纯钛表面载LAMA3涂层在种植体—牙龈生物学封闭形成中的作用

发布时间：2018-01-02 10:19

本文关键词：纯钛表面载LAMA3涂层在种植体—牙龈生物学封闭形成中的作用　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：种植义齿要在口腔环境中获得长期的稳定,不仅需要稳固的种植体-骨结合,种植体-牙龈上皮结合界面作为生物屏障在抵御病原体入侵中也极为重要。与天然牙-结合上皮界面相似,种植体周围上皮主要依靠内基板和半桥粒附着在种植体表面。然而,种植体与牙龈的结合较为薄弱,上皮的附着仅局限在根方1/3。内基板中的重要组份为laminin 332,其中的α3链在细胞行为的调控以及半桥粒的形成中起重要作用。本研究通过在种植体表面构建载LAMA3基因的生物涂层,利用体内、体外实验进行生物学评价,并探讨该涂层在种植体周围上皮附着形成中的作用及机制。本研究中首先利用层层自组装技术在纯钛表面制备由壳聚糖(CS)、透明质酸(HA)和IV型胶原(COL)组装的生物涂层,通过扫描电镜(SEM)、原子力显微镜(AFM)、X射线光电子能谱(XPS)和接触角测试仪对不同组装层数以及交联前后的涂层进行检测,并评估各组样品对上皮细胞活性的影响。综合表面形貌以及XPS的检测结果,表明CS/(HA/COL)n多层膜组装至5层时经化学交联后的涂层较为稳定,因此选取5层作为后续研究中的组装层数。细胞活性检测结果也表明CS/(HA/COL)n涂层在化学交联前后都具有良好的细胞相容性。在构建完成CS/(HA/COL)5涂层的基础上,将抗腺病毒抗体偶联至涂层表面,利用抗原-抗体的特异性结合将载LAMA3重组腺病毒固定在CS/(HA/COL)5涂层上,从而实现纯钛表面LAMA3基因原位转染体系的构建。通过条件优化,偶联抗体的最佳浓度为30μg/孔,每个样品腺病毒使用量为1×108PFU。然后利用人永生化表皮细胞HaCaT对纯钛表面载LAMA3基因涂层进行细胞学评价。结果表明该涂层有利于上皮细胞的早期粘附和伸展,能有效提高lamininα3的表达,并能诱导结合界面成熟半桥粒结构的形成。最后通过体内实验,利用免疫组织形态学、透射电镜观察和辣根过氧化物酶(HRP)浸润性检测等研究纯钛表面载LAMA3涂层在种植体-牙龈生物学封闭形成中的作用。结果表明该涂层在创伤早期可以有效提高种植体周围上皮laminin a3的表达,加快种植体周围上皮的形成,促进种植体-牙龈结合界面半桥粒的整合,相比于光滑表面种植体组可以明显减少HRP在种植体周围牙龈组织中的浸润。本研究在纯钛表面构建载LAMA3基因的生物涂层,能有效实现种植体表面原位基因转染,提高上皮细胞中laminin a3链的表达。体内、外的研究均表明该涂层有利于上皮细胞的早期粘附和伸展,促进结合界面上半桥粒结构的形成,并有效改善种植体-牙龈上皮的生物学封闭。本文为促进种植体周围上皮附着形成和改善种植体-牙龈生物学封闭的研究提供了新的思路以及实验依据。
[Abstract]:To achieve long-term stability of implant denture in the oral environment, not only need to implant bone solid combination implant gingival epithelial interface as a biological barrier against the pathogens that invade are also extremely important. With natural teeth with epithelial interface are similar, around the implant on the skin mainly depends on the substrate and in hemidesmosome attachment on the surface of implant. However, combination of implant and gingival epithelial attachment is relatively weak, only in the root 1/3. in the substrate in an important component for the laminin 332, the alpha 3 chain in the regulation of cell behavior and hemidesmosome formation plays an important role. This study constructed by in vivo biological the coating, carrying LAMA3 gene on implant surface, biological evaluation in vitro, and to investigate the effect and mechanism of adhesion formation in the coating in the epithelium around the implant. This study firstly by LBL In the surface of pure titanium was prepared by chitosan (CS), hyaluronic acid (HA) and type IV collagen (COL) biological coating assembly, by scanning electron microscopy (SEM), atomic force microscopy (AFM), X ray photoelectron spectroscopy (XPS) and contact angle tester for detection of different coatings the number of the layers and before and after crosslinking, and evaluate the influence of each sample on the epithelial cell activity. Surface morphology and XPS detection results, showed that CS/ (HA/COL) n multilayer assembly to 5 layer coating by chemical crosslinking in the relatively stable, so as to select 5 layer assembly layers in the follow-up research. Cell activity the test results also show that the CS/ (HA/COL) n coatings have good biocompatibility in chemical crosslinking. Before and after in the construction of CS/ (HA/COL) based on the 5 coating, the surface of anti adenovirus antibody coupled to the coating, the use of specific antigen antibody binding to LAMA3 recombinant adenovirus Fixed in CS/ (HA/COL) 5 coating, so as to realize the construction of LAMA3 pure titanium surface in situ gene transfection system. By optimizing the conditions, the optimal concentration of antibody coupled with 30 g/ holes, each sample of adenovirus usage was 1 * 108PFU. and then use the immortalized human epidermal cells of HaCaT on the surface of pure titanium coating containing LAMA3 gene cytological evaluation. The results show that the coating is conducive to early adhesion of epithelial cells and stretch, can effectively improve the laminin alpha 3 expression, and can induce the interface formed by the combination of mature hemidesmosome structure. Finally, in vivo, using immunohistochemical morphology, transmission electron microscopy and horseradish peroxidase (HRP) invasive detection etc. study on the surface of pure titanium LAMA3 coating on the implant gingival biology closed formation. The results show that the coating can effectively improve the expression of peri implant epithelium laminin A3 in the early stage of trauma, Accelerate the formation of peri implant epithelium, promote implant junctional interface integration hemidesmosomes, compared to the smooth surface of the implant group can significantly reduce HRP infiltration in gingival tissue around the implants. The establishment of biocoatings carrying LAMA3 gene on the surface of pure titanium implant surface can effectively achieve the in situ gene transfection to improve the expression of laminin, A3 chain in epithelial cells. In vivo, the results show that the coating is conducive to the early adhesion of epithelial cells and stretch, promoting the combination of hemidesmosomes structure on the interface, and effectively improve the closed biological implant gingival epithelium. In order to promote implant epithelial attachment formation and improvement research body - gum biology closed planter provides ideas and new experimental evidence.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R783.6

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