羧甲基壳聚糖钙与骨髓基质细胞生物相容性的初步实验

A preliminary experimental study on bone marrow stromal cell biocompatibility of carboxymethyl chitosan calcium

目的通过体外小鼠骨髓基质细胞培养,从细胞形态和细胞增殖方面评价新型生物材料羧甲基壳聚糖钙的细胞生物相容性。方法合成羧甲基壳聚糖钙,进行红外光谱分析,并用DMEM制备浸提液。分别用含羧甲基壳聚糖钙浸提成分的培养液(实验组)、DMEM培养液(阴性对照组)和含6.4g/L苯酚培养液(阳性对照组)培养骨髓基质细胞,通过倒置相差显微镜观察和MTT比色法评价细胞生长及增殖情况。结果红外光谱分析提示,羧甲基壳聚糖与钙离子发生了络合反应。镜下观察,细胞培养1、2、3d,实验组与阴性对照组细胞数量明显增加,细胞形态正常;阳性对照组细胞数量明显减少,细胞固缩甚至崩解。MTT比色法,实验组与阴性对照组吸光度(A)值间无统计学差异(P>0.05),实验组各时间点的毒性分级为0级;阳性对照组A值明显低于其他组(P<0.01),毒性分级为4级。结论羧甲基壳聚糖钙复合材料对小鼠骨髓基质细胞有良好细胞生物相容性,是一种可用于组织工程的新型生物材料。

Objective To evaluate the cellular biocompatibility of the carboxymethyl chitosan calcium （CCC）composite by observing the morphology and proliferation of murine bone marrow stromal cells（BMSCs） in vitro. Methods CCC was synthesized and then analyzed by an infrared spectrometer （IR）. CCC extraction prepared with DMEM （experimental group）,DMEM culture medium （negative control group） and 6.4g/L phenol culture medium （positive control group） were used for BMSCs culture. The morphology and proliferation of BMSCs were evaluated by inverted phase contrast microscopic observation and MTT assay. Results Infrared spectrum analysis indicated that CCC had been formed by complex reaction between carboxymethyl chitosan and calcium ions. On culture day 1,2 and 3,the cells disaggregated and the cell number noticeably decreased in the positive control group; however,the cell number in the experimental group and negative control group remarkably increased,with normal cell appearance. The absorbance （A） values of the experimental group and the negative control group were increased with time,and there was no statistically significant difference between the two groups （P0.05）. While the A value in the positive control group was significantly decreased compared with the other two groups （P0.01）. At each time point of culture,the cytotoxicitygrade was 0 in the experimental group and 4 in the positive control group. Conclusion The CCC composite has potential application in tissue engineering because of good cellular compatibility with BMSCs.