陰極弧沉積二氧化鈦摻硅涂層對(duì)成骨細(xì)胞生物活性影響的實(shí)驗(yàn)研究
本文選題:陰極弧沉積 + 硅元素 ; 參考:《蘇州大學(xué)》2013年博士論文
【摘要】:第一部分鈦基關(guān)節(jié)假體陰極弧沉積二氧化鈦摻硅涂層的制備及表征 目的:利用過(guò)濾陰極弧沉積技術(shù)制備的涂層具有結(jié)合力強(qiáng)的特性,采用該方法在鈦基假體表面沉積含有硅元素的二氧化鈦涂層及不含硅的單純二氧化鈦涂層,制備二氧化鈦摻硅涂層及二氧化鈦涂層,并檢測(cè)其表征。方法:將硅片和鈦片作為陰極等離子源,共沉積于鈦基關(guān)節(jié)假體表面,制備含有硅元素的二氧化鈦涂層,將該涂層與以雙鈦片為陰極源沉積的純二氧化鈦涂層進(jìn)行對(duì)照研究。以掃描電鏡觀察兩種涂層的表面形貌,X射線光電子能譜儀測(cè)定兩組涂層的元素組成,X射線衍射儀測(cè)定兩組涂層的相組成,自動(dòng)化接觸角測(cè)量?jī)x測(cè)量?jī)山M涂層表面的水接觸角、對(duì)兩組涂層的可濕性進(jìn)行分析,并檢測(cè)兩組涂層的表面能。結(jié)果:X射線光電子能譜分析顯示,陰極弧沉積二氧化鈦摻硅涂層中含有4.6%的硅元素;X射線衍射結(jié)果顯示這兩種涂層可能是無(wú)定形態(tài),鈦基質(zhì)峰顯示清晰;掃描電鏡結(jié)果顯示已摻入的硅元素未顯著改變涂層的表面形貌。二氧化鈦摻硅涂層及二氧化鈦涂層接觸角分別為83±0.74°、94±0.78°,二氧化鈦摻硅涂層的親水性改善。二氧化鈦涂層的表面能明顯低于二氧化鈦摻硅涂層組。結(jié)論:摻硅未明顯改變過(guò)濾陰極弧沉積二氧化鈦摻硅涂層的表面形貌,但涂層的化學(xué)組成發(fā)生改變,改善了涂層的親水性,增加了涂層的表面能;結(jié)合力強(qiáng)的陰極弧沉積二氧化鈦摻硅涂層若具有良好的細(xì)胞生物活性,將為未來(lái)植入物表面改性提供新的選擇,該新型植入物涂層值得進(jìn)一步研究。 第二部分鈦基關(guān)節(jié)假體陰極弧沉積二氧化鈦摻硅涂層對(duì)成骨細(xì)胞粘附活性的影響 目的:研究陰極弧沉積二氧化鈦摻硅涂層對(duì)成骨細(xì)胞MG63粘附活性的影響。方法:以二氧化鈦摻硅涂層作為實(shí)驗(yàn)組,單純二氧化鈦涂層作為對(duì)照組,以相同的密度在兩組涂層表面分別種植MG63細(xì)胞。采用細(xì)胞計(jì)數(shù)CCK8法分別在1、4、12、24小時(shí)檢測(cè)粘附于兩組涂層表面的細(xì)胞數(shù)目;掃描電鏡于第4、12、24小時(shí)觀察成骨細(xì)胞在兩組材料表面的鋪展情況;培養(yǎng)2、12小時(shí),進(jìn)行細(xì)胞肌動(dòng)蛋白張力纖維、細(xì)胞核兩重?zé)晒馊旧,培養(yǎng)12、24小時(shí),進(jìn)行粘著斑蛋白、肌動(dòng)蛋白張力纖維、細(xì)胞核三重?zé)晒馊旧^察兩組涂層表面細(xì)胞局部粘著斑及細(xì)胞骨架的分布形態(tài)。結(jié)果:MG63細(xì)胞在兩組涂層表面培養(yǎng)1、4小時(shí),CCK8檢測(cè)結(jié)果顯示粘附在兩組涂層表面的細(xì)胞數(shù)未見(jiàn)顯著差異(P0.05);當(dāng)培養(yǎng)時(shí)間延長(zhǎng)至12、24小時(shí),CCK8檢測(cè)結(jié)果顯示粘附于二氧化鈦摻硅涂層表面的細(xì)胞數(shù)量較二氧化鈦涂層表面的細(xì)胞數(shù)量顯著增多(P 0.05);培養(yǎng)2小時(shí),,兩組涂層表面細(xì)胞肌動(dòng)蛋白張力纖維熒光染色形態(tài)相似,當(dāng)培養(yǎng)時(shí)間延長(zhǎng)到12、24小時(shí),Si-TiO2涂層表面MG63細(xì)胞肌動(dòng)蛋白和粘著斑蛋白的分布、表達(dá)較二氧化鈦涂層組增強(qiáng);培養(yǎng)4小時(shí),電鏡觀察兩組涂層表面細(xì)胞形態(tài)未見(jiàn)差別;培養(yǎng)12小時(shí),MG63細(xì)胞在二氧化鈦摻硅涂層表面?zhèn)巫闵L(zhǎng)較二氧化鈦涂層表面細(xì)胞偽足更長(zhǎng)、更密;培養(yǎng)24小時(shí),二氧化鈦摻硅涂層表面細(xì)胞覆蓋面積較二氧化鈦摻硅涂層表面細(xì)胞覆蓋面積顯著增加(P 0.05)。結(jié)論:在陰極弧沉積二氧化鈦摻硅涂層表面培養(yǎng)的MG63細(xì)胞肌動(dòng)蛋白張力纖維和粘著斑蛋白明顯增強(qiáng),這促進(jìn)了細(xì)胞骨架重構(gòu)及局部焦點(diǎn)粘附的形成,鈦基關(guān)節(jié)假體陰極弧沉積二氧化鈦摻硅涂層可以有效促進(jìn)成骨細(xì)胞MG63的粘附。 第三部分鈦基關(guān)節(jié)假體陰極弧沉積二氧化鈦摻硅涂層對(duì)成骨細(xì)胞增殖、凋亡的影響 目的:研究陰極弧沉積二氧化鈦摻硅涂層對(duì)成骨細(xì)胞MG63增殖、凋亡的影響。方法:以二氧化鈦摻硅涂層作為實(shí)驗(yàn)組,單純二氧化鈦涂層作為對(duì)照組,分別以相同的密度種植MG63細(xì)胞進(jìn)行培養(yǎng)。細(xì)胞培養(yǎng)1、5天后,使用場(chǎng)發(fā)射電鏡分別觀察兩組涂層表面細(xì)胞的生長(zhǎng)情況;流式細(xì)胞儀分析細(xì)胞在兩組涂層表面生長(zhǎng)1、5天后的細(xì)胞存活和凋亡百分比。培養(yǎng)1、3、5天,采用細(xì)胞計(jì)數(shù)CCK8法分別檢測(cè)兩組涂層表面MG63細(xì)胞增殖數(shù)量。結(jié)果:電鏡觀察結(jié)果表明,培養(yǎng)1、5天,二氧化鈦摻硅涂層表面細(xì)胞增殖數(shù)量較單純二氧化鈦涂層明顯增多。流式細(xì)胞術(shù)分析結(jié)果顯示:培養(yǎng)1、5天,在兩組涂層表面培養(yǎng)的MG63細(xì)胞存活和凋亡比例未見(jiàn)明顯差異(P0.05)。CCK8檢測(cè)結(jié)果表明,細(xì)胞培養(yǎng)1、3、5天,二氧化鈦摻硅涂層表面MG63細(xì)胞增殖數(shù)顯著多于單純二氧化鈦涂層表面細(xì)胞增殖數(shù)(P 0.05)。結(jié)論:陰極弧沉積二氧化鈦摻硅涂層表面培養(yǎng)的成骨細(xì)胞MG63增殖數(shù)目較二氧化鈦涂層表面細(xì)胞數(shù)目明顯增多,顯示二氧化鈦摻硅涂層能夠有效促進(jìn)細(xì)胞在其表面增殖,而二氧化鈦摻硅涂層與單純二氧化鈦涂層對(duì)于在其表面培養(yǎng)的成骨細(xì)胞MG63的凋亡未見(jiàn)明顯差異,表明陰極弧沉積二氧化鈦摻硅涂層是一種細(xì)胞生物活性良好的涂層材料。 第四部分鈦基關(guān)節(jié)假體陰極弧沉積二氧化鈦摻硅涂層對(duì)成骨細(xì)胞分化活性的影響 目的:研究陰極弧沉積二氧化鈦摻硅涂層對(duì)成骨細(xì)胞MG63分化活力的影響。方法:以二氧化鈦摻硅涂層作為實(shí)驗(yàn)組,單純二氧化鈦涂層作為對(duì)照組,表面種植相同密度的MG63細(xì)胞進(jìn)行培養(yǎng)。分別于第1、3、5天三個(gè)時(shí)間點(diǎn)收集標(biāo)本,檢測(cè)兩組涂層表面MG63細(xì)胞堿性磷酸酶活性。MG63細(xì)胞在兩組涂層表面分別培養(yǎng)3、5天,收集標(biāo)本進(jìn)行細(xì)胞I型膠原熒光染色,熒光顯微鏡觀察兩組涂層表面成骨細(xì)胞MG63的分化標(biāo)志物I型膠原的表達(dá)。細(xì)胞在兩組涂層表面培養(yǎng)1、3、5天三個(gè)時(shí)間點(diǎn)收集標(biāo)本,采用實(shí)時(shí)定量RT-PCR法檢測(cè)兩組涂層表面MG63細(xì)胞I型膠原及骨鈣素的基因表達(dá);采用Western blot法檢測(cè)兩組涂層表面MG63細(xì)胞I型膠原的蛋白表達(dá)。結(jié)果:檢測(cè)結(jié)果顯示兩組涂層表面MG63細(xì)胞堿性磷酸酶活性隨培養(yǎng)時(shí)間的增加均逐漸增高。在培養(yǎng)1天時(shí),兩組涂層表面細(xì)胞堿性磷酸酶活性無(wú)明顯差異(P0.05),但在培養(yǎng)時(shí)間延長(zhǎng)至第3、5天時(shí)二氧化鈦摻硅涂層表面細(xì)胞的堿性磷酸酶活性明顯高于單純二氧化鈦涂層組(P 0.05)。熒光顯微鏡觀察,在兩組涂層表面細(xì)胞培養(yǎng)至第3、5天時(shí)I型膠原染色結(jié)果顯示,二氧化鈦摻硅涂層表面MG63細(xì)胞I型膠原表達(dá)豐富,分布廣泛,而單純二氧化鈦涂層表面MG63細(xì)胞I型膠原表達(dá)量少且稀疏。實(shí)時(shí)定量RT-PCR檢測(cè)結(jié)果顯示,二氧化鈦摻硅涂層表面MG63細(xì)胞I型膠原及骨鈣素基因表達(dá)在培養(yǎng)第1天時(shí)與對(duì)照組無(wú)明顯差異(P0.05),但在培養(yǎng)第3、5天時(shí)二氧化鈦摻硅涂層表面MG63細(xì)胞I型膠原及骨鈣素基因表達(dá)明顯高于單純二氧化鈦涂層組(P 0.05);Western blot檢測(cè)結(jié)果顯示兩組涂層表面MG63細(xì)胞I型膠原蛋白表達(dá)在培養(yǎng)第1天時(shí),兩組之間無(wú)明顯差異(P0.05),但培養(yǎng)至第3、5天時(shí),二氧化鈦摻硅涂層表面MG63細(xì)胞I型膠原蛋白表達(dá)量明顯較單純二氧化鈦涂層組增高(P 0.05)。結(jié)論:與陰極弧沉積二氧化鈦涂層相比,二氧化鈦摻硅涂層通過(guò)增加堿性磷酸酶活性,上調(diào)骨鈣素基因、Ⅰ型膠原纖維基因和蛋白表達(dá)促進(jìn)MG63細(xì)胞分化,表明陰極弧沉積二氧化鈦摻硅涂層是一種生物活性良好的涂層材料。 第五部分鈦基關(guān)節(jié)假體陰極弧沉積二氧化鈦摻硅涂層促進(jìn)成骨細(xì)胞粘附活性的信號(hào)轉(zhuǎn)導(dǎo)通道 目的:研究陰極弧沉積二氧化鈦摻硅涂層對(duì)成骨細(xì)胞MG63粘附活性影響的可能信號(hào)轉(zhuǎn)導(dǎo)通道。方法:以二氧化鈦摻硅涂層作為實(shí)驗(yàn)組,單純二氧化鈦涂層作為對(duì)照組,表面種植相同密度的MG63細(xì)胞進(jìn)行培養(yǎng)。分別于細(xì)胞培養(yǎng)后第1、4、12、24小時(shí)四個(gè)時(shí)間點(diǎn)收集標(biāo)本,采用實(shí)時(shí)定量RT-PCR法檢測(cè)兩組涂層表面MG63細(xì)胞整合素β1、粘著斑激酶基因表達(dá);細(xì)胞培養(yǎng)后于第4、12、24小時(shí)三個(gè)時(shí)間點(diǎn)收集標(biāo)本,Western blot法檢測(cè)兩組涂層表面MG63細(xì)胞粘著斑激酶、粘著斑激酶磷酸化蛋白表達(dá)。結(jié)果:實(shí)時(shí)定量RT-PCR法檢測(cè)顯示,細(xì)胞培養(yǎng)1、4小時(shí),兩組涂層表面MG63細(xì)胞整合素β1基因表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),細(xì)胞培養(yǎng)12、24小時(shí),二氧化鈦摻硅涂層表面MG63細(xì)胞整合素β1基因表達(dá)顯著高于單純二氧化鈦涂層組(P 0.05);細(xì)胞培養(yǎng)1小時(shí),兩組涂層表面MG63細(xì)胞粘著斑激酶基因表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),細(xì)胞培養(yǎng)4、12、24小時(shí),二氧化鈦摻硅涂層表面MG63細(xì)胞粘著斑激酶基因表達(dá)顯著高于單純二氧化鈦涂層組(P 0.05);細(xì)胞培養(yǎng)4小時(shí),Western blot法檢測(cè)顯示兩組涂層表面MG63細(xì)胞粘著斑激酶、粘著斑激酶磷酸化蛋白表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),細(xì)胞培養(yǎng)時(shí)間延長(zhǎng)至12、24小時(shí),二氧化鈦摻硅涂層表面MG63細(xì)胞粘著斑激酶、粘著斑激酶磷酸化蛋白表達(dá)顯著高于單純二氧化鈦涂層組(P 0.05)。結(jié)論:陰極弧沉積二氧化鈦摻硅涂層與單純二氧化鈦涂層相比能促進(jìn)MG63粘附活性,二氧化鈦摻硅涂層對(duì)細(xì)胞骨架和粘著斑的調(diào)節(jié)可能受整合素途徑和粘著斑激酶磷酸化的介導(dǎo),并通過(guò)整合素β1-粘著斑激酶信號(hào)轉(zhuǎn)導(dǎo)途徑影響MG63細(xì)胞的粘附活性。
[Abstract]:Preparation and characterization of titanium dioxide - doped silicon dioxide coating on the cathodic arc of the first part of titanium - based joint prosthesis
Objective : To prepare a titanium dioxide coating and a titanium dioxide coating with silicon element on the surface of titanium - based prosthesis by using the coating of titanium dioxide coating and titanium dioxide coating containing silicon element on the surface of titanium - based prosthesis by using the method of filtering cathodic arc deposition .
X - ray diffraction shows that the two coatings may be amorphous , and the titanium matrix peaks are clear ;
The results showed that the contact angle of titanium dioxide coating was 83 鹵 0.74 擄 , 94 鹵 0.78 擄 , and the hydrophilicity of TiO _ 2 - doped silicon coating was improved .
If the titanium dioxide - doped silicon dioxide coating with strong binding force has good cell biological activity , it will provide a new choice for surface modification of implants in the future , which is worth further study .
Effect of Ti - based joint prosthesis cathodic arc deposition on the adhesion of osteoblast in the second part of titanium - based joint prosthesis
Objective : To study the effect of titanium dioxide doped silicon dioxide coating on the adhesion of osteoblast MG63 . Methods : MG63 cells were grown on the surface of two groups of coating with titanium dioxide as experimental group and titanium dioxide coating as control group . The number of cells adhered to the surface of two groups of coating was measured at 1 , 4 , 12 and 24 hours by cell counting method .
Scanning electron microscope ( SEM ) was used to observe the spreading of osteoblast on the surface of two groups of materials at the 4th , 12th and 24th hours ;
The results showed that MG63 cells were cultured for 1 , 4 hours on the surface of two coats of coating . The results showed that MG63 cells had no significant difference in the number of cells adhered to the two groups of coated surfaces ( P0.05 ) .
When the culture time was prolonged to 12 and 24 hours , the results showed that the number of cells adhering to the surface of titania - doped coating increased significantly ( P 0.05 ) .
The morphology of actin filament was similar to that of the two groups of coated surface cells . When the culture time was prolonged to 12 and 24 hours , the distribution of actin and adhesion protein of MG63 cells was observed in the surface MG63 of Si - TiO2 coating .
The morphology of the surface cells of the two groups was observed by electron microscope for 4 hours .
After 12 hours of culture , MG63 cells were pseudopolytically grown on the surface of titanium dioxide doped silica coating , which was longer and denser than the surface cells of titanium dioxide coating .
The coverage area of the surface cells of the titanium dioxide - doped silica coating increased significantly compared with that of the titanium dioxide - doped coating ( P 0.05 ) . Conclusion : MG63 cells cultured on the surface of titanium dioxide doped with titanium dioxide on the surface of the cathode arc enhanced significantly , which promoted the formation of cytoskeletal remodeling and local focal adhesion , and the titanium - based joint prosthesis cathodic arc deposition titania - doped silica coating can effectively promote the adhesion of the osteoblast MG63 .
Effect of Ti - based joint prosthesis cathodic arc deposition on proliferation and apoptosis of osteoblast in third part of titanium - based joint prosthesis
Objective : To study the effect of titania - doped silica coating on proliferation and apoptosis of osteoblast MG63 cells .
The results showed that the proliferation of MG63 cells cultured for 1 , 5 days and 5 days in the coated surface of the two groups was significantly higher than that of the pure titanium dioxide coating ( P0.05 ) . The results showed that the proliferation of MG63 cells on the surface of the two groups was significantly higher than that of the pure titanium dioxide coating ( P 0.05 ) . Conclusion : The proliferation of osteoblasts MG63 on the surface of TiO _ 2 - doped TiO _ 2 coating is obviously increased compared with that of TiO _ 2 coating . It shows that the silica - doped coating can effectively promote the proliferation of the cells on its surface , while the silica - doped coating and the pure titanium dioxide coating have no obvious difference to the apoptosis of the osteoblasts MG63 cultured on the surface . It is shown that the titanium dioxide - doped coating is a kind of coating material with good biological activity .
Effect of Ti - based joint prosthesis cathodic arc deposition on osteoblast differentiation activity in the fourth part of titanium - based joint prosthesis
Objective : To study the effect of titanium dioxide doped silicon dioxide coating on the differentiation and activity of MG63 cells . Methods : Using titanium dioxide as experimental group , the MG63 cells with the same density were cultured on the surface of the two groups . The MG63 cells were cultured for 3 and 5 days at the 1st , 3rd and 5th day respectively . The samples were collected from three time points on the surface of the two groups . The expression of type I collagen and bone calcitrin in MG63 cells were detected by real time quantitative RT - PCR .
Results : The results showed that the alkaline phosphatase activity of MG63 cells increased gradually with the increase of culture time , but the activity of alkaline phosphatase in the surface cells of the two groups was significantly higher than that in the pure titanium dioxide coating group ( P 0.05 ) . The results showed that the expression of type I collagen of MG63 cells and the expression of type I collagen of MG63 cells were less and sparse in the surface MG63 cells . The results showed that the expression of type I collagen and bone calcium gene in MG63 cells was significantly higher than that in the control group ( P0.05 ) .
Western blot showed that there was no significant difference between the two groups ( P0.05 ) , but the expression of type I collagen of MG63 cells was significantly higher than that of pure titanium dioxide coating group ( P 0.05 ) . Conclusion : Compared with the cathodic arc deposition titanium dioxide coating , the silica - doped coating can promote the differentiation of MG63 cells by increasing the activity of alkaline phosphatase , up - regulation of bone - calvin gene , type I collagen fiber gene and protein expression , suggesting that the cathodic arc - deposited titanium dioxide - doped coating is a good biological active coating material .
Signal transduction pathway for promoting osteoblast adhesion activity by deposition of titanium dioxide - doped silicon dioxide coating on the cathodic arc of the fifth part of titanium - based joint prosthesis
Objective : To study the possible signal transduction pathways of titanium dioxide - doped silica coating on the adhesion of osteoblasts MG63 . Methods : Using titania - doped silicon coating as experimental group , the MG63 cells with the same density were cultured on the surface . The samples were collected at 4 , 12 and 24 hours after cell culture . The expression of integrin 尾1 and adhesion kinase gene was detected by real - time quantitative RT - PCR .
The expression of MG63 cell adhesion kinase and focal adhesion kinase phosphorylation protein was detected by Western blot at the 4th , 12th and 24th hours after cell culture . The results showed that the expression of integrin 尾1 gene in MG63 cells was significantly higher than that of titanium dioxide coating group ( P 0.05 ) .
There was no statistical difference between MG63 cell adhesion kinase gene expression in the two groups of coated surface MG63 cells ( P0.05 ) . The expression of adhesion kinase gene of MG63 cell was significantly higher than that of pure titanium dioxide coating group ( P 0.05 ) .
After 4 hours of cell culture , Western blot assay showed no statistical difference ( P0.05 ) . The expression of phosphorylated protein in MG63 cell was significantly higher than that of titanium dioxide coating group ( P 0.05 ) . Conclusion : The adhesion activity of MG63 can be promoted as compared with pure titanium dioxide coating by cathodic arc deposition . The regulation of the cytoskeletal and focal spot by titania - doped silica coating may be mediated by integrin pathway and phosphorylation of focal adhesion kinase , and the adhesion activity of MG63 cells can be influenced by integrin 尾1 - adhesion kinase signal transduction pathway .
【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】:2013
【分類號(hào)】:R318.17
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