【作者】 杨帆；

【导师】 赵士芳；

【作者基本信息】 浙江大学 ， 口腔医学， 2011， 博士

【摘要】 缩短种植体骨结合的时间,达到种植体早期负载甚至即刻负载是种植体表面改性研究的主要目标。除了种植体的表面形貌和化学性质的影响,患者种植部位的骨量和骨质也是影响种植体成功的重要因素。我国现已进入老龄化社会,骨质疏松症患者增多,对牙种植体的需求不断上升,然而另一方面,系统性骨质疏松影响颌骨的骨量和骨密度,成为了牙种植手术的一个相对禁忌症。所以,让钛种植体和骨质疏松症患者周围骨组织之间快速牢固的结合是牙种植手术现今急需克服的一个难题。近年来研究发现他汀类药物具有促进骨形成、抑制骨吸收的双重作用,并且能诱导骨形成蛋白-2(BMP-2),——一类已知的具有最强的骨诱导能力的生长因子的表达。本研究采用物理吸附法和仿生沉积法,在纯钛种植体表面设计、构建辛伐他汀涂层,通过体外细胞实验筛选合适浓度,然后建立骨质疏松症大鼠种植体动物模型,研究含药涂层对骨质疏松症大鼠种植体骨结合的影响。研究分两部分：第一部分吸附辛伐他汀纯钛表面对骨质疏松症大鼠种植体骨结合作用的研究实验方法和结果将纯钛钛片表面打磨抛光,再喷砂酸蚀处理,得到我们所需要的基底面。然后将辛伐他汀溶解在75%乙醇溶液中,制备成终浓度为0(对照组),10-7M,10-6M,10-5M和10-4 M的辛伐他汀乙醇溶液,将钛片浸泡在上述溶液中48小时,通过物理吸附作用在钛片表面吸附上辛伐他汀。通过场发射扫描电镜FSEM对钛片表面进行表征。然后在上述各组钛片上培养小鼠前成骨细胞系MC3T3-E1,观察涂层对细胞成骨分化能力的影响。FSEM显示该涂层基本保留了原来的粗糙表面形貌。细胞实验的结果表明低浓度的辛伐他汀处理组(10-7，10-6M,尤其是10-7M)能显著促进MC3T3-E1的分化。基于体外细胞实验的结果,我们设计螺纹型种植钉,分为对照组,实验组1(10-7M)和实验组2(10-6M)三个组,植入骨质疏松症大鼠。在术后1周、2周、4周和12周分别处死一批动物,制作硬组织切片。采用Olympus光镜显微镜和Image-Pro PlusR半自动图像处理软件对种植体-骨接触率和种植体螺纹内骨密度进行测量,并进行统计学分析。结果显示,在各个观察点,2个实验组表面形成新骨的时间早于对照组,形成新骨的数量也高于对照组,并有统计学差异,两个实验组之间没有统计学差异。第二部分纯钛表面磷酸钙盐仿生沉积法制备的辛伐他汀涂层对骨质疏松症大鼠种植体骨结合作用的研究实验方法和结果纯钛钛片基底面处理同实验第一部分。然后通过仿生沉积法在纯钛表面构建仿生涂层,这其中,我们在仿生液B中加入不同浓度的辛伐他汀(终浓度分别为0,10-7M,10-6M和10-5M),这样得到了含有不同浓度含药涂层。采用FSEM、XRD和FTIR观察分析涂层的表面形貌、晶体结构和化学组成,在上述各组钛片上培养小鼠前成骨细胞系MC3T3-E1,观察涂层对细胞增殖和成骨分化能力的影响.FSEM显示,低浓度组(10-7M组和10-6M组)的晶体的排列结构与对照组并没有显著区别；高浓度组(10-5M,10-4M和10-3M)表面的晶体形态和尺寸均有较大的改变：片状的晶体随着浓度升高而变得越薄,边缘也不规则；花状结构减少,绒毛样球状的结构出现；浓度越大,这种变化就越明显,10-3M组表面片状结构完全消失,表面呈现大小不一的球状结构。XRD和FTIR的结果显示所有涂层表面都含有磷酸八钙OCP,而且涂层内含有辛伐他汀,并且随着仿生液中辛伐他汀浓度的增高,涂层内辛伐他汀浓度也随之升高。细胞实验的结果表明低浓度的辛伐他汀处理组10-6M能增强MC3T3-E1细胞ALP活性,OC分泌,同时对细胞增殖也没明显影响。基于体外细胞实验的结果,我们设计螺纹型种植钉,分为对照组,实验组(10-6M)两个组,植入骨质疏松症大鼠。在术后4周和12周分别处死一批动物,制作硬组织切片。采用Olympus光镜显微镜和Image-Pro PlusR半自动图像处理软件对种植体骨接触率和种植体螺纹内骨密度进行测量,并进行统计学分析。结果显示,在各个观察点,实验组表面形成新骨的时间和数量均早于或高于对照组,并有统计学差异。结论在纯钛种植体表面构建含辛伐他汀药物涂层能促进骨质疏松症大鼠的种植体表面新骨的形成,从而增强种植体-骨的结合强度,为临床开发适合骨质疏松症患者的新型种植体提供了科学实验依据。更多还原

【Abstract】 BACKGROUNDShortening the period of implant-bone osseointegration to achieve early loading even instant loading, is the main goal of research of surface modification of dental implants. Besides the effects of surface morphology and chemistry of implant surface, the recipient bone quantity and quality is also crucial factors. Since China is an aging society now, the population of osteoporotic people is increasing, as well as the need for dental implants. However, systematic osteoporosis affects the quantity and quality of jaw bones, which is considered as a contraindication of dental implant surgery. Thus, the urgent task of modern implantology is to achieve fast and solid osseointegration of titanium implants and surrounding osteoporotic bone tissue.Recently, it was reported that a kind of liposoluble statin, simvastatin, could induce the expression of bone morphogenetic protein (BMP)-2 mRNA and that, as a result, it promoted bone formation on the calvaria of mice. And some researchers demonstrated the topical application of statins improved bone formation around implants. In this context, we designed simvastatin-loaded porous titanium implant surface by immersion method and biomimetic method. The aim of this study was to investigate the feasibility of local delivery of simvastatin using these two methods and the effects of the delivery on implant osseointegration by in vitro and in vivo experiments. The study is divided into two parts:Part I Preparation of simvastatin-loaded porous titanium surfaces by immersion method on Ti-based dental implant surface and its effects on implant osseointegration in osteoporotic ratsMETHODS AND RESULTSWe got our substrate Ti surfaces by polishing, sandblasting and etching. The control group consisted of cells cultured on titanium disks without any intervention for different time intervals (4 d,7 d and 14 d), whereas the experimental groups (simvastatin-loaded groups) consisted of cells cultured on titanium disks that were pre-incubated in varying concentration (10-7M,10-6M, 11-5M and 10-4M) of simvastatin for the same time intervals of the control. Alkaline phosphatase activity (ALP), Type I collagen synthesis and osteocalcin release were used to measure the cellular osteoblastic activities.All simvastatin-loaded groups showed increased ALP activity compared to control group at every time point, especially the 10-7 M group significantly increased the activity by almost 4-fold at 4 days (P＜0.05). In the Type I collagen synthesis assay, all simvastatin-loaded groups showed an increase and the effect was inverse dose dependent (maximal at 10-1M). Furthermore, this stimulatory effect of simvastatin was also observed in osteocalcin release assay (P＜0.05, at 10-7M,10-6M, maximal at 10-7 M). Based on the in vitro results, roughened implants were divided into control groups (n=32), test 1 group (n=32), and test 2 group (n=32). Test implants were immersed into 10"7 M (Test 1 implants) or 10-6M (Test 2 implants) simvastatin solutions for drug adsorption onto implant surfaces.48 ovariectomized rats randomly received two implants in both tibiae. After 1,2,4, and 12 weeks of implantation, tibias were retrieved and prepared for histomorphometric evaluation. Bone-implant contact (BIC) and bone area around implant, as well as histological findings, were obtained. Results:New bone formation on test implant surfaces was seen after 1 week while it was seen on the control implant surface after 2 weeks. There were more bone tissue and bone-implant contact along the test implant surfaces than which along the control implant surface. The test 1 and 2 implants showed a significantly greater bone area and BIC compared to the control implant during the observation periods (p<0.05). No differences were found between the test 1 and test 2 implants after 1,2,4, and 12 weeks (p>0.05).Part II Preparation of simvastatin-incorporated coating by biomimetic method on Ti-based dental implant surface and its effects on implant osseointegration in osteoporotic ratsMETHODS AND RESULTSWe got our substrate Ti surfaces by polishing, sandblasting and etching as previously described. Simvastatin was prepared onto titanium porous surfaces by biomimetic calcium phosphate coating by adding a series of concentrations simvastation (the final concentration is 10-7M,10-6M and 10-5M) in SBF-2. FSEM, XRD and FTIR were utilized to observe the surface morphology and the crystal structure of the coatings. MC3T3-E1 cells were cultured on these disks with varying concentration of simvatatin. The total protein content, ALP activity and osteocalcin production were measured to examine the effects of coatings to the proliferation and differentiation ability.FSEM results showed that sharp crystal flakes were deposited on the control plate surface. The crystalline coating entirely covered the substrate surface. All the crystal plates were oriented more or less perpendicularly to the surface of the plates. The flake morphology and orientation did not obviously change at the low concentration of simvastatin (10-7M and 10-6M). However, the morphology and size of crystal flakes changed at the high concentration. At the 10-5M and10-4M concentration, flake-like crystals combined into sphere-like structures. The size of flakes dramaticly decreased. At the 10-3M concentration, the sphere-like structures were seen on the sample surfaces while the flake crystals disappeared. XRD and FTIR results indicated the presence of OCP in all the coatings. Simvastatin has been incorporated into the synthetic OCP coatings. What’s more, the content of simvastatin in the coating also increased with the increasing concentration of simvastatin in SBF-2 accordingly.Based on the in vitro results,10-6 M (Test implants) was chosen for the treatment of implant screws in osteoporotic animal experiments. After 4 and 12 weeks of implantation, tibias were retrieved and prepared for histomorphometric evaluation. Bone-implant contact (BIC) and bone area around implant, as well as histological findings, were obtained. New bone formation on test implant surfaces was seen earlier than control group. Also there were more bone tissue and bone-implant contact along the test implant surfaces than which along the control implant surface at 4 and 12 weeks. (p<0.05).Conclusion:These results indicate that simvastatin-loaded porous implant surface has the potential to improve implant osseointegration in the ovariectomized model, which provide scientific proof for develop novel dental implants for osteoprotic patients clinically.更多还原