微米—纳米钛合金表面对成骨细胞生物学行为的影响

发布时间：2018-06-18 00:30

本文选题：骨整合 + 微米形貌　；参考：《吉林大学》2011年硕士论文

【摘要】：钛和钛合金由于其良好的机械性能、生物相容性、抗腐蚀性，从而广泛应用于医学生物材料领域，如牙科种植体等植入材料。种植体植入体内后和宿主组织直接接触，与组织体液发生一系列的细胞-细胞反应、细胞-蛋白作用后最终形成稳定的骨整合。研究表明种植体的表面特性，决定骨对种植体的生物反应。因此许多学者通过改变种植体表面形貌或化学成分，来探讨成骨细胞在生物材料表面的生物学行为，促进成骨细胞的功能。种植体表面形貌的研究主要集中在微米形貌，一致认为粗糙的微米形貌与光滑表面相比，具有更好的促进成骨的作用，但目前研究发现微米表面可以促进成骨细胞的分化但是抑制成骨细胞的增殖。近年来种植体表面的纳米形貌结构越来越受到关注，纳米形貌能为细胞提供仿生环境，促进成骨细胞的粘附、增殖、分化、基质合成，因此许多学者开始研究兼具微米、纳米结构的生物材料。然而微米-纳米表面对表面的具体作用尚不清楚，其应用与临床还是挑战。为了进一步理解表面微米-钠米结构是如何影响成骨细胞生物活性的现象。本实验通过电化学方法在钛合金表面形成兼具微米和纳米形貌的双级孔微结构，并评价该表面微结构对成骨细胞吸附、增殖、形态及分化的影响。将MG-63细胞分别于电化学（Electrochemical，EC）、喷砂和酸蚀组（sandblasted and acid-etching，SLA）和机械加工组(machined，M)表面培养，显微镜下计活细胞数，计算1-24小时（hour，h）细胞在不同表面的贴壁率;采用四甲基偶氮唑蓝(3-(4,5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazoliumbromide ,MTT)法评价细胞在第1d、3d、5d和7d的细胞增殖率；场发射扫描电镜观察细胞接种后1天（day，d）、3天、5天、7天胞形态的变化；并用碱性磷酸酶试剂盒检测1d、3d、5d和7d碱性磷酸酶的活性变化。结果显示：MG-63细胞接种各表面后1-24h，随着时间延长，贴壁率逐渐增加，接种1h电化学（EC）组细胞贴壁率明显高于SLA组和M组的细胞贴壁率（P＜0.05），其中M组细胞贴壁率最低；各组细胞在2h、6h、12h和24h贴壁率较1h细胞贴壁率明显增加（P＜0.05）。且仍然以EC贴壁率最大，M组贴壁率最小。随着时间的延长，培养于EC、SLA和M表面的MG-63细胞都进行分裂增殖，第3d、第7d，EC组细胞增殖能力强于SLA组（P＜0.05）；第3d、第5d，EC组细胞增殖能力强于M组（P＜0.05），M组细胞增值率在第1d出现增殖抑制，而第5d和第7d则继续增加。扫描电镜结果显示：第1d，EC表面碗型凹内有MG-63细胞附着，呈梭形，伸展良好，细胞表面有大量微绒毛和细小突起，并且伸出许多伪足附着在侧壁。SLA表面呈现不规则，附着的MG-63细胞主要为球形，伪足和微绒毛均较少。M表面的MG-63细胞呈球形，紧贴表面，绒毛和伪足不明显。第3d，，EC表面布满了MG-63细胞，细胞紧密的贴在EC表面，细胞更加扁平，伸出的大量微绒毛，细胞表面有很多突起。SLA表面微孔形态被MG-63细胞遮盖，细胞呈球形，梭形及不规则形态。M表面的MG-63细胞呈现球形，与表面附着的不牢固，少许微绒毛，未见伪足。第5d和7d，EC表面MG-63细胞大多已经展开，紧密的贴在EC钛表面，细胞更加扁平，少量细胞呈现球形；SLA表面的MG-63细胞也已经呈现片状，细胞扁平，少量的梭形和球形细胞；M表面的细胞仍旧呈现球形，但已经展开，紧密的贴在表面，微绒毛已经连成片，伪足少见。EC、SLA和M组的ALP活性在第1d、3d、5d和第7d时，EC组和SLA组的ALP活性明显高于M组钛合金表面ALP活性；第1d、第5d和第7d，EC组ALP活性明显高于SLA组ALP活性（P＜0.05）。上述结果表明，用电化学方法可以在钛合金表面形成兼具微米和纳米双级微结构的表面形貌，该表面有利于促进成骨细胞相容性，提高成骨细胞的粘附、增殖和ALP活性，为研究细胞-种植体的相互作用以及进一步优化微米和纳米的协调作用提供了理论依据。
[Abstract]:Titanium and titanium alloys are widely used in the field of medical biomaterials, such as dental implants, because of their good mechanical properties, biocompatibility and corrosion resistance. The implant is implanted in the body and directly contact with the host tissue, and occurs a series of cell cell reactions with the tissue fluid. After the action of the cell protein, the implant is eventually stable. The study shows that the surface characteristics of the implant determine the biological response of the bone to the implant. So many scholars study the biological behavior of osteoblasts on the surface of the biomaterial by changing the surface morphology or chemical composition of the implants to promote the function of the osteoblasts.
The surface morphology of the implant is mainly focused on the micron morphology. It is agreed that the rough micromorphology has a better role in promoting osteogenesis compared with the smooth surface. However, it is found that the micron surface can promote the differentiation of osteoblasts but inhibit the proliferation of osteoblasts. The more concerned, the nano morphology can provide biomimetic environment for cells and promote the adhesion, proliferation, differentiation and matrix synthesis of osteoblasts. Therefore, many scholars have begun to study the biomaterials with both micron and nanostructures. However, the specific effect of micron nano surface on the surface is still unclear, and its application and clinical challenge are still not clear.
In order to further understand how the surface micrometer sodium meter structure affects the bioactivity of osteoblasts, this experiment forms a two-stage microstructure with both micron and nano morphology on the surface of titanium alloy by electrochemical method, and evaluates the effect of the surface microstructures on the adsorption, colonization, morphology and differentiation of osteoblasts.
The MG-63 cells were treated with electrochemical (Electrochemical, EC), sandblasted and acid-etching, SLA, and mechanical processing group (machined, M) surface culture. Under microscope, the number of living cells was counted, and the adhesion rate of 1-24 hours (hour, H) cells on different surfaces was calculated. Four methyl azazolium blue (3-) was used. Phenyltetrazoliumbromide, MTT) method was used to evaluate cell proliferation rate of cells in 1D, 3D, 5D and 7d; field emission scanning electron microscopy was used to observe the changes of cell morphology at 1 days (day, d), 3 days, 5 days, 7 days, and the activity changes of 1D, 3D, 5D and 7d alkaline phosphatase were detected with alkaline phosphatase kits.
The results showed that after MG-63 cells were inoculated on each surface, the adherence rate of cell adherence increased with time, and the adherence rate of cells in 1H electrochemistry (EC) group was significantly higher than that in group SLA and M group (P < 0.05). The cell adherence rate of M group was the lowest, and the rate of cell adherence in 2H, 6h, 12h and 24h was significantly increased in each group (EC < 0.05). And still with the maximum EC adherence rate, the M group adhered to the smallest wall rate. With the prolongation of time, MG-63 cells cultured on the surface of EC, SLA and M proliferated, and the proliferation ability of 3D, 7d and EC groups was stronger than that of the SLA group (P < 0.05). The results of 5D and 7d continued to increase. The scanning electron microscope showed that the MG-63 cells were attached to the surface of the bowl type of the surface of the EC surface, with a spindle shape, a good extension, a large number of microvilli and small protuberances on the surface of the cell, and a lot of pseudo feet attached to the side wall of the wall to appear irregular on the surface of the.SLA surface, and the adherent MG-63 cells were mainly spherical, and the pseudo foot and microvilli were compared. The MG-63 cells on the surface of less.M are spherical, close to the surface, and the villus and the pseudo foot are not obvious. The surface of 3D, EC is covered with MG-63 cells, the cells are tightly attached to the surface of EC, the cells are more flat, and a large number of microvilli protruded, and the surface of the cells is covered with MG-63 cells on the surface of the surface of the.SLA, and the cells are spherical, shuttle and irregular form of.M form. The surface of MG-63 cells appeared globular, not firmly attached to the surface, a little microvilli and no pseudo foot. 5D and 7d, MG-63 cells on the surface of EC were mostly unfolded, tightly attached to the surface of EC titanium, the cells were more flat, and a small number of cells were spherical, and the MG-63 cells on the SLA surface had also been flattened, flat, and a small number of spindle and spherical cells. The cells on the surface of M are still spherical, but they have been expanded, closely attached to the surface, the microvilli have been rejoined, and the pseudo foot is rare.EC, and the ALP activity of the SLA and M groups in the 1D, 3D, 5D and 7d is significantly higher than that of the titanium alloy surface. < 0.05).
The above results show that the surface morphology of both micron and nanoscale microstructures can be formed on the surface of titanium alloy by electrochemical method. The surface is beneficial to promote the compatibility of osteoblasts, increase the adhesion, proliferation and ALP activity of osteoblasts, to study the phase interaction of cell implant and to further optimize the coordination of micron and nanoscale. The effect provides a theoretical basis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329;TG146.23

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