本文選題：種植體穿齦部分　切入點(diǎn)：微溝槽　出處：《福建醫(yī)科大學(xué)》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的為了增強(qiáng)種植體穿齦部分的軟組織愈合,阻止細(xì)菌入侵,保證種植體長(zhǎng)期穩(wěn)定性,制作合適的微溝槽尺寸和抗菌涂層,確定“接觸誘導(dǎo)”信號(hào)傳導(dǎo)機(jī)制。進(jìn)一步尋找細(xì)胞生物相容性好,“接觸誘導(dǎo)”性能優(yōu)越,同時(shí)具有抗菌性能的種植體穿齦部分材料表面。方法第一部分:原代培養(yǎng)人牙齦成纖維細(xì)胞(HGF),針對(duì)細(xì)胞大小設(shè)計(jì)不同溝槽尺寸,利用光刻技術(shù)制作微溝槽形貌,溝槽寬度等于間隔分別為15μm,30μm,60μm,溝槽的深度為5μm和10μm,分組為T(mén)15/5,T15/10,T30/5,T30/10,T60/5,T60/10,光滑鈦(T0)表面為對(duì)照組,測(cè)量各組材料表面形貌,粗糙度,親水性等理化性能,通過(guò)通過(guò)免疫熒光和掃描電鏡觀察細(xì)胞“接觸誘導(dǎo)”的形態(tài),利用CCK-8,流式細(xì)胞檢測(cè)細(xì)胞增殖和細(xì)胞周期推進(jìn)的差異,通過(guò)Realtime-PCR和Western blotting比較粘著斑附著(FAs)相關(guān)蛋白(Intergrina5,Intergrinβ1,vinculin)的基因和蛋白水平的表達(dá),進(jìn)一步比較對(duì)血漿和細(xì)胞來(lái)源FN吸附和分泌的差異,對(duì)比不同溝槽上方“接觸誘導(dǎo)”相關(guān)信號(hào)蛋白活化(p-FAK及GTP-RhoA/ROCK)的差異,綜合研究結(jié)果選擇最優(yōu)的溝槽尺寸通過(guò)相應(yīng)的信號(hào)蛋白活化抑制劑,最終明確“接觸誘導(dǎo)”的機(jī)制及信號(hào)通路之間的串話。第二部分:通過(guò)第一部分選擇的溝槽尺寸利用磁控濺射制備納米氮化鈦(TiN)和銀涂層(Ag),與鈦光滑表面(Ti-S)相比,實(shí)驗(yàn)組為光滑表面制備抗菌涂層:Ag-S,TiN-S,溝槽表面制備抗菌涂層:Ti-MG,Ag-MG,TiN-MG。通過(guò)檢測(cè)材料表面微溝槽形貌(SEM),納米涂層形貌和粗糙度(AFM),親水性測(cè)量,元素分析以及Ag+釋放進(jìn)一步明確材料表面理化性能,進(jìn)一步研究抗菌納米涂層與微溝槽形貌結(jié)合后對(duì)HGF的細(xì)胞生物相容性,“接觸誘導(dǎo)”效應(yīng)的影響以及對(duì)牙齦卟啉單胞菌(Pg)的抗菌作用差異。結(jié)果1.光刻技術(shù)成功制備具有溝槽形貌的鈦表面,各組寬度和深度呈典型的各向異性平行排列,微米級(jí)粗糙度均高于T0,其中T60/10最高,T15/5最低;T60/5表面親水性最好,T15/10疏水性最大甚至大于光滑組T0;2.不同尺寸溝槽表面對(duì)HGF的增殖和細(xì)胞周期推進(jìn)不同,T60/10具有促進(jìn)早期粘附作用,細(xì)胞增殖的幅度最大,增殖的時(shí)間最長(zhǎng),細(xì)胞周期S期占有的細(xì)胞比例最大;3.不同尺寸溝槽對(duì)HGF均成功的起到了“接觸誘導(dǎo)”的作用,但誘導(dǎo)程度不同,細(xì)胞排列一致性T60最高,T15最低,細(xì)胞骨架形變率T60/10最高;4.與FAs相關(guān)的重要蛋白(Intergrina5,Intergrinβ1,vinculin)于T60/10溝槽上方表達(dá)最高。5.T60對(duì)血漿FN的吸附最差,對(duì)細(xì)胞FN分泌刺激作用最好;6.與“接觸誘導(dǎo)”相關(guān)的信號(hào)蛋白的活化:p-FAK,GTP-RhoA/ROCK于T60/10組表達(dá)最高,p-FAK于T30與T15沒(méi)有差異,GTP-RhoA在T15表達(dá)高于T30,ROCK在T30組表達(dá)高于T15組;7.選擇T60/10為最優(yōu)的溝槽尺寸,此時(shí)抑制p-FAK對(duì)細(xì)胞排列一致性沒(méi)有太大的影響,對(duì)RhoA-GTP的活化影響無(wú)統(tǒng)計(jì)學(xué)意義,分別對(duì)RhoA-GTP/ROCK信號(hào)蛋白進(jìn)行抑制均不同程度影響了“接觸誘導(dǎo)”效應(yīng),以ROCK抑制更為明顯,對(duì)RhoA-GTP抑制的同時(shí)也減少了FAK蛋白的磷酸化。8.通過(guò)磁控濺射技術(shù)成功實(shí)現(xiàn)在光滑和選定溝槽上方制作抗菌TiN,Ag涂層,能夠保持材料原有形貌且賦予材料新的理化表面,其中納米粗糙度TiN最小,Ag涂層表面最大,親水性TiN-MG最好,Ag-S最差,XPS可見(jiàn)TiN涂層上方含間隙氮和氧,Ag涂層上方含氧量最少是導(dǎo)致親水性下降的原因,溝槽表面的Ag+釋放高于光滑表面,且釋放量持續(xù)7d均高于0.1ppb(最低抗菌濃度);9.不同涂層表面影響細(xì)胞增殖和細(xì)胞周期的推進(jìn),Ag涂層表面細(xì)胞增殖和細(xì)胞周期S期占有的比例低于其他涂層表面,TiN-MG表面細(xì)胞增殖和細(xì)胞周期S期占有的比例最高,不同涂層上方細(xì)胞FAs相關(guān)蛋白的表達(dá)存在差異,Ag涂層上方Intergrina5,Intergrinβ1,FN,Vinculin的表達(dá)均比其他涂層表面少,而TiN-MG表達(dá)最高。Ag-MG,TiN-MG都能成功誘導(dǎo)細(xì)胞順著溝槽排列,細(xì)胞排列一致性比例無(wú)差異,不同涂層溝槽表面“接觸誘導(dǎo)”相關(guān)的信號(hào)蛋白p-FAK以及ROCK表達(dá)均高于光滑表面,ROCK活化與表面化學(xué)成分無(wú)關(guān),而Ag涂層的溝槽表面p-FAK相對(duì)較低于Ti-MG和TiN-MG。10.Ag涂層的抗菌作用最強(qiáng),TiN涂層的抗菌能力居中,相同涂層下溝槽形貌并沒(méi)有增加細(xì)菌的粘附量。結(jié)論1.T60/10作為各向異性微溝槽的表面親水性好,最有利于HGF粘附增殖和細(xì)胞周期推進(jìn),對(duì)HGF的“接觸誘導(dǎo)”效應(yīng)最好:細(xì)胞排列一致性和細(xì)胞骨架形變率最高,此外FAs相關(guān)的蛋白和“接觸誘導(dǎo)”相關(guān)的信號(hào)蛋白在基因和蛋白水平上表達(dá)和活化度均最高,雖然對(duì)血漿FN的吸附弱于其他溝槽表面,但對(duì)細(xì)胞FN的分泌刺激作用最好,由于細(xì)胞來(lái)源的FN能彌補(bǔ)血漿來(lái)源FN的功能,最終T60/10建議為種植體穿齦部分最合適的溝槽尺寸。2.利用磁控濺射技術(shù)成功的實(shí)現(xiàn)了同時(shí)具有微溝槽形貌(T60/10)又具有抗菌功能的納米涂層,其中微溝槽上方制作Ag涂層細(xì)胞相容性較差,而抗菌性最高,而微溝槽上方制作納米TiN涂層具有一定的抗菌性能及最優(yōu)的細(xì)胞生物相容性亦保持了“接觸誘導(dǎo)”效應(yīng),因此納米TiN涂層推薦與T60/10的微溝槽結(jié)合作為具有抗菌性能的種植體穿齦部分的材料表面。
[Abstract]:In order to enhance the soft tissue implant transgingival part of healing, prevent bacterial invasion, ensure the long-term stability of the implant, making micro groove size and antibacterial coating, determine the contact induced signal transduction mechanism. Further search for cells with good biocompatibility, "contact induced" superior performance, also has the antibacterial properties of implants the transgingival part of the material surface. Methods: the first part: the primary cultured human gingival fibroblasts (HGF), different groove size designed for cell size, making the micro groove morphology using photolithography technique, the groove width is equal to the interval of 15 m, 30 m, 60 m, the depth of the groove is 5 m and 10 m, T15/5 T15/10, T30/5 group, T30/10, T60/5, T60/10, and smooth surface of titanium (T0) as the control group, each group of materials measuring surface topography, roughness, hydrophilicity and other physicochemical properties through immunofluorescence and scanning electron microscopy of thin "Cell contact induced" form, using CCK-8, differences in cell proliferation and cell cycle flow cytometry to promote, through Realtime-PCR and Western blotting (FAs) attachment of focal adhesion related proteins (Intergrina5, Intergrin beta 1, vinculin) gene and protein expression level, further comparison of the differences in absorption and secretion of plasma and the cell source of FN, comparing the different groove above the "contact induced" related signal protein activation (p-FAK and GTP-RhoA/ROCK) differences, comprehensive research results to choose the optimal size of groove through signal corresponding protein activation inhibitors, finally clear "crosstalk between mechanism and signal pathway induced by contact". The second part: the first part of the trench the size of the choice of preparing nano titanium nitride by magnetron sputtering (TiN) and silver coating (Ag), and the smooth surface of titanium (Ti-S) compared to the experimental group for the preparation of smooth surface Bacteria: Ag-S, TiN-S, coating groove surface preparation of antibacterial coating: Ti-MG, Ag-MG, TiN-MG. by detecting the material surface micro groove morphology (SEM), nano coating morphology and surface roughness (AFM), hydrophilicity measurement, elemental analysis and Ag+ release to further clarify the material surface physicochemical properties, further study on antibacterial and micro nano coating after binding of HGF groove morphology cell biocompatibility, the effect of contact induced effect of Porphyromonas gingivalis (Pg) antibacterial effect. The differences of 1. lithography prepared with groove morphology of titanium surface, the width and depth of a typical anisotropic parallel arrangement, micron roughness the degree of T60/10 was higher than T0, the highest, lowest T15/5; T60/5 surface hydrophilic hydrophobic T15/10 best, even greater than the maximum smooth group T0; 2. different sizes of groove surface HGF proliferation and cell cycle promoting different T60/10 To promote the early adhesion effect, the maximum amplitude of cell proliferation, the proliferation of most of the time, the proportion of cells in the S phase of the cell cycle has the largest; 3. different sizes of groove on the HGF were successfully played "induce" role, but induced by different degrees, cells arranged in the same T60 T15 the highest, lowest, cytoskeleton the deformation rate of T60/10 is the highest; 4. important proteins associated with FAs (Intergrina5, Intergrin beta 1, vinculin) on the T60/10 expression of.5.T60 on adsorption groove above the highest plasma FN worst on cell secretion of FN stimulation and "6. best; activation induced contact related signaling proteins: p-FAK, GTP-RhoA/ROCK expression in the T60/10 group the highest p-FAK in T30 and T15 did not differ between the expression of GTP-RhoA in T15 was higher than that of T30 and ROCK expression in T30 group than in T15 group; 7. T60/10 is selected as the optimal groove size, the inhibition of p-FAK is not much effect on cell arrangement consistency Ring, no significant influence on the activation of RhoA-GTP, respectively, were inhibitory effect of different degree of the contact induced effect on RhoA-GTP/ROCK signal protein with ROCK inhibition was more obvious for RhoA-GTP inhibition also reduced the phosphorylation of.8. FAK protein by magnetron sputtering technology of Ag coating on the smooth and successful implementation of the selected channel above making antibacterial TiN, can keep the original shape, material and surface physicochemical gives new materials, including nano roughness TiN minimum, Ag coating, hydrophilic TiN-MG was the best, the worst Ag-S, XPS visible TiN coated layer above with clearance of nitrogen and oxygen, the oxygen content of at least Ag coating above is the cause of hydrophilic fall, the groove surface of the release of Ag+ is higher than that of the smooth surface, and the release amount of sustained 7d was higher than that of 0.1ppb (minimum antibacterial concentration); 9. different coating effects to promote cell proliferation and cell cycle, Ag coating Cell proliferation and cell cycle of surface cells in the S phase proportion was lower than that of the other surface of the coating, cell proliferation and cell cycle of TiN-MG surface S occupy the highest proportion of differentially expressed proteins related to FAs cells above the different coating, Ag coating above Intergrina5, Intergrin beta 1, FN, Vinculin expression was less than the other surface of the coating, and the highest expression of TiN-MG.Ag-MG, TiN-MG can successfully induce cell along the groove arranged, cells arranged in the consistency ratio between different coating groove surface contact induced signal related protein p-FAK and ROCK expression were higher than that of the smooth surface, the activation of ROCK has nothing to do with the surface chemical composition, and the groove surface of Ag coating is relatively lower than p-FAK and Ti-MG TiN-MG.10.Ag coating is the strongest antibacterial effect, middle antibacterial ability of TiN coating, coating the same trench and did not increase the amount of bacteria adhesion morphology. Conclusion 1.T60/10 The surface hydrophilicity as anisotropic micro grooves, the most conducive to the proliferation and cell cycle of HGF adhesion to HGF forward, "contact induced effect": the best arrangement of cells consistency and cytoskeleton deformation rate is the highest, the addition of FAs related proteins and contact induced related signal proteins in gene and protein level the expression and activation degree were the highest, while the adsorption of plasma FN is weaker than other grooved surface, but the secretion of stimulation to cell FN is the best, because the cells derived from the FN plasma source can make up for the function of FN, the final T60/10 recommendations for implant transgingival part of the most appropriate groove size.2. by magnetron sputtering technology successfully at the same time with the groove surface morphology (T60/10) and nano coating antibacterial function, the micro channel above the production of coated Ag cell compatibility is poor, and the highest antibacterial, and making nano micro grooves above TiN coating has certain antibacterial properties and optimal cell biocompatibility, and also maintains the "contact induction" effect. Therefore, nano TiN coating is recommended to be combined with T60/10 micro grooves as the surface material of gingival part with antibacterial properties.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R783.1
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本文編號(hào)：1590427