| Objective: Bone marrow stromal cells (BMSCs) are commonly used in bone tissue engineering as osteogenic stem cells. The culture conditions that stimulating expansion and osteoblastic differentiation of BMSCs and the appropriate extracellular matrixes that composite with BMSCs to repair bone defects are the key fields of research in BMSCs. We choice basic fibroblast growth factor(bFGF) as bone anabolic substance to observe its effect on the proliferation and osteoblastic differentiation of rabbit BMSCs, and use bovine type 1 collagen matrix gel as extracellular matrix for the BMSCs expanded by bFGF to repair rabbit bone defects.Materials and Methods: The experiment is divided into three sections:Part I : Osteobalstic differentiation of rabbit bone marrow stromal cells cultured in experimental culture medium. Bone marrow were collected with an 18-gauge syringe needle from four rabbits tibia, and diluted by Liquid Hanks. After centrifugal separation by Ficoll's liquid, the nucleolar cell layers were colleted, and washed and cultured in RMPI1640 supplement with 20% fetal bovine serum (standard medium). Next, the passage cells were separated into two part, one is cultured in standard medium alone and the other cultured in the standard medium with the presence of dexamethasone(Dex 10nmol), ascrobic acid(AA 50ug/ml) and B -glycerophosphate(B GP 10mmol). Cell morphology, ALP activity, ability to form osteocalin (BGP) and the formation of mineralized nodules were observed and determined. Part II :The effect of bFGF on the proliferation andosteoblastic differentiation of bone marrow stromal cell. Bone marrow from three rabbits was collected and culture in the same way as mentioned above. The bFGF was added into the cultured medium with a varied density of 0, 0.1, 1.0, 10,100ng/ml. After 7 days' culture, the number of cfu-f were counted and cell proliferation (MTT), protein content were measured in 3 days and 6 days individually. Meanwhile ALP activity and the formation of mineralized nodules were detected.Part III: Repair of rabbit cranial defects using type I collagen matrix gels/BMSCs composition. BMSCs from 4 rabbit were cultured and expanded in vitro, mixed with type I collagen matrix gels. A full-thicked cranial defect with 1.5cm in diameter was created in 10 rabbit, without damage of duramater. Cranial defects were painted with collagen gels mixed with BMSCs of his own in four rabbits(group I ,n=4), four were painted with collagen gels alone(group II ,n=4), and two defects left were unpainted(group III,n=2). The animals were sacrificed in 4 or 8 weeks and new bone regeneration in the defects was investigated by gross specimen inspection, histological analysis and the proportion of dry weight and wet weight.Results:Part I : After 24 hours inoculation, adhered cells were detected. At 72 hours, the cultured cells were spindle-shaped or lozenge. 7 days later, discrete colonies of different sizes were formed and the cells appeared different shapes-squama, polygon, long shuttle, etc. with different amounts of long or short protuberances. Approximately 14 days later, the colonies amalgamated into one layer or multi-layered, and no contact inhibition was observed. ALP activity and BGP were higher in the experimental medium. Exeperiment groups showed strong positive effects in Von Kossa,whereas comparative group negative.Part II: At 7 days, the number of cfu-f of BMSCs showed that when bFGF was 0-10ng/ml, the number of cfu-f increased along with the increase of bFGF density. There was no distinct difference between 100ng/ml and Ing/ml. Cells protein content cultured for 6 days were higher than those cultured for 3 days in all specimen. It was founded that when bFGF density was 0-10ng/ml, the protein content increased along with the increase of bFGF density; when the bFGF density reached lOOng/ml, protein content was lower than that of in 10ng/ml and no distinct difference between lOOng/ml and Ing/ml. The cell proliferation by MTT showed that the value of cells cultured for 6 days were higher tha...
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