羟基磷灰石球形颗粒表面微形貌构建及其对干细胞生物学行为的调控

doi:10.15541/jim20160260

无机材料学报 ›› 2017, Vol. 32 ›› Issue (3): 319-325.DOI: 10.15541/jim20160260

羟基磷灰石球形颗粒表面微形貌构建及其对干细胞生物学行为的调控

智伟¹, 匙峰¹, 李金雨¹, 周腾¹, 屈树新¹, 汪建新¹, 张聪², 翁杰¹

(1. 西南交通大学材料科学与工程学院, 材料先进技术教育部重点实验室, 成都 610031; 2. 中国人民解放军第309医院, 北京 100091)

收稿日期:2016-04-18 修回日期:2016-07-07 出版日期:2017-03-20 网络出版日期:2017-02-24
作者简介:智伟(1977–), 女, 工程师. E-mail: zhiwei@home.swjtu.edu.cn
基金资助:
国家重点研发计划(2016YFC1102000, 2016YFB0700800);国家自然科学基金(31400809);中央高校基本科研业务费专项资金(2682015CX006)

Surface Microstructure on Hydroxyapatite Spherules and Its Regulation on Stem Cells

ZHI Wei¹, SHI Feng¹, LI Jing-Yu¹, ZHOU Teng¹, QU Shu-Xin¹, WANG Jian-Xin¹, ZHANG Cong², WENG Jie¹

(1. Key Laboratory of Advanced Technologies of Materials (Ministry of Education), School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China; 2. The 309st Hospital of Chinese PLA, Beijing 100091, China)

Received:2016-04-18 Revised:2016-07-07 Published:2017-03-20 Online:2017-02-24
About author:ZHI Wei. E-mail: zhiwei@home.swjtu.edu.cn
Supported by:
National Key Research and Development Program of China (2016YFC110200, 2016YFB07000800);National Natural Science Foundation of China (31400809);Fundamental Research Funds for the Central Universities (2682015CX006)

摘要/Abstract

摘要：

通过调节溶胶-凝胶体系中羟基磷灰石(HA)粉末和甲壳素(Chitin)的质量比, 制备具有不同表面微形貌的HA球形颗粒。扫描电子显微镜(SEM)表征结果显示: 随着HA/Chitin质量比从4/1增加到35/1, 球形颗粒的表面微形貌发生了明显变化, 由粗糙渐趋平滑, 微米级皱褶逐渐减少至消失, 微孔隙率从(35%±0.8%)减少到(10.4%±0.7%)。体外细胞培养的结果表明具有微米级皱褶, 微孔隙率较高的粗糙表面具有引导干细胞铺展和增殖的作用, 微孔隙率低的平滑表面则具有引导干细胞轴向延伸及骨向分化的趋势。同时, HA球形颗粒表面微形貌对干细胞表面特征性抗原标志物的表达具有调控作用。

关键词: 羟基磷灰石, 表面微形貌, 干细胞, 生物学行为

Abstract:

HA spheres with different surface microstructures were prepared by a Sol-Gel route with various hydroxyapatite (HA)/chitin ratios. The surface morphologies, phase compositions, and biomimetic mineralization ability of samples were respectively analyzed with scanning electron microscopy (SEM), X-ray diffraction (XRD), and biomimetic mineralization test. Furthermore, the influences of the surface microstructure on biological behavior of bone marrow-derived mesenchymal stem cells were examined by Alamar blue, SEM, alkaline phosphatase assay and flow cytometry. When the mass ratio of HA/Chitin was increased from 4/1 to 35/1, the surface microstructure of HA spheres changed substantially, as showed by a reduction of micro-porosity from (35%±0.8%) to (10.4%±0.7%), decrease of surface roughness, and gradual disappearance of micro-creases. The result of biomimetic mineralization test showed that the surface microstructure had important influence to the ability of biomimetic mineralization for HA spheres. In vitro culture of bone marrow-derived mesenchymal stem cells demonstrated that the rougher sphere surface, with abundant micro-creases and micro-pores, supported cell spreading and proliferation. In comparison, the smoother sphere surface with fewer micro-pores effectively induced cell elongation and up-regulated ALP expression, which suggested stem cells osteogenic differentiation. Meanwhile, the surface microstructure of spherules also modulated the expression of characteristic antigen makers on the surface of stem cells.

Key words: hydroxyapatite, surface microstructure, stem cell, biological behavior

中图分类号:

R318
TQ174

智伟, 匙峰, 李金雨, 周腾, 屈树新, 汪建新, 张聪, 翁杰. 羟基磷灰石球形颗粒表面微形貌构建及其对干细胞生物学行为的调控[J]. 无机材料学报, 2017, 32(3): 319-325.

ZHI Wei, SHI Feng, LI Jing-Yu, ZHOU Teng, QU Shu-Xin, WANG Jian-Xin, ZHANG Cong, WENG Jie. Surface Microstructure on Hydroxyapatite Spherules and Its Regulation on Stem Cells[J]. Journal of Inorganic Materials, 2017, 32(3): 319-325.

图/表 8

图1 HA4和HA35球形颗粒烧结后的XRD图谱

Fig. 1 XRD patterns of HA4 spherules and HA35 spherules

图2 不同HA/Chitin比例制备HA球形颗粒表面SEM照片

Fig. 2 SEM images of surface HA spheres with different initial ratios of Chitin/HAa1, b1: HA4; a2, b2: HA10; a3, b3: HA20; a4, b4: HA30; a5, b5: HA35

表1 不同表面微形貌的HA球形颗粒的微孔隙率

Table 1 Microporosity and specific surface area of HA spheres with different ratios of HA/Chitin

HA/Chitin	Micro-porosity/%	BET/(m²·g^-1)
4/1	35±0.8	152.49
10/1	30.1±0.9	/
20/1	25.7±0.4	86.39
30/1	19.6±0.5	/
35/1	10.4±0.7	17.25

图3 矿化3 d后HA球形颗粒表面的SEM照片

Fig. 3 SEM images of the surface of HA spheres (a, b) mineralized for 3 da1, b1: HA4; a2, b2: HA20; a3, b3: HA35

图4 骨髓来源间充质干细胞与不同表面微形貌的HA球形颗粒共培养3 d后的粘附和生长情况

Fig. 4 SEM images of BM-MSCs cultured on HA spehres with different surface microstructures after 3 da, d: HA4; b, e: HA20; c, f: HA35

图5 不同表面微形貌对细胞增殖的影响

Fig. 5 Proliferation of BM-MSCs cultured on tissue culture plates and HA spheres with different surface microstructures at 1, 3, 5 and 7 d(* represents statistical difference, P<0.05)

表2 BM-MSCs与不同表面微形貌的HA球形颗粒共培养7 d后表面特异性抗原标志物表达

Table 2 Surface characteristic markers expression of BM- MSCs cultured on HA spheres with different surface microstructures after 7 d

	CD29	CD44	CD90
Control	99.2%	93%	98.9%
HA₄	89.4%	67%	90.9%
HA₂₀	74.6%	56%	76.0%
HA₃₅	66.9%	40%	59.7%

图6 不同表面微形貌对细胞分化的影响

Fig. 6 Expression of ALP of BM-MSCs cultured on HA spheres with different surface microstructures at 7 d(* represents statistical difference, P<0.05)

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羟基磷灰石球形颗粒表面微形貌构建及其对干细胞生物学行为的调控

Surface Microstructure on Hydroxyapatite Spherules and Its Regulation on Stem Cells

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