不同钛表面大鼠成骨细胞膜片成骨效应的初步研究
发布时间：2018-11-25 22:03
【摘要】：目的:初步观察不同钛表面大鼠成骨细胞膜片的成骨效应,以期分析细胞膜片骨组织工程技术与钛表面改性的联合运用是否能对钛种植体骨整合界面的形成具有明显的促进作用。方法:取24h新生SD大鼠颅骨体外分离、提取原代细胞,培养传至第3代,定期观察细胞形态并通过苏木精—伊红(hematoxylin-eosin staining,HE)染色、碱性磷酸酶(alkaline phosphatase staining,ALP)染色、茜素红(alizarin red staining,ARS)染色、I型胶原免疫组化染色鉴定成骨细胞。静态水接触角测试不同钛表面润湿性;电镜扫面(SEM)观察不同钛表面形貌特征及负载细胞后生长特性;再将已鉴定的第三代成骨细胞以0.5x106cells/ml密度接种于不同表面钛片上制备成骨细胞膜片,定期观察膜片形态并采用ALP活性测试膜片分化;通过实时荧光定量PCR(RT-qPCR)及免疫蛋白印记法(Western blotting)检测OPN,OPG,RANKL成骨标志性因子基因及蛋白的表达水平;将体外培养14天的成骨细胞膜片植入裸鼠皮下,分别于术后第8周、16周取出植入物行HE染色、Vonkossa染色和I型胶原免疫组化染色,评价在不同钛表面成骨细胞膜片体内异位成骨的能力。结果:原代细胞经培养及鉴定,细胞形态学复合成骨细胞特性;三种钛表面静态水接触角均呈大于5°,小于90°,呈亲水性表面(小于5°为超亲水性,大于90°为疏水性,介于5°-90°之间为亲水性),实验组较对照组更接近超亲水性,而实验组之间比较无显著差异(P0.05);电镜扫描显示机械抛光组表面较光滑,成骨细胞附着生长不连续,基质衔接有断裂且多为单层生长;阳极氧化组表面形成较为整齐均匀的微孔阵列结构,细胞生长密集有序,基质延孔边缘连接、爬行;喷砂酸蚀组表面形成“蜂窝状”多级微孔,凹凸不平,细胞粘附后呈三维方向叠层生长;ALP检测,各时间点,实验组活性均高于对照组(P0.05),实验组之间ALP活性表达无显著差异(P0.05);各组成骨细胞膜片在第14天ALP活性均低于第7天(P0.05)。RT-qPCR及Western blot检测:组间比较,实验组中细胞膜片OPN、OPG mRNA和蛋白的表达量在各时间点均高于对照组(P0.05),实验组之间比较在各时间点均无显著差异(P0.05);组内比较,实验组及对照组OPN、OPG mRNA和蛋白从第7天到第14天呈上调趋势(实验组及对照组OPNmRNA和蛋白:P0.05,对照组OPG mRNA及蛋白:P0.05,实验组OPG mRNA及蛋白:P0.05);不同钛表面对RANKL mRNA及蛋白的表达没有显著影响(P0.05)。体内异位成骨实验染色结果显示,在第8周对照组未能形成骨样基质,实验组均能形成骨样基质,但实验组间差异不明显;第16周,三组均能形成类骨样组织,可见成骨组织相关结构,相比对照组,实验组形态及成熟度相对更佳,实验组之间比较,综合三种染色分析,阳极氧化组及喷砂酸蚀组形成骨组织无明显差异。结论:(1)相比机械抛光表面,大鼠成骨细胞膜片在阳极氧化及喷砂酸蚀钛表面具有更佳的成骨效应,阳极氧化与喷砂酸蚀组间差异无显著性。(2)钛表面大鼠成骨细胞膜片成骨效应的调控可能主要通过影响OPG的表达来实现,对RANKL的影响不显著。(3)阳极氧化及喷砂酸蚀表面成骨细胞膜片在8周时可见骨样基质形成,机械抛光组膜片未见骨样基质,在16周后均能在体内形成骨组织。
[Abstract]:Objective: To observe the bone-forming effect of the rat osteoblast membrane in different titanium surface, in order to analyze whether the combined application of the cell membrane fragment bone tissue engineering technology and the surface modification of the titanium can promote the formation of the bone complete interface of the titanium implant. Methods: 24 h neonatal SD rats were isolated in vitro, the primary cells were extracted, and the cells were cultured to the third generation. The morphology of the cells was observed on a regular basis and stained with hematoxylin-eosin (HE), alkaline phosphatase (ALP) staining, and alizarin red (ARS) staining. Osteoblasts were identified by immunohistochemical staining of type I collagen. the wettability of different titanium surfaces is tested by the contact angle of the static water; the morphology and the growth characteristics of the different titanium surfaces are observed by an electron microscope scanning surface (SEM); and the cultured third-generation osteoblasts are inoculated on the titanium sheets with different surfaces at a density of 0.5x106cells/ ml to prepare the osteoblast membrane, OPN, OPG, RANKL were detected by real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting to detect the expression level of OPN, OPG, RANKL into bone marker gene and protein. At the 8th and 16th weeks of the operation, HE staining, Vonkossa staining and immunohistochemical staining of type I collagen were taken out, and the ability of ectopic bone formation in different titanium surface osteoblast membranes was evaluated. Results: The primary cells were cultured and identified, and the morphology of the cells was characterized by the cell morphology. The contact angle of the three titanium surfaces was more than 5 掳, less than 90 掳, the hydrophilic surface (less than 5 掳 is super-hydrophilic, more than 90 掳 is hydrophobic, in that experiment group, the surface of the mechanical polishing group was smooth and the adhesion of the osteoblast was not continuous. the surface of the anode oxidation group is formed with a relatively orderly and uniform micropore array structure, The activity of ALP in the experimental group was higher than that in the control group (P0.05). The ALP activity in the experimental group was lower than that of the control group (P0.05). The ALP activity of each group was lower than that of the 7th day (P0.05). RT-qPCR and Western blot analysis: The results showed that the expression of OPN, OPG mRNA and protein in the cell membrane in the experimental group was higher than that in the control group at all time points (P0.05). There was no significant difference between the experimental group and the control group (P0.05). The expression of OPG mRNA and protein from day 7 to day 14 was up-regulated (OPPNmRNA and protein in the experimental group and control group: P 0.05, control group OPG mRNA and protein: P0.05). The expression of OPG mRNA and protein in the experimental group was not significantly affected (P0.05). In the experimental group, the bone-like matrix could not be formed in the control group at the 8th week, but the experimental group could form the bone-like matrix, but the difference between the experimental groups was not obvious; in the 16th week, the three groups can form the bone-like tissue, and it can be seen as the related structure of the bone tissue, compared with the control group, The morphology and maturity of the experimental group were relatively better, and there were no significant differences between the experimental group and the experimental group. Conclusion: (1) Compared with the mechanical polishing surface, the rat osteoblast membrane has better bone-forming effect on the surface of the anodic oxidation and the sandblasted titanium, and there is no significant difference between the anodic oxidation and the sandblasted acid etching group. (2) The regulation of the osteogenic effect of the rat osteoblast membrane in the titanium surface may be mainly realized by influencing the expression of OPG, and the effect on RANKL is not significant. (3) The bone-like matrix was formed at 8 weeks after the anodic oxidation and the surface of the acid-treated osteoblast, and the bone-like matrix was not found in the membrane of the mechanical polishing group, and the bone tissue could be formed in the body after 16 weeks.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.1
本文编号：2357498
[Abstract]:Objective: To observe the bone-forming effect of the rat osteoblast membrane in different titanium surface, in order to analyze whether the combined application of the cell membrane fragment bone tissue engineering technology and the surface modification of the titanium can promote the formation of the bone complete interface of the titanium implant. Methods: 24 h neonatal SD rats were isolated in vitro, the primary cells were extracted, and the cells were cultured to the third generation. The morphology of the cells was observed on a regular basis and stained with hematoxylin-eosin (HE), alkaline phosphatase (ALP) staining, and alizarin red (ARS) staining. Osteoblasts were identified by immunohistochemical staining of type I collagen. the wettability of different titanium surfaces is tested by the contact angle of the static water; the morphology and the growth characteristics of the different titanium surfaces are observed by an electron microscope scanning surface (SEM); and the cultured third-generation osteoblasts are inoculated on the titanium sheets with different surfaces at a density of 0.5x106cells/ ml to prepare the osteoblast membrane, OPN, OPG, RANKL were detected by real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting to detect the expression level of OPN, OPG, RANKL into bone marker gene and protein. At the 8th and 16th weeks of the operation, HE staining, Vonkossa staining and immunohistochemical staining of type I collagen were taken out, and the ability of ectopic bone formation in different titanium surface osteoblast membranes was evaluated. Results: The primary cells were cultured and identified, and the morphology of the cells was characterized by the cell morphology. The contact angle of the three titanium surfaces was more than 5 掳, less than 90 掳, the hydrophilic surface (less than 5 掳 is super-hydrophilic, more than 90 掳 is hydrophobic, in that experiment group, the surface of the mechanical polishing group was smooth and the adhesion of the osteoblast was not continuous. the surface of the anode oxidation group is formed with a relatively orderly and uniform micropore array structure, The activity of ALP in the experimental group was higher than that in the control group (P0.05). The ALP activity in the experimental group was lower than that of the control group (P0.05). The ALP activity of each group was lower than that of the 7th day (P0.05). RT-qPCR and Western blot analysis: The results showed that the expression of OPN, OPG mRNA and protein in the cell membrane in the experimental group was higher than that in the control group at all time points (P0.05). There was no significant difference between the experimental group and the control group (P0.05). The expression of OPG mRNA and protein from day 7 to day 14 was up-regulated (OPPNmRNA and protein in the experimental group and control group: P 0.05, control group OPG mRNA and protein: P0.05). The expression of OPG mRNA and protein in the experimental group was not significantly affected (P0.05). In the experimental group, the bone-like matrix could not be formed in the control group at the 8th week, but the experimental group could form the bone-like matrix, but the difference between the experimental groups was not obvious; in the 16th week, the three groups can form the bone-like tissue, and it can be seen as the related structure of the bone tissue, compared with the control group, The morphology and maturity of the experimental group were relatively better, and there were no significant differences between the experimental group and the experimental group. Conclusion: (1) Compared with the mechanical polishing surface, the rat osteoblast membrane has better bone-forming effect on the surface of the anodic oxidation and the sandblasted titanium, and there is no significant difference between the anodic oxidation and the sandblasted acid etching group. (2) The regulation of the osteogenic effect of the rat osteoblast membrane in the titanium surface may be mainly realized by influencing the expression of OPG, and the effect on RANKL is not significant. (3) The bone-like matrix was formed at 8 weeks after the anodic oxidation and the surface of the acid-treated osteoblast, and the bone-like matrix was not found in the membrane of the mechanical polishing group, and the bone tissue could be formed in the body after 16 weeks.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.1
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