微沟槽形貌和纳米抗菌涂层运用于牙种植体穿龈部分材料表面的基础性研究

发布时间：2018-03-09 21:59

  本文选题：种植体穿龈部分　切入点：微沟槽　出处：《福建医科大学》2014年博士论文　论文类型：学位论文

【摘要】：目的为了增强种植体穿龈部分的软组织愈合,阻止细菌入侵,保证种植体长期稳定性,制作合适的微沟槽尺寸和抗菌涂层,确定“接触诱导”信号传导机制。进一步寻找细胞生物相容性好,“接触诱导”性能优越,同时具有抗菌性能的种植体穿龈部分材料表面。方法第一部分:原代培养人牙龈成纤维细胞(HGF),针对细胞大小设计不同沟槽尺寸,利用光刻技术制作微沟槽形貌,沟槽宽度等于间隔分别为15μm,30μm,60μm,沟槽的深度为5μm和10μm,分组为T15/5,T15/10,T30/5,T30/10,T60/5,T60/10,光滑钛(T0)表面为对照组,测量各组材料表面形貌,粗糙度,亲水性等理化性能,通过通过免疫荧光和扫描电镜观察细胞“接触诱导”的形态,利用CCK-8,流式细胞检测细胞增殖和细胞周期推进的差异,通过Realtime-PCR和Western blotting比较粘着斑附着(FAs)相关蛋白(Intergrina5,Intergrinβ1,vinculin)的基因和蛋白水平的表达,进一步比较对血浆和细胞来源FN吸附和分泌的差异,对比不同沟槽上方“接触诱导”相关信号蛋白活化(p-FAK及GTP-RhoA/ROCK)的差异,综合研究结果选择最优的沟槽尺寸通过相应的信号蛋白活化抑制剂,最终明确“接触诱导”的机制及信号通路之间的串话。第二部分:通过第一部分选择的沟槽尺寸利用磁控溅射制备纳米氮化钛(TiN)和银涂层(Ag),与钛光滑表面(Ti-S)相比,实验组为光滑表面制备抗菌涂层:Ag-S,TiN-S,沟槽表面制备抗菌涂层:Ti-MG,Ag-MG,TiN-MG。通过检测材料表面微沟槽形貌(SEM),纳米涂层形貌和粗糙度(AFM),亲水性测量,元素分析以及Ag+释放进一步明确材料表面理化性能,进一步研究抗菌纳米涂层与微沟槽形貌结合后对HGF的细胞生物相容性,“接触诱导”效应的影响以及对牙龈卟啉单胞菌(Pg)的抗菌作用差异。结果1.光刻技术成功制备具有沟槽形貌的钛表面,各组宽度和深度呈典型的各向异性平行排列,微米级粗糙度均高于T0,其中T60/10最高,T15/5最低;T60/5表面亲水性最好,T15/10疏水性最大甚至大于光滑组T0;2.不同尺寸沟槽表面对HGF的增殖和细胞周期推进不同,T60/10具有促进早期粘附作用,细胞增殖的幅度最大,增殖的时间最长,细胞周期S期占有的细胞比例最大;3.不同尺寸沟槽对HGF均成功的起到了“接触诱导”的作用,但诱导程度不同,细胞排列一致性T60最高,T15最低,细胞骨架形变率T60/10最高;4.与FAs相关的重要蛋白(Intergrina5,Intergrinβ1,vinculin)于T60/10沟槽上方表达最高。5.T60对血浆FN的吸附最差,对细胞FN分泌刺激作用最好;6.与“接触诱导”相关的信号蛋白的活化:p-FAK,GTP-RhoA/ROCK于T60/10组表达最高,p-FAK于T30与T15没有差异,GTP-RhoA在T15表达高于T30,ROCK在T30组表达高于T15组;7.选择T60/10为最优的沟槽尺寸,此时抑制p-FAK对细胞排列一致性没有太大的影响,对RhoA-GTP的活化影响无统计学意义,分别对RhoA-GTP/ROCK信号蛋白进行抑制均不同程度影响了“接触诱导”效应,以ROCK抑制更为明显,对RhoA-GTP抑制的同时也减少了FAK蛋白的磷酸化。8.通过磁控溅射技术成功实现在光滑和选定沟槽上方制作抗菌TiN,Ag涂层,能够保持材料原有形貌且赋予材料新的理化表面,其中纳米粗糙度TiN最小,Ag涂层表面最大,亲水性TiN-MG最好,Ag-S最差,XPS可见TiN涂层上方含间隙氮和氧,Ag涂层上方含氧量最少是导致亲水性下降的原因,沟槽表面的Ag+释放高于光滑表面,且释放量持续7d均高于0.1ppb(最低抗菌浓度);9.不同涂层表面影响细胞增殖和细胞周期的推进,Ag涂层表面细胞增殖和细胞周期S期占有的比例低于其他涂层表面,TiN-MG表面细胞增殖和细胞周期S期占有的比例最高,不同涂层上方细胞FAs相关蛋白的表达存在差异,Ag涂层上方Intergrina5,Intergrinβ1,FN,Vinculin的表达均比其他涂层表面少,而TiN-MG表达最高。Ag-MG,TiN-MG都能成功诱导细胞顺着沟槽排列,细胞排列一致性比例无差异,不同涂层沟槽表面“接触诱导”相关的信号蛋白p-FAK以及ROCK表达均高于光滑表面,ROCK活化与表面化学成分无关,而Ag涂层的沟槽表面p-FAK相对较低于Ti-MG和TiN-MG。10.Ag涂层的抗菌作用最强,TiN涂层的抗菌能力居中,相同涂层下沟槽形貌并没有增加细菌的粘附量。结论1.T60/10作为各向异性微沟槽的表面亲水性好,最有利于HGF粘附增殖和细胞周期推进,对HGF的“接触诱导”效应最好:细胞排列一致性和细胞骨架形变率最高,此外FAs相关的蛋白和“接触诱导”相关的信号蛋白在基因和蛋白水平上表达和活化度均最高,虽然对血浆FN的吸附弱于其他沟槽表面,但对细胞FN的分泌刺激作用最好,由于细胞来源的FN能弥补血浆来源FN的功能,最终T60/10建议为种植体穿龈部分最合适的沟槽尺寸。2.利用磁控溅射技术成功的实现了同时具有微沟槽形貌(T60/10)又具有抗菌功能的纳米涂层,其中微沟槽上方制作Ag涂层细胞相容性较差,而抗菌性最高,而微沟槽上方制作纳米TiN涂层具有一定的抗菌性能及最优的细胞生物相容性亦保持了“接触诱导”效应,因此纳米TiN涂层推荐与T60/10的微沟槽结合作为具有抗菌性能的种植体穿龈部分的材料表面。
[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.

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R783.1
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本文编号：1590427