Characteristics of bone marrow mesenchymal stem cells in a rat model of retinoic acid-induced osteoporosis

doi:10.3969/j.issn.2095-4344.1703

Abstract

Abstract:

BACKGROUND: Retinoic acid has been directly used to intervene with bone marrow mesenchymal stem cells in vitro; however, there is still a lack of research on in vitro characteristics of bone marrow mesenchymal stem cells in an osteoporosis rat model induced by retinoic acid.
OBJECTIVE: To establish the osteoporosis rat model induced by retinoic acid and to observe the characteristics of bone marrow mesenchymal stem cells from the rat model in vitro.
METHODS: Sprague-Dawley rats were randomly divided into two groups. The experimental group was treated with retinoic acid, 70 mg/kg per day for 2 weeks, in order to quickly establish osteoporosis models, while the control group was given an equal volume of normal saline. Bone marrow mesenchymal stem cells were cultured using the whole bone marrow culture method in vitro and their characteristics (cell morphology, growth curve and surface antigens) were observed. After osteogenic induction, the in vitro mineralization capacity and alkaline phosphatase secretion capacity of the cells were measured. After adipogenic induction, the adipogenic ability of cells was detected.
RESULTS AND CONCLUSION: (1) Morphology of bone marrow mesenchymal stem cells was basically the same in the two groups. The proliferation capacity of the cells in the experimental group was higher than that in the control group at 9 days after culture. (2) After osteogenic induction, the alkaline phosphatase activity in the experimental group was lower than that in the control group (P < 0.05), which was consistent with the results of alizarin red staining and alkaline phosphatase staining. (3) After adipogenic induction, larger number of lipid droplets and higher absorbance value for oil red O could be observed in the experimental group than the control group (P < 0.05). To conclude, bone marrow mesenchymal stem cells from the osteoporosis rat model induced by retinoic acid have lower osteogenic capacity but higher adipogenic capacity than the normal cells.

Key words: retinoic acid, osteoporosis model, bone marrow mesenchymal stem cells, osteogenic differentiation, adipogenic differentiation, rat, Natural Science Foundation of Guangxi Zhuang Autonomous Region

CLC Number:

Long Yanming, Xie Mengsheng, Xue Wenli, Huang Jiajie, Li Xiaojie. Characteristics of bone marrow mesenchymal stem cells in a rat model of retinoic acid-induced osteoporosis[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(17): 2637-2643.

Figures/Tables 2

2.1 实验动物数量分析参加实验的20只雌性SD大鼠，实验组10只，对照组10只，进入结果分析的数量仍为20只，两组大鼠均无脱落。 2.2 大鼠体质量变化实验组灌胃第2天后出现体质量下降，对照组大鼠体质量稳定上升，灌胃14 d时，实验组大鼠体质量明显低于对照组(P < 0.05)，差异有显著性意义，见图1。 2.3 大鼠骨密度值变化大鼠灌胃2周后测量实验组和对照组全身骨密度值，实验组的骨密度值低于对照组(P < 0.05)，差异有显著性意义，见图2。 2.4 苏木精-伊红染色结果实验组大鼠股骨干骺端骨小梁纤细，骨髓腔增宽，骨小梁分离度大，见图3A；对照组骨小梁粗大，结构清晰完整，排列规则而较致密，交织成网状，见图3B；从苏木精-伊红染色结果得出实验组骨量明显少于对照组。 2.5 骨髓间充质干细胞形态实验组和对照组骨髓间充质干细胞原代培养第5天，镜下观察细胞多呈星形、多角形，细胞集落样生长，见图4A，B；传3代骨髓间充质干细胞姬姆萨染色显示实验组细胞以短梭形为主，排列成漩涡状和放射状，见图4C；对照组姬姆萨染色结果显示细胞形态与实验组基本一致，见图4D。 2.6 生长曲线两组骨髓间充质干细胞都呈S形生长，1-3 d处于潜伏期，3-7 d为快速生长期，前7 d两组细胞生长速度无明显差异，第7天后对照组细胞逐渐进入平台期，实验组细胞继续生长，第9天实验组细胞数量明显大于对照组，差异有显著性意义(P < 0.05)，见图5。 2.7 细胞表面抗原表达实验组和对照组骨髓间充质干细胞均一表达CD90、CD44，表达率均在85%以上，阴性表达CD34，表达率不足1%，见图6。 2.8 茜素红染色结果骨髓间充质干细胞成骨诱导21 d后，细胞呈铺石路样生长，可见钙盐沉积形成钙结节，茜素红染色后钙结节呈橘红色；实验组钙结节体积小，见图7A；对照组钙结节体积大，染色深，见图7B。 2.9 碱性磷酸酶染色结果骨髓间充质干细胞成骨诱导14 d，碱性磷酸酶染色可见胞膜和胞浆颗粒染色呈蓝黑色，实验组块状深染颗粒较少，见图8A，对照组块状深染颗粒较多，染色深，见图8B。 2.10 碱性磷酸酶活力骨髓间充质干细胞成骨诱导7 d和14 d检测碱性磷酸酶活力，第7天实验组和对照组细胞碱性磷酸酶活力无明显差异；第14天实验组碱性磷酸酶活力低于对照组(P < 0.05)，差异有显著性意义，见图9。 2.11 油红O染色结果骨髓间充质干细胞成脂诱导28 d后，经油红O染色，脂滴被染成鲜红色，细胞核蓝色深染，实验组脂肪细胞数量多，合并成串珠状，见图10A；对照组脂肪细胞散在，数量少，见图10B；实验组油红O吸光度值高于对照组(P < 0.05)，差异有显著性意义，见图11。"

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