LRP5修饰的BMSC膜片在种植体骨结合中的作用及种植体失败率临床研究

发布时间：2018-05-17 08:02

  本文选题：纯钛种植体 + 骨髓间充质干细胞膜片　； 参考：《浙江大学》2017年博士论文

【摘要】：种植牙被认为是人类的第三副牙齿,种植义齿修复后,能最大程度地提高缺牙患者的咀嚼效率,延缓牙槽骨吸收,提高生活质量。大量的研究报道了种植体表面改性的方法,然而相较于种植体表面处理,对于种植体骨结合的形成机理的研究较少。本研究通过在种植体表面构建低密度脂蛋白受体-5(Low-density lipoprotein receptor-related protein 5,LRP5)过表达和 LRP5 干扰的细胞膜片,利用体内、体外实验进行生物学评价,并探讨该基因改性的细胞膜片在种植体骨结合中的作用。本研究中首先在二氧化钛(Ti02)纳米点石英晶片表面预铺层黏连蛋白-521(Laminin-521,LN-521),利用光控细胞薄层技术制备出骨髓间充质干细胞膜片(Bone marrow mesenchymal stem cell sheet,BMSC sheet),通过粘附、增殖、活性、三向分化等实验证实了BMSC膜片的成功构建,并且LN-521明显促进了BMSCs的粘附,365nm紫外线(ultraviolet ray,UvV)对BMSC膜片的获取是安全的,获得的BMSC膜片具有良好的细胞活性,呈叠层生长状态,并保持着细胞干性,可以向成骨、成脂、成软骨方向分化。粘附实验的结果显示当LN-521的浓度为1200ng/mL时,对BMCSs促粘附作用较强,能够稳定的构建BMSC膜片,因此后续实验中将选取1200ng/mL作为LN-521的预铺浓度。在构建完成BMSC膜片的基础上,利用慢病毒转染技术制备载绿色荧光蛋白(GFP)和荧光素酶报告基因的BMSC膜片,从而实现膜片种植体的体内追踪。结果显示,通过小动物活体成像系统检测,携带荧光素酶报告基因的BMSC膜片种植体在植入后1个月内可以检测到信号,2个月后信号消失,但GFP示踪方法并不适合在体内追踪BMSC膜片种植体。然后在构建完成BMSC膜片种植体的基础上,制备Ti02纳米点种植体。体内外实验结果表明Ti02纳米点表面能够显著促进BMSC膜片的成骨向分化,有利于BMSCs的粘附和增殖,能有效提高成骨相关基因碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)、Ⅰ 型胶原(CollagenⅠ),骨形态发生蛋白 2(bonemorphogenicprotein2,BMP2)、Runx2、Osterix 的表达,并能促进种植体骨结合。最后通过腺病毒和慢病毒转染技术,以BMSC膜片为载体,本研究构建了LRP5过表达和LRP5干扰型的BMSC膜片种植体。体内外实验结果表明LRP5过表达后能够明显地促进BMSCs向成骨细胞分化,经典的Wnt信号通路激活,成骨相关基因Runx2、Osterix、ALP、OCN、BMP2表达增加,加快种植体骨结合的形成;相反地,LRP5干扰后会显著降低BMSCs向成骨细胞分化,抑制经典的Wnt信号通路,成骨相关基因Runx2、Osterix、ALP、OCN、BMP2表达减少,种植体骨结合形成减慢。临床研究通过回顾浙江大学医学院附属口腔医院2006-2011年间的种植患者资料,结果显示种植体失败发生率跟患者的年龄、性别、种植牙位、种植术式、种植医生资历等多种因素相关,种植体周围炎是种植体发生失败的主因,种植医师应在治疗过程中预防种植体周围炎的发生发展,降低种植失败率。
[Abstract]:Implant tooth is considered to be the third pair of human teeth. Implant denture can improve the masticatory efficiency, delay alveolar bone resorption and improve the quality of life. A large number of studies have reported the methods of surface modification of implants. However, compared with implant surface treatment, there are few studies on the formation mechanism of implant bone bonding. The aim of this study was to construct over expressed low density lipoprotein receptor low density lipoprotein receptor-related protein 5 LRP5 (LRP5) and LRP5 interference cell membranes on the surface of the implants for biological evaluation in vivo and in vitro. The role of the modified cell membrane in the osseous binding of implants was investigated. In this study, bone marrow mesenchymal stem cell sheettte (BMSCs) membranes were prepared by photo-controlled cell thin-layer technique, and bone marrow mesenchymal stem cell sheettes were prepared by adhesion, proliferation, and activity of bone mesenchymal stem cells (BMSCs), which were precoated with laminin-521 (laminin-521) and LN-521 on the surface of quartz wafers of titanium dioxide (Ti02). Three-way differentiation experiments confirmed the successful construction of BMSC membrane, and LN-521 significantly promoted the BMSCs adhesion to UV ultraviolet UV V). The obtained BMSC membrane had good cellular activity and was in a laminated growth state, and the obtained BMSC membrane was safe to obtain the BMSC membrane. And maintain the dry nature of the cell, can be osteogenic, adipogenic, cartilage differentiation. The results of adhesion experiment showed that when the concentration of LN-521 was 1200ng/mL, the adhesion to BMCSs was stronger, and the BMSC membrane could be constructed stably. Therefore, 1200ng/mL was chosen as the concentration of LN-521 in subsequent experiments. Based on the construction of BMSC membrane, the BMSC membrane carrying green fluorescent protein (GFP) and luciferase reporter gene was prepared by lentivirus transfection technique, so that the membrane implants could be traced in vivo. The results showed that the BMSC membrane implants carrying luciferase reporter gene could detect the signal within one month of implantation and then disappeared after 2 months. However, GFP tracer method is not suitable for tracking BMSC membrane implants in vivo. Then, on the basis of constructing BMSC film implant, Ti02 nano-dot implant was prepared. The results of in vitro and in vivo experiments showed that the surface of Ti02 nanoparticles could significantly promote the osteogenic differentiation of BMSC membranes and facilitate the adhesion and proliferation of BMSCs. It can effectively increase the expression of ALP, osteocalcinin, Collagen 鈪，

本文编号：1900596