应用纳米晶胶原基骨体外构建组织工程骨  

In vitro construction of tissue-engineered bone using human mesenchymal stem cells and nano-hydroxyapatite/collagen composite scaffold

作　　者：夏青[1] 沈铁城[1] 黄永辉[1] 许文荣[2] 

机构地区：[1]江苏大学附属医院骨科,江苏省镇江市212001 [2]江苏大学医学技术学院,江苏省镇江市212001

出　　处：《中国组织工程研究与临床康复》2007年第23期4492-4495,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:应用组织工程技术,建立体外诱导人骨髓间充质干细胞,与纳米晶羟基磷灰石/胶原骨共培养的人工骨模型,探求细胞与纳米晶胶原基骨结合的最佳模式。方法:实验于2005-09/2006-09在江苏大学医学技术学院中心实验室完成。①实验材料:纳米晶胶原基骨材料由清华大学材料系崔福斋等人研制。骨髓取自骨科取髂骨患者(男性,30岁,患者知情同意)。②实验干预:全骨髓法体外培养骨髓间充质干细胞并扩增,应用成骨诱导剂诱导向成骨细胞表型转化。取纳米晶胶原基骨材料经60Co照射灭菌,无水乙醇疏水化,三蒸水洗去乙醇等处理制备支架材料。细胞与支架材料复合培养。③实验分组:骨髓间充质干细胞培养至第3代后分为两组:复合材料后加成骨诱导剂组:骨髓间充质干细胞先与纳米晶胶原基骨复合培养3d后再加入成骨诱导剂;成骨细胞复合材料组:骨髓间充质干细胞在诱导成骨细胞后与纳米晶胶原基骨复合培养。④实验评估:记录第3代骨髓间充质干细胞生长曲线。将诱导后细胞分别进行碱性磷酸酶染色、Von Kossa染色。扫描电镜比较两组复合物经体外孵育2周后,细胞在其中生长情况。结果:①细胞生长情况:倒置显微镜下观察原代培养的骨髓细胞增殖迅速,基本都呈形态均一成纤维样细胞,10～12d左右即可长满,并可稳定传代,传代细胞7～9d即可传代。诱导培养后的细胞呈现典型的成骨细胞形态和生物学特征。构建出骨髓间充质干细胞与骨组织共培养的模型。②体外复合后细胞生长及基质分泌情况:细胞可在纳米晶胶原基骨内表面良好贴壁,复合材料后加成骨诱导剂组,细胞贴附,但成骨细胞少见;成骨细胞复合材料组,细胞数量明显较前者多。复合培养8d,分布于支架材料上的细胞大量增殖、分泌细胞外基质。第14天,大量细胞在材料表面和孔隙中生长。细胞之间广泛存AIM： To establish a model for in vitro inducing human mesenchymal stem cells （hMSCs） differentiation using nano-hydroxyapatite ceramic/collagen （nHAC） scaffold, so as to explore the best method of integrating hMSCs into nHAC bone. METHODS： The experiment was carded out in the central laboratory of School of Medical Tech＇nology. Jiangsu University from September 2005 to September 2006. The hMSCs were harvested from the whole bone marrow of iliac bone of a male patient （30 years old） with informed consent, and amplified. Then they were induced and differentiated into the phenotype of osteoblast by the revulsant, nHAC material was radiated by ^60Co and water drained by absolute alcohol, then washed by distilled water for three times to remove the alcohol. The cells and scaffold material were co-cultured. The 3^rd generation MSCs were divided into two groups： Group A： The revulsant was added after 3 days of MSCs culture on nHAC; Group B： MSCs were induced into osteoblasts, and then combined with the scaffold. The growth curve of the third generation MSCs was drawn, and the induced cells were alkaline phosphatase stained and Von Kossa stained. After 2 weeks of culturing, cell viability in two groups were evaluated through scanning electron microscope. RESULTS：①By the microscope observation, the cultured primarily MSCs rapidly differentiated and proliferated into uniform fibroblast-like cells, and reached confluence and passaged stably on about the tenth and twelfth days. MSCs after induced culture displayed the traditional morphological and biological characteristics of osteoblasts, indicating the co-culture models of MSCs and bone tissue was constructed. ②MSCs could attach to the inner surface of nHAC after combination： In group A. the cells attached to the materials, but osteoblasts were rarely found, whereas more MSCs and osteoblasts were found in group B. Eight days after co-culture, the osteoblasts proliferated in the scaffold material, and secreted extracellular matrix. A plenty o

关 键 词：纳米晶胶原基骨 间质干细胞 组织工程 组织构建 

分 类 号：R318.08[医药卫生—生物医学工程]