Dopamine-induced biomimetic hydroxyapatite coating: preparation and biological effect on bone marrow mesenchym stem cells

doi:10.3969/j.issn.2095-4344.1525

Abstract

Abstract:

BACKGROUND: Hydroxyapatite (HA) coating can be formed on different kinds of metals assisted by polydopamine (PDA). However, the preparation of HA coating with biomimetic method assisted by PDA on artificial synthetic materials is rarely reported.
OBJECTIVE: To prepare the HA coating on HA/P66 surface assisted by PDA and to evaluate its effect on bone marrow mesenchymal stem cells.
METHODS: First HA coating was prepared on the HA/P66 surface using the biomimetic method assisted by PDA to obtain HA-PDA-HA/P66 substrate and then verified by X-ray photoelectron spectroscopy and scanning electron microscopy. Its surface properties such as hydrophilicity and surface roughness were also characterized. Then C3H10T1/2 cells were co-cultured with HA/P66, PDA-HA/P66 and HA-PDA-HA/P66. After 6 and 12 hours of cultivation, the initial cell adhesion was observed by cell counting kit-8. After 1 day of cultivation, cell morphology was observed by scanning electron microscope. After 1 and 3 days of cultivation, cell proliferation was assessed by 4,6-diamino-2-phenyl indole staining. After osteogenic induction for 7 and 10 days, intracellular alkaline phosphatase activity was detected. After osteogenic induction for 14 days, mineralization of the extracellular matrix was detected by alizarin red staining.
RESULTS AND CONCLUSION: (1) HA coating was successfully prepared on the HA/P66 substrate which was verified by X-ray photoelectron spectroscopy and scanning electron microscope. HA coating greatly increased the hydrophilicity and surface roughness of HA/P66. (2) After 6 and 12 hours of cultivation, the number of adherent cells on the surface of HA-PDA-HA/P66 was significantly higher than that in the other groups (P < 0.05). After 1 day of cultivation, the cells on the surface of HA-PDA-HA/P66 and PDA-HA/P66 showed polygonal shape with a large number of pseudopodia. (3) After 1 and 3 days of cultivation, the cell proliferation on the surface of HA-PDA-HA/P66 was significantly higher than that in the other two groups (P < 0.05). (4) After 7 and 10 days of osteogenic induction, the alkaline phosphatase activity of cells on the surface of HA-PDA-HA/P66 was significantly higher than that of the other groups. After 14 days of osteogenic induction, there were more calcium nodules on the surface of HA-PDA-HA/P66 than in the other groups. These findings indicate that HA coating can be successfully prepared on the surface of HA/P66 through the biomimetic process assisted by PDA and has good biocompatibility and bioactivity.

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: Hydroxyapatites, Nylons, Cell Biology, Tissue Engineering

CLC Number:

Xu Yanan, Yang Qiming, Li Hong, Jiang Dianming, Qiao Bo. Dopamine-induced biomimetic hydroxyapatite coating: preparation and biological effect on bone marrow mesenchym stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(1): 47-54.

Figures/Tables 3

2.2 不同材料表面细胞黏附和增殖图4所示为细胞与材料共培养6，12 h的细胞黏附情况，共培养6，12 h后，材料表面的细胞数量均为HA-PDA-HA/P66 > 聚多巴胺-纳米羟基磷灰石/聚酰胺66 > 纳米羟基磷灰石/聚酰胺66，说明羟基磷灰石及聚多巴胺涂层均有利于细胞黏附。图5所示为共培养1，3 d后通过DAPI染色观察材料表面细胞增殖情况，随时间延长，各个样本表面的细胞数均明显增加，HA-PDA-HA/P66和聚多巴胺-纳米羟基磷灰石/聚酰胺66材料同一时间点表面的细胞数明显多于纳米羟基磷灰石/聚酰胺66。通过定量分析进一步证实材料表面细胞的增殖能力：HA-PDA-HA/P66 >聚多巴胺-纳米羟基磷灰石/聚酰胺66 >纳米羟基磷灰石/聚酰胺66，见图6，说明羟基磷灰石及聚多巴胺涂层均能促进细胞增殖。 2.3 不同材料表面细胞形态图7所示为材料与细胞共培养1 d后使用扫描电镜观察材料表面的细胞形态，纳米羟基磷灰石/聚酰胺66材料表面细胞呈长条形；在羟基磷灰石-聚多巴胺-纳米羟基磷灰石/聚酰胺66和聚多巴胺-纳米羟基磷灰石/聚酰胺66材料表面可见细胞铺展的更大，且可见较多丝状伪足伸出，这说明相比于纳米羟基磷灰石/聚酰胺66材料，此两种材料更有利于C3H10T1/2细胞的伸展。 2.4 不同材料表面细胞成骨分化结果碱性磷酸酶活性检测：图8所示为成骨诱导培养7，10 d后不同材料表面细胞内碱性磷酸酶活性，HA-PDA-HA/P66材料表面细胞内碱性磷酸酶活性明显高于聚多巴胺-纳米羟基磷灰石/聚酰胺66和纳米羟基磷灰石/聚酰胺66材料(P < 0.05)，而聚多巴胺-纳米羟基磷灰石/聚酰胺66材料表面的细胞内碱性磷酸酶活性高于纳米羟基磷灰石/聚酰胺66材料。茜素红染色：图9所示为成骨诱导培养14 d后使用不同材料表面细胞内钙结节形成情况，HA-PDA-HA/P66材料周围的钙结节明显多于其他组，聚多巴胺-纳米羟基磷灰石/聚酰胺66材料周围的钙结节又多于纳米羟基磷灰石/聚酰胺66材料。"

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