Callus Formation Enhanced By BMP-7 Gene Modified Autologous Bone Marrow MSCs In Rat Mandibular Distraction Osteogenesis

Distraction osteogenesis (DO) is a well established Ilizarov technique for correction of limb discrepancy, as well as many craniofacial deformities and bone defects. As a technique of endogenous bone tissue engineering, DO have many advantages for treatment of above diseases over traditional orthopaedic surgery. However, the long consolidation period the patients must face during this procedure and fibrous union or even nonunion of the bone callus under some special circumstances remain the major problems that impede its wide clinical application.Various attempts have been made to accelerate callus formation and shorten the consolidation phase. These include applications of low velocity ultrasound, electrical and electromagnetic stimulation, hyperbaric oxygen therapy, growth hormone and growth factors. Some of these approaches have yielded promising results, but none of them have achieved wide clinical application.Although many cytokines. growth factors, hormones and extracellular components are capable of regulating bone regeneration and remodeling during bone growth and repair, bone morphogenetic proteins (BMPs) are among themost potent osteogenic factors. BMPs belong to TGF-beta superfamily and their osteogenic function is mediated by special cell surface type I/II receptors and intracellular Smads signal cascades. BMPs regional gene therapy has been successfully used in tissue engineering for reconstruction of bone and cartilage defects and great progresses have been achieved. Recently, several studies demonstrated that there were special temporospatial expression of BMPs in the distraction gap, which suggested that BMPs ma)' play an important role in bone regeneration during DO as they do in bone and cartilage tissue engineering However, the physiological effects of BMPs may not be maximized by direct deliver of recombinant proteins because of rapid diffusion in vivo and relatively short-term bioactivity. Contradictory results were obtained in previous studies using this technique for callus stimulation. The use of BMPs regional gene therapy, however, has the potential to induce long-term, high-level BMP production at sites requiring bone formation. Therefore the application of BMPs gene therapy may overcome the shortages of direct recombinant protein deliver} and better results are expected.Mesenchymal stem cells (MSCs) are a heterogenous cell population which can differentiate into osteoblasts and chondroblasts both in vitro and in vivo. Because of their mutidifferentiation potential. MSCs has been widely utilized in tissue engineering and gene therapy for reconstruction of bone and cartilage. By using MSCs as a cell vector. BMPs ex vivo gene therapy can not only guarantee the stable expression of BMP for bone induction, but also offer enough osteogenic cells derived from MSCs for osteoregeneration. Therefore, MSCs is the best candidate cell vector for BMPs gene therapy to enhance callus formation during DO.Based on above knowledges, a new BMP-7 gene modified MSCs transplantation approach is used in this study to accelerate callus formation in rat mandibular distraction osteogenesis. To our knowledge, this is the first report to explore the efficiency of BMP ex vivo gene therapy on callus acceleration during DO. Rat mandibular distraction osteogenesis model was first established and recombinant plasmid pHGFP-BMP7 was constructed in vitro. Then pEGFP-BMP7 was transiently transfected into rat bone marrow MSCs and BMP-7 modified autologuous MSCs were thereafter introduced into rat mandibular distraction gap. The effect of BMP-7 gene therapy on callus formation were evaluated by radiographic, histological and histomorphometric examinations. The results of this study will provide valuable informations for clinical application of BMP-7 gene therapy to accelerate callus formation during distraction osteogenesis.The results of this study show that:1. Rat mandibular distraction osteogenesis was established by creating osteotomy line from middle of anterior ramus ridge to inferior border of the mandible and this was confirmed by both radiographic and histological examinations.2. Rat bone marrow MSCs was successfully isolated by density gradient centrifugation and they can differentiate into osteoblasts in vitro under bone supplements. The elevated expression of alkaline phosphotase (ALP) and the ability to mineralize extracellular matrix confirmed their osteoblast differentiation.3. Construct of recombinant eukaryote pHGFP-BMP7 by gene clone technique was successful and their transient transfection into rat bone marrow MSCsdemonstrated that the expression of BMP-7 lasted about 4 weeks, with the highest expression within 2 weeks.4. Transplantation of BMP-7 gene modified autologuous MSCs accelerated callus formation and shortened consolidation phase in rat mandibular distraction osteogenesis, and this was confirmed by radiographic, histomorphometric examinations, and detection of BMP-7 in vivo expression.5. The approach of BMP-7 gene modified autologuous MSCs transplantation utilized in this study offers a new strategy for callus acceleration in distracton osteogenesis, as well as for bone regeneration in other orthopaedic reconstruction areas.