人工关节假体阴极弧沉积氧化锆膜对成骨细胞生物活性影响的实验研究

发布时间：2018-06-19 16:11

本文选题：真空阴极弧沉积 + 氧化锆　；参考：《苏州大学》2013年博士论文

【摘要】：[摘要]目的：试图采用磁过滤真空阴极弧沉积法在钛金属假体表面制备氧化锆膜，并检测其表征。方法：采用磁过滤真空阴极弧沉积法在纯钛片表面制备氧化锆膜，然后对氧化锆膜的形貌特征、化学组成、相组成等进行表征检测。结果：检测显示真空阴极弧沉积制备的氧化锆膜比较均匀、光滑、致密，具有纳米结构的表面。结论：采用磁过滤真空阴极弧沉积系统可在纯钛表面制备均匀、致密的氧化锆膜，制备的氧化锆膜具有纳米结构的表面，阴极弧沉积氧化锆膜的纳米表面结构可能与它的生物活性有关。 [摘要]目的：探讨阴极弧沉积氧化锆膜对成骨样MG63细胞粘附及增殖活性的影响。方法：以阴极弧沉积ZrO_2膜作为实验组，纯Ti作为对照组，分别按一定的密度接种MG63细胞进行培养。MTT法检测1、6、12、24小时两组材料表面附着的成骨细胞数；扫描电镜观察12和24小时成骨细胞在两组材料表面的铺展情况；鬼笔环肽荧光染色倒置荧光显微镜观察12和24小时两组材料表面细胞骨架蛋白的表达。CCK-8法检测1、4、7、10天时两组材料表面成骨细胞的增殖活性。结果：MTT结果显示：1小时后，两组样品表面MG63细胞附着的数量差异无统计学意义（P＞0.05）；6、12和24小时后，ZrO_2膜表面的细胞数显著多于纯Ti表面，且差异具有统计学意义（P＜0.05）。扫描电镜观察结果显示12和24小时后ZrO_2组的细胞铺展优于Ti组，荧光显微镜观察结果显示12和24小时后ZrO_2组表面细胞骨架蛋白的表达多于Ti组；CCK结果显示：培养4天及7天后，ZrO_2膜表面的细胞增殖活性显著高于纯Ti表面，且差异具有统计学意义（P＜0.05）。结论：人工关节假体表面阴极弧沉积制备的氧化锆膜具有促进成骨细胞粘附及增殖的能力。 [摘要]目的：探讨阴极弧沉积氧化锆膜对成骨样MG63细胞分化表型标志物的影响。方法：以阴极弧沉积ZrO_2膜作为实验组，纯Ti作为对照组，分别按一定的密度接种MG63细胞进行培养。分别于第1、4、7、10天收集标本，检测碱性磷酸酶（ALP）活性；以定量RT-PCR法检测成骨细胞分化标志物碱性磷酸酶、I型胶原（COLI）及骨钙素（OC）的基因表达情况；荧光染色倒置荧光显微镜观察4天时COLI蛋白的表达；ELISA检测1、4、7、10天时成骨细胞分泌OC蛋白量。结果：在培养第1天时，两组碱性磷酸酶活性及基因表达差异无统计学意义（p＞0.05）；随着培养时间的延长，两组碱性磷酸酶活性及基因表达均呈现出逐渐增加的趋势，第4、7、10天时，ZrO_2组碱性磷酸酶活性及基因表达显著高于纯Ti组（p＜0.05）。第1天时，且两组COLI基因表达差异无统计学意义（p＞0.05）；在第4天及7天时，ZrO_2组COLI基因表达显著高于纯Ti组（p＜0.05），在第10天时，两组COLI基因表达无统计学差异（p＞0.05）。在第1天和第4天培养时，两组OC基因表达无统计学差异（p＞0.05）；但在第7天和第10天时，ZrO_2组的OC基因表达显著高于纯Ti组（p＜0.05）。第4天时，ZrO_2组表面的COLI蛋白合成量多于Ti组。两组OC蛋白分泌量在第1、4天时无统计学差异（p＞0.05）；但在第7、10天时，ZrO_2组OC蛋白分泌量均显著高于Ti组（p＜0.05）。结论：阴极弧沉积氧化锆膜具有提高MG63细胞分化表型标志物水平的能力，表明阴极弧沉积氧化锆膜是一种具有良好生物活性的涂层材料。 [摘要]目的：探讨人工关节假体表面阴极弧沉积氧化锆膜对成骨细胞破骨细胞分化相关基因表达及蛋白分泌的影响。方法：以阴极弧沉积ZrO_2膜作为实验组，纯Ti作为对照组，分别按一定的密度接种MG63细胞进行培养。分别于第1、4、7、10天收集标本，以定量RT-PCR法检测成骨细胞破骨细胞分化相关的骨保护素（OPG）和核因子κ B受体活化因子配体（RANKL）基因表达情况；ELISA检测1、4、7、10天时成骨细胞分泌OPG和RANKL蛋白量。结果：第1天及第4天时，两组OPG基因表达无统计学差异（p＞0.05）；第7天及第10天时，ZrO_2组OPG基因表达显著高于Ti组（p＜0.05）。第1天及第4天时，两组RANKL基因表达无显著差异（p＞0.05）；在第7天及第10天时，ZrO_2组的基因表达显著低于Ti组（p＜0.05）。培养第1天及第4天时，两组样品表面的成骨细胞OPG蛋白分泌无统计学差异（p＞0.05）；第7天及第10天时，ZrO_2组OPG蛋白分泌显著高于Ti组（p＜0.05）。第1天及第4天时，两组样品表面的成骨细胞RANKL蛋白分泌无显著差异（p＞0.05）；第7天及第10天时，ZrO_2组的RANKL蛋白分泌显著低于Ti组（p＜0.05）。结论：人工关节假体表面阴极弧沉积氧化锆膜能够上调成骨细胞OPG水平，同时下调RANKL水平，提高OPG/RANKL相对比率，从而抑制破骨细胞的分化和活性。［摘要］目的：探讨人工关节假体表面阴极弧沉积氧化锆膜对成骨样MG63细胞活性影响的可能信号传导通路。方法：以阴极弧沉积ZrO_2膜作为实验组，纯Ti作为对照组，分别按照一定的密度在表面接种MG63细胞进行培养。分别于第6小时，24小时，4天，7天后四个时间点收集标本，检测整合素β1、FAK、ERK1、ERK2、c-fos及c-jun的基因表达。结果：6、24小时及4天后，ZrO_2组整合素β1基因表达显著高于Ti组（p＜0.05）；7天后，两组整合素β1表达无统计学差异（p＞0.05）。6、24小时及4天后，ZrO_2组FAK基因表达显著高于Ti组（p＜0.05）；7天后，两组FAK基因表达无统计学差异（p＞0.05）；6小时及24小时后，ZrO_2组ERK1基因表达显著高于Ti组（p＜0.05）；4天及7天后，两组ERK1基因表达无显著差异（p＞0.05）。6、24小时及4天后，ZrO_2组ERK2基因表达显著高于T组（p＜0.05）；7天后，，两组样品表面的ERK2基因表达无显著差异（p＞0.05）。6、24小时、4天及7天后，ZrO_2组c-fos基因表达显著高于Ti组（p＜0.05）。6、24小时及4天后，ZrO_2组c-jun表达显著高于Ti组（p＜0.05）；7天后，两组样品表面的c-jun基因表达无统计学差异（p＞0.05）。结论：人工关节假体表面阴极弧沉积氧化锆膜可能通过整合素介导的MAPK/ERK信号传导途径影响细胞活性。
[Abstract]:[Abstract] Objective: to prepare zirconia film on the surface of titanium metal prosthesis by magnetic filtration vacuum cathodic arc deposition, and to detect its characterization. Method: the zirconium oxide film was prepared on the surface of pure titanium by vacuum cathodic arc deposition by magnetic filtration, and then the morphology, composition and phase composition of the zirconia film were detected. It is found that the zirconia films prepared by vacuum cathodic arc deposition are homogeneous, smooth, compact, and have nanoscale surfaces. Conclusion: a homogeneous, compact zirconia film can be prepared on pure titanium surface by magnetic filtration vacuum cathodic arc deposition system. The prepared zirconia film has the surface of nanoscale structure, and the cathode arc deposited nano zirconia film The surface structure may be related to its biological activity.
[Abstract] Objective: To investigate the effect of cathodic arc deposition of zirconium oxide film on the adhesion and proliferation of osteoid MG63 cells. Methods: the cathode arc deposition ZrO_2 membrane was used as the experimental group and the pure Ti was used as the control group. The.MTT method was used to detect the number of osteoblasts attached to the two groups of 1,6,12,24 hours by inoculating MG63 cells at a certain density. The spread of osteoblasts on the surface of the two groups of 12 and 24 hours was observed by scanning electron microscope; the expression of cytoskeleton protein on the surface of material surface of 12 and 24 hours two groups was observed by the fluorescence staining of phreous cyclic peptide fluorescent staining microscope. The proliferation activity of the osteoblasts on the surface of two groups of materials at 1,4,7,10 days was detected by.CCK-8. Results: the results of MTT showed that: 1 After hours, there was no significant difference in the number of MG63 cell adhesion on the surface of the two groups (P > 0.05). After 6,12 and 24 hours, the number of cells on the surface of the ZrO_2 membrane was significantly more than that of the pure Ti surface, and the difference was statistically significant (P < 0.05). The results of scanning electron microscopy showed that the cell spreading of the ZrO_2 group was better than the Ti group and the fluorescence microscope view after 12 and 24. The results showed that the expression of cytoskeleton protein on the surface of ZrO_2 group was more than that of Ti group after 12 and 24 hours. The results of CCK showed that the cell proliferation activity on the surface of ZrO_2 membrane was significantly higher than that on pure Ti surface at 4 and 7 days, and the difference was statistically significant (P < 0.05). Conclusion: the zirconia membrane prepared by the cathode arc deposition on the surface of artificial joint prosthesis It has the ability to promote osteoblast adhesion and proliferation.
[Abstract] Objective: To investigate the effect of cathodic arc deposition of zirconium oxide film on the differentiation phenotype markers of osteoid MG63 cells. Methods: the cathode arc deposition ZrO_2 membrane was used as the experimental group and the pure Ti was used as the control group. The cultured MG63 cells were inoculated at a certain density, and the alkaline phosphatase (ALP) activity was detected on day 1,4,7,10. Quantitative RT-PCR assay was used to detect the gene expression of alkaline phosphatase, I type collagen (COLI) and Osteocalcin (OC), and the expression of COLI protein at 4 days by fluorescence staining; ELISA was used to detect the secretion of OC protein in 1,4,7,10 days. Results: two groups of alkaline phosphatase activity at first days of culture. There was no significant difference in sex and gene expression (P > 0.05). With the prolongation of culture time, the activity of alkaline phosphatase and gene expression in the two groups increased gradually. At the time of 4,7,10, the alkaline phosphatase activity and gene expression in group ZrO_2 were significantly higher than those in the pure Ti group (P < 0.05). At first days, the two groups of COLI gene expression were no different. Study significance (P > 0.05); at fourth and 7 days, the expression of COLI gene in group ZrO_2 was significantly higher than that in pure Ti group (P < 0.05). At tenth days, there was no statistical difference between the two groups (P > 0.05). There was no statistical difference in the expression of OC gene in two groups at first and fourth days (P > 0.05), but the OC gene table in ZrO_2 group and tenth days was on seventh and tenth days. It was significantly higher than the pure Ti group (P < 0.05). On the fourth day, the COLI protein synthesis on the surface of the ZrO_2 group was more than that in the Ti group. There was no statistical difference between the two groups of OC protein secretion at day 1,4 (P > 0.05), but at day 7,10, the secretory amount of OC protein in the ZrO_2 group was significantly higher than that of the Ti group (0.05). The ability of the level of phenotypic markers indicates that the zirconia film deposited by cathodic arc deposition is a good coating material with good bioactivity.
[Abstract] Objective: To investigate the effect of the cathodic arc deposition of zirconium oxide film on the osteoblast differentiation related gene expression and protein secretion on the surface of the artificial joint prosthesis. Methods: the cathode arc deposition ZrO_2 membrane was used as the experimental group, the pure Ti was used as the control group, and the cultured MG63 cells were cultured on a certain density, respectively, on day 1,4,7,10. The expression of osteoprotegerin (OPG) and nuclear factor kappa B receptor activating factor ligand (RANKL) gene expression of osteoclast differentiation in osteoblasts was detected by quantitative RT-PCR method. The secretion of OPG and RANKL protein in osteoblasts at 1,4,7,10 days was detected by ELISA. The results showed that there was no statistical difference between two groups of OPG gene expression at first days and fourth days (P > > 0.05); the expression of OPG gene in group ZrO_2 was significantly higher than that in group Ti (P < 0.05). The expression of RANKL gene in two groups was not significantly different (P > 0.05) at first and 4 days, and at seventh days and 10 days, the gene expression in group ZrO_2 was significantly lower than that in Ti group (P < 0.05). During incubation of first and 4 days, the OPG protein of the osteoblasts on the surface of the 10 groups There was no statistical difference (P > 0.05); the secretion of OPG protein in group ZrO_2 was significantly higher than that in group Ti (P < 0.05) on day seventh and 10 days. On the first day and the 4 day, there was no significant difference in the secretion of RANKL protein on the surface of the two samples (P > 0.05); at seventh days and 10 days, the secretion of RANKL protein in ZrO_2 group was significantly lower than that of the Ti group (P < 0.05). Conclusion: Human The cathode arc deposition of zirconium oxide film on the surface of the joint prosthesis can increase the OPG level of osteoblast, decrease the RANKL level and increase the relative ratio of OPG/RANKL, thus inhibiting the differentiation and activity of osteoclast.
Objective: To explore the possible signal transduction pathway of the effect of zirconium oxide film on the osteoid MG63 cells on the surface of the artificial joint prosthesis. Methods: the cathode arc deposition ZrO_2 membrane was used as the experimental group, and the pure Ti was used as the control group, and the cultured MG63 cells were cultured on the surface according to a certain density. Sixth hours, 24 respectively. The expression of integrin beta 1, FAK, ERK1, ERK2, c-fos and c-jun was collected for hours, 4 days and 7 days after four time points. Results: the expression of integrin beta 1 gene in ZrO_2 group was significantly higher than that in Ti group (P < 0.05) after 6,24 hours and 4 days, and there was no statistical difference in the expression of integrin beta 1 (P > 0.05).6,24 hours and 4 days after 7 days. The expression of FAK gene was significantly higher than that of the Ti group (P < 0.05). The expression of FAK gene in the two groups was not statistically significant (P > 0.05). After 6 hours and 24 hours, the expression of ERK1 gene in ZrO_2 group was significantly higher than that in the Ti group (P < 0.05). There was no significant difference in the expression of ERK1 gene in two groups (P > 0.05).6,24 hours and 4 days after 4 and 7 days, and the expression of the ZrO_2 group was significantly higher than that of the Ti group. (P < 0.05); after 7 days, there was no significant difference in the expression of ERK2 gene on the surface of the two groups (P > 0.05).6,24 hours, 4 days and 7 days later, the expression of c-fos gene in ZrO_2 group was significantly higher than that in Ti group (P < 0.05).6,24 hours and 4 days, c-jun expression in ZrO_2 group was significantly higher than that of Ti group (0.05). 7 days later, there was no statistical difference in the expression of gene expression on the surface of group two. 0.05) conclusion: the surface of cathodic arc deposited zirconia membrane on artificial joint prosthesis may affect cell activity through integrin mediated MAPK/ERK signaling 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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