Osteochondral Composite Graft Constructed By Growth Factor-induced Bone Marrow Mesenchymal Stem Cells And "Two-phase" Allogenic Bone Matrix Gelatin.

Objective:To explore the potential of the tessue engineered osteochondral composite graft which was formed with induced marrow derived stormal cell (MSC) and "two-phase" bone martix gelatin (BMG)Methods: This experiments was performed in the laboratory of the second affiliated hospital of Dalian Medical University from June,2008 to Jun ,2009.①The experimental animals were Japan big ear rabbits(2.5-3.5kg ) aged 2-3months after birth, of either sex,which were provided by Animal Cultivation Center in Dalian Medical University. MSC were isolated from the bone marrow in aseptic conditions and purified and cultured in vitro by density gradient centrifagation and adherent culture. After subculturing the third or forth generation MSC, were seperately induced to osteoblasts and lipoblasts by osteogenic inductor including dexamethasone,vitamin C,β-phosphoglycerol and to lipoblasts by chonrogenesis inductour including dexamethsone,Vitamin C ,TGF-β1,ITS-3146.②To scaffold "two-phase " allogeneic BMG (one side of wholly decalcidication and the other side of semi decalcidication)③The osteoblast precursor cells and the chondrocyte precursors cells were seeded into the "two-phase" allogeneic BMG to constructing tissue engineering osteochondral composite graft.Masson's trichrome staining,ALP staining and CollagenⅡimmunohistochemical staining,were used for identification.Results:①The features of MSC were as follows: the shape of primary cells was as shotspindled,grew slowly,the 7th daysand into plateau later. Passage cells grew slowly for 3 days after being passaged and went into long-growth during the 3rd and the 4th .The cell of feasiform shape became polygon,trigangular fater osteogenic inucation, The fears from mesen- chymal stem cells and become round or oval after chondrogenesis induction.②The construction of the allogeneic BMG is theri-dimension and the cavernous. It has the good quality of intensity and flexibility,the intensity of the side of semi decalcification is higher than that of the side of wholly decalcification have lower intensity than the other.③The osteochondral composite graft of osteogenic induction presented the red in the 21th day and the other presented the blue.The cells did not express calcium nodus in the 14th day after osteogenic induction and the cytoplasm of the most cells presented brownish-black in the 21th day after alkaline phasphatase calcium-cobalt staining. CollagenⅡstaining:the chondrogenesis CollagenⅡwas expressed mainly in cytoplasm of the chondrogensis in the 21th day .Conculsion:The two phase allogeneic BMG scaffold (one side of wholly decalcification and the other side of semi decalcification) can be used as the osteochondral composite graft for Tissue engineered to repair the defect articular cartilage.