【摘要】：[目的]探討微弧氧化硅-二氧化鈦微孔涂層材料的表面特征，及其對(duì)成骨細(xì)胞系MC3T3-E1細(xì)胞粘附、增殖活性的影響。 [方法]配制含硅酸鈉的電解液，采用微弧氧化法制備含硅離子的硅-二氧化鈦微孔涂層材料（Si-TiO_2），對(duì)該涂層的表面特征、化學(xué)組成等進(jìn)行檢測(cè)。以Si-TiO_2微孔涂層材料作為實(shí)驗(yàn)組，以二氧化鈦（TiO_2）和純鈦（Ti）作為對(duì)照組，按一定的密度在三種材料表面接種成骨細(xì)胞系MC3T3-E1細(xì)胞。接種24小時(shí)后鬼筆環(huán)肽染色，激光共聚焦顯微鏡觀察各種材料表面MC3T3-E1細(xì)胞骨架蛋白的分布情況；12和24小時(shí)掃描電鏡觀察細(xì)胞在三種材料表面的伸展?fàn)顩r；第1，4，7和14天MTT法檢測(cè)三種材料表面的細(xì)胞活力。 [結(jié)果]采用微弧氧化法制備的Si-TiO_2微孔涂層表面已形成多孔的納米級(jí)涂層，已成功引入微量元素硅（Si），相組成大多為銳鈦礦TiO_2。將含硅離子的微孔涂層（Si-TiO_2）與未含硅離子的微孔涂層（TiO_2）相比，結(jié)果顯示硅離子的引入并未明顯改變微孔涂層的形貌特征和相組成。而Si-TiO_2組在接種MC3T3-E1細(xì)胞24小時(shí)后骨架蛋白的分布較對(duì)照組明顯密集，12和24小時(shí)后掃描電鏡顯示Si-TiO_2組表面細(xì)胞的伸展明顯優(yōu)于對(duì)照組；MTT法檢測(cè)結(jié)果表明，，接種后第4和7天Si-TiO_2組表面的細(xì)胞活力較對(duì)照組均顯著增高，有統(tǒng)計(jì)學(xué)差異（p0.05），但在接種后第14天時(shí)三組無(wú)明顯差別。 [結(jié)論]微弧氧化法不僅能在鈦金屬表面形成納米級(jí)微孔涂層，且能在不改變涂層原有形貌特征和相組成的情況下引入硅離子。Si-TiO_2微孔涂層能有效促進(jìn)成骨細(xì)胞系MC3T3-E1細(xì)胞在其表面的粘附和增殖活性。 [目的]探討微弧氧化硅-二氧化鈦微孔涂層材料對(duì)成骨細(xì)胞MC3T3-E1早期分化及成骨特異性基因表達(dá)的影響。 [方法]以硅-二氧化鈦微孔涂層材料（Si-TiO_2）作為實(shí)驗(yàn)組，以二氧化鈦涂層材料（TiO_2）和純鈦（Ti）作為對(duì)照組，按一定的密度在三種材料表面接種成骨細(xì)胞系MC3T3-E1細(xì)胞，分別于接種后第1、4、7和14天四個(gè)時(shí)間點(diǎn)收集細(xì)胞，并檢測(cè)成骨細(xì)胞早期分化特異性因子堿性磷酸酶(ALP)活性；實(shí)時(shí)定量PCR（Real-time PCR）檢測(cè)早期分化特異性基因的表達(dá)，包括ALP、成骨細(xì)胞特異轉(zhuǎn)錄因子Runx2、型膠原(Coll-1)。 [結(jié)果]細(xì)胞接種后第4和7天，Si-TiO_2組ALP活性均明顯高于其余兩組，而在接種后第14天時(shí)，各組間ALP活性無(wú)明顯差異，但Si-TiO_2組ALP活性較第7天有下降趨勢(shì)。Real-time PCR檢測(cè)顯示，Si-TiO_2組ALP基因表達(dá)在第4和7天均明顯高于其余兩組（p0.05），而第14天時(shí)各組間ALP基因表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異，但Si-TiO_2組ALP基因表達(dá)較第7天有下降趨勢(shì)；各組Runx2基因表達(dá)隨細(xì)胞培養(yǎng)時(shí)間的延長(zhǎng)呈現(xiàn)出逐漸增加的趨勢(shì)，Si-TiO_2組Runx2基因的表達(dá)于接種后第7天時(shí)明顯高于其余兩組（p0.05），第14天時(shí)Si-TiO_2和TiO_2組Runx2基因的表達(dá)明顯高于Ti組（p0.05），而Si-TiO_2組稍高于TiO_2組，但無(wú)顯著性差異；Coll-1基因表達(dá)結(jié)果顯示，接種后第1,4天兩個(gè)時(shí)間點(diǎn)各組之間無(wú)明顯差異，但第7、14天時(shí)，Si-TiO_2組的表達(dá)量明顯高于其余兩組（p0.05）。 [結(jié)論] Si-TiO_2微孔涂層材料能在早期促進(jìn)成骨細(xì)胞系MC3T3-E1的分化，上調(diào)成骨細(xì)胞早期分化特異性基因的表達(dá)，加速骨的形成。表明Si-TiO_2涂層材料具有良好的生物活性和生物相容性。 [目的]探討微弧氧化硅-二氧化鈦微孔涂層材料對(duì)成骨細(xì)胞系MC3T3-E1細(xì)胞礦化及特異性蛋白表達(dá)的影響。 [方法]以硅-二氧化鈦微孔涂層材料（Si-TiO_2）為實(shí)驗(yàn)組，以二氧化鈦涂層材料（TiO_2）和純鈦（Ti）作為對(duì)照組，按一定的密度在三種材料表面接種成骨細(xì)胞系MC3T3-E1細(xì)胞。分別于接種后第1、4、7、14天個(gè)時(shí)間點(diǎn)收集標(biāo)本，實(shí)時(shí)定量PCR（Real-time PCR）檢測(cè)成骨細(xì)胞晚期礦化的蛋白標(biāo)志物骨唾液蛋白(BSP)、骨鈣素(OCN)的mRNA表達(dá)；Western-blot法檢測(cè)BSP、OCN接種后1、7、14天的蛋白表達(dá)。 [結(jié)果]各組BSP的mRNA表達(dá)隨培養(yǎng)時(shí)間延長(zhǎng)呈現(xiàn)出逐漸增加的趨勢(shì)，接種后第1、4天兩個(gè)時(shí)間點(diǎn)在各組之間無(wú)明顯差異，但第7、14天時(shí)，Si-TiO_2組BSP表達(dá)明顯高于其余兩組（p0.05）。OCN的mRNA表達(dá)于第4、7、14天三個(gè)時(shí)間點(diǎn)（p0.05），Si-TiO_2組的表達(dá)量均高于其余兩組（p0.05）。Western-blot結(jié)果顯示，各組BSP和OCN蛋白表達(dá)隨培養(yǎng)時(shí)間的延長(zhǎng)也呈現(xiàn)出逐漸增加的趨勢(shì)，在接種后第1天時(shí)，各組的BSP和OCN蛋白表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異,而在第7、14天時(shí)，Si-TiO_2組BSP和OCN的蛋白表達(dá)均明顯高于其余兩組（p0.05）。 [結(jié)論] Si-TiO_2微孔涂層材料能通過(guò)上調(diào)成骨細(xì)胞系MC3T3-E1細(xì)胞礦化期特異性基因和蛋白的表達(dá)，促進(jìn)成骨細(xì)胞系MC3T3-E1細(xì)胞的礦化�？赡芘c該涂層表面的多孔粗糙和引入硅離子有關(guān)。 [目的]探討硅-二氧化鈦微孔涂層材料對(duì)成骨細(xì)胞系MC3T3-E1細(xì)胞凋亡的影響。 [方法]以硅-二氧化鈦微孔涂層材料（Si-TiO_2）作為實(shí)驗(yàn)組，以二氧化鈦（TiO_2）和純鈦（Ti）作為對(duì)照組，按一定的密度在三種材料表面接種成骨細(xì)胞MC3T3-E1，分別于接種后的第1、4、7和14天檢測(cè)成骨細(xì)胞Caspase-3的活性，Hoechst染色檢測(cè)細(xì)胞的凋亡數(shù)量、流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率。 [結(jié)果]各組細(xì)胞凋亡數(shù)量隨細(xì)胞培養(yǎng)時(shí)間的延長(zhǎng)先增加后減少，接種第1天時(shí)細(xì)胞凋亡少見(jiàn)；第4天時(shí)，細(xì)胞凋亡增加，而第7天細(xì)胞凋亡最明顯，至第14天有所減輕，而以Si-TiO_2組凋亡最顯著，其它兩組間無(wú)顯著性差異；各組材料表面的細(xì)胞內(nèi)Caspase-3活性第一天無(wú)明顯差別。第4、7、14天時(shí)，Si-TiO_2組細(xì)胞內(nèi)Caspase-3活性明顯高于Ti和TiO_2組，有顯著性差異（p0.05）。與第7天相比，Si-TiO_2組第14天Caspase-3活性有所降低；第1天時(shí)，三組細(xì)胞凋亡率均較低，第4、7天，三組細(xì)胞凋亡率均增高，第7天時(shí)，Si-TiO_2組增高最明顯。第14天時(shí)，三組凋亡率較前均降低。第4、7、14天Si-TiO_2組細(xì)胞凋亡率明顯高于Ti和TiO_2組，有顯著性差異（p0.05）。 [結(jié)論] Si-TiO_2微孔涂層材料能在早、中期顯著誘導(dǎo)成骨細(xì)胞凋亡。 [目的]探討微弧氧化硅-二氧化鈦微孔涂層材料對(duì)成骨細(xì)胞系MC3T3-E1細(xì)胞生物活性調(diào)控的可能的信號(hào)通路。 [方法]以硅-二氧化鈦微孔涂層材料（Si-TiO_2）作為實(shí)驗(yàn)組，以二氧化鈦（TiO_2）和純鈦（Ti）作為對(duì)照組，按一定的密度在三種材料表面接種成骨細(xì)胞系MC3T3-E1細(xì)胞，分別于接種后的第1、4、7和14天收集細(xì)胞，實(shí)時(shí)定量PCR（Real-time PCR）法檢測(cè)Wnt信號(hào)通路分子低密度脂蛋白受體相關(guān)蛋白（Lrp5）、Dickkopf1（Dkk1）及EPRK信號(hào)通路分子ERK1/2及其下游轉(zhuǎn)錄因子c-fos的mRNA表達(dá)。 [結(jié)果]各組Lrp5基因表達(dá)隨細(xì)胞培養(yǎng)時(shí)間的延長(zhǎng)逐漸增加，接種后第7天時(shí)，Si-TiO_2組Lrp5的表達(dá)顯著高于其余兩組（p 0.05），第14天時(shí)，Si-TiO_2組Lrp5的表達(dá)也明顯高于Ti涂層組（p 0.05）。Dkk1基因表達(dá)結(jié)果卻相反，Si-TiO_2、TiO_2組Dkk1基因表達(dá)隨著培養(yǎng)時(shí)間的延長(zhǎng)而逐漸降低，而Ti組Dkk1基因表達(dá)隨著培養(yǎng)時(shí)間的延長(zhǎng)而增高，第4、7、14天，Si-TiO_2組Dkk1基因的表達(dá)顯著低于其于兩組，差別有顯著意義（p 0.05）。各組ERK1mRNA表達(dá)隨細(xì)胞培養(yǎng)時(shí)間的延長(zhǎng)逐漸增加，至第4天時(shí)，各組ERK1的表達(dá)均增高，而Si-TiO_2組增高與Ti組相比有顯著性差異（p0.05）；至第7、14天時(shí)，Si-TiO_2組ERK1基因表達(dá)顯著高于其于兩組（p0.05）。而ERK2mRNA表達(dá)在各時(shí)間點(diǎn)各組間無(wú)明顯差別。接種第1、4、7、14天，Si-TiO_2組c-fos的表達(dá)均顯著高于Ti和TiO_2組（p0.05）；第4天時(shí)，Si-TiO_2組c-fos的表達(dá)水平最高，至第7、14天時(shí)有所降低。 [結(jié)論] Si-TiO_2涂層材料可能通過(guò)Wnt和EPRK信號(hào)通路的活化調(diào)控成骨細(xì)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.08

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