| Tissue engineering is a newly rising science of co-development and combination of modern cell biology, biomaterial science and engineering. Bone tissue engineering is a major branch. Main problems are seed cells and its carriage. Mostly scholar' s inclination is applicate marrow stromal cells as seed cells, because MSCs have some superiorityes, such as abundant source, little damage and conveniently obtain. The mainly factor which restrict bone tissue engineering developing is scaffold. There isn' t any biomaterial completely fit for bone tissue engineering. So the trend of biomaterial study is improve its characteristics? structure or surface modification, complex biomaterial come into being because of demand. Ceramic bovine bone/hydrogel was used as scaffold in this experiment, hope to full play the superiorityes of ceramic bovine bone and hydrogenl, to construct a new scaffold of bone tissue engineering.Part I: To observe the biological compatibility of ceramic bovine bone and hydrogel and Marrow stromal cells.Frist Marrow stromal cells were separated, cultured in vitro and induced. Rat' s MSCs which cultured in vitro were induced byminerlization induce liquor (10%FBS, lOnmol Dexamethasone,450mg/L L-Vitamin C and lOmmol?桽odium Glycerophosphate DMEM culture medium ). The change of Cells characteristics were observed by inverted phase contrast microscope, the alkaline phosphatase level of cells, MTT chromometry, von-Kossa staining and immunohistochemistry stains. Results: 5 days later, some marrow stromal cells have been differentiated into osteoblast. 20 days later, many mineralized nodules formed.Second, we investigate the effect of compound pattern of ceramic bovine bone and hydrogel on bone marrow stromal cell attachment, proliferation and differentiation. Find the best way of constructing tissue-engineering bone. Then we observe the effect of ceramic bovine bone and hydrogel complex (CBB/HG), 3 -tricalcium phosphate and hydrogel complex(?TCP/HG) on bone marrow stromal cells (MSC) osteogenesis potentiality. Observed marrow stromal cells attachment and morphology by scanning electronic microscope, the proliferation of marrow stromal cells was evaluated by measuring the cell number, Alkaline phosphatase (ALP) activity was examined by histochemistry, type I collagen synthesis was examined by immunohistochemistry stains. Results: the compound pattern of CBB/HG/MSC is the best. CBB/HG, ?TCP /HG promote bone marrow stromal cell proliferation, different! -ation and extracellular matrix synthesis.Part ?: We investigate the osteogenesis of MSCs on ceramicbovine bone/hydrogel. Methods: inoculating MSCs on ceramic bovine5bone (lump and particle) /hydrogel and ?TCP/HG, drop rhBMP-2? TGF?P in complexes, then the complexes were embedded evenly in nude mice or autogenous subcutaneous. To observe the osteogenesis by HE Staining? modified Mallory' s trichrome staining, image analysis. Result: there are form new bones in nude mice or autogenous subcutaneous. Osteogenesis potentiality of associable apply rhBMP-2 and TGF-P is better than apply rhBMP-2 and TGF-P respectively.Tissue engineering technique was utilized to compose MSCs ?ceramic bovine bone (lump or particle) /hydrogel hybrid for repair of a Sprague 桪awley rat model of critical size calvarial defects. To observe the osteogenesis by HE StainingN modified Mallory' s trichrome staining, and image analysis. New bone was formed around scaffold. As bone tissue engineering scaffold, CBB/HG is better than ?TCP/HG after investigating structure of new bone , the area of new bone matrix and mature bone matrix.Through above experiments we can make a conclusion that marrow stromal cells can be induced in vitro into osteoblasts, it can be applied in bone engineering. Once MSCs are complxed with proper carriage such as CBB/HG, ?TCP/HG, the new complex indicates obvious bone formation effects and has an excellent application prospect in bone tissue engineering.
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