Abstract
Critical size defects of bone and delayed fracture healing due to metabolic disorders are still problems in orthopaedic surgery. Adenoviral vectors encoding bone morphogenetic protein-2 (Ad.BMP-2) have been used to stimulate bone formation in small animals. The present study evaluated the use of direct adenoviral gene transfer for inducing bone formation in a large animal. Standardized iliac crest defects were created surgically on both sides of the pelvic bone of white mountain sheep. The efficiency of gene transfer was evaluated using recombinant adenoviruses carrying the cDNA for luciferase. High levels of transgene expression, restricted to the site of injection, were found for the 1st week. Transgene expression then fell considerably, but could still be detected for up to 5 weeks. To investigate the effect on bone healing, Ad.BMP-2 (1011 particles in 200 μl saline) was unilaterally injected into iliac crest defects and into tibial osteotomies. The contralateral defects remained untreated to evaluate possible systemic effects. The controls were treated with saline solution. Bone formation within the defect, assessed by micro-computed tomography (CT) measurement at 8 weeks, and callus formation after osteotomy were significantly reduced following direct application of Ad.BMP-2. The retardation compared to untreated control animals was additionally found at the contralateral iliac crest indicating a systemic inhibitory effect. Histological analysis confirmed the CT measurement and showed an increased number of inflammatory cells within both defects. Antibodies against the adenovirus and the transgene product were detected in all treated animals. These data show a systemic retardation of bone formation following a single local injection of Ad.BMP-2 in sheep. This finding stands in contrast to the data obtained from small animal models. Further studies are needed to determine the contribution of the immune response to these results, and whether a lower dose of Ad.BMP-2 would be advantageous.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
References
Sudkamp NP, Haas NP, Sinnig M, Sottmann G, Tscherne H . Incidence of pseudarthroses in open fractures: analysis of 948 open fractures. Aktuelle Traumatol 1993; 23: 59–67.
Arrington ED, Smith WJ, Chambers HG, Bucknell AL, Davino NA . Complications of iliac crest bone graft harvesting. Clin Orthop 1996; 329: 300–309.
Banwart JC, Asher MA, Hassanein RS . Iliac crest bone graft harvest donor site morbidity. A statistical evaluation. Spine 1995; 20: 1055–1060.
Summers BN, Eisenstein SM . Donor site pain from the ilium. A complication of lumbar spine fusion. J Bone Joint Surg Br 1989; 71: 677–680.
Urist MR . Bone: formation by autoinduction. Science 1965; 150: 893–899.
Reddi AH . Bone morphogenetic proteins: from basic science to clinical applications. J Bone Joint Surg Am 2001; 83-A Suppl 1 (Part 1): S1–S6.
Schmitt JM, Hwang K, Winn SR, Hollinger JO . Bone morphogenetic proteins: an update on basic biology and clinical relevance. J Orthop Res 1999; 17: 269–278.
Friedlaender GE, Perry CR, Cole JD, Cook SD, Cierny G, Muschler GF et al. Osteogenic protein-1 (bone morphogenetic protein-7) in the treatment of tibial nonunions. J Bone Joint Surg Am 2001; 83-A Suppl 1 (Part 2): S151–S158.
Govender S, Csimma C, Genant HK, Valentin-Opran A, Amit Y, Arbel R et al. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am 2002; 84-A: 2123–2134.
Valentin-Opran A, Wozney J, Csimma C, Lilly L, Riedel GE . Clinical evaluation of recombinant human bone morphogenetic protein-2. Clin Orthop 2002; 395: 110–120.
Baltzer AW, Lattermann C, Whalen JD, Ghivizzani S, Wooley P, Krauspe R et al. Potential role of direct adenoviral gene transfer in enhancing fracture repair. Clin Orthop 2000; 379 (Suppl): S120–S125.
Egermann M, Schneider E, Evans CH, Baltzer AW . The potential of gene therapy for fracture healing in osteoporosis. Osteoporos Int 2005; 16 (Suppl 2): S120–S128.
Baltzer AW, Lattermann C, Whalen JD, Braunstein S, Robbins PD, Evans CH . A gene therapy approach to accelerating bone healing. Evaluation of gene expression in a New Zealand white rabbit model. Knee Surg Sports Traumatol Arthrosc 1999; 7: 197–202.
Crystal RG . Transfer of genes to humans: early lessons and obstacles to success. Science 1995; 270: 404–410.
Wilson JM . Adenoviruses as gene-delivery vehicles. N Engl J Med 1996; 334: 1185–1187.
Yang Y, Li Q, Ertl HC, Wilson JM . Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol 1995; 69: 2004–2015.
Harvey BG, Hackett NR, El Sawy T, Rosengart TK, Hirschowitz EA, Lieberman MD et al. Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs. J Virol 1999; 73: 6729–6742.
Yang Y, Jooss KU, Su Q, Ertl HC, Wilson JM . Immune responses to viral antigens versus transgene product in the elimination of recombinant adenovirus-infected hepatocytes in vivo. Gene Therapy 1996; 3: 137–144.
Chen P, Kovesdi I, Bruder JT . Effective repeat administration with adenovirus vectors to the muscle. Gene Therapy 2000; 7: 587–595.
Baltzer AW, Lattermann C, Whalen JD, Wooley P, Weiss K, Grimm M et al. Genetic enhancement of fracture repair: healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene. Gene Therapy 2000; 7: 734–739.
Bertone AL, Pittman DD, Bouxsein ML, Li J, Clancy B, Seeherman HJ . Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit ulnar osteotomy model. J Orthop Res 2004; 22: 1261–1270.
Betz OB, Betz VM, Nazarian A, Pilapil CG, Vrahas MS, Bouxsein ML et al. Direct percutaneous gene delivery to enhance healing of segmental bone defects. J Bone Joint Surg Am 2006; 88: 355–365.
Martinek V, Latterman C, Usas A, Abramowitch S, Woo SL, Fu FH et al. Enhancement of tendon-bone integration of anterior cruciate ligament grafts with bone morphogenetic protein-2 gene transfer: a histological and biomechanical study. J Bone Joint Surg Am 2002; 84-A: 1123–1131.
Food and Drug Administration. Guidelines for Preclinical and Clinical Evaluation of Agents Used in the Prevention or Treatment of Postmenopausal Osteoporosis. FDA Division of Metablism and Endocrine Drug Products: Washington, DC, 1994.
Michou AI, Santoro L, Christ M, Julliard V, Pavirani A, Mehtali M . Adenovirus-mediated gene transfer: influence of transgene, mouse strain and type of immune response on persistence of transgene expression. Gene Therapy 1997; 4: 473–482.
Baltzer AW, Whalen JD, Stefanovic-Racic M, Ziran B, Robbins PD, Evans CH . Adenoviral transduction of human osteoblastic cell cultures: a new perspective for gene therapy of bone diseases. Acta Orthop Scand 1999; 70: 419–424.
Krebsbach PH, Gu K, Franceschi RT, Rutherford RB . Gene therapy-directed osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo. Hum Gene Ther 2000; 11: 1201–1210.
Lee JY, Peng H, Usas A, Musgrave D, Cummins J, Pelinkovic D et al. Enhancement of bone healing based on ex vivo gene therapy using human muscle-derived cells expressing bone morphogenetic protein 2. Hum Gene Ther 2002; 13: 1201–1211.
Bishop NE, Schneider E, Ito K . An experimental two degrees-of-freedom actuated external fixator for in vivo investigation of fracture healing. Med Eng Phys 2003; 25: 335–340.
Lill CA, Hesseln J, Schlegel U, Eckhardt C, Goldhahn J, Schneider E . Biomechanical evaluation of healing in a non-critical defect in a large animal model of osteoporosis. J Orthop Res 2003; 21: 836–842.
Ruter A, Mayr E . Pseudarthrosis. Chirurg 1999; 70: 1239–1245.
Southwood LL, Frisbie DD, Kawcak CE, Ghivizzani SC, Evans CH, McIlwraith CW . Evaluation of Ad-BMP-2 for enhancing fracture healing in an infected defect fracture rabbit model. J Orthop Res 2004; 22: 66–72.
Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM . Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci USA 1994; 91: 4407–4411.
Yang Y, Ertl HC, Wilson JM . MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity 1994; 1: 433–442.
Alden TD, Pittman DD, Hankins GR, Beres EJ, Engh JA, Das S et al. In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector. Hum Gene Ther 1999; 10: 2245–2253.
Lehmkuhl HD, Cutlip RC, Brogden KA . Seroepidemiologic survey for adenovirus infection in lambs. Am J Vet Res 1993; 54: 1277–1279.
Viljanen VV, Gao TJ, Lindholm TC, Lindholm TS, Kommonen B . Xenogeneic moose (Alces alces) bone morphogenetic protein (mBMP)-induced repair of critical-size skull defects in sheep. Int J Oral Maxillofac Surg 1996; 25: 217–222.
Kandziora F, Pflugmacher R, Scholz M, Knispel C, Hiller T, Schollmeier G et al. Comparison of BMP-2 and combined IGF-I/TGF-ss1 application in a sheep cervical spine fusion model. Eur Spine J 2002; 11: 482–493.
Kirker-Head CA, Gerhart TN, Armstrong R, Schelling SH, Carmel LA . Healing bone using recombinant human bone morphogenetic protein 2 and copolymer. Clin Orthop 1998; 349: 205–217.
Magin MN, Delling G . Improved lumbar vertebral interbody fusion using rhOP-1: a comparison of autogenous bone graft, bovine hydroxylapatite (Bio-Oss), and BMP-7 (rhOP-1) in sheep. Spine 2001; 26: 469–478.
Sandhu HS, Toth JM, Diwan AD, Seim III HB, Kanim LE, Kabo JM et al. Histologic evaluation of the efficacy of rhBMP-2 compared with autograft bone in sheep spinal anterior interbody fusion. Spine 2002; 27: 567–575.
Gao TJ, Lindholm TS, Kommonen B, Ragni P, Paronzini A, Lindholm TC et al. The use of a coral composite implant containing bone morphogenetic protein to repair a segmental tibial defect in sheep. Int Orthop 1997; 21: 194–200.
Lill CA, Gerlach UV, Eckhardt C, Goldhahn J, Schneider E . Bone changes due to glucocorticoid application in an ovariectomized animal model for fracture treatment in osteoporosis. Osteoporos Int 2002; 13: 407–414.
Acknowledgements
The study was supported by NIH grant RO1 AR 0520243 as well as the AO-Research-Institute which is partly funded by royalties from licenses granted to Synthes-Stratec and Mathys Medical LTD. We receive nothing of value, and wish to thank Ronald Wieling and Heide Kloeppel for assistance with surgery and animal care, and Mauro Alini for cell culture support.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Egermann, M., Lill, C., Griesbeck, K. et al. Effect of BMP-2 gene transfer on bone healing in sheep. Gene Ther 13, 1290–1299 (2006). https://doi.org/10.1038/sj.gt.3302785
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.gt.3302785
Keywords
This article is cited by
-
Enhanced Bone Regeneration by Bone Morphogenetic Protein-2 after Pretreatment with Low-Intensity Pulsed Ultrasound in Distraction Osteogenesis
Tissue Engineering and Regenerative Medicine (2022)
-
Current Trends in Viral Gene Therapy for Human Orthopaedic Regenerative Medicine
Tissue Engineering and Regenerative Medicine (2019)
-
Gene therapy approaches to regenerating the musculoskeletal system
Nature Reviews Rheumatology (2015)
-
Bone regenerative medicine: classic options, novel strategies, and future directions
Journal of Orthopaedic Surgery and Research (2014)
-
Using genes to facilitate the endogenous repair and regeneration of orthopaedic tissues
International Orthopaedics (2014)