硅—二氧化钛人工关节假体微孔涂层材料促成骨能力及其机理的体外实验研究

发布时间：2018-09-11 09:22

【摘要】：[目的]探讨微弧氧化硅-二氧化钛微孔涂层材料的表面特征，及其对成骨细胞系MC3T3-E1细胞粘附、增殖活性的影响。 [方法]配制含硅酸钠的电解液，采用微弧氧化法制备含硅离子的硅-二氧化钛微孔涂层材料（Si-TiO_2），对该涂层的表面特征、化学组成等进行检测。以Si-TiO_2微孔涂层材料作为实验组，以二氧化钛（TiO_2）和纯钛（Ti）作为对照组，按一定的密度在三种材料表面接种成骨细胞系MC3T3-E1细胞。接种24小时后鬼笔环肽染色，激光共聚焦显微镜观察各种材料表面MC3T3-E1细胞骨架蛋白的分布情况；12和24小时扫描电镜观察细胞在三种材料表面的伸展状况；第1，4，7和14天MTT法检测三种材料表面的细胞活力。 [结果]采用微弧氧化法制备的Si-TiO_2微孔涂层表面已形成多孔的纳米级涂层，已成功引入微量元素硅（Si），相组成大多为锐钛矿TiO_2。将含硅离子的微孔涂层（Si-TiO_2）与未含硅离子的微孔涂层（TiO_2）相比，结果显示硅离子的引入并未明显改变微孔涂层的形貌特征和相组成。而Si-TiO_2组在接种MC3T3-E1细胞24小时后骨架蛋白的分布较对照组明显密集，12和24小时后扫描电镜显示Si-TiO_2组表面细胞的伸展明显优于对照组；MTT法检测结果表明，，接种后第4和7天Si-TiO_2组表面的细胞活力较对照组均显著增高，有统计学差异（p0.05），但在接种后第14天时三组无明显差别。 [结论]微弧氧化法不仅能在钛金属表面形成纳米级微孔涂层，且能在不改变涂层原有形貌特征和相组成的情况下引入硅离子。Si-TiO_2微孔涂层能有效促进成骨细胞系MC3T3-E1细胞在其表面的粘附和增殖活性。 [目的]探讨微弧氧化硅-二氧化钛微孔涂层材料对成骨细胞MC3T3-E1早期分化及成骨特异性基因表达的影响。 [方法]以硅-二氧化钛微孔涂层材料（Si-TiO_2）作为实验组，以二氧化钛涂层材料（TiO_2）和纯钛（Ti）作为对照组，按一定的密度在三种材料表面接种成骨细胞系MC3T3-E1细胞，分别于接种后第1、4、7和14天四个时间点收集细胞，并检测成骨细胞早期分化特异性因子碱性磷酸酶(ALP)活性；实时定量PCR（Real-time PCR）检测早期分化特异性基因的表达，包括ALP、成骨细胞特异转录因子Runx2、型胶原(Coll-1)。 [结果]细胞接种后第4和7天，Si-TiO_2组ALP活性均明显高于其余两组，而在接种后第14天时，各组间ALP活性无明显差异，但Si-TiO_2组ALP活性较第7天有下降趋势。Real-time PCR检测显示，Si-TiO_2组ALP基因表达在第4和7天均明显高于其余两组（p0.05），而第14天时各组间ALP基因表达无统计学差异，但Si-TiO_2组ALP基因表达较第7天有下降趋势；各组Runx2基因表达随细胞培养时间的延长呈现出逐渐增加的趋势，Si-TiO_2组Runx2基因的表达于接种后第7天时明显高于其余两组（p0.05），第14天时Si-TiO_2和TiO_2组Runx2基因的表达明显高于Ti组（p0.05），而Si-TiO_2组稍高于TiO_2组，但无显著性差异；Coll-1基因表达结果显示，接种后第1,4天两个时间点各组之间无明显差异，但第7、14天时，Si-TiO_2组的表达量明显高于其余两组（p0.05）。 [结论] Si-TiO_2微孔涂层材料能在早期促进成骨细胞系MC3T3-E1的分化，上调成骨细胞早期分化特异性基因的表达，加速骨的形成。表明Si-TiO_2涂层材料具有良好的生物活性和生物相容性。 [目的]探讨微弧氧化硅-二氧化钛微孔涂层材料对成骨细胞系MC3T3-E1细胞矿化及特异性蛋白表达的影响。 [方法]以硅-二氧化钛微孔涂层材料（Si-TiO_2）为实验组，以二氧化钛涂层材料（TiO_2）和纯钛（Ti）作为对照组，按一定的密度在三种材料表面接种成骨细胞系MC3T3-E1细胞。分别于接种后第1、4、7、14天个时间点收集标本，实时定量PCR（Real-time PCR）检测成骨细胞晚期矿化的蛋白标志物骨唾液蛋白(BSP)、骨钙素(OCN)的mRNA表达；Western-blot法检测BSP、OCN接种后1、7、14天的蛋白表达。 [结果]各组BSP的mRNA表达随培养时间延长呈现出逐渐增加的趋势，接种后第1、4天两个时间点在各组之间无明显差异，但第7、14天时，Si-TiO_2组BSP表达明显高于其余两组（p0.05）。OCN的mRNA表达于第4、7、14天三个时间点（p0.05），Si-TiO_2组的表达量均高于其余两组（p0.05）。Western-blot结果显示，各组BSP和OCN蛋白表达随培养时间的延长也呈现出逐渐增加的趋势，在接种后第1天时，各组的BSP和OCN蛋白表达无统计学差异,而在第7、14天时，Si-TiO_2组BSP和OCN的蛋白表达均明显高于其余两组（p0.05）。 [结论] Si-TiO_2微孔涂层材料能通过上调成骨细胞系MC3T3-E1细胞矿化期特异性基因和蛋白的表达，促进成骨细胞系MC3T3-E1细胞的矿化。可能与该涂层表面的多孔粗糙和引入硅离子有关。 [目的]探讨硅-二氧化钛微孔涂层材料对成骨细胞系MC3T3-E1细胞凋亡的影响。 [方法]以硅-二氧化钛微孔涂层材料（Si-TiO_2）作为实验组，以二氧化钛（TiO_2）和纯钛（Ti）作为对照组，按一定的密度在三种材料表面接种成骨细胞MC3T3-E1，分别于接种后的第1、4、7和14天检测成骨细胞Caspase-3的活性，Hoechst染色检测细胞的凋亡数量、流式细胞仪检测细胞凋亡率。 [结果]各组细胞凋亡数量随细胞培养时间的延长先增加后减少，接种第1天时细胞凋亡少见；第4天时，细胞凋亡增加，而第7天细胞凋亡最明显，至第14天有所减轻，而以Si-TiO_2组凋亡最显著，其它两组间无显著性差异；各组材料表面的细胞内Caspase-3活性第一天无明显差别。第4、7、14天时，Si-TiO_2组细胞内Caspase-3活性明显高于Ti和TiO_2组，有显著性差异（p0.05）。与第7天相比，Si-TiO_2组第14天Caspase-3活性有所降低；第1天时，三组细胞凋亡率均较低，第4、7天，三组细胞凋亡率均增高，第7天时，Si-TiO_2组增高最明显。第14天时，三组凋亡率较前均降低。第4、7、14天Si-TiO_2组细胞凋亡率明显高于Ti和TiO_2组，有显著性差异（p0.05）。 [结论] Si-TiO_2微孔涂层材料能在早、中期显著诱导成骨细胞凋亡。 [目的]探讨微弧氧化硅-二氧化钛微孔涂层材料对成骨细胞系MC3T3-E1细胞生物活性调控的可能的信号通路。 [方法]以硅-二氧化钛微孔涂层材料（Si-TiO_2）作为实验组，以二氧化钛（TiO_2）和纯钛（Ti）作为对照组，按一定的密度在三种材料表面接种成骨细胞系MC3T3-E1细胞，分别于接种后的第1、4、7和14天收集细胞，实时定量PCR（Real-time PCR）法检测Wnt信号通路分子低密度脂蛋白受体相关蛋白（Lrp5）、Dickkopf1（Dkk1）及EPRK信号通路分子ERK1/2及其下游转录因子c-fos的mRNA表达。 [结果]各组Lrp5基因表达随细胞培养时间的延长逐渐增加，接种后第7天时，Si-TiO_2组Lrp5的表达显著高于其余两组（p 0.05），第14天时，Si-TiO_2组Lrp5的表达也明显高于Ti涂层组（p 0.05）。Dkk1基因表达结果却相反，Si-TiO_2、TiO_2组Dkk1基因表达随着培养时间的延长而逐渐降低，而Ti组Dkk1基因表达随着培养时间的延长而增高，第4、7、14天，Si-TiO_2组Dkk1基因的表达显著低于其于两组，差别有显著意义（p 0.05）。各组ERK1mRNA表达随细胞培养时间的延长逐渐增加，至第4天时，各组ERK1的表达均增高，而Si-TiO_2组增高与Ti组相比有显著性差异（p0.05）；至第7、14天时，Si-TiO_2组ERK1基因表达显著高于其于两组（p0.05）。而ERK2mRNA表达在各时间点各组间无明显差别。接种第1、4、7、14天，Si-TiO_2组c-fos的表达均显著高于Ti和TiO_2组（p0.05）；第4天时，Si-TiO_2组c-fos的表达水平最高，至第7、14天时有所降低。 [结论] Si-TiO_2涂层材料可能通过Wnt和EPRK信号通路的活化调控成骨细胞的生物活性。
[Abstract]:[Objective] To investigate the surface characteristics of micro-arc silica-titanium dioxide microporous coating material and its effect on the adhesion and proliferation of osteoblast MC3T3-E1 cells.
[Methods] Silicon-titanium dioxide microporous coating material (Si-TiO_2) containing silicon ion was prepared by micro-arc oxidation method in the electrolyte containing sodium silicate. The surface characteristics and chemical composition of the coating were tested. The Si-TiO_2 microporous coating material was used as the experimental group, and the titanium dioxide (TiO_2) and pure titanium (Ti) as the control group. MC3T3-E1 cells were inoculated on the surface of the three materials. The distribution of cytoskeleton proteins on the surface of MC3T3-E1 cells was observed by laser confocal microscopy after 24 hours of inoculation. The cell elongation on the surface of the three materials was observed by scanning electron microscopy at 12 and 24 hours. MTT assay was used on the 1st, 4th, 7th and 14th days. Surface cell viability.
[Results] Porous nano-coatings were formed on the surface of Si-TiO_2 micro-porous coatings prepared by micro-arc oxidation method. Trace element silicon (Si) was successfully introduced into the coatings. The phase composition of the coatings was mostly anatase titanium dioxide. The distribution of cytoskeleton protein in the Si-TiO_2 group was significantly denser than that in the control group 24 hours after inoculation. Scanning electron microscopy after 12 and 24 hours showed that the cell extension of the Si-TiO_2 group was significantly better than that of the control group. MTT assay showed that the cells on the surface of the Si-TiO_2 group on the 4th and 7th days after inoculation were significantly denser than that of the control group. Compared with the control group, the activity increased significantly (p0.05), but there was no significant difference among the three groups on the 14th day after inoculation.
[Conclusion] Micro-arc oxidation can not only form nano-porous coatings on titanium surface, but also introduce silicon ions without changing the original morphology and phase composition of the coatings.
[Objective] To investigate the effect of micro-arc silica-titanium dioxide microporous coating material on the early differentiation of osteoblasts MC3T3-E1 and the expression of osteoblast-specific genes.
[Methods] Silicon-titanium dioxide microporous coating material (Si-TiO_2) was used as the experimental group, titanium dioxide coating material (TiO_2) and pure titanium (Ti) as the control group. The osteoblast line MC3T3-E1 cells were inoculated on the surface of the three materials at a certain density. The cells were collected at the first, fourth, seventh and fourteenth days after inoculation, and the early osteoblasts were detected. Differentiation-specific factor alkaline phosphatase (ALP) activity; Real-time PCR (Real-time PCR) was used to detect the expression of early differentiation-specific genes, including ALP, osteoblast-specific transcription factor Runx2 and collagen type 1.
[Results] On the 4th and 7th day after inoculation, the ALP activity in Si-TiO_2 group was significantly higher than that in the other two groups, but on the 14th day after inoculation, there was no significant difference in ALP activity among the three groups, but the ALP activity in Si-TiO_2 group was decreased compared with that in the 7th day. Real-time PCR showed that the expression of ALP gene in Si-TiO_2 group was significantly higher than that in the other two groups on the 4th and 7th day (p0.05). There was no significant difference in the expression of ALP gene between the groups on the 14th day, but the expression of ALP gene in the Si-TiO_2 group decreased compared with the 7th day. The expression of Runx2 gene in the Si-TiO_2 group increased gradually with the prolongation of cell culture time. The expression of Runx2 gene in the Si-TiO_2 group was significantly higher than that in the other two groups on the 7th day after inoculation (p0.05). The expression of Runx2 gene in the two groups was significantly higher than that in the Ti group (p0.05), while that in the Si-TiO_2 group was slightly higher than that in the Ti_2 group, but there was no significant difference. The expression of Coll-1 gene showed no significant difference between the two groups at the 1st and 4th day after inoculation, but at the 7th and 14th day, the expression of Runx2 gene in the Si-TiO_2 group was significantly higher than that in the other two groups (p0.05).
[Conclusion] Si-TiO_2 microporous coating material can promote the differentiation of osteoblast line MC3T3-E1 in the early stage, up-regulate the expression of early differentiation-specific genes of osteoblasts, and accelerate the formation of bone.
[Objective] To investigate the effect of micro-arc silica-titanium dioxide microporous coating material on mineralization and specific protein expression of osteoblast cell line MC3T3-E1.
[Methods] Osteoblast line MC3T3-E1 cells were seeded on the surface of silicon-titanium dioxide microporous coating material (Si-TiO 2) as experimental group, titanium dioxide coating material (TiO 2) and pure titanium (Ti) as control group at a certain density. Samples were collected at 1,4,7,14 days after inoculation and detected by real-time quantitative PCR (Real-time PCR). Bone salivary protein (BSP), osteocalcin (OCN), a protein marker of osteoblast late mineralization, was expressed in mRNA. The expression of BSP and OCN was detected by Western blot at 1,7,14 days after inoculation.
[Results] The expression of BSP mRNA in each group increased gradually with the prolongation of culture time. There was no significant difference between the two groups at 1 and 4 days after inoculation, but at 7 and 14 days, the expression of BSP in Si-TiO_2 group was significantly higher than that in the other two groups (p0.05). The expression of OCN mRNA in Si-TiO_2 group was higher than that in the other two groups (p0.05). Western-blot results showed that the expression of BSP and OCN protein in each group also showed a gradual increase with the prolongation of culture time. At the first day after inoculation, the expression of BSP and OCN protein in each group had no statistical difference, but at the 7th and 14th day, the expression of BSP and OCN protein in Si-TiO_2 group was significantly higher than that in the other two groups (p0.05).
[Conclusion] Si-TiO_2 microporous coating materials can promote the mineralization of osteoblast MC3T3-E1 cells by up-regulating the expression of mineralization-specific genes and proteins in osteoblast MC3T3-E1 cells, which may be related to the porous surface roughness of the coating and the introduction of silicon ions.
[Objective] to investigate the effect of silica titania microporous coating material on the apoptosis of osteoblast MC3T3-E1 cells.
[Methods] Osteoblasts MC3T3-E1 were inoculated on the surface of silicon-titanium dioxide microporous coating material (Si-TiO 2) as experimental group, titanium dioxide (TiO 2) and pure titanium (Ti) as control group at a certain density. The activity of osteoblasts Caspase-3 was detected on the first, fourth, seventh and fourteenth days after inoculation, and the apoptosis was detected by Hoechst staining. The cell apoptosis rate was detected by flow cytometry.
[Results] The number of apoptotic cells in each group increased first and then decreased with the prolongation of cell culture time, apoptosis was rare on the first day of inoculation, increased on the fourth day, and the apoptosis was most obvious on the seventh day, and decreased on the fourteenth day. The apoptosis in Si-TiO_2 group was the most significant, and there was no significant difference between the other two groups. Caspase-3 activity was significantly higher in Si-TiO_2 group than in Ti and TiO_2 group on the 4th, 7th and 14th day (p0.05). Compared with the 7th day, Caspase-3 activity in Si-TiO_2 group decreased on the 14th day, and the apoptosis rate in the 3rd group was lower on the 1st day, and increased on the 4th and 7th day. On the 7th day, the apoptosis rate of Si-TiO_2 group was significantly higher than that of Ti and TiO_2 group. On the 14th day, the apoptosis rate of Si-TiO_2 group was significantly lower than that of Ti and TiO_2 group (p0.05).
[Conclusion] Si-TiO_2 microporous coating material can significantly induce osteoblast apoptosis in the early and middle stages.
[Objective] To investigate the possible signaling pathway of microarc silicon oxide-titanium dioxide microporous coating material in regulating the biological activity of osteoblast cell line MC3T3-E1.
[Methods] Osteoblast line MC3T3-E1 cells were seeded on the surface of silicon-titanium dioxide microporous coating material (Si-TiO 2) as experimental group, titanium dioxide (TiO 2) and pure titanium (Ti) as control group. The cells were collected on the 1st, 4th, 7th and 14th day after inoculation, and the Wnt signal was detected by real-time quantitative PCR (Real-time PCR). Expression of low density lipoprotein receptor-related protein (Lrp5), Dickkopf1 (Dkk1) and EPRK signaling pathway molecule ERK1/2 and its downstream transcription factor c-fos mRNA.
[Results] Lrp5 gene expression increased gradually with the prolongation of cell culture time. On the 7th day after inoculation, the expression of Lrp5 in Si-TiO_2 group was significantly higher than that in the other two groups (p 0.05). On the 14th day, the expression of Lrp5 in Si-TiO_2 group was also significantly higher than that in Ti-coated group (p 0.05). On the contrary, the expression of Dkk1 gene in Si-TiO_2 and TiO_2 groups was significantly higher than that in Ti-coated group (p 0.05). The expression of Dkk1 gene in Ti group increased with the prolongation of culture time. On the 4th, 7th and 14th day, the expression of Dkk1 gene in Si-TiO_2 group was significantly lower than that in the two groups (p The expression of ERK1 gene in Si-TiO_2 group was significantly higher than that in Ti group (p0.05) on the 7th and 14th day, but there was no significant difference in the expression of ERK2 mRNA between each group at each time point. On the 1st, 4th, 7th and 14th day of inoculation, the expression of c-fos in Si-TiO_2 group was significantly higher than that in Ti and TiO_2 group (p0.05); on the 4th day, the expression of c-fos in Si-TiO_2 group was significantly higher than that in Ti and Ti_2 group (p0.05). The expression level of -fos was the highest, and decreased to 7,14 days.
[Conclusion] Si-TiO_2 coating materials may regulate the biological activity of osteoblasts through activation of Wnt and EPRK signaling pathways.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08

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