不同鈦表面大鼠成骨細胞膜片成骨效應的初步研究
發(fā)布時間：2018-11-25 22:03
【摘要】：目的:初步觀察不同鈦表面大鼠成骨細胞膜片的成骨效應,以期分析細胞膜片骨組織工程技術與鈦表面改性的聯(lián)合運用是否能對鈦種植體骨整合界面的形成具有明顯的促進作用。方法:取24h新生SD大鼠顱骨體外分離、提取原代細胞,培養(yǎng)傳至第3代,定期觀察細胞形態(tài)并通過蘇木精—伊紅(hematoxylin-eosin staining,HE)染色、堿性磷酸酶(alkaline phosphatase staining,ALP)染色、茜素紅(alizarin red staining,ARS)染色、I型膠原免疫組化染色鑒定成骨細胞。靜態(tài)水接觸角測試不同鈦表面潤濕性;電鏡掃面(SEM)觀察不同鈦表面形貌特征及負載細胞后生長特性;再將已鑒定的第三代成骨細胞以0.5x106cells/ml密度接種于不同表面鈦片上制備成骨細胞膜片,定期觀察膜片形態(tài)并采用ALP活性測試膜片分化;通過實時熒光定量PCR(RT-qPCR)及免疫蛋白印記法(Western blotting)檢測OPN,OPG,RANKL成骨標志性因子基因及蛋白的表達水平;將體外培養(yǎng)14天的成骨細胞膜片植入裸鼠皮下,分別于術后第8周、16周取出植入物行HE染色、Vonkossa染色和I型膠原免疫組化染色,評價在不同鈦表面成骨細胞膜片體內(nèi)異位成骨的能力。結(jié)果:原代細胞經(jīng)培養(yǎng)及鑒定,細胞形態(tài)學復合成骨細胞特性;三種鈦表面靜態(tài)水接觸角均呈大于5°,小于90°,呈親水性表面(小于5°為超親水性,大于90°為疏水性,介于5°-90°之間為親水性),實驗組較對照組更接近超親水性,而實驗組之間比較無顯著差異(P0.05);電鏡掃描顯示機械拋光組表面較光滑,成骨細胞附著生長不連續(xù),基質(zhì)銜接有斷裂且多為單層生長;陽極氧化組表面形成較為整齊均勻的微孔陣列結(jié)構(gòu),細胞生長密集有序,基質(zhì)延孔邊緣連接、爬行;噴砂酸蝕組表面形成“蜂窩狀”多級微孔,凹凸不平,細胞粘附后呈三維方向疊層生長;ALP檢測,各時間點,實驗組活性均高于對照組(P0.05),實驗組之間ALP活性表達無顯著差異(P0.05);各組成骨細胞膜片在第14天ALP活性均低于第7天(P0.05)。RT-qPCR及Western blot檢測:組間比較,實驗組中細胞膜片OPN、OPG mRNA和蛋白的表達量在各時間點均高于對照組(P0.05),實驗組之間比較在各時間點均無顯著差異(P0.05);組內(nèi)比較,實驗組及對照組OPN、OPG mRNA和蛋白從第7天到第14天呈上調(diào)趨勢(實驗組及對照組OPNmRNA和蛋白:P0.05,對照組OPG mRNA及蛋白:P0.05,實驗組OPG mRNA及蛋白:P0.05);不同鈦表面對RANKL mRNA及蛋白的表達沒有顯著影響(P0.05)。體內(nèi)異位成骨實驗染色結(jié)果顯示,在第8周對照組未能形成骨樣基質(zhì),實驗組均能形成骨樣基質(zhì),但實驗組間差異不明顯;第16周,三組均能形成類骨樣組織,可見成骨組織相關結(jié)構(gòu),相比對照組,實驗組形態(tài)及成熟度相對更佳,實驗組之間比較,綜合三種染色分析,陽極氧化組及噴砂酸蝕組形成骨組織無明顯差異。結(jié)論:(1)相比機械拋光表面,大鼠成骨細胞膜片在陽極氧化及噴砂酸蝕鈦表面具有更佳的成骨效應,陽極氧化與噴砂酸蝕組間差異無顯著性。(2)鈦表面大鼠成骨細胞膜片成骨效應的調(diào)控可能主要通過影響OPG的表達來實現(xiàn),對RANKL的影響不顯著。(3)陽極氧化及噴砂酸蝕表面成骨細胞膜片在8周時可見骨樣基質(zhì)形成,機械拋光組膜片未見骨樣基質(zhì),在16周后均能在體內(nèi)形成骨組織。
[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 擄, less than 90 擄, the hydrophilic surface (less than 5 擄 is super-hydrophilic, more than 90 擄 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.
【學位授予單位】：貴州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R783.1
本文編號：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 擄, less than 90 擄, the hydrophilic surface (less than 5 擄 is super-hydrophilic, more than 90 擄 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.
【學位授予單位】：貴州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R783.1
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