| Bone defect resulting from various trauma infection and tumor resection is a difficult problem in orthopaedics field. Autologous bone graft is considered to be the gold standard of bone graft material, but there is only a limited quantity of bone available to harvest. Moreover, harvesting of autologous bone grafts has significant morbidity, including persistent donor site pain and local infection. Allogenic bone graft is another alternative to the autograft. However, there is risk of possible rejection and potential pathogens transmission. From 1990s, investigators have paid more attention to exploit and research the tissue engineering bone. In general, the seeding cells cultured in vitro are seeded in native or synthetic extracellular matrix which could be biocompatible and biodegradable, and the complex of seeding cells with scaffold was implanted in the body for expecting cell events such as adhesion, proliferation, differentiation and so on. At present, the focus of bone tissue engineering has been principally in cells, biomaterials and repairing of the bone defect.Mesenchymal stem cells(MSCs) are remarkable, having the capacity to self-renew and differentiate along multiple lineages including osteogenesis and to contribute to ongoing tissue maintenance and regeneration after injury in the adult, which are easily to be obtained and expanded. MSCs are thus ideal cellular vehicles for the transduction and expression of the therapeutic gene. For these advantandges, MSCs have become the extensive seeding cells in bone tissue engineering. Compared with periosteum-derived osteoblasts, manipulation of isolated bone marrow MSCs is conveniently and little injury. Although various experiments have indicated that lot of growth factors such as bone morphogenetic protein-2 and osteogenic protein-1 could promote the osteogenic differentiation, the exact mechanism of osteogenic differentiation directly from MSCs has not been well clarity. To make clear this mechanism will benefit to construct the engineering bone.The expression of Cbfa1, the osteoblasts-specific transcription factor, only be detected in osteoblasts and bone during adulthood and the condensation area of mesenchymal cellsduring embryogenesis. Cbfal gene knockout mice have been proved that Cbfal played a crucial role in osteoblast differentiation and bone formation. It was not clearly that MSCs can be differentiated into osteoblasts directly modified by Cbfal.Due to the reasons mentioned above and on the base of the feature of MSCs, we transfected the Cbfal recombinant adenovirus into MSCs and then induced the cells differentiate into osteoblasts. The genetically modified MSCs and acellular bone extracellular matrix, which prepared the bones of inbred Banna mini-pig, were compounded and cultured in a flow perfusion bioreactor. At last, the constructed engineering bones were grafted into the rabbit radius defects. The main results are as follows.1. The Cbfal recombinant adenovirus and control adenovirus pAdEasy- GFP-gal were constructed using the pAdEasy system. The favorite gene was detected by fluorescent microscope and polymerase chain reaction. The liter of Cbfal adenovirus was 1.6 1012. The adenovirus can mediate the expression of Cbfal in NIH3T3 cells by infection study.2. The Cbfal adenovirus were transfected into MSCs and the expression of Cbfal was veritified by Western blot, immunohistochemical stain and fluorescent microscope. The osteoblast marker genes, such as ALP, OCN, OPN, type I collagen, and series of others gene including BMP-2, MyoD, PPAR Y 2, OPG, were detected by semiquantify RT-PCR. Moreover, ALP activity and osteocalcin production were measured by the 4-nitrophenylphosphate method and radioimmunoassay, respectively. The results showed that all but type I collagen genes were up-regulated. ALP activity and osteocalcin production were markable increased. On the contrast, the genes represent differentiation into adipose or muscle were down-regulated. All of these indicated that MSCs were induced to differentiate into osteo... |
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