Chondrogenic differentiation of bone marrow stem cells on a hydroxyapatite coated titanium plate

doi:10.3969/j.issn.2095-4344.1523

Abstract

Abstract:

BACKGROUND: Hydroxyapatite has good biocompatibility and bioactivity and can promote adhesion, proliferation and chondrogenic differentiation of stem cells.
OBJECTIVE: To investigate the effect of hydroxyapatite coated titanium plate on chondrogenic differentiation of bone marrow mesenchymal stem cells.
METHODS: Hydroxyapatite coating was fabricated on the titanium surface by electrochemical deposition technique (deposition time: 3 minutes and 1.5 hours) and characterized by field emission scanning electron microscopy for coating morphology and by X-ray diffraction for the chemical composition of the coating. Bone marrow samples from four Sprague-Dawley rats of 3 weeks old (purchased from Hubei Provincial Laboratory Animal Center, Wuhan, China) were taken to culture bone marrow mesenchymal stem cells using whole bone marrow adherent method. Passage 3 cells were inoculated onto the titanium plate with or without coating followed by 4 weeks chondrogenic induction. For assessing the ability of chondrogenesis, cell counting kit-8 assay was applied to test the cell proliferation; dimethylmethylene blue colorimetric assay was used to detect the content of glycosaminoglycans; and RT-PCR was used to test the expression of chondrocyte specific genes.
RESULTS AND CONCLUSION: (1) The hydroxyapatite coating was evenly distributed on the titanium plate and its structure was uniform. The X-ray diffraction peaks of hydroxyapatite powder generally matched the characteristic X-ray diffraction pattern of hydroxyapatite. (2) The hydroxyapatite-coated titanium plate improved the proliferation of bone marrow mesenchymal stem cells and showed better bioactivity compared to the non-coated titanium plate. (3) The glycosaminoglycan content was significantly higher in the hydroxyapatite coating group than the non-coated group (P < 0.01). (4) The RT-PCR results showed higher expression levels of chondrocyte specific genes (SOX9, type II collagen, type X collagen, aggrecan) in the hydroxyapatite coating group than the non-coated group (P < 0.05). The expression of type II collagen was significantly higher in the 1.5-hour group than the 0.5-hour group and non-coated group (P < 0.05). To conclude, the hydroxyapatite coating could evenly distribute on the titanium plate via electrophoretic deposition. Hydroxyapatite coated titanium surface has good biocompatibility and can promote proliferation and chondrogenic differentiation of rat bone marrow mesenchymal stem cells in vitro.

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: Bone Marrow, Mesenchymal Stem Cells, Titanium, Hydroxyapatites, Electrochemical Techniques, Tissue Engineering

CLC Number:

Qin Liling, Yan Yan, Yu Zhuo, Qin Qiaoling, Li Wei, Liu Li. Chondrogenic differentiation of bone marrow stem cells on a hydroxyapatite coated titanium plate[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(1): 35-40.

Figures/Tables 1

2.1 纯钛片及两种不同电沉积时间的羟基磷灰石涂层外观见图1。空白纯钛片表面粗糙，无涂层；短时间电沉积组钛片呈灰白色，表面有粉末状涂层均匀分布；长时间电沉积组钛片呈白色，表面有粉末状涂层均匀分布。 2.2 涂层的场发射扫描电子显微镜形貌及X射线衍射分析 2.2.1 钛片表面及羟基磷灰石涂层的微观形貌图2为喷酸酸蚀后钛片及其羟基磷灰石涂层的场发射扫描电子显微镜形貌。图2A可见钛片表面经过酸蚀过后的蜂窝状结构，图2B，2C可见羟基磷灰石涂层比较均匀的分布在钛片上，高倍镜显示羟基磷灰石为头部稍尖的棒状。短时间电沉积组棒长度大小为(190±25) nm，长时间电沉积组棒长度大小为(460±28) nm，结构比较一致，呈花瓣状散开。 2.2.2 X射线衍射分析涂层粉末的化学组成图3的X射线衍射图谱显示了羟基磷灰石在2θ=25.9°和31°到33°的几个特征峰，与标准的羟基磷灰石衍射图谱进行比较后，两者的特征峰基本一致，且Jade.6软件对其进行物相分析显示该粉末主要的结晶形态为Ca10(PO4)6(OH)2，说明通过此电化学沉积法制备出了较纯的羟基磷灰石。该样品其(002)和(222)衍射峰高于标准羟基磷灰石样品的衍射峰，这是由于晶体生长中晶面择优取向不同造成的。 2.3 骨髓间充质干细胞增殖及其形态 2.3.1 CCK-8检测细胞增殖能力图4显示在第3天时，纯钛片组和有羟基磷灰石涂层钛片组细胞数目差异无显著性意义，在第7天时，有羟基磷灰石涂层钛片组的细胞数超过了纯钛片组，但是短时间电沉积组和长时间电沉积组之间差异无显著性意义，说明此羟基磷灰石涂层具有较好的细胞相容性，并在长期培养时可以促进细胞生长。 2.3.2 免疫荧光染色观察骨髓间充质干细胞形态细胞经过鬼笔环肽及DAPI染色后，图5可以清楚看到细胞核及细胞骨架的形态，各组细胞均生长良好，并呈现出一个具有多伪足的伸展状的细胞形态，说明该涂层具有良好的生物活性，并有利于细胞的增殖。 2.4 糖胺多糖含量由图6可见，经过4周的成软骨诱导分化后，羟基磷灰石涂层组的糖胺多糖含量略高于纯钛片组，短时间电沉积组和长时间电沉积组之间差异无显著性意义(P > 0.05)，说明羟基磷灰石涂层可以促进软骨外基质糖胺多糖的分泌，并间接证明骨髓间充质干细胞已成功诱导分化成了软骨细胞。 2.5 RT-PCR分析软骨相关基因表达由图7可见，在体外经过4周成软骨诱导，羟基磷灰石涂层组促进了骨髓间充质干细胞软骨相关基因的表达，且均高于纯钛片组，差异有显著性意义(P < 0.05)。长时间电沉积组的Ⅱ型胶原基因表达量明显高于短时间电沉积组和纯钛片组，作为软骨标志性基因，说明较大颗粒涂层能很好的促进干细胞诱导分化成软骨细胞。"

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