Effects of platelet-rich plasma on the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.17.005

Abstract

Abstract:

BACKGROUND: Studies have demonstrated that platelet-rich plasma can promote osteogenesis and proliferation of bone marrow mesenchymal stem cells, adipose-derived stem cells and skeletal muscle satellite cell, but whether it can promote the proliferation and osteogenesis of human umbilical cord mesenchymal stem cells (hUC-MSCs) is unclear.

OBJECTIVE: To investigate the effects of different concentrations of platelet-rich plasma on the proliferation and osteogenic differentiation of hUC-MSCs.

METHODS: Passage 5 hUC-MSCs were cultured in medium containing different concentrations of platelet-rich plasma (0, 250, 500, 750, 1 000, 2 000 ng/L), respectively. Cell proliferation was detected at 1, 3, 5, 7 days after culture, and the best platelet-rich plasma mass concentration was screened. Afterwards, passage 5 hUC-MSCs were divided and cultured in complete medium (blank control group), optimal concentration of platelet-rich plasma (platelet rich plasma group), osteogenesis induction medium (osteogenic induction group), or the osteogenesis induction medium containing the optimal concentration of platelet-rich plasma (combined group). Cell activity of alkaline phosphatase was detected after cultivated for 3, 7, 14 days. Osteopontin, bone specific transcription factor, and osteocalcin mRNA relative expression levels were detected after cultivated for 7, 14, 21 days. Mineralization of the extracellular matrix was detected after cultivated for 21 days.

RESULTS AND CONCLUSION: After 5 and 7 days of culture, the cell proliferation was higher in 500, 750, 1 000 ng/L platelet-rich plasma groups than 0 ng/L group (P < 0.05), and 750 ng/L platelet-rich plasma showed the best effect on cell proliferation, which was used in the following experiments. Compared with the other groups, the combined group had significantly increased alkaline phosphatase activity (P < 0.05), and up-regulated osteopontin, bone specific transcription factor and osteocalcin mRNA relative expression levels (P < 0.05) at different culture times. In addition, the degree of extracellular matrix mineralization in the combined group was also higher than that in the other three groups (P < 0.05). To conclude, 750 ng/L platelet-rich plasma can promote hUC-MSCs proliferation and osteogenic 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: Platelet-Rich Plasma, Umbilical Cord, Mesenchymal Stem Cells, Tissue Engineering

CLC Number:

R394.2

Yang Kai, Cheng Ya-dong, Wang Peng, Wu Tong, Li Shu-shan, Min You-hui. Effects of platelet-rich plasma on the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(17): 2652-2658.

Figures/Tables 2

2.2 各组hUC-MSCs细胞形态学表现选取最佳质量浓度的富血小板血浆(750 ng/L)对hUC-MSCs细胞进行处理，空白对照组第3，7，14天的细胞形态无明显改变；富血小板血浆组第3天细胞增殖速度加快，第7天时可见部分细胞呈梭形改变，第14天约有20%细胞由梭形转变成多边形；成骨诱导组在第3天细胞呈多边形改变，数量未见明显变化，在第14天细胞数量明显增多且80%细胞呈多边形改变；联合在第14天时已有明显钙结节沉积，见图1。 2.3 各组hUC-MSCs内碱性磷酸酶活性的比较空白对照组不同时间点的碱性磷酸酶活性较低，且无明显变化；随着培养时间的增加，富血小板血浆组、成骨诱导组、联合组碱性磷酸酶活性有着不同程度的提高，富血小板血浆组不同时间点的碱性磷酸酶活性均高于空白对照组(P < 0.05)，成骨诱导组不同时间点的碱性磷酸酶活性均高于富血小板血浆组(P < 0.05)，联合组不同时间点的碱性磷酸酶活性均高于成骨诱导组(P < 0.05)，见表2。 2.4 各组hUC-MSCs细胞内成骨分化标志基因表达量的比较空白对照组培养7，14，21 d的成骨分化标志基因骨桥蛋白、骨特异性转录因子、骨钙蛋白只有微量表达，且无明显变化。与空白对照组相比，富血小板血浆组培养7，14，21 d的各成骨分化标志基因表达均有不同程度增加(P < 0.05)；与富血小板血浆组相比，成骨诱导组培养7，14，21 d的各成骨分化标志基因表达均有不同程度增加(P < 0.05)；与成骨诱导组相比，联合组培养7，14，21 d的各成骨分化标志基因表达均有不同程度上调(P < 0.05)，见表3。 2.5 各组hUC-MSCs细胞内钙与茜素红结合物的比较与空白对照组相比，富血小板血浆组钙与茜素红结合物水平升高(8.73±3.12，26.19±5.95，P < 0.05)；与富血小板血浆组相比，成骨诱导组钙与茜素红结合物水平(45.65±4.98)升高(P < 0.05)；与成骨诱导组相比，联合组钙与茜素红结合物水平(65.48±4.00)升高(P < 0.05)。"

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