Chinese Journal of Tissue Engineering Research ›› 2020, Vol. 24 ›› Issue (31): 4941-4947.doi: 10.3969/j.issn.2095-4344.2137
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Calcined bone/chitosan composite promotes osteogenic differentiation of bone marrow mesenchymal stem cells in Sprague-Dawley rats
Liao Jian1, Huang Xiaolin2, Zhou Qian1, Huo Hua1, Qi Yuhan1, Wu Chao1, Shi Qianhui1, Yang Tongjing1,Liao Yunmao3, Liang Xing3
1School/Hospital of Stomatology, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 2Department of Dental Implant, Zhongshan People’s Hospital, Zhongshan 528403, Guangdong Province, China; 3West China School of Stomatology, Sichuan University, Chengdu 610041, Sichuan Province, China
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Received:2020-02-27Revised:2020-03-05Accepted:2020-04-15Online:2020-11-08Published:2020-09-03 -
Contact:Liang Xing, Professor, Doctoral supervisor, West China School of Stomatology, Sichuan University, Chengdu 610041, Sichuan Province, China -
About author:Liao Jian, MD, Associate professor, Associate chief physician, Master’s supervisor, School/Hospital of Stomatology, Guizhou Medical University, Guiyang 550004, Guizhou Province, China -
Supported by:the National Natural Science Foundation of China, No. 81660179 ; the Natural Science Foundation of Guizhou Province, No. [2016]1124 ; the Joint Fund of Natural Science Foundation of Guizhou Province, No. LH[2016]7257
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Cite this article
Liao Jian, Huang Xiaolin, Zhou Qian, Huo Hua, Qi Yuhan, Wu Chao, Shi Qianhui, Yang Tongjing, Liao Yunmao, Liang Xing. Calcined bone/chitosan composite promotes osteogenic differentiation of bone marrow mesenchymal stem cells in Sprague-Dawley rats [J]. Chinese Journal of Tissue Engineering Research, 2020, 24(31): 4941-4947.
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2.1 大鼠骨髓间充质干细胞的形态 在倒置相差显微镜下观察可见：刚接种的细胞绝大多数呈圆形，以悬浮状分散在培养基中，在24 h半量换液时，部分细胞已贴壁，细胞呈不规则分布，在细胞培养瓶底呈分散片集状；48 h换液时可见绝大多数细胞均已贴壁，细胞开始呈放射状排列，以梭形和星形细胞居多；3-5 d后放射状排列的细胞形成细胞集落，细胞以梭形为主，伸出长短不一的突起，大部分为单核、胞浆丰富，有清晰的核仁，见图1A，B；1周后，细胞呈集落状生长，漩涡状，融合度达到80%左右，在9 d左右细胞片状融合，见图1C，即可传代培养；细胞传代1 d后就会完全贴壁生长，此时可见细胞形态一致，呈梭形，轮廓清楚，生长比较旺盛，见图1D；1周左右可以传代，细胞形态变得越来越均一，无显著变化。 "
2.2 大鼠骨髓间充质干细胞的生长曲线 从图2可以看出，第3代培养的骨髓间充质干细胞在1-3 d为细胞生长潜伏期，4-6 d为细胞对数增殖期，6 d后进入平台期。 "
2.3 大鼠骨髓间充质干细胞的成骨诱导能力鉴定 成骨诱导液诱导培养后，细胞形态变化较大，逐渐由梭形转变为多边形，胞质中出现钙化斑点；诱导14 d后碱性磷酸酶染色可见细胞的胞核被染成蓝色，见图3A；诱导21 d后，细胞呈多层性、重叠性排列，形成多层白色的钙化结节，见图3B，茜素红染色矿化结节呈现红色，见图3C，D。 "
2.4 大鼠骨髓间充质干细胞表面标志物的表达 流式细胞术检测结果显示CD29、CD90为阳性表达，阳性表达率分别为99.9%和99.1%；CD34、CD45为阴性表达，符合骨髓间充质干细胞的表面标记，见图4。 "
2.5 骨髓间充质干细胞在复合材料周围的生长情况 倒置相差显微镜下可见：骨髓间充质干细胞与材料共培养1 d后生长良好，许多细胞从材料边缘爬出到培养板中，见图5A-C；骨髓间充质干细胞与材料共培养3 d后细胞增殖明显，复合材料边缘已经铺满细胞，见图5D-F。 "
2.6 锻烧骨/壳聚糖复合材料对成骨诱导的骨髓间充质干细胞成骨分化相关基因表达的影响 图6A-C分别显示碱性磷酸酶、Ⅰ型胶原和骨钙素在不同时间的表达。在诱导第1天，碱性磷酸酶、Ⅰ型胶原和骨钙素在每组材料中几乎都没有表达，与对照组比差异无显著性意义(P > 0.05)。 图6A可以看出：碱性磷酸酶从诱导的第1天开始到第14天表达呈上升趋势，14 d后碱性磷酸酶的表达量逐渐下降；在第7天和第14天，3种复合材料组碱性磷酸酶的表达均高于对照组(P < 0.05)，锻烧骨/壳聚糖=2/1组碱性磷酸酶表达略高于另外2种复合材料组，但单因素方差分析结果显示3组不同比例材料之间差异无显著性意义(P > 0.05)。 从图6B可以看出：Ⅰ型胶原从诱导的第1天开始到第14天表达呈上升趋势，14 d后Ⅰ型胶原的表达量逐渐下降，但第21天的表达明显高于第1天；在第7天和第14天，3种复合材料组Ⅰ型胶原的表达均高于对照组(P < 0.05)，锻烧骨/壳聚糖=2/1组Ⅰ型胶原表达仍略高于另外2种复合材料组，但单因素方差分析结果显示3组不同比例材料之间差异无显著性意义(P > 0.05)。 从图6C可以看出：骨钙素从诱导后的第1天到第7天几乎没有表达，从第7天开始到第21天表达呈上升趋势；在第14天和第21天，3种复合材料组骨钙素的表达均高于对照组(P < 0.05)，锻烧骨/壳聚糖=2/1组骨钙素表达仍略高于另外2种复合材料组，但单因素方差分析结果显示3组不同比例材料之间差异无显著性意义(P > 0.05)。 "
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