骨髓间充质干细胞与不同基质修饰的纳米晶胶原基骨体外生物相容性研究

研究大鼠骨髓间充质干细胞(MSCs)诱导培养后与不同基质修饰的纳米晶胶原基骨(nanoHydroxyapatite/collagen,nHAC)的生物相容性,为骨组织工程提供一种新型复合支架材料。SD大鼠MSCs经成骨诱导培养、扩增,进行成骨细胞表征后,种植与支架材料体外复合培养。实验分为4组:实验组A,纤维蛋白(FB)和纤维连接蛋白(FN)修饰的纳米晶胶原基骨((FB FN)-nHAC);实验组B,纤维蛋白修饰的纳米晶胶原基骨(FB-nHAC);实验组C,纤维连接蛋白修饰的纳米晶胶原基骨(FN-nHAC);对照组D,单纯的纳米晶胶原基骨(nHAC)。通过检测支架材料的细胞黏附率、不同时间点(3、7、10、14d)支架材料中细胞数、碱性磷酸酶活性以及扫描电镜观察细胞在材料上的生长状况,比较分析不同支架材料与细胞生物相容性差异。大鼠MSCs经诱导培养14d后,碱性磷酸酶细胞化学染色、I型胶原免疫荧光染色及矿化沉积茜素红染色均为阳性;细胞与支架材料黏附率A组最高为74.4%;支架材料中细胞数量均随培养时间延长而增长,且A组细胞数增加较快,与相同时相点其他各组材料中细胞数差异有显著性(P<0.05);各时相点细胞碱性磷酸酶活性表达A组最高,差异亦有显著性(P<0.05)。电镜观察发现4组材料上均有细胞生长,但A组的细胞生长状况明显好于其他组。大鼠MSCs经成骨诱导培养,可表达成骨细胞表型,(FB FN)-nHAC在体外实验中表现出与细胞优良的生物相容性,可作为较理想的新型复合支架应用于骨组织工程。

In vitro Biocompatibility between Bone Marrow Mesenchymal Stem Cells and Nanohydroxyapatite/collagen Modified by Different Matrixes

The aims of this study are to investigate the in vitro biocompatibility between bone marrow mesenchymal stem cells (MSCs) that have been induced and nanohydroxyapatite/collagen (nHAC) modified by different matrixes, and to explore a new scaffold for bone tissue engineering. MSCs obtained from SD rat bone marrow were induced and proliferated. After their osteoblastic phenotype was demonstrated, MSCs were seeded onto the nHAC scaffolds modified by fibrin (FB) and fibronectin(FN) (experiment group A), FB(experiment group B) and FN(experiment group C) as well as common nHAC(control group D). The adhesive rates were calculated. At different time points (3, 7, 10,14d), the cells on the scaffolds was counted and alkaline phosphatase (ALP) activity was detected. The cell growth on scaffolds were also observed by scanning electron micrographe (SEM).The differences among groups were compared and analyzed. The differentiation of MSCs to osteoblastic phenotype was demonstrated by the positive stainings of alkaline phosphatase (ALP), collagen type I and mineralized node. The adhesive rates of group A was 74.4%, higher than those of any other groups. The cell numbers in the scaffolds increased with cultivating time for the four groups, and the speed of group A was the fastest. At the same time point compared group A with other groups, the difference in the cell number had statistical significance (P < 0.05). The expression of the ALP in group A was the highest at every time point (P < 0.05). Cells could be observed on every scaffold by SEM, and the cells in group A grew obviously better than those in other groups. MSCs can be induced to express osteoblastic phenotype. (FB FN)-nHAC has good biocompatibility to the cells, which showed the potential of novel scaffold for bone tissue engineering.