本文選題：骨整合 + 微米形貌��； 參考：《吉林大學(xué)》2011年碩士論文

【摘要】：鈦和鈦合金由于其良好的機械性能、生物相容性、抗腐蝕性，從而廣泛應(yīng)用于醫(yī)學(xué)生物材料領(lǐng)域，如牙科種植體等植入材料。種植體植入體內(nèi)后和宿主組織直接接觸，與組織體液發(fā)生一系列的細(xì)胞-細(xì)胞反應(yīng)、細(xì)胞-蛋白作用后最終形成穩(wěn)定的骨整合。研究表明種植體的表面特性，決定骨對種植體的生物反應(yīng)。因此許多學(xué)者通過改變種植體表面形貌或化學(xué)成分，來探討成骨細(xì)胞在生物材料表面的生物學(xué)行為，促進成骨細(xì)胞的功能。 種植體表面形貌的研究主要集中在微米形貌，一致認(rèn)為粗糙的微米形貌與光滑表面相比，具有更好的促進成骨的作用，但目前研究發(fā)現(xiàn)微米表面可以促進成骨細(xì)胞的分化但是抑制成骨細(xì)胞的增殖。近年來種植體表面的納米形貌結(jié)構(gòu)越來越受到關(guān)注，納米形貌能為細(xì)胞提供仿生環(huán)境，促進成骨細(xì)胞的粘附、增殖、分化、基質(zhì)合成，因此許多學(xué)者開始研究兼具微米、納米結(jié)構(gòu)的生物材料。然而微米-納米表面對表面的具體作用尚不清楚，其應(yīng)用與臨床還是挑戰(zhàn)。 為了進一步理解表面微米-鈉米結(jié)構(gòu)是如何影響成骨細(xì)胞生物活性的現(xiàn)象。本實驗通過電化學(xué)方法在鈦合金表面形成兼具微米和納米形貌的雙級孔微結(jié)構(gòu)，并評價該表面微結(jié)構(gòu)對成骨細(xì)胞吸附、增殖、形態(tài)及分化的影響。 將MG-63細(xì)胞分別于電化學(xué)（Electrochemical，EC）、噴砂和酸蝕組（sandblasted and acid-etching，SLA）和機械加工組(machined，M)表面培養(yǎng)，顯微鏡下計活細(xì)胞數(shù)，計算1-24小時（hour，h）細(xì)胞在不同表面的貼壁率;采用四甲基偶氮唑藍(lán)(3-(4,5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazoliumbromide ,MTT)法評價細(xì)胞在第1d、3d、5d和7d的細(xì)胞增殖率；場發(fā)射掃描電鏡觀察細(xì)胞接種后1天（day，d）、3天、5天、7天胞形態(tài)的變化；并用堿性磷酸酶試劑盒檢測1d、3d、5d和7d堿性磷酸酶的活性變化。 結(jié)果顯示：MG-63細(xì)胞接種各表面后1-24h，隨著時間延長，貼壁率逐漸增加，接種1h電化學(xué)（EC）組細(xì)胞貼壁率明顯高于SLA組和M組的細(xì)胞貼壁率（P＜0.05），其中M組細(xì)胞貼壁率最低；各組細(xì)胞在2h、6h、12h和24h貼壁率較1h細(xì)胞貼壁率明顯增加（P＜0.05）。且仍然以EC貼壁率最大，M組貼壁率最小。隨著時間的延長，培養(yǎng)于EC、SLA和M表面的MG-63細(xì)胞都進行分裂增殖，第3d、第7d，EC組細(xì)胞增殖能力強于SLA組（P＜0.05）；第3d、第5d，EC組細(xì)胞增殖能力強于M組（P＜0.05），M組細(xì)胞增值率在第1d出現(xiàn)增殖抑制，而第5d和第7d則繼續(xù)增加。掃描電鏡結(jié)果顯示：第1d，EC表面碗型凹內(nèi)有MG-63細(xì)胞附著，呈梭形，伸展良好，細(xì)胞表面有大量微絨毛和細(xì)小突起，并且伸出許多偽足附著在側(cè)壁。SLA表面呈現(xiàn)不規(guī)則，附著的MG-63細(xì)胞主要為球形，偽足和微絨毛均較少。M表面的MG-63細(xì)胞呈球形，緊貼表面，絨毛和偽足不明顯。第3d，，EC表面布滿了MG-63細(xì)胞，細(xì)胞緊密的貼在EC表面，細(xì)胞更加扁平，伸出的大量微絨毛，細(xì)胞表面有很多突起。SLA表面微孔形態(tài)被MG-63細(xì)胞遮蓋，細(xì)胞呈球形，梭形及不規(guī)則形態(tài)。M表面的MG-63細(xì)胞呈現(xiàn)球形，與表面附著的不牢固，少許微絨毛，未見偽足。第5d和7d，EC表面MG-63細(xì)胞大多已經(jīng)展開，緊密的貼在EC鈦表面，細(xì)胞更加扁平，少量細(xì)胞呈現(xiàn)球形；SLA表面的MG-63細(xì)胞也已經(jīng)呈現(xiàn)片狀，細(xì)胞扁平，少量的梭形和球形細(xì)胞；M表面的細(xì)胞仍舊呈現(xiàn)球形，但已經(jīng)展開，緊密的貼在表面，微絨毛已經(jīng)連成片，偽足少見。EC、SLA和M組的ALP活性在第1d、3d、5d和第7d時，EC組和SLA組的ALP活性明顯高于M組鈦合金表面ALP活性；第1d、第5d和第7d，EC組ALP活性明顯高于SLA組ALP活性（P＜0.05）。 上述結(jié)果表明，用電化學(xué)方法可以在鈦合金表面形成兼具微米和納米雙級微結(jié)構(gòu)的表面形貌，該表面有利于促進成骨細(xì)胞相容性，提高成骨細(xì)胞的粘附、增殖和ALP活性，為研究細(xì)胞-種植體的相互作用以及進一步優(yōu)化微米和納米的協(xié)調(diào)作用提供了理論依據(jù)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329;TG146.23

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