Hydroxyapatite Fiber Material With BMP-2 Gene Induces Ectopic Bone Formation Mitsumasa Oda, Shinji Kuroda, Hisatomo Kondo, Shohei Kasugai Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo 113-8549, Japan Received 8 February 2008; revised 9 July 2008; accepted 8 August 2008 Published online 4 November 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31258 Abstract: Collagen containing bone morphogenetic protein-2 (BMP-2) expression vector, which is called ‘‘gene-activated matrix,’’ promotes bone regeneration when transplanted to the bone defect. We speculated that hydroxyapatite fiber (HF) would be an ideal matrix for ‘‘gene- activated matrix’’ especially for bone regeneration, because it is oseteoconductive and has high affinity to DNA. The purpose of this study is to clarify whether HF containing BMP-2 expression vector induces ectopic bone formation. We prepared HF containing 0, 10, 50, and 100 lg BMP-2 expression vector. Wistar male rats (8 weeks) were used and each rat received two HF implants in the left and right dorsal muscle. The rats were sacrificed 4, 8, and 12 weeks after the operation, and implants were analyzed radiographically by softex, dual-energy X-ray absorptiometry, and they were histologically examined. At 4 weeks, HF containing 50 or 100 lg BMP-2 expression vector showed high bone mineral contents and large radiopaque volume compared to the other implants. At 8 and 12 weeks, HF containing 50 lg BMP-2 expression vector exerted the highest values in the radiographic analyses. Bonelike tissue was histologically observed in HF containing 50 and 100 lg BMP-2 expression vector groups but not detected in the other implants. The present results suggest that HF is potential as a matrix for ‘‘gene-activated matrix’’ for bone tissue engineering. ' 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 90B: 101–109, 2009 Keywords: bone morphogenetic protein 2; gene therapy; hydroxyapatite fiber; ectopic bone formation; calcium phosphate INTRODUCTION Although autologous bone graft is recognized as a ‘‘gold standard’’ for bone augmentation, limitation of the harvest- able bone volume and morbidity of the donor site are the problems. Thus, development of a simple and effective method for bone augmentation is required in orthopedic and dental fields. Bone morphogenetic protein-2 (BMP-2), a member of the BMP family, which is a potent osseoin- ductive factor, 1 induces osteogenic differentiation of mes- enchymal cells, and local administration of recombinant BMP-2 protein in vivo induces orthotopic and ectopic bone formation. 2,3 Although clinical application of recombinant human BMP-2 or BMP-7 have been expected for bone aug- mentation, high cost in clinical applications is a problem, because relatively high doses of these recombinant proteins are necessary. One of the main reasons for high dose of these recombinant proteins is short half life of the proteins in vivo. 4 On the other hand, bone regeneration with BMP gene transfer has been reported. 5–16 When BMP genes are trans- ferred to some cells, these cells produce BMP proteins sub- sequently stimulating bone regeneration. There are several gene transfer techniques used for bone regeneration, which are generally categorized in ex vivo 5,8,9,11–13,16 and in vivo 6,7,10,14,15,17–19 gene transfers with viral 5,8,9,11,13,15–17,19 or nonviral 6,7,10,12,14,18,19 transfection. ‘‘In vivo gene trans- fer with nonviral transfection’’ is simple and safe compared to other gene transfers, although its gene transfection effi- ciency is low. Bonadio et al. have developed a unique gene transfer method. 6,7,10 They used a naked plasmid vector encoding a protein stimulating osteogenesis, combined it with collagen, and transplanted that complex to bone defects in rats and dogs. They demonstrated that this com- bination stimulated bone regeneration in bone defects and termed this formula as ‘‘gene-activated matrix (GAM).’’ However, the efficiency of gene transfer of the original GAM system is still low, because a large amount of naked plasmids is required: for instance, 1 mg plasmid DNA for Correspondence to: M. Oda (e-mail: mrmasa.irm@tmd.ac.jp) Contract grant sponsor: Ministry of Education and Science in Japan; Contract grant numbers: 15659475, 16390579 Contract grant sponsor: Tokyo Medical and Dental University (Grant for Center of Excellence Program for Frontier Research on Molecular Destruction and Recon- struction of Tooth and Bone) ' 2008 Wiley Periodicals, Inc. 101