阴极弧沉积二氧化钛掺硅涂层对成骨细胞生物活性影响的实验研究

发布时间：2018-06-08 00:16

本文选题：阴极弧沉积 + 硅元素　；参考：《苏州大学》2013年博士论文

【摘要】：第一部分钛基关节假体阴极弧沉积二氧化钛掺硅涂层的制备及表征目的：利用过滤阴极弧沉积技术制备的涂层具有结合力强的特性，采用该方法在钛基假体表面沉积含有硅元素的二氧化钛涂层及不含硅的单纯二氧化钛涂层，制备二氧化钛掺硅涂层及二氧化钛涂层，并检测其表征。方法：将硅片和钛片作为阴极等离子源，共沉积于钛基关节假体表面，制备含有硅元素的二氧化钛涂层，将该涂层与以双钛片为阴极源沉积的纯二氧化钛涂层进行对照研究。以扫描电镜观察两种涂层的表面形貌，X射线光电子能谱仪测定两组涂层的元素组成，X射线衍射仪测定两组涂层的相组成，自动化接触角测量仪测量两组涂层表面的水接触角、对两组涂层的可湿性进行分析，并检测两组涂层的表面能。结果：X射线光电子能谱分析显示，阴极弧沉积二氧化钛掺硅涂层中含有4.6%的硅元素；X射线衍射结果显示这两种涂层可能是无定形态，钛基质峰显示清晰；扫描电镜结果显示已掺入的硅元素未显著改变涂层的表面形貌。二氧化钛掺硅涂层及二氧化钛涂层接触角分别为83±0.74°、94±0.78°，二氧化钛掺硅涂层的亲水性改善。二氧化钛涂层的表面能明显低于二氧化钛掺硅涂层组。结论：掺硅未明显改变过滤阴极弧沉积二氧化钛掺硅涂层的表面形貌，但涂层的化学组成发生改变，改善了涂层的亲水性，增加了涂层的表面能；结合力强的阴极弧沉积二氧化钛掺硅涂层若具有良好的细胞生物活性，将为未来植入物表面改性提供新的选择，该新型植入物涂层值得进一步研究。第二部分钛基关节假体阴极弧沉积二氧化钛掺硅涂层对成骨细胞粘附活性的影响目的：研究阴极弧沉积二氧化钛掺硅涂层对成骨细胞MG63粘附活性的影响。方法：以二氧化钛掺硅涂层作为实验组，单纯二氧化钛涂层作为对照组，以相同的密度在两组涂层表面分别种植MG63细胞。采用细胞计数CCK8法分别在1、4、12、24小时检测粘附于两组涂层表面的细胞数目；扫描电镜于第4、12、24小时观察成骨细胞在两组材料表面的铺展情况；培养2、12小时，进行细胞肌动蛋白张力纤维、细胞核两重荧光染色，培养12、24小时，进行粘着斑蛋白、肌动蛋白张力纤维、细胞核三重荧光染色，观察两组涂层表面细胞局部粘着斑及细胞骨架的分布形态。结果：MG63细胞在两组涂层表面培养1、4小时，CCK8检测结果显示粘附在两组涂层表面的细胞数未见显著差异(P0.05)；当培养时间延长至12、24小时，CCK8检测结果显示粘附于二氧化钛掺硅涂层表面的细胞数量较二氧化钛涂层表面的细胞数量显著增多(P 0.05)；培养2小时，，两组涂层表面细胞肌动蛋白张力纤维荧光染色形态相似，当培养时间延长到12、24小时，Si-TiO2涂层表面MG63细胞肌动蛋白和粘着斑蛋白的分布、表达较二氧化钛涂层组增强；培养4小时，电镜观察两组涂层表面细胞形态未见差别；培养12小时，MG63细胞在二氧化钛掺硅涂层表面伪足生长较二氧化钛涂层表面细胞伪足更长、更密；培养24小时，二氧化钛掺硅涂层表面细胞覆盖面积较二氧化钛掺硅涂层表面细胞覆盖面积显著增加(P 0.05)。结论：在阴极弧沉积二氧化钛掺硅涂层表面培养的MG63细胞肌动蛋白张力纤维和粘着斑蛋白明显增强，这促进了细胞骨架重构及局部焦点粘附的形成，钛基关节假体阴极弧沉积二氧化钛掺硅涂层可以有效促进成骨细胞MG63的粘附。第三部分钛基关节假体阴极弧沉积二氧化钛掺硅涂层对成骨细胞增殖、凋亡的影响目的：研究阴极弧沉积二氧化钛掺硅涂层对成骨细胞MG63增殖、凋亡的影响。方法：以二氧化钛掺硅涂层作为实验组，单纯二氧化钛涂层作为对照组，分别以相同的密度种植MG63细胞进行培养。细胞培养1、5天后，使用场发射电镜分别观察两组涂层表面细胞的生长情况；流式细胞仪分析细胞在两组涂层表面生长1、5天后的细胞存活和凋亡百分比。培养1、3、5天，采用细胞计数CCK8法分别检测两组涂层表面MG63细胞增殖数量。结果：电镜观察结果表明，培养1、5天，二氧化钛掺硅涂层表面细胞增殖数量较单纯二氧化钛涂层明显增多。流式细胞术分析结果显示：培养1、5天，在两组涂层表面培养的MG63细胞存活和凋亡比例未见明显差异(P0.05)。CCK8检测结果表明，细胞培养1、3、5天，二氧化钛掺硅涂层表面MG63细胞增殖数显著多于单纯二氧化钛涂层表面细胞增殖数(P 0.05)。结论：阴极弧沉积二氧化钛掺硅涂层表面培养的成骨细胞MG63增殖数目较二氧化钛涂层表面细胞数目明显增多，显示二氧化钛掺硅涂层能够有效促进细胞在其表面增殖，而二氧化钛掺硅涂层与单纯二氧化钛涂层对于在其表面培养的成骨细胞MG63的凋亡未见明显差异，表明阴极弧沉积二氧化钛掺硅涂层是一种细胞生物活性良好的涂层材料。第四部分钛基关节假体阴极弧沉积二氧化钛掺硅涂层对成骨细胞分化活性的影响目的：研究阴极弧沉积二氧化钛掺硅涂层对成骨细胞MG63分化活力的影响。方法：以二氧化钛掺硅涂层作为实验组，单纯二氧化钛涂层作为对照组，表面种植相同密度的MG63细胞进行培养。分别于第1、3、5天三个时间点收集标本，检测两组涂层表面MG63细胞碱性磷酸酶活性。MG63细胞在两组涂层表面分别培养3、5天，收集标本进行细胞I型胶原荧光染色，荧光显微镜观察两组涂层表面成骨细胞MG63的分化标志物I型胶原的表达。细胞在两组涂层表面培养1、3、5天三个时间点收集标本，采用实时定量RT-PCR法检测两组涂层表面MG63细胞I型胶原及骨钙素的基因表达；采用Western blot法检测两组涂层表面MG63细胞I型胶原的蛋白表达。结果：检测结果显示两组涂层表面MG63细胞碱性磷酸酶活性随培养时间的增加均逐渐增高。在培养1天时，两组涂层表面细胞碱性磷酸酶活性无明显差异(P0.05)，但在培养时间延长至第3、5天时二氧化钛掺硅涂层表面细胞的碱性磷酸酶活性明显高于单纯二氧化钛涂层组(P 0.05)。荧光显微镜观察，在两组涂层表面细胞培养至第3、5天时I型胶原染色结果显示，二氧化钛掺硅涂层表面MG63细胞I型胶原表达丰富，分布广泛，而单纯二氧化钛涂层表面MG63细胞I型胶原表达量少且稀疏。实时定量RT-PCR检测结果显示，二氧化钛掺硅涂层表面MG63细胞I型胶原及骨钙素基因表达在培养第1天时与对照组无明显差异(P0.05)，但在培养第3、5天时二氧化钛掺硅涂层表面MG63细胞I型胶原及骨钙素基因表达明显高于单纯二氧化钛涂层组(P 0.05)；Western blot检测结果显示两组涂层表面MG63细胞I型胶原蛋白表达在培养第1天时，两组之间无明显差异(P0.05)，但培养至第3、5天时，二氧化钛掺硅涂层表面MG63细胞I型胶原蛋白表达量明显较单纯二氧化钛涂层组增高(P 0.05)。结论：与阴极弧沉积二氧化钛涂层相比，二氧化钛掺硅涂层通过增加碱性磷酸酶活性，上调骨钙素基因、Ⅰ型胶原纤维基因和蛋白表达促进MG63细胞分化，表明阴极弧沉积二氧化钛掺硅涂层是一种生物活性良好的涂层材料。第五部分钛基关节假体阴极弧沉积二氧化钛掺硅涂层促进成骨细胞粘附活性的信号转导通道目的：研究阴极弧沉积二氧化钛掺硅涂层对成骨细胞MG63粘附活性影响的可能信号转导通道。方法：以二氧化钛掺硅涂层作为实验组，单纯二氧化钛涂层作为对照组，表面种植相同密度的MG63细胞进行培养。分别于细胞培养后第1、4、12、24小时四个时间点收集标本，采用实时定量RT-PCR法检测两组涂层表面MG63细胞整合素β1、粘着斑激酶基因表达；细胞培养后于第4、12、24小时三个时间点收集标本，Western blot法检测两组涂层表面MG63细胞粘着斑激酶、粘着斑激酶磷酸化蛋白表达。结果：实时定量RT-PCR法检测显示，细胞培养1、4小时，两组涂层表面MG63细胞整合素β1基因表达无统计学差异(P0.05)，细胞培养12、24小时，二氧化钛掺硅涂层表面MG63细胞整合素β1基因表达显著高于单纯二氧化钛涂层组(P 0.05)；细胞培养1小时，两组涂层表面MG63细胞粘着斑激酶基因表达无统计学差异(P0.05)，细胞培养4、12、24小时，二氧化钛掺硅涂层表面MG63细胞粘着斑激酶基因表达显著高于单纯二氧化钛涂层组(P 0.05)；细胞培养4小时，Western blot法检测显示两组涂层表面MG63细胞粘着斑激酶、粘着斑激酶磷酸化蛋白表达无统计学差异(P0.05)，细胞培养时间延长至12、24小时，二氧化钛掺硅涂层表面MG63细胞粘着斑激酶、粘着斑激酶磷酸化蛋白表达显著高于单纯二氧化钛涂层组(P 0.05)。结论：阴极弧沉积二氧化钛掺硅涂层与单纯二氧化钛涂层相比能促进MG63粘附活性，二氧化钛掺硅涂层对细胞骨架和粘着斑的调节可能受整合素途径和粘着斑激酶磷酸化的介导，并通过整合素β1-粘着斑激酶信号转导途径影响MG63细胞的粘附活性。
[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 卤 0.74 掳 , 94 卤 0.78 掳 , 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 .
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.17

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