Series Studies On Repairing Bone Defect With Methods Of Tissue-engineering And BMP-2 Gene Transfection In Osteoporotic Rats

Bone defect due to tumor,trauma, or other disorders is one of the most common problem to Oral&Maxillary Surgeon,the morbidity of failed bone healing is significant,especially in those patients with osteoporosis.As such defects can not heal spontaneously ,several therapeutic approaches including autologous bone transplantation have been used,but the amounts of autogenous bone is finite,and sometimes may lead to severe complications just like infection, nonunion in donor and recipient site.With additional understanding of the molecular mechanism during the bone regeneration process,it now is possible to intervene in an attempt to augment bone healing.Bone morphogenetic protein-2(BMP-2) is a pleiotropic regulator during fracture repair, governing the key steps in bone induction cascade such as chemotaxis,mitosis,and differentiation of bone marrow stromal cells(BMSCs),stimulation of extracellular matrix synthesis, binding to matrix components,maintenance of phenotype.With the rapid development of bone tissue engineering and gene therapy,we have new approach to create bioartificial bone tissue.Human BMP-2(hBMP-2) gene is transferred into BMSCs,stimulates themselves todifferentiate into osteoblasts,the cells are seeded in macroporous biomaterials in vitro and then this newly grown bone tissue may be implanted in the defect site.While the cells expand in vivo,they express specific growth factors at high levels,stimulates themselves and other cells to differentiate into osteoblasts continuosly.This system is apparently very attractive for bone tissue engineering applications.Based on the adventage of tissue engineering in conjunction with gene therapy,the research has done by 4 serial experiments.1.Cultivation and induced differentiation of BMSCs of SD rats with type I osteoporosis in vitro.The modle of Type I osteoporosis was established,BMSCs were harvested and cultured for 14 days in a-MEM medium supplemented with osteogenic or adipogenic inducer respectively.Gomori and Von Kossa's staining demonstrated positive osteoblast phenotypes of alkaline phosphatase (ALP )and mineralized nods by osteogenic inducer, while Oil Red O staining identified MSCs treated with adipogenic medium resulted in adipocyte formation,and there is no significant difference in the percentage of positive stained cells between two groups.It showes BMSCs from SD rats with osteoporosis maintain their differentiation potential.2. Expression of BMP-2 during fracture healing of tibia in osteoporotic rats. Standardized secondary fracture healing in osteoporotic rats was induced using a modified technique.In a series of intervals of the fracture healing , radiographical,histological ,immuhistochemistry and RT-PCR analysis showed the expression of BMP-2 declined and delayed significantly in osteoporotic rats.lt indicated that BMP-2 plays an important role in boneregeneration.3. Transfection of BMSCs from osteoporotic rats with hBMP-2 gene in vitro. To optimize the rate of transfection mediated by cationic liposome , pEGFP-Nl plasmid was used. 3 days after transfection,positive result were confirmed by immunohistochemistry,in situ hybridization,and Western-blot essay in hBMP-2 gene transferred BMSCs.4. hBMP-2 gene modified tissue-engineered bone repairing mandibular bone defects in osteoporotic ratsAutogenous transfected or untransfected BMSCs were seeded in macroporous coral hydroxyapatite(CHA) or poly-DL- latic acid/ hydroxyapatite(PDLLA/HA) scaffolds respectively,then implanted in the defect site created in the ramus of mandible of osteoporotic rats.At 4-8 th weeks after implantation,histomorphornetric analysis revealed that the tissue-engineered bone seeded with rhBMP-2 gene transfected BMSCs has a mature bone matrix , but in untreated BMSCs group, just minor bone regeneration and some adipocyte at the margins of the defect could be seen.In this paper,after BMSCs were successfully cultured,osteoblast phenotype differentiation of BMSCs were induced by rhBMP-2 transfection in vitro and hBMP-2 gene modified tissue-engineered bone implanted in mandibular bone defects in osteoporotic rats promoting bone formation in vivo,indicate BMSCs-mediated rhBMP-2 gene therapy in conjunction with bone tissue engineering may allow for successful treatment of large bone defects inosteoporosis patients.