钛纳米管及不同化学诱导条件对骨髓间充质干细胞成骨分化的影响

Influences of TiO2 nanotubes and chemical stimulation on osteogenetic differentiation of MSCs

摘要:

目的：在不同培养基中，对比研究 SD大鼠骨髓间充质干细胞（mesenchymal stem cells，MSCs）在 TiO2纳米管表面的成骨向分化特点。方法：分离并纯化 MSCs，电化学阳极氧化法制备 TiO2纳米管。使用 4 种培养体系：常规培养体系、抗坏血酸+β-甘油磷酸钠培养体系、抗坏血酸+β-甘油磷酸钠+1α，25-双羟维生素 D3 培养体系、抗坏血酸+β-甘油磷酸钠+地塞米松培养体系，分别在光滑面纯钛片，管径 30、70、100 nm 的 TiO2纳米管表面培养 MSCs，3、7、14 d 后检测碱性磷酸酶活性；21 d 后检测钙沉积量；14 d 后real-time PCR检测成骨相关基因Runx2及OSX的表达。结果：同一种 TiO2纳米管结构表面，抗坏血酸+β-甘油磷酸钠+地塞米松组碱性磷酸酶活性（F=338.542，P=0.000）、钙沉积量（F=417.012，P=0.000）及成骨相关转录因子 Runx2（F=14.419，P=0.000）和 OSX（F=42.011，P=0.000）的表达均较其他组高，差异具有统计学意义；同一种培养体系下，管径 30 nm 的 TiO2纳米管结构表面碱性磷酸酶活性（F=53.170，P=0.000）、钙沉积量（F=264.268，P=0.000）及成骨相关转录因子 Runx2（F=3.196，P=0.037）及 OSX（F=5.895，P=0.003）的表达均较其他组高，差异具有统计学意义。培养体系与材料结构有交互作用（碱性磷酸酶活性（F=6.322，P=0.000）、钙沉积量（F=33.330，P=0.000）、OSX的表达量（F=2.825，P=0.015），其中抗坏血酸+β-甘油磷酸钠+地塞米松培养体系和管径 30 nm的 TiO2纳米管结构是最佳组合。结论：MSCs的成骨向分化与材料的表面结构和诱导药物的化学刺激相关。在相同的 TiO2纳米管结构表面，MSCs在抗坏血酸+β-甘油磷酸钠+地塞米松培养体系下成骨向分化能力较好；在相同培养体系下，管径 30 nm的 TiO2纳米管结构更有利于MSCs的成骨向分化；抗坏血酸+β-甘油磷酸钠+地塞米松培养体系和管径 30 nm 的 TiO2纳米管结构的组合条件最有利于MSCs 的成骨向分化。

Abstract:

Objective：To investigate the osteogenic differentiation of bone marrow mesenchymal stem cells（MSCs） derived from Sprague Dawley rats on TiO2 nanotubes under different inducing conditions. Methods：Isolated and purified MSCs were cultured on the surface structures of plane titalium and TiO2 nanotubes anodized of 30，70，100 nm diameter. Four osteogenic differentiation methods were ap－proached：regular culture medium（control group），regular medium with L-Ascorbic acid and β-glycerophosphate，regular medium with L-ascorbic acid，β-glycerophosphate and 1α，25-dihydroxyvitamin D3 and regular medium with L-ascorbic acid，β-glycerophosphate and dexamethasone. Alkaline phosphatase（ALP） activities of all groups were analyzed on 3，7，14 d after the culture，respectively. Calcium deposition was detected on 21 d after the culture. Real-time PCR was applied to quantitate the expression of osteoblast related gene Runx2 and OSX on 14 d after the culture. Results：MSCs cultured on the same surface structure demonstrated higher ALP activity（F=338.542，P=0.000），calcium deposition（F=417.012，P=0.000） as well as Runx2（F=14.419，P=0.000） and OSX（F=42.011，P=0.000） gene expression in the L-ascorbic acid + β-glycerophosphate + dexamethasone group than in the others，with statistically significant differences. Within the same culture condition，ALP activity（F=53.170，P=0.000），calcium deposition（F=264.268，P=0.000） and Runx2（F=3.196，P=0.037） and OSX（F=5.895，P=0.003） gene expression were much higher on the surface structure of 30 nm diameter TiO2 nanotubes，with statistically significant differences. Culture condition and material structure had interactions（ALP activity（F=6.322，P=0.000），calcium deposition（F=33.330，P=0.000） and gene expression of OSX（F=2.825，P=0.015）），in which L-ascorbic acid + β-glycerophosphate + dexamethasone culture and 30 nm diameter TiO2 nanotubes was the best combination. Conclusion：The osteogenic differentiation of MSCs can be influenced by both the structure of material surface and chemical stimulation. On the same surface structure，MSCs in the L-ascorbic acid + β-glycerophosphate + dexamethasone culture group has greater potential of osteogenic differentiation than the others. Under the same chemical stimulation，MSCs on 30 nm diameter TiO2 nanotubes are more apt to differentiating towards osteoblasts than the way on other surface structures. The combination condition of L-ascorbic acid + β-glycerophosphate + dexamethasone culture and 30 nm diameter TiO2 nanotubes is most conductive to the os－teogenic differentiation of MSCs.