骨髓基质干细胞与负载去细胞基质的壳聚糖/纳米羟基磷灰石复合微球联合培养的生物相容性研究  

Biocompatibility of bone marrow mesenchymal stem cells and composite microspheres of decellularized matrix loaded chitosan/nanohydroxyapatite in co-culture

作　　者：乔瑞红[1] 王东[1] 孙海钰[1] 高敏[1] 

机构地区：[1]山西医科大学第二医院骨科,太原030001

出　　处：《中华创伤骨科杂志》2014年第8期710-714,共5页Chinese Journal of Orthopaedic Trauma

基　　金：山西省专利推广项目（2012.3）

摘　　要：目的 探讨负载去细胞基质的壳聚糖（CS）/纳米羟基磷灰石（nHA）复合微球与骨髓基质干细胞（BMSCs）联合培养的生物相容性.方法 制备负载去细胞基质的CS/nHA复合微球,分离、提纯SD大鼠的BMSCs.实验分为4组（每组每个时间点6个样本）：空白组：单纯BMSCs培养,对照组1：去细胞基质与BMSCs共培养,对照组2：nHA与BMSCs共培养,实验组：负载去细胞基质的CS/nHA与BMSCs共培养.于培养14、21 d,采用茜素红染色观察BMSCs的成骨分化能力.于培养3、7、10、14、21 d,应用酶联免疫吸附测定试剂盒检测各组碱性磷酸酶（ALP）活性,应用逆转录聚合酶链式反应检测Ⅰ型胶原和骨桥蛋白的表达. 结果 负载去细胞基质的CS/nHA复合微球呈球形,粒径范围为3.52 ～ 29.65 μm.培养第21天,实验组内可见大量钙质沉积,茜素红染色呈阳性;对照组有少量钙质沉积;空白组钙质沉积更少.从培养第7天开始,实验组的ALP活性、Ⅰ型胶原表达量明显高于其他3组,对照组1、对照组2的ALP活性、Ⅰ型胶原表达量又明显高于空白组,差异均有统计学意义（P＜0.05）.从培养第10天开始,实验组的骨桥蛋白表达量明显高于其他3组,对照组1、对照组2的骨桥蛋白表达量又明显高于空白组,差异均有统计学意义（P＜0.05）.培养第21天,实验组和对照组ALP活性、Ⅰ型胶原及骨桥蛋白的表达均达到高峰. 结论 负载去细胞基质的CS/nHA复合微球与BMSCs具有良好的生物相容性.Objective To explore the biocompatibility of bone marrow mesenchymal stem cells （BMSCs） and composite microspheres of decellularized matrix loaded chitosan/nanohydroxyapatite （DMCS/nHA） in co-culture.Methods After preparation of composite microspheres of DMCS/nHA and isolation and purification of BMSCs from SD rats,the culture was conducted in 4 groups （6 specimens at each time point for each group）.BMSCs were cultured with the composite microspheres in the experimental group,with decellularized matrix in control group 1,with nHA in control group 2,and with nothing in the blank control group.The osteogenic differentiation capability of BMSCs in vitro was assessed by alizarin red staining at 14 and 21 days after culture.The activity of alkaline phosphate （ALP） was assessed by ELISA and the expression levels of type Ⅰ collagen and oesteopontin by RT-PCR at 3,7,10,14 and 21 days after culture.Results The composite microspheres were spherical in shape and the particle diameters ranged from 3.52 to 29.65 μm.At 21 days,the experimental group had a greater amount of calcified tubercle with positive alizarin red staining than both control groups and the blank control group.After 7 days,the expression levels of ALP and type Ⅰ collagen were significantly higher in the experimental group than in the other 3 groups,and in both control groups than in the blank control group （P 〈 0.05）.After 10 days,the expression of oesteopontin was significantly higher in the experimental group than in the other 3 groups,and in both control groups than in the blank control group （P 〈 0.05）.The expression of ALP,collagen Ⅰ and oesteopontin reached the peak values at 21 days in the experimental group and both control groups.Conclusion The composite microspheres of DMCS/nHA may have excellent biocompatibility with BMSCs.

关 键 词：间充质干细胞 骨髓 壳聚糖 羟基磷灰石类 材料试验 

分 类 号：R318.08[医药卫生—生物医学工程]