Experimental Ex Vivo Gene Therapy in Autologous Critical- Size Craniofacial Bone Regeneration Sophia Chia-Ning Chang, M.D., Ph.D., Fu Chan Wei, M.D., Huoli Chuang, M.D., Yu Ray Chen, M.D., Jan Kan Chen, Ph.D., Kuei C. Lee, B.S., Philip K. T. Chen, M.D., Ching Lung Tai, M.Eng., and Jueren Lou, M.D. Taipei and Taoyuan, Taiwan; and St. Louis, Mo. In therapeutic bone repairs, autologous bone grafts, conventional or vascularized allografts, and biocompat- ible artificial bone substitutes all have their shortcomings. The bone formed from peptides [recombinant human bone morphogenetic proteins (BMPs)], demineralized bone powder, or a combination of both is small in size. Tissue engineering may be an alternative for cranial bone repair. In this study, the authors developed an animal model to test the hypothesis that replication-defective, adenovirus-mediated human BMP-2 gene transfer to bone marrow stromal cells enhances the autologous bone for- mation for repairing a critical-size craniofacial defect. The mesenchymal stromal cells of miniature swine were sep- arated from the iliac crest aspirate and expanded in mono- layer culture 1 month before implantation. The cultured mesenchymal stromal cells were infected with recombi- nant, replication-defective human adenovirus BMP-2,7 days before implantation. Bilateral 2  5-cm 2 cranial de- fects were created, leaving no osteogenic periosteum and dura behind. Mesenchymal stromal cells at 5  10 7 /ml were mixed with collagen type I to form mesenchymal stromal cell/polymer constructs. Mesenchymal stromal cells used for the control site were infected with adeno- virus -Gal under the same conditions. After 6 weeks and 3 months, 10 miniature swine were euthanized and the cranium repair was examined. Near-complete repair of the critical-size cranial defect by tissue-engineered mes- enchymal stromal cell/collagen type I construct was ob- served. The new bone formation area (in square centi- meters) measured by three-dimensional computed tomography demonstrated that the improvement from 6 weeks to 3 months was significantly greater on the exper- imental side than on the control side (2.15 cm 2 versus 0.54 cm 2 , p  0.001) and significantly greater at 3 months than at 6 weeks (2.13 cm 2 versus 0.52 cm 2 , p  0.001). The difference between the experimental and control groups was significant at 3 months (mean difference, 2.13 cm 2 ; p  0.001). The maximal compressive strength of the new bone was similar to that of the normal cranial bone when evaluated by biomechanical testing (cranium bone versus tissue-engineered bone, 88.646  5.121 MPa versus 80.536  19.302 MPa; p = 0.227). Adenovirus was absent from all constructs by immunochemical staining at 6 weeks and 3 months after implantation. The successful repair of cra- nial defects in this experiment demonstrates the efficacy of the integration of the autologous stem cell concept, gene medicine, and polymers in producing tissue-engi- neered bone. (Plast. Reconstr. Surg. 112: 1841, 2003.) More than 1 million surgical procedures per- formed in the United States each year involve bone and cartilage replacement. 1 Autogenous vascularized bone grafts can be harvested from temporal, scapular, radial, rib, iliac, fibular, and metatarsal bones but may cause sequelae in donor sites. Allografts are limited in usage because of immunologic rejections, transmis- sion of infectious diseases, premature resorp- tion, and donor shortage. The new bone gen- erated from peptides [recombinant human bone morphogenetic protein (BMP)], 2 demin- eralized bone powder, 3 or a combination of both 4 is small in size. Biocompatible bone sub- stitutes 5 are also not suitable for critical-size defects. In recent years, the development of tissue-engineering techniques has enabled us to create functional tissues with biocompatible, biodegradable polymers seeded with living cells. 1 It has been proved that osteoblasts are de- From the Departments of Plastic Surgery and Neurosurgery, Chang-Gung Memorial Hospital; Department of Physiology, Chang Gung University; Department of Orthopedic Surgery, Biomechanics Center; and Department of Orthopedic Surgery, Washington University. Received for publication June 5, 2002; revised May 19, 2003. Presented in part at the Ninth International Society of Craniofacial Surgery, in Visby, Gotland, Sweden, June 18, 2001; the Inaugural Congress of the World Society for Reconstructive Microsurgery, in Taipei, Taiwan, October 30 to November 3, 2001; and the 47th Annual Meeting of Plastic Surgery Research Council, in Boston, Massachusetts, April 20, 2002. Dr. Sophia Chia-Ning Chang received the Young Investigator Award at the First Biennial Meeting of the European Tissue Engineering Society, in Freiburg, Germany, November 8, 2001. DOI: 10.1097/01.PRS.0000091168.73462.1A 1841