Directed differentiation of bone marrow mesenchymal stem cells under regulation by polyacrylamide with different stiffness

doi:10.3969/j.issn.2095-4344.0956

Abstract

Abstract:

BACKGROUND: The hardness of substrate materials can directly affect the differentiation fate of stem cells, but the sensitivity and regulatory mechanism of stem cells differentiating into different somatic cells are still unclear.
OBJECTIVE: To explore the effect of substrate mechanical properties on the differentiation of bone marrow mesenchymal stem cells and its mechanism.
METHODS: In this study, the mechanical strength of polyacrylamide was modulated to be similar to that of nerve tissue (0.5 kPa), cartilage tissue (10 kPa), myocardial tissue (20 kPa) and bone tissue (40 kPa), respectively. Differentiation of bone marrow mesenchymal stem cells on the substrate with different elastic moduli was detected using ELISA, and cytoskeleton morphology and vinculin expression were detected using cytoskeleton staining and immunofluorescence staining.
RESULTS AND CONCLUSION: (1) Bone marrow mesenchymal stem cells on the substrate with low stiffness trended to differentiate into nerve and cartilage, while the cells on the substrate with high stiffness had no obvious trend to differentiate into cardiocytes and osteoblasts. This indicated that bone marrow mesenchymal stem cells were sensitive to the mechanical properties of the material during nerve and cartilage differentiation, while were insensitive to the mechanical properties of the material during cardiocyte and osteoblast differentiation. (2) The expression level of vinculin increased as the substrate stiffness increased, indicating that the mechanical signals induced by bone marrow mesenchymal stem cells on low stiffness substrates are relatively low, which can activate certain signaling pathways related to cartilage and neural differentiation.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Acrylamides, Bone Marrow, Mesenchymal Stem Cells, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Guo Jiang-long, He Jing, Wu Fang. Directed differentiation of bone marrow mesenchymal stem cells under regulation by polyacrylamide with different stiffness[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 3981-3986.

Figures/Tables 2

2.1 动态力学分析弹性模量通过动态力学分析的数据，可以发现最终聚丙烯酰胺的弹性模量与作者最初设计的弹性模量相近，实际的弹性模量分别为0.75，9，21，36 kPa，与神经组织、软骨组织、心肌组织和骨组织模量类似，见图1。 2.2 细胞增殖结果细胞在弹性模量10，20，40 kPa的材料上培养时，细胞数量随着时间的增加而增加，而细胞在弹性模量0.5 kPa的材料上培养时，细胞数量始终处于较低的水平。总体来看，细胞的增殖结果为10 kPa>40 kPa>20 kPa>0.5 kPa，见图2。 2.3 细胞骨架与黏着斑蛋白复染结果细胞在材料上第1天时，总体来看都偏弱，细胞在弹性模量0.5，10，40 kPa的材料上培养时，相对于20 kPa与对照组，骨架较为明显，这说明在第1天时细胞铺展不完全。从鬼笔环肽的染色结果来看，第3天时细胞在弹性模量10 kPa与40 kPa的材料上培养时，细胞骨架较为清晰，细胞在弹性模量20 kPa和0.5 kPa的材料上培养时，骨架较弱，且细胞形态呈圆形。从细胞骨架的清晰程度来看，10 kPa>40 kPa>对照组>20 kPa>0.5 kPa。从黏着斑蛋白的染色结果看，细胞在弹性模量40 kPa的材料上培养时，黏着斑蛋白的表达量最高。细胞在弹性模量20 kPa的材料上培养时，虽然骨架形态较弱，但是黏着斑蛋白的表达呈现较高的水平。在弹性模量10 kPa的材料上培养时，虽然细胞骨架最为清晰，但是黏着斑蛋白的表达量较低。细胞在弹性模量0.5 kPa的材料上培养时，黏着斑蛋白的表达量最低。总体来看，黏着斑蛋白的表达量为：对照组>40 kPa>20 kPa>10 kPa>0.5 kPa，见图3。 2.4 细胞分化相关标志物表达微管蛋白、巢蛋白是神经细胞分化晚期的标志物。Ⅱ型胶原与聚焦蛋白聚糖广泛存在于软骨细胞外基质中，是软骨细胞分化的标志物。缝隙连接蛋白是一种表达在心肌细胞中的跨膜蛋白；心肌肌钙蛋白I存在于心肌细胞胞浆中，是心肌细胞分化的标志物。骨钙素在骨钙代谢调节中起重要作用；碱性磷酸酶是骨组织中合成的一类功能性酶，是成骨分化的标志物，见图4。 BMSCs在0.5 kPa的基底上培养时，随着培养天数的增加，神经分化相关蛋白微管蛋白、巢蛋白的表达量增多，巢蛋白的表达量在第14天时明显高于其他组，微管蛋白的表达量在第7天和第14天明显高于其他组的基底材料。 BMSCs在0.5 kPa与10 kPa的基底上培养时，软骨分化标志物Ⅱ型胶原的表达量在第14天时要显著高于其他组，而聚焦蛋白聚糖的表达量在10 kPa的基底上较高，尤其是在第3天与第5天时，表达量远高于其他组。成骨分化的标志物碱性磷酸酶和骨钙素的表达量在4组基底上没有表现出明显的差别。心肌细胞分化的特征蛋白缝隙连接蛋白和心肌肌钙蛋白I的表达，虽然在各组之间存在一定的差别，但无明显规律。"

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