种植体微纳米形貌对成骨细胞行为影响的分子机制研究

发布时间：2018-09-15 19:40

【摘要】：纯钛表面微纳米形貌能够很好地模拟人体骨组织和细胞外微环境的结构，因此可能具有更好成骨细胞生物学效应，但材料形貌对细胞调控的具体分子机制尚不清楚。本研究采用酸蚀和阳极氧化的方法，在纯钛表面构建微纳米形貌，以人成骨肉瘤细胞系MG63为研究对象，全面系统评价了微纳米形貌对成骨细胞生物学行为的影响，并采用分子生物学方法，深入研究参与材料形貌调控细胞行为的信号通路途经，以阐明骨植入材料的表面形貌对骨组织形成的影响的分子生物学机制。本研究的方法及结果不仅为骨植入材料的表面结构的优化设计及基因修饰提供依据以达到提高骨植入材料的骨结合效率的目的，而且将为探明其它材料表面因素如表面化学成分或其它骨植入材料与细胞及组织相互作用的分子机制研究提供参考，从而更加全面指导骨植入材料的表面设计。第一部分纯钛表面微纳米形貌的制备【目的】采用课题组前期实验中已建立的纯钛表面微纳米形貌的制备方案，制备含有不同管径TiO2纳米管的微纳米复合梯度形貌，为后续的实验提供研究模型。【方法】将纯钛试样抛光至镜面，采用5%的氢氟酸酸蚀试样30min，试样超声清洗后，采用稳压直流阳极氧化电源在5V和20V两个电压下分别对试样进行阳极氧化处理1h，采用场发射扫描电镜观察试样表面形貌。【结果】纯钛表面经酸蚀和阳极氧化处理后形成微米坑表面复合不同管径TiO2纳米管的微纳米复合结构。TiO2纳米管管径的大小与阳极氧化电压成正比，5V和20V电压下分别形成管径约为30nm和100nm的纳米管阵列。【结论】酸蚀和阳极氧化方法能够在纯钛表面形成微米坑复合不同管径TiO2纳米管的微纳米形貌。第二部分微纳米形貌对成骨细胞生物学行为的影响【目的】评价微纳米形貌对成骨细胞生物学行为的影响。【方法】将人骨肉瘤细胞系MG63细胞接种于材料表面，采用扫描电镜观察试样表面细胞的伸展形态，MTT方法观察细胞增殖水平，实时定量PCR方法检测细胞在材料表面成骨相关基因的表达水平，采用天狼星红染色观察试样表面成骨细胞的胶原分泌能力，采用茜苏红染色观察材料表面成骨细胞的细胞外基质矿化能力。【结果】成骨细胞MG63在微纳米表面充分伸展，细胞随着TiO2纳米管管径的增大而呈现逐渐伸长的形态。微纳米形貌对细胞的增殖没有显著性影响，在30nm管径纳米管表面，增殖能力稍有下降。微纳米形貌显著上调了细胞成骨相关基因的表达水平，促进了成骨细胞的胶原分泌能力和细胞外基质矿化能力，并且这种促进作用在含有100nm纳米管的微纳米形貌表面更加明显。【结论】微纳米形貌能够促进成骨细胞MG63的成骨分化功能，含有大管径纳米管（100nm）的微纳米形貌对成骨分化的促进作用更加明显。第三部分微纳米形貌表面Wnt/-catenin信号通路对成骨细胞的影响【目的】检测Wnt/-catenin信号通路是否参与材料对细胞行为的影响，评价Wnt/-catenin信号通路在微纳米形貌调控成骨细胞功能中的作用。【方法】将MG63细胞接种于材料表面，采用实时定量PCR方法检测试样表面MG63细胞中Wnt信号通路中受体、激活因子和抑制因子的表达水平，采用Westernblot检测细胞核内-catenin水平；将外源性Wnt信号通路激活剂Wnt3a和抑制剂Dkk1分别加入到细胞培养液中，随后采用Western blot检测细胞核内-catenin水平，并采用实时定量PCR方法检测试样表面成骨相关基因的表达水平，采用商业试剂盒检测成骨细胞碱性磷酸酶表达水平，采用天狼星红染色检测成骨细胞胶原分泌能力的变化，采用MTT方法检测细胞增殖能力，采用流式细胞术检测细胞凋亡的变化。【结果】微纳米形貌上调了Wnt信号通路受体--低密度脂蛋白相关蛋白6（LRP6）和Wnt信号通路激活剂Wnt3a的表达，下调了Wnt通路抑制因子Dkk1/2和分泌型卷曲蛋白相关蛋白1/2（sFRP1/2）的表达，Wnt/-catenin信号在微纳米形貌表面被激活。在光滑表面加入外源性Wnt信号激活剂Wnt3a上调Wnt/-catenin信号活性后，成骨分化相关指标如：成骨基因的表达、碱性磷酸酶水平和胶原分泌能力都随之增高。在微纳米形貌表面加入外源性Wnt信号抑制剂Dkk1后，微纳米表面原本增高的Wnt/-catenin活性受到明显的抑制，成骨分化相关指标的表达水平也随之受到明显的抑制。材料表面改变Wnt/-catenin信号活性后对细胞增殖和凋亡没有明显的影响。【结论】微纳米形貌通过调控Wnt信号通路中Wnt因子的表达，，激活Wnt/-catenin信号活性，被激活的Wnt/-catenin信号促进了成骨细胞在微纳米形貌表面的成骨分化。第四部分微纳米形貌表面ILK/-catenin信号通路对成骨细胞的影响【目的】检测整合素连接酶/-catenin（ILK/-catenin）信号通路在微纳米形貌调控成骨细胞生物学行为过程中的作用。【方法】将MG63细胞接种于试样表面，采用实时定量PCR方法检测-catenin、ILK、整合素1和3亚基的表达水平，采用western blot方法检测胞浆和胞核内-catenin、胞浆内磷酸化糖原合成激酶3（p-GSK3）和ILK的表达水平；采用小干扰RNA转染方法沉默ILK的表达后，检测试样表面细胞成骨基因表达水平的改变、胶原分泌能力和细胞外基质矿化水平的变化，并再一次采用western blot检测胞浆和胞核内-catenin、胞浆内GSK3和p-GSK3表达水平的变化。【结果】微纳米形貌促进了整合素1和3亚基和ILK的表达。在微纳米表面高表达的ILK一方面通过促进-catenin mRNA的表达，另一方面通过磷酸化GSK3进而抑制-catenin在胞浆内的降解，两方面共同作用激活了-catenin信号活性。采用小干扰RNA下调ILK表达后，微纳米形貌表面的成骨相关基因的表达水平、胶原分泌能力和细胞外基质矿化水平也随之显著下降。【结论】ILK/-catenin信号通路参与调控微纳米形貌对成骨分化功能的影响。第五部分微纳米形貌表面ILK/ERK1/2信号通路对成骨细胞的影响【目的】检测整合素连接酶/细胞外调节蛋白激酶1/2（ILK/ERK1/2）信号通路在微纳米形貌调控成骨细胞生物学行为过程中的作用。【方法】将MG63细胞接种到材料表面，采用western blot方法检测试样表面总ERK1/2、磷酸化ERK1/2和ILK的表达水平；采用小干扰RNA转染的方法沉默ILK表达后，再次采用western blot检测试样表面总ERK1/2和磷酸化ERK1/2表达水平的变化。将ERK1/2信号通路抑制剂U0126加入到细胞培养液中处理细胞后，采用MTT方法检测试样表面细胞增殖能力的变化，采用实时定量PCR方法检测细胞成骨分化相关基因表达水平的变化，采用商业试剂盒染色检测成骨细胞碱性磷酸酶分泌水平的变化，采用天狼星红染色检测细胞胶原分泌能力的变化，采用茜苏红染色检测成骨细胞细胞外基质矿化水平的变化。【结果】微纳米形貌显著性上调了ILK和磷酸化ERK1/2的活性。通过小干扰RNA转染下调ILK表达之后，微纳米形貌表面磷酸化ERK1/2的活性也显著降低。U0126显著性抑制了微纳米形貌表面细胞的增殖水平、成骨基因表达水平、胶原分泌能力和细胞外基质矿化水平。【结论】ILK/ERK1/2信号通路参与调控微纳米形貌对成骨增殖和分化的影响。
[Abstract]:Pure titanium surface micro-and nano-morphology can well simulate the structure of human bone tissue and extracellular microenvironment, so it may have better biological effect on osteoblasts, but the specific molecular mechanism of cell regulation by material morphology is still unclear. Osteosarcoma cell line MG63 was studied. The effects of micro-and nano-morphology on the biological behavior of osteoblasts were comprehensively and systematically evaluated. The signal pathways involved in the morphological regulation of osteosarcoma cells were studied by molecular biological methods to clarify the effects of surface morphology of bone implants on bone formation. The methods and results of this study not only provide the basis for optimizing the surface structure and gene modification of bone implant materials to improve the bone-binding efficiency of bone implant materials, but also reveal the surface factors of other materials such as surface chemical composition or the interaction between other bone implant materials and cells and tissues. Sub mechanism research provides a reference for more comprehensive guidance on the surface design of bone implant materials.
Part one
Preparation of micro and nano morphology on pure titanium surface
[Objective] The Gradient Morphology of titanium nanotubes with different diameters was prepared by using the preparation method of the surface micro-nano morphology of pure titanium which had been established in the previous experiment of our research group.
[Methods] The pure titanium sample was polished to the mirror surface, and the sample was etched with 5% hydrofluoric acid for 30 minutes. After ultrasonic cleaning, the samples were anodized for 1 h at 5V and 20V respectively by a voltage regulated DC anodizing power supply. The surface morphology of the samples was observed by field emission scanning electron microscopy.
[Results] The surface of pure titanium was treated by acid etching and anodic oxidation to form micro-and nano-composite structure with different diameter of nanotubes. The diameter of nanotubes was proportional to anodic oxidation voltage, and nanotube arrays with diameter of about 30 nm and 100 nm were formed at 5V and 20V respectively.
[Conclusion] Acid etching and anodic oxidation can form micro-pits on the surface of pure titanium and form micro-and nano-morphologies of TiO2 nanotubes with different diameters.
The second part
Effects of micro and nano morphology on biological behavior of osteoblasts
[Objective] to evaluate the effect of micro and nano morphology on the biological behavior of osteoblasts.
[Methods] Human osteosarcoma cell line MG63 was inoculated on the surface of the material. The morphology of osteoblasts was observed by scanning electron microscopy, the proliferation of osteoblasts was observed by MTT, the expression of osteogenic related genes on the surface of the material was detected by real-time quantitative PCR, and the osteoblasts on the surface of the sample were observed by Sirius red staining. The ability of collagen secretion was observed by Alizarin staining.
[Results] Osteoblasts MG63 fully extended on the surface of micro-nano-tubes and gradually elongated with the increase of the diameter of the nano-tubes. The morphology of micro-nano-tubes had no significant effect on the proliferation of the cells. On the surface of the nano-tubes with 30 nm diameter, the proliferation ability of osteoblasts decreased slightly. At the same level, the collagen secretion ability and extracellular matrix mineralization ability of osteoblasts were promoted, and this effect was more obvious on the surface of the micro-nano morphology containing 100 nm nanotubes.
[Conclusion] Micronano-morphology can promote osteogenic differentiation of osteoblasts MG63, and the effect of micronano-morphology with large diameter nanotubes (100 nm) on osteogenic differentiation is more obvious.
The third part
Effects of Wnt/-catenin signal pathway on osteoblasts on micro and nano morphology surfaces
[Objective] To investigate the role of Wnt /-catenin signaling pathway in the regulation of osteoblast function by micro-and nano-morphology.
[Methods] MG63 cells were seeded on the surface of the material, and the expression levels of Wnt receptor, activator and inhibitor in MG63 cells were detected by real-time quantitative PCR. The levels of intranuclear-catenin were detected by Western blot, and the exogenous Wnt signal pathway activator Wnt3a and inhibitor Dkk1 were added to MG63 cells respectively. In the culture medium, Western blot was used to detect the level of - Catenin in the nucleus, real-time quantitative PCR was used to detect the expression of osteogenic related genes, commercial kit was used to detect the expression of alkaline phosphatase in osteoblasts, and Sirius red staining was used to detect the change of collagen secretion ability of osteoblasts. Cell proliferation was detected by MTT, and apoptosis was detected by flow cytometry.
[Results] Micronano-morphology up-regulated the expression of Wnt signaling pathway receptor, low density lipoprotein-related protein 6 (LRP6) and Wnt signaling pathway activator Wnt3a, down-regulated the expression of Wnt pathway inhibitor Dkk1/2 and secretory convolution protein-related protein 1/2 (sFRP1/2), and activated the Wnt/-catenin signal on the surface of micronano-morphology. When the exogenous Wnt signal activator Wnt3a was added to the surface of micronano-particles, the expression of osteogenic genes, the level of alkaline phosphatase and the ability of collagen secretion were increased. The expression levels of osteogenic differentiation-related indicators were also significantly inhibited by the obvious inhibition. There was no significant effect on the proliferation and apoptosis of the cells by altering the Wnt/catenin signal activity on the surface of the material.
[Conclusion] Micron-nano morphology can activate Wnt /-catenin signal activity by regulating the expression of Wnt factor in Wnt signaling pathway, and the activated Wnt /-catenin signal promotes osteogenic differentiation of osteoblasts on the surface of micron-nano morphology.
The fourth part
Effects of ILK/-catenin signal pathway on osteoblasts on micro and nano morphology surfaces
[Objective] To investigate the role of integrin ligase /-catenin (ILK /-catenin) signaling pathway in the regulation of osteoblast biological behavior by micro-and nano-morphology.
[Methods] MG63 cells were inoculated on the surface of the sample, and the expression levels of - catenin, ILK, integrin 1 and 3 subunits were detected by real-time quantitative PCR, and the expression levels of - catenin, phosphorylated glycogen synthase kinase 3 (p-GSK3) and ILK in the cytoplasm and nucleus were detected by Western blot. After expression, the changes of osteogenic gene expression, collagen secretion and extracellular matrix mineralization were detected, and the expressions of intracytoplasmic and nuclear-catenin, GSK3 and p-GSK3 were detected by Western blot.
[Results] Integrin-1 and integrin-3 subunits and IL-K were enhanced by micro-and nano-morphology. High expression of IL-K on the micro-and nano-surface activated the activity of-catenin signal by promoting the expression of-catenin mRNA on the one hand and inhibiting the degradation of-catenin in the cytoplasm by phosphorylating GSK3 on the other. After K expression, the expression level of osteogenesis-related genes, collagen secretion ability and extracellular matrix mineralization on the surface of micro-and nano-morphology decreased significantly.
[Conclusion] ILK/-catenin signaling pathway is involved in regulating the effect of micro and nano morphology on osteogenic differentiation.
The fifth part
Effects of ILK/ERK1/2 signal pathway on osteoblasts on micro and nano morphology surfaces
[Objective] To investigate the role of integrin ligase/extracellular regulated protein kinase 1/2 (ILK/ERK1/2) signaling pathway in the morphological regulation of osteoblasts.
[Methods] MG63 cells were seeded onto the surface of the material, and the total ERK1/2, phosphorylated ERK1/2 and ILK expression levels were detected by Western blot. After the ILK expression was silenced by small interfering RNA transfection, the total ERK1/2 and phosphorylated ERK1/2 expression levels were detected by Western blot. After the cells were treated with U0126, MTT was used to detect the changes of cell proliferation, real-time quantitative PCR was used to detect the expression of osteogenic differentiation-related genes, and commercial kit staining was used to detect the changes of alkaline phosphatase secretion in osteoblasts. Sirius red staining was used to detect the changes of collagen secretion and alizarin staining was used to detect the mineralization of extracellular matrix of osteoblasts.
[Results] The activity of ILK and phosphorylated ERK1/2 were significantly up-regulated by micro-and nano-morphology. The activity of phosphorylated ERK1/2 on the surface of micro-and nano-morphology was also significantly down-regulated by small interfering RNA transfection. U0126 significantly inhibited the proliferation level of cells on the surface of micro-and nano-morphology, osteogenic gene expression level, collagen secretion ability and fineness. Extracellular matrix mineralization level.
[Conclusion] ILK/ERK1/2 signaling pathway is involved in regulating the effect of micro and nano morphology on the proliferation and differentiation of osteoblasts.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R783.1

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