Dermal Fibroblast-Mediated BMP2 Therapy to Accelerate Bone Healing in an Equine Osteotomy Model Akikazu Ishihara, 1,2 Lisa J. Zekas, 3 Alan S. Litsky, 4 Steven E. Weisbrode, 2 Alicia L. Bertone 1,2,3 1 Comparative Orthopedic Research Laboratories, Department of Veterinary Clinical Sciences, The Ohio State University, 601 Tharp Street, Columbus, Ohio 43210, 2 Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, 3 Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, Ohio, 4 Orthopaedic BioMaterials Laboratory, Departments of Orthopaedics and Biomedical Engineering, The Ohio State University, Columbus, Ohio Received 27 February 2009; accepted 7 July 2009 Published online 23 September 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20978 ABSTRACT: This study evaluated healing of equine metacarpal/metatarsal osteotomies in response to percutaneous injection of autologous dermal fibroblasts (DFbs) genetically engineered to secrete bone morphogenetic protein-2 (BMP2) or demonstrate green fluorescent protein (GFP) gene expression administered 14 days after surgery. Radiographic assessment of bone formation indicated greater and earlier healing of bone defects treated with DFb with BMP2 gene augmentation. Quantitative computed tomography and biomechanical testing revealed greater mineralized callus and torsional strength of DFb-BMP2-treated bone defects. On the histologic evaluation, the bone defects with DFb-BMP2 implantation had greater formation of mature cartilage and bone nodules within the osteotomy gap and greater mineralization activity on osteotomy edges. Autologous DFbs were successfully isolated in high numbers by a skin biopsy, rapidly expanded without fastidious culture techniques, permissive to adenoviral vectors, and efficient at in vitro BMP2 protein production and BMP2-induced osteogenic differentiation. This study demonstrated an efficacy and feasibility of DFb-mediated BMP2 therapy to accelerate the healing of osteotomies. Skin cell-mediated BMP2 therapy may be considered as a potential treatment for various types of fractures and bone defects. ß 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:403– 411, 2010 Keywords: bone healing; BMP2; dermal fibroblast; adenovirus Transplantation of genetically engineered autologous cells, known as cell-mediated gene therapy or ex vivo gene therapy, has a great potential for the treatment of orthopedic disorders to accelerate bone healing and restore the loss of bony structure and function. 1 Cell- mediated delivery of an osteogenic gene has the advantages of controlling cell transduction efficiency compared to direct gene delivery, increasing a cellular- ity of the recipient injury site, permitting surgeon selection of cell type for implantation, and capitalizing on an autocrine and paracrine effect in which the transplanted cells not only differentiate into desired cell types but also secrete osteogenic growth factor into the treatment site. Pluripotent stem cells such as embryonic stem cells (ESCs) or adult mesenchymal stem cells (MSCs) have been extensively studied for cell-mediated therapy for osteogenic gene augumentation. 2,3 How- ever, use of ESC involves an ethical issue and potential risk of adverse immune reaction by allogenic implan- tation, and application of MSC requires an invasive biopsy procedure as the cells are commonly isolated from bone marrow or adipose tissue. In addition, stem cells have shown to require fastidious culture conditions during cell expansion in order to maintain their pluripotentiality and to grow to high numbers without morbidity. 4 While ESCs and MSCs are strong candidates for cell- mediated gene therapy, dermal fibroblasts (DFbs) have received scientific attention as a carrier cell because they can be easily isolated by a relatively less painful harvest technique with less risk of infection or donor site morbidity. In recent years, the plasticity and reprogram- ming capacity of DFbs have been shown by deriving DFb into induced pluripotent stem cells with defined genes such as Oct3/4, Sox2, Klf4, and c-Myc. 5 Such DFb- derived pluripotent cells have been differentiated into several functional phenotypes including neurons, car- diomyocytes, and hematopoeitic cells. 6–8 In addition, DFbs have been demonstrated to undergo osteogenic differentiation and convert to bone-forming cells with a single osteogenic gene transduction, including bone morphogenetic protein-2 (BMP2), 9,10 BMP7, 11,12 Runt- related gene-2, 10 and Lim mineralization protein-3. 13 Autologous implantation of DFb with osteogenic gene augumentation has also been shown to induce ectopic bone formation 12,13 and contribute to the bone healing in rodent models based on radiography, computed tomog- raphy, and histology. 10,11,13 These encouraging results support additional work to investigate skin cell-medi- ated gene delivery, improvement of mechanical strength of repaired bone, and efficacy for bone healing in larger animal species to demonstrate a clinical relevance of enhanced healing in people. The efficacy of molecular therapy in small animal experiments may not straight- forwardly transfer to large animal models. 14 Use of a large animal model will also evaluate the practicality in cost- and time-efficiency for cell transplantation using large numbers of genetically engineered cells in an immunocompetent adult animal. We have previously shown that the delayed percuta- neous delivery of adenoviral vectors encoding the BMP2 gene, as well as the BMP6 gene, into the equine osteotomy model induced an acceleration of bone healing and up-regulation of genes associated with JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2010 403 Additional Supporting Information may be found in the online version of this article. Correspondence to: Alicia L. Bertone (T: 614-292-6661; F: 614-292- 7599; E-mail: bertone.1@osu.edu) ß 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.