不同组织源性细胞作为大动物骨缺损修复研究用骨组织工程种子细胞的增殖及成骨分化比较

Proliferation of and differentiation of osteoblastic cells as bone tissue engineering seed cells for the repairing of segmental bone defect:comparison of different source

【目的】比较源自同一大动物个体不同组织的成骨性种子细胞的体外增殖及成骨分化能力,寻找和筛选更符合大动物骨缺损修复研究要求的种子细胞来源和分离方法。【方法】选用中国青山羊模型,参照常规方法,分离骨膜、骨髓、脂肪源性细胞进行体外培养,倒置显微镜观察,记录原代细胞的汇合生长时间;细胞传代后成骨诱导培养21 d,四甲基偶氮唑蓝(MTT)比色法检测细胞增殖活力;细胞内碱性磷酸酶(ALP)活性及细胞分泌骨桥蛋白测定、钙结节茜素红S染色检测细胞的成骨分化能力。【结果】骨膜、脂肪、骨髓源性原代细胞汇合生长时间分别为14、11和7 d;传代后MTT法检测显示细胞增殖能力从强到弱依次为骨髓、脂肪、骨膜源性细胞。细胞内ALP活性、细胞分泌骨桥蛋白及钙结节染色显示细胞进入成骨分化的先后顺序依次为骨膜、骨髓、脂肪源性细胞。【结论】在体外培养的各个阶段,来自同一大动物个体的不同组织源性细胞的增殖及成骨分化能力存在明显差异,其中骨髓及脂肪源性细胞更适于大动物骨缺损研究的需要。

【Objective】To compare proliferation and osteogenic differentiation of osteoblastic cells from three different large animal locations for tissue-engineered bone used to repair the segmental bone defect in large animal models.【Methods】Osteoblastic cells were derived from three different locations：periosteum,bone marrow and fat of goat.The morphology and confluent culture time of primary cultures were compared.The in vitro proliferation and osteogenic potential of subcultures was studied by culturing them in osteogenic media for 21 days and compared with respect to MTT detection,alkaline phosphatase（ALP） activity,amount of osteopontin secreted in the media,and nodule formation.【Results】The confluent culture time of primary cultures derived from periosteum,bone marrow,and fat was 14,11,and 7 days respectively.The expansion of the cells in subcultures was highest for bone marrow cells as compared with fat mesenchymal stem cells and periosteum cells.The onset of osteogenic differentiation in chronologicaol order was periosteum cells,bone marrow stem cells,and fat mesenchymal stem cells as revealled by alkaline phosphatase activity,amount of osteopontin secreted in the media,and nodule formation.【Conclusions】The proliferation and differentiation of osteoblastic cells derived from different locations of a single animal was extremely different at the same phase of in vitro culture.The present study demonstrates that bone marrow stem cells and fat mesenchymal stem cells are more satisfy to repair the segmental bone defect in large animal models.