response leading to a delayed union Therefore, novel treatment approaches for atrophic non- or nonunion after a fracture still poses a challenge to or- unions are desirable to reduce the morbidity and costs for thopaedic surgeons. It is estimated that approximately 5.6 patients. Experimental studies have reported the delivery million fractures occur in the United States each year of of growth hormone (GH), transforming growth factor- (TGF- ), or bone morphogenetic proteins (BMP) into the Received: February 26, 2003 healing site of fresh fractures.4,6,22,32 The direct delivery Revised: August 14, 2003 of growth factors has major drawbacks because of their Accepted: November 6, 2003 short biologic half-lives, which typically are in the range From the *Department of Orthopaedic Surgery and †Department of Molecu- lar Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA; and of minutes to hours.7 To overcome this problem BMP-2 ‡Department of Orthopaedic Surgery, Heinrich-Heine-University, Dussel- and OP-1 have been delivered in a collagen sponge yield- dorf, Germany. ing very high local concentrations to the fracture site.8,9,12 This work was supported by a grant from the Albert B. Ferguson Orthopae- dic Fund of the Pittsburgh Foundation. Another option to overcome this problem may be to de- Correspondence to: Christian Lattermann, MD, Department of Orthopaedic liver the genes encoding these growth factors, rather than Surgery, University of Pittsburgh Medical Center, Kaufmann Building, Suite delivering the growth factors. 1010, 3471 Fifth Avenue, Pittsburgh, PA 15213. Phone: 412-648-1090; Fax: 412-648-8412; E-mail: email@example.com. To study traditional and novel treatment options for DOI: 10.1097/01.blo.0000137292.25504.22 atrophic nonunions it is essential to test these techniques in 237 Clinical Orthopaedics 238 Lattermann et al and Related Research a reliable animal model. No reliable animal model for an fracture site to hold the silastic tubing in place (Fig 1D). After atrophic nonunion has yet been reported and validated. We routine irrigation, the wound was closed. After 4 weeks, the therefore decided to investigate an observation of Oni,24 cerclage wires and the Silastic tubings were surgically removed who studied fracture healing and observed that some rab- through two small incisions. For radiologic analysis, anteroposterior and lateral views bits did not achieve healing of a tibial fracture when the from each rabbit were immediately obtained postoperatively and bone was devascularized and revascularization of the frac- consecutively 1, 2, 3, 4, 8, 16, and 64 weeks after surgery. The ture was prevented for 4 weeks. The first goal of the cur- radiographs were analyzed for nonunion, callus, and ectopic rent study therefore was to validate an atrophic nonunion bone formation. At the time the radiograph was obtained, the model in a rabbit tibia based on the observations of Oni.24 tibia was clinically assessed for rotational stability. In the second part of this study we investigated the feasi- At 8, 16, and 64 weeks after surgery, four rabbits each were bility of delivering a marker gene percutaneously into the euthanized for histologic analysis. The right tibia was harvested nonunion site, as a prelude to developing a percutaneous in and the muscle tissue was stripped. The fibrous capsule of the vivo gene therapy approach to the treatment of atrophic nonunion site was left in place. The tibia was cut 2 cm proximal nonunions. Several methods are available for the delivery and 2 cm distal from the nonunion site and the intramedullary of transgenes into musculoskeletal tissues. We chose to Steinmann pin was removed. The samples were fixed in 4% paraformaldehyde (pH 7.5) for 7–10 days. After fixation, the test the feasibility of a percutaneous gene transfer to the tibial samples were decalcified for 4–6 weeks in 50 mL conical atrophic nonunion site using an adenoviral vector carrying tubes containing 20% EDTA. Paraffin sections were cut at a a marker gene. thickness of 5–7 m and stained with hematoxylin and eosin. The remaining 12 rabbits were used for the gene transfer MATERIALS AND METHODS feasibility study. The first generation adenoviral vector ( E1, E3) used in this study is replication deficient because of a We used 24 skeletally mature New Zealand White rabbits weigh- deletion of the E1 gene. The lacZ marker gene is inserted in ing 4.5–5 kg. All experimental animals were housed and oper- place of the E1 gene. Gene expression is driven by the human ated in the Central Animal Facility of the University of Pitts- cytomegalovirus early promoter. The Ad/CMV/lacZ virus is burgh Medical Center, and the treatment protocol for animal grown in 293 cells (ATCC, Bethesda, MD), a human embryonic subjects was approved by the Institutional Animal Care and Use kidney cell line that constitutively expresses the E1-encoded Committee of the University of Pittsburgh Medical Center. proteins E1a and E1b. Viral titers were determined by optical All 24 rabbits included in this study had the same surgical density at 260 nm. Eight weeks after the initial surgery, these 12 procedure. The rabbits were tranquilized with an intramuscular rabbits were tranquilized using 2 mg/kg xylazine and 20 mg/kg dose of 4 mg/kg xylazine and 40 mg/kg ketamine preoperatively. ketamine intramuscularly. The nonunion was observed fluoro- Anesthesia was maintained using 1.5–2.5% isoflurane delivered scopically. After sterilization of the tibia, a syringe with a 27- through a mask. The animals were monitored with electrocardi- gauge needle was inserted percutaneously into the fibrous gap ography and pulse oximetry throughout the procedure. Postop- under fluoroscopic control. After the needle was placed cor- eratively, a dose of 0.1 mg/kg Torbugesic (Wyeth, Philadel- rectly, 1 × 107 pfu (plaque forming units) of Ad/CMV-lacZ virus phia, PA) was administered subcutaneously twice a day for 2 diluted in 50 L saline solution was injected into the nonunion days. After sterilization and preparation of the right tibia, an site. The rabbits were divided into four groups of three animals anteromedial incision of approximately 7–8 cm was made. The each. In the first group, the histologic analysis of the marker tibia was exposed using a periosteal elevator. In rabbits, the gene expression was done 1 week after viral injection; in the distal fibulotibial insertion usually is located in the upper 1⁄2 of second group, the histologic analysis was done 2 weeks after the tibial shaft (Fig 1A). This structure was used as a bony viral injection; in the third group, the histologic analysis was landmark, and the tibia was cut using a high-speed dental burr done 3 weeks after viral injection; and in the fourth group, the with a 2-mm burr bit 1 cm distal from the fibulotibial insertion. histologic analysis was done 4 weeks after viral injection. The The periosteum was thoroughly stripped 1.5 cm proximal and expression of the lacZ marker gene was detected histologically distal from the fracture site. Then the marrow cavity was reamed by X-gal staining. using the dental burr and a 2.5-mm drill bit. A hole was drilled retrogradely through the marrow cavity into the anteromedial aspect of the tibial head. An exactly fitted Steinmann pin was RESULTS placed antegradely through this drill hole (Fig 1B). The pin Twenty-three of the 24 rabbits had a radiologically docu- diameter in our series ranged from 3–4.5 mm. Afterward, silastic mented nonunion (Fig 2). One rabbit had a soft tissue tubing (Nalgene 180 clear PVC tubing; Fisher Scientific, Pitts- burgh, PA) was placed over the proximal end of the fracture and infection after the primary procedure and had to be ex- advanced proximally for 1 cm as described by Oni (Fig 1C).24 cluded from the study. Radiographic analysis showed no The distal fracture end was reduced into the silastic tubing and callus formation in the original fracture site for as many as the Steinmann pin was advanced into the distal marrow cavity. 4 weeks when the silastic tubing was removed. After 2 The Steinmann pin was cut to its appropriate lengths and two weeks, in three of the rabbits there was minor callus for- cerclage wires were positioned proximally and distally to the mation at the interface of the distal edge of the silastic Number 425 August 2004 Atrophic Nonunion in a Rabbit 239 Fig 1A–D. The tibial bone was exposed and the tibia was cut 1 cm distal from the fibulotibial insertion. (A) The proximal stump with the distal fibulotibial insertion is shown after periosteal stripping. (B) The marrow cavity was reamed and a Steinmann pin was inserted. (C) Silastic tubing was placed over the proximal end of the fracture. The distal fracture end was reduced into the silastic tubing and the Steinmann pin was advanced into the distal marrow cavity. (D) The silastic tubing was fixed and sealed using two cerclage wires. tubing to the distal tibia. However, this was a minor reac- nonunion site. Within 200 m proximal to and distal from tion that did not interfere with percutaneous removal of the the nonunion site no viable osteocytes were detectable. silastic tubing. After removal of the silastic tube, no ad- From the periphery we detected a fibrous ingrowth into the ditional callus formation was detected. Radiologic healing nonunion site, which was intimately associated with the was not seen in any of the rabbits at any time for as many ends of the bone. Although scar tissue was found 8 and 16 as 64 weeks. The bone originally covered by the silastic weeks after silastic tube removal, very little inflammatory tubing showed a slightly reduced diameter after 16 weeks activity was detected in the fibrous scar tissue. in some animals. The clinical examination showed unre- Expression of the lacZ marker gene was detected his- stricted rotational instability of the distal part of the right tologically by X-gal staining at all times. The most striking tibia in all 23 rabbits until euthanasia. Because of this finding was distribution of the transfected cells in the non- obvious clinical instability we did not do a biomechanical union (Fig 4). We did not see lacZ + cells in the bony parts analysis on the specimen. of the nonunion. The fibrous scar tissue, however, showed The paraffin sections showed normal eosin staining of marked expression of -galactosidase. The transfected the bone with regular bony architecture and fibrous in- cells were distributed throughout the fibrous scar with no- growth into the nonunion site (Fig 3). There was progres- ticeable accumulation in the fibrous capsule that had sive loss of viable osteocytes in their osteons toward the formed around the nonunion. We detected marker gene Clinical Orthopaedics 240 Lattermann et al and Related Research Fig 2A–D. (A) This anteroposterior radio- graph shows establishment of the atrophic nonunion. (B) There was no ectopic bone formation around the silastic tubing after 2 weeks. At 4 weeks, the Silastic tube was removed. After (C) 8 and (D) 16 weeks there was no callus formation. The bone ends round up and a visible gap persists. expression for as many as 28 days. There was no sign of cularized providing an ideal environment for bone healing. monocyte or neutrophil infiltration, hyperemia, new blood In contrast, an osseous nonunion in humans usually is vessel formation, or any other indications of an inflamma- caused by hypermobility of the fracture or decreased blood tory reaction directed against virally infected cells. The supply to the fracture.21 From the biologic standpoint, the samples taken from the lung, liver, and spleen did not segmental defect mimics a different biologic situation than show any cells with -galactosidase activity at any time. a common hypertrophic or atrophic nonunion. Therefore, we tested a new approach to establish an atrophic tibial DISCUSSION nonunion in a rabbit model. Our procedure was based on the observations of Oni, who reported that tibial fractures Throughout the last decade, numerous animal studies have in a rabbit eventually fail to heal in a devascularized situ- been done which assessed the healing potential of non- ation.24 An additional focus of this study was to test the unions after certain operative and nonoperative pro- feasibility of a novel therapeutic approach to promote cedures.14,17,20,22,27,29,30,32 All of these studies used a healing of atrophic nonunions using a percutaneous gene critical-size defect in long bones of different animal spe- transfer technique. Atrophic nonunions usually are treated cies (segmental defect) to mimic a nonunion. The segmen- with techniques involving extensive surgery.21,28 How- tal defect model creates a gap in a long bone, which cannot ever, atrophic nonunions frequently are the result of an be bridged by fracture callus and therefore the fracture unfavorable soft tissue envelope which may complicate cannot heal.26 At the fracture ends, however, a normal additional invasive procedures. The available nonopera- fracture healing response showing granulation tissue and tive or minimally invasive techniques have not produced formation of a fracture callus, with spontaneous filling of reliable results in the treatment of atrophic nonunions.5,31 15–20% of the defect can be observed.14 In addition, there Bone growth factors, such as BMP-2, have been shown to is no increased micromotion between the bone ends and increase fracture healing significantly in animal mod- the surrounding healthy soft tissue usually is highly vas- els16,32,33 and in human clinical trials.8,12 One of the major Number 425 August 2004 Atrophic Nonunion in a Rabbit 241 investigated the feasibility of percutaneous gene delivery into the nonunion site, using an adenoviral vector carrying the lacZ marker gene. The current study has strengths and limitations. Our operative technique created an atrophic nonunion as de- termined by radiologic and histologic criteria. The oppos- ing ends of the bone seemed atrophic with focal necrosis and no radiologic signs of healing were recorded. How- ever, the validity of our data is limited since the radiologic and histologic evaluations were qualitative and therefore subject to interobserver and intraobserver variations. Moreover, no detailed investigation of the immunologic response to the adenoviral gene transfer was done. Al- though the histologic sections did not show any significant neutrophil or lymphocyte infiltration, we cannot com- pletely rule out an early inflammatory response because we did not do immunohistochemical analysis for CD4/CD8 or antibody titer measurements for specific neu- tralizing antibodies against the adenoviral vector or the transgene. At distant organ sites, we did not find any cells with -galactosidase activity at any time. However, a real- time PCR was not done and therefore we are unable to determine whether adenoviral particles were present at dis- tant organ sites. In addition, no quantification of new ves- sel formation has been done in the current study. We at- tempted to establish an atrophic nonunion model using several steps including periosteal stripping, intramedullary reaming, and application of a silastic tube for 4 weeks. Because no control groups were used, we are unable to determine whether it was the periosteal stripping, the in- tramedullary reaming, the silastic tube, or the combination Fig 3A–B. (A) The nonunion site was repopulated by fibrous of these agents that led to the atrophic nonunion. Using scar tissue that aligned intimately with the bone ends (arrow). this suggested model, it was shown that healing of the (B) At 8 weeks after removal of the silastic tubing the bone fracture did not occur in any animal in our study, therefore, ends seemed atrophic with empty osteocyte lacunae (arrows). study provides investigators with a reliable animal model of an atrophic nonunion. Experimental methods for heal- ing of atrophic nonunions can be tested, using our sug- drawbacks of single delivery of growth factors is the short gested nonunion model. The study showed that percuta- biologic half-life of most growth factors. We know from neous in vivo gene delivery into a nonunion site is fea- work on chronic skin wounds that one application of sible. Additional studies should focus on gene transfer of growth factors in a fibrous scar tissue is not sufficient to bone growth factors or angiogenic factors to test their reestablish a healing response.18 Sustained delivery of healing potential. growth factors, such as BMP-2, may be necessary for In contrast to segmental defect models described ear- treatment of atrophic nonunions, and a delivery system lier,14,17,20,22,27,29,30,32 this model is based on periosteal that can provide a continuous local release of growth fac- stripping, intramedullary reaming, and decreased vascular tors is desirable. The suggested delivery of BMP-2 or supply to the fractured bone. These etiologic factors are OP-1 in a collagen sponge8,9,12 still requires an extensive consistent with the clinical risk factors for development of debridement and surgical approach for growth factor de- an atrophic nonunion.21,24,28 livery. Using adenoviral vectors, gene expression has been Novel treatment approaches in orthopaedic surgery in- seen for as many as 6 weeks in various soft tissues.10,11,15 clude application of bone growth factors. Several growth In addition, it was shown that gene delivery to bone and factors, such as TGF- , BMP-2, and BMP-7 have been fractures using viral vectors is feasible and leads to an tested in different animal models and have been reported accelerated healing response.1,2,19,23 Therefore, we also to enhance fracture healing.2,6,17,19,32 The treatment of Clinical Orthopaedics 242 Lattermann et al and Related Research Fig 4A–B. (A) After 1 week, a lon- gitudinal section (magnification × 100) showed transgene expression in the surrounding tissue of the non- union (arrows). (B) In a cross section (magnification × 60), gene expres- sion can be seen 3 weeks after gene delivery (arrows). atrophic fracture nonunions may benefit from such ap- of an adenovirus expressing BMP7. J Cell Biochem 78:476–486, 2000. proaches. We suggest a reliable and reproducible atrophic 8. Friedlaender GE, Perry CR, Cole JD, et al: Osteogenic protein-1 nonunion model in a rabbit. Novel treatment approaches, (bone morphogenetic protein-7) in the treatment of tibial nonunions. such as injection of bone growth factors or gene transfer, J Bone Joint Surg 83A(Suppl):S151–S158, 2001. 9. Geesink RG, Hoefnagels NH, Bulstra SK: Osteogenic activity of can be tested using our suggested nonunion model. OP-1 bone morphogenetic protein (BMP-7) in a human fibular de- fect. J Bone Joint Surg 81B:710–718, 1999. 10. Gerich TG, Kang R, Fu FH, Robbins PD, Evans CH: Gene transfer Acknowledgments to the patellar tendon. Knee Surg Sports Traumatol Arthrosc 5:118– We thank Kurt Weiss and Joan Rosenberger for help with animal 123, 1997. care and surgery, and Dr. Jonny Huard for use of his imaging 11. Ghivizzani SC, Lechman ER, Tio C, et al: Direct retrovirus- mediated gene transfer to the synovium of the rabbit knee: Impli- facilities. We thank Dr. Freddie H. Fu for support of this project. cations for arthritis gene therapy. Gene Ther 4:977–982, 1997. 12. Govender S, Csimma C, Genant HK, et al: Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: References A prospective, controlled, randomized study of four hundred and 1. Baltzer AW, Lattermann C, Whalen JD, et al: A gene therapy ap- fifty patients. J Bone Joint Surg 84A:2123–2134, 2002. proach to accelerating bone healing: Evaluation of gene expression 13. Heckmann JD, Sarasohn-Kahn J: The economics of treating tibia frac- in a New Zealand White rabbit model. Knee Surg Sports Traumatol tures: The cost of delayed unions. Bull Hosp Jt Dis 56:63–72, 1997. Arthrosc 7:197–202, 1999. 14. Hietaniemi K, Peltonen J, Paavolainen P: An experimental model 2. Baltzer AWA, Lattermann C, Whalen JD, et al: Genetic enhance- for nonunion in rats. Injury 26:681–686, 1995. ment of fracture repair: Healing of an experimental segmental de- 15. Huard J, Krisky D, Oligino T, et al: Gene transfer to muscle using fect by adenoviral transfer of the BMP-2 gene. Gene Ther 7:734– herpes simplex virus-based vectors. Neuromuscul Disord 7:299– 739, 2000. 313, 1997. 3. Beaver R, Brinker MR, Barrack RL: An analysis of the actual cost 16. Johnson EE, Urist MR, Finerman GAM: Repair of segmental de- of tibial nonunions. J La State Med Soc 149:200–206, 1997. fects of the tibia with cancellous bone grafts augmented with human 4. Bostrom M, Lane JM, Tomin E, et al: Use of bone morphogenetic bone morphogenetic protein: A preliminary report. Clin Orthop protein-2 in the rabbit ulnar nonunion model. Clin Orthop 327:272– 236:249–257, 1988. 282, 1996. 17. Johnson EE, Urist MR, Schmalzried TP, et al: Autogeneic cancel- 5. Brighton CT, Black J, Friedenberg ZB, et al: A multicenter study of lous bone grafts in extensive segmental ulnar defects in dogs: Ef- the treatment of nonunion with constant direct current. J Bone Joint fects of xenogeneic bovine bone morphogenetic protein without and Surg 63A:2–13, 1981. with interposition of soft tissues and interruption of blood supply. 6. Cook SD, Wolfe MW, Salkeld SL, Rueger DC: Effect of recombi- Clin Orthop 249:254–265, 1989. nant human osteogenic protein-1 on healing of segmental defects in 18. Knighton DR, Ciresi KF, Fiegel VD, Austin LL, Butler L: Classi- non-human primates. J Bone Joint Surg 77A:734–750, 1995. fication and treatment of chronic nonhealing wounds: Successful 7. Franceschi RT, Wang D, Krebsbach PH, Rutherford RB: Gene treatment with autologous platelet-derived wound healing factors therapy for bone formation: In vitro and in vivo osteogenic activity (PDWHF). Ann Surg 204:322–330, 1986. Number 425 August 2004 Atrophic Nonunion in a Rabbit 243 19. Liebermann JR, Le LQ, Wu L, et al: Regional gene therapy with a 27. Sciadini MF, Dawson JM, Johnson KD: Bovine-derived bone pro- BMP-2-producing murine stromal cell line induces heterotopic and tein as a bone graft substitute in a canine segmental defect model. orthotopic bone formation in rodents. J Orthop Res 16:330–339, J Orthop Trauma 11:496–508, 1997. 1988. 28. Taylor C: Delayed Union and Nonunion in Fractures. In Crenshaw 20. Miclau T, Lindsey RW, Probe R, Rahn BA, Perren SM: Autogenous AH (ed). Campbell’s Operative Orthopaedics. St Louis, Mosby cancellous bone graft incorporation in a gap defect in the canine Year Book Inc 85-124, 1992. femur. J Orthop Trauma 10:108–113, 1996. 29. Tiedeman JJ, Connolly JF, Strates BS, Lippiello L: Treatment of 21. Moeller ME, Thomas RJ: Treatment of nonunion in fractures of nonunion by percutaneous injection of bone marrow and deminer- long bones. Clin Orthop 138:141–153, 1979. alized bone matrix: An experimental study in dogs. Clin Orthop 22. Moxham JP, Kibblewhite DJ, Dvorak M, et al: TGF-beta 1 forms 268:294–302, 1991. functionally normal bone in a segmental sheep tibial diaphyseal 30. Toombs JP, Wallace LJ: Evaluation of autogeneic and allogeneic defect. J Otolaryngol 25:388–392, 1996. cortical chip grafting in a feline tibial nonunion model. Am J Vet 23. Niyibizi C, Baltzer AWA, Lattermann C, et al: Potential role for Res 46:519–528, 1985. gene therapy in the augmentation of fracture healing. Clin Orthop 31. Vogel J, Hopf C, Eysel P, Rompe JD: Application of extracorporal 355(Suppl):S148–S153, 1998. shock-waves in the treatment of pseudarthrosis of the lower extrem- 24. Oni OO: A nonunion model of the rabbit tibial diaphysis. Injury ity. Arch Orthop Trauma Surg 116:480–483, 1997. 26:619–622, 1995. 32. Yasko AW, Lane JM, Fellinger EJ, et al: The healing of segmental 25. Praemer A, Furner S, Rice D: Musculoskeletal Conditions in the bone defects, induced by recombinant human bone morphogenetic United States. Park Ridge, IL, American Academy of Orthopaedic protein (rhBMP-2): A radiographic, histological, and biomechanical Surgeons 1992. study in rats. J Bone Joint Surg 74A:659–670, 1992. 26. Schmitz JP, Hollinger JO: The critical size defect as an experimen- 33. Zegzula HD, Buck DC, Brekke J, Wozney JM, Hollinger JO: Bone tal model for craniomandibulofacial nonunions. Clin Orthop formation with use of rhBMP-2. J Bone Joint Surg 79A:1778–1790, 205:299–308, 1986. 1997.
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