改良扩孔介孔硅介导大鼠骨髓间充质干细胞成骨向分化

doi:10.3877/cma.j.issn.1674-1366.2018.03.001

目的

探讨自制的扩孔介孔硅纳米粒子（LPMSNs）的材料性能，及其对大鼠骨髓间充质干细胞（BMSCs）增殖和成骨向分化的影响。

方法

溶胶凝胶法合成介孔硅纳米颗粒（MSNs），使用扩孔剂1，3，5-三甲苯（TMB）对初始合成的小孔径高温扩孔。透射电镜（TEM）、N₂吸附解吸附、孔径分布、孔容及比表面积分析、傅里叶红外光谱（FTIR）、热重分析（TGA）检测其形貌结构和理化性能。体外分离培养BMSCs，并对其增殖能力和多向分化潜能进行鉴定，取第3代细胞用于实验。配置不同浓度LPMSNs培养液，采用细胞计数试剂盒（CCK-8）检测LPMSNs的细胞毒性。分为对照组、LPMSNs组（10 μg/mL），培养21 d后，茜素红染色法检测各组细胞矿化结节。培养7、14 d后，实时荧光定量聚合酶链反应（PCR）法检测成骨相关基因碱性磷酸酶（ALP）、Runx相关转录因子（Runx2）、骨钙素（OCN）表达。培养14 d后，蛋白免疫印迹法（Western blot）检测成骨相关蛋白水平表达。使用SPSS 20.0对数据进行单因素方差分析（One-Way ANOVA）和t检验比较，以P<0.05认为差异有统计学意义。

结果

TEM显示LPMSNs粒径约为200 nm，其N₂吸附解吸附曲线为Ⅳ型等温线，有吸附的迟滞环；LPMSNs的孔径分布图显示其孔径分布峰值为7.39 nm，BET比表面积图计算出来的BET比表面积为（340.5 ± 2.8）m²/g，BJH孔容为1.8 cm³/g。在LPMSNs的FTIR的数据中，可观察到位于460 cm^-1的Si-O-Si横向摇摆振动峰（TO1）；位于800 cm^-1的Si-O-Si对称伸缩振动峰（TO2）；位于1070 cm^-1的Si-O-Si非对称伸缩振动峰（TO3），位于940 ~ 960 cm^-1的Si-OH键振动峰，位于3000 ~ 3700 cm^-1的-OH振动峰。TGA显示LPMSNs在28~1000 ℃之间整个失重所占百分比约为1.61%。CCK-8法结果显示低浓度（<20 μg/mL）的LPMSNs对细胞活力无明显影响。茜素红染色法显示与对照组相比，LPMSNs组的细胞矿化结节形成数量较多，体积较大。实时荧光定量PCR结果表明，LPMSNs组7 d时ALP表达量（6.2 ± 1.9）与对照组相比显著提高（t = 4.83，P = 0.0085），Runx2表达量（4.3 ± 0.6）与对照组相比显著提高（t = 9.76，P = 0.0006）；14 d时ALP表达量（19.2 ± 1.4）与对照组相比显著提高（t = 4.86，P = 0.0083）、Runx2表达量（4.9 ± 0.7）与对照组相比显著提高（t = 7.73，P = 0.0203）、OCN表达量（17.2 ± 3.6）与对照组相比显著提高（t = 5.52，P = 0.0052）。Western blot的结果显示，成骨分化的第14天，LPMSNs组的Runx2相对表达量（1.7 ± 0.3）与对照组相比呈上升趋势（t = 3.48，P = 0.0254）；OCN相对表达量（1.69 ± 0.21）与对照组相比显著提高，差异有统计学意义（t = 5.71，P = 0.0047）。

结论

本研究制备LPMSNs具有较大的比表面积、孔容和亲水性，适用于负载较大分子量的蛋白或细胞因子，细胞毒性小，可促进大鼠BMSCs成骨向分化，作为骨缺损的修复材料具备一定的应用价值。

关键词: 间质干细胞, 骨生成, 纳米粒, 扩孔介孔硅

Objective

To study the material properties and effect of the synthetic large pore mesoporous silica nanoparticles (LPMSNs) on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats.

Methods

Mesoporous silica nanoparticles (MSNs) was synthetised with the method of sol gel. The expanding agent 1, 3, 5-Trimethylbenzene was applied to expand the porous of the initial synthesis MSNs with high temperature. Transmission electron microscopy (TEM) , N₂ adsorption-desorption, pore size distribution, pore volume and specific surface area analysis, Fourier infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) were used to detect the morphological structure as well as physical and chemical properties of LPMSNs. BMSCs were cultured in vitro and their proliferative capacity and multidirectional differentiation potential were identified. The third generation of the cultured cells were selected for subsequent experiment. Different concentrations of LPMSNs were configured and a CCK-8 kit was applied to detect the cytotoxicity of LPMSNs. The osteogenic induction cell medium contained LPMSNs (10 μg/mL) was incubated with the BMSCs cells. After 21 days, alizarin red staining was used to detect the cell mineralized nodules and compared with the control group. The expression of bone related genes ALP, Runx2 and OCN were detected by qPCR method after 7 and 14 days incubation. After 14 days of cultivation, the expression of bone related gene Runx2 and OCN at protein level was detected by Western blot. The data were analyzed by One-Way ANOVA and t test by SPSS 20.0 software package. The difference was statistically significant with P<0.05.

Results

TEM showed that LPMSNs particle diameter was about 200 nm, and the Nitrogen sorption isotherms of LPMSNs exhibited typical type Ⅳ isotherm and a hysteresis loop. The pore width, Brunauer-Emmett-Teller (BET) specific surface area and Barrett-Joyner-Halenda (BJH) pore volume were 7.39 nm, (340.5 ± 2.8) m²/g and 1.8 cm³/g respectively. As shown in FTIR spectra of LPMSNs, 460 cm^-1 represents the transverse optical rocking motions of Si-O-Si, 800 cm^-1 represents the symmetric stretching of Si-O-Si; 1070 cm^-1 represents the antisymmetric stretching of Si-O-Si. The peak of 940-960 cm^-1 represents the Si-OH bond, 3000-3700 cm^-1 represents the-OH bond. TGA showed the weight loss percentage of LPMSNs in 28-1000 ℃ was about 1.61%. The results indicated that low concentration (<20 μg/mL) LPMSNs had no significant effect on the cell viability. Alizarin red staining showed more mineralized nodules in LPMSNs group compared with the control group. PCR results showed that at the 7th day, the expression of ALP (6.2 ± 1.9) and Runx2 (4.3 ± 0.6) in LPMSNs group were significantly higher than the control group (t_ALP= 4.83, P_ALP = 0.0085; t_Runx2 = 9.76, P_Runx2 = 0.0006) respectively. At the 14th day, the expression of ALP (19.2±1.4) , Runx2 (4.9 ± 0.7) and OCN (17.2 ± 3.6) in LPMSNs group were significantly higher than control group (P<0.05) . The results of Western blot showed that the expression of Runx2 in LPMSNs group (1.7 ± 0.3) was significantly higher than control group (t = 3.48, P = 0.0254) together with the expression of OCN (1.69±0.21) in LPMSNs group (t = 5.71, P = 0.0047) at the 14th day.

Conclusions

The modified LPMSNs had large specific surface area, pore volume and hydrophilic surface, which is suitable for loading large molecular weight proteins or cytokines. LPMSNs could induce the osteogenic differentiation of rat BMSCs without cytotoxicity. Therefore, LPMSNs is a promising biomaterial for bone regeneration.

Key words: Mesenchymal stem cells, Osteogenesis, Nanoparticles, Large pore mesoporous silica nanoparticles

表1 实时荧光定量聚合酶链反应分析的引物序列

基因	引物序列
ALP	F:5′-GAAGTCCGTGGGCATCGT-3′
?	R:5′-CAGTGCGGTTCCAGACATAG-3′
Runx2	F:5′-CCCAACTTCCTGTGCTCC-3′
?	R:5′-AGTGAAACTCTTGCCTCGTC-3′
OCN	F:5′-GGCAGTAAGGTGGTGAATAG-3′
?	R:5′-GTCCTGGAAGCCAATGTG-3′
GAPDH	F:5′-TGCTGAGTATGTCGTGGAG-3′
?	R:5′-GTCTTCTGAGTGGCAGTGAT-3′

表1 实时荧光定量聚合酶链反应分析的引物序列

基因	引物序列
ALP	F:5′-GAAGTCCGTGGGCATCGT-3′
?	R:5′-CAGTGCGGTTCCAGACATAG-3′
Runx2	F:5′-CCCAACTTCCTGTGCTCC-3′
?	R:5′-AGTGAAACTCTTGCCTCGTC-3′
OCN	F:5′-GGCAGTAAGGTGGTGAATAG-3′
?	R:5′-GTCCTGGAAGCCAATGTG-3′
GAPDH	F:5′-TGCTGAGTATGTCGTGGAG-3′
?	R:5′-GTCTTCTGAGTGGCAGTGAT-3′

图1 扩孔介孔硅纳米粒子（LPMSNs）透射电镜图

图2 扩孔介孔硅纳米粒子（LPMSNs）的N₂吸附解吸附和孔容孔径分析图

图3 扩孔介孔硅纳米粒子（LPMSNs）的傅里叶红外光谱（FTIR）图

图4 扩孔介孔硅纳米粒子（LPMSNs）热重分析重量-温度变化曲线

图5 骨髓间充质干细胞（BMSCs）细胞鉴定结果

图6 不同浓度扩孔介孔硅纳米粒子（LPMSNs）细胞毒性细胞计数试剂盒（CCK-8）法检测结果图

图7 各组成骨诱导矿化结节茜素红染色结果图

图8 各组成骨相关基因表达

图9 各组成骨分化相关基因蛋白水平的表达

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Stimulation of osteogenesis of rat bone marrow mesenchymal stem cells by modified large pore mesoporous silica nanoparticles

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Stimulation of osteogenesis of rat bone marrow mesenchymal stem cells by modified large pore mesoporous silica nanoparticles