负载bFGF或BMP-2的脱细胞真皮基质屏障膜引导骨再生的作用探讨

发布时间：2018-06-13 23:11

本文选题：脱细胞真皮基质 + 碱性成纤维细胞生长因子　；参考：《山东大学》2017年硕士论文

【摘要】：背景与目的:引导组织再生(GTR)或引导骨再生(GBR)是牙周/骨组织再生医学领域的重要技术,当今医学的发展不仅要求GTR膜作为屏障,而且希望其能作为载体释放药物,如抗炎药,生长因子,粘附因子等促进缺损区域的愈合。碱性成纤维细胞生长因子(bFGF)对干细胞有明显促增殖作用,并有促血管形成及神经再生等功能。骨形态发生蛋白-2(BMP-2)以促成骨分化为特色。我们认为BMP-2和bFGF均可作为GTR和GBR屏障膜材料的备选负载因子。另外,脱细胞真皮基质(ADM)来自于天然皮肤,是一种被去除细胞结构的细胞外基质,并且其本身具有一定的促进组织再生的生物活性,目前商品化的ADM膜已在临床广泛应用但是,是否负载BMP-2或bFGF的ADM膜材料更能有效加速骨缺损愈合有待探索,本实验的目的是比较负载bFGF、BMP-2的ADM屏障膜材料在骨缺损修复中的作用,并探讨其可能的不同机制。材料与方法:(1)膜材料负载因子。无菌生理盐水分别配置200ng/ml的bFGF和800ng/ml的BMP-2,取大小相等的1cm2的膜材料,0.5ml溶液浸泡过夜,真空冻干机冻干膜材料备用。采用ELISA和体外缓释系统检测载药膜材料的释药情况。(2)实验分为四组,分别为空白对照组(control),单纯膜材料组(ADM),负载BMP-2的膜材料组(ADM+BMP-2),负载bFGF的膜材料组(ADM+bFGF)。在8w龄的雌性Wistar大鼠的颅骨左侧顶骨上制备8mm×7mm椭圆全厚骨缺损,分别将上述膜材料覆盖于缺损表面,并设置空白对照组。于术后1周,2周和8周处死实验动物、取材。(3)术后1周和2周,采用CD34-/CD90+免疫荧光双染观察骨缺损愈合过程中材料对间充质干细胞的募集情况。(4)术后8周,通过HE染色,Micro CT扫描分析比较四个实验组骨缺损的整体愈合情况。(5)术后8周,免疫组化检测骨桥蛋白(OPN)的表达以检测干细胞成骨分化的情况。所得数据全部采用均数±标准差(Mean±SD)表示。应用SPSS 17.0软件包,对相关数据进行单因素方差分析,α=0.05。结果:(1)负载因子并冻干的膜材料在形态、结构上未发生明显改变。(2)术后实验动物术后成活率约90%,缺损处无明显炎症,并无明显免疫排斥反应发生。(3)CD34-/CD90+免疫荧光双染结果显示ADM+bFGF在1周和2周时对干细胞的募集作用很强,明显高于其他三组(p0.01)。(4)组织学HE染色观察发现,ADM组、ADM+BMP-2组及ADM+bFGF组缺损处修复组织量明显多于空白对照组;8w时MicroCT扫描分析结果显示,所有植有ADM膜的实验组骨愈合情况明显好于空白对照组,ADM+BMP-2组、ADM+bFGF组骨缺损基本完全愈合,新生骨体积明显多于其他两组。(5)骨桥蛋白(OPN)免疫组化染色显示8周时ADM+BMP-2组、ADM+bFGF组的平均光密度(IOD)无明显差异,但显著高于空白对照组和单纯ADM组(p0.05)。结论:与传统生长因子BMP-2相比,bFGF对干细胞有早期募集和促增殖的作用,同时保留了其成骨向分化能力,其与GTR膜材料的结合不仅发挥了屏障膜的空间保持功能,而且可有效加速骨缺损的愈合。
[Abstract]:Background & objective: guided tissue Regeneration (GTRR) or guided Bone Regeneration (GBR) is an important technology in periodontal / bone regeneration medicine. The development of modern medicine not only requires GTR membrane to act as a barrier, but also hopes that it can be used as a carrier to release drugs, such as anti-inflammatory drugs. Growth factor, adhesion factor and so on promote the healing of defect area. Basic fibroblast growth factor (bFGF) can promote the proliferation of stem cells and promote angiogenesis and nerve regeneration. Bone morphogenetic protein-2 (BMP-2) is characterized by bone differentiation. We consider that BMP-2 and bFGF can be used as alternative load factors for GTR and GBR barrier membrane materials. In addition, acellular dermal matrix (ADM) comes from natural skin and is an extracellular matrix with removed cellular structure, and it has certain biological activity to promote tissue regeneration. At present, commercial ADM membrane has been widely used in clinical practice. Whether ADM membrane material loaded with BMP-2 or bFGF can accelerate bone defect healing more effectively remains to be explored. The purpose of this study was to compare the role of ADM barrier membrane loaded with bFGF- BMP-2 in bone defect repair and to explore its different mechanisms. Materials and methods 1) membrane material loading factor. 200ng/ml bFGF and 800ng/ml BMP-2 were prepared with aseptic saline respectively. The membrane material of 1cm2 of equal size was soaked in 0.5ml solution for the night. The vacuum freeze-drying machine was used for freeze-drying membrane material. Elisa and in vitro slow-release system were used to detect the drug release of drug-loaded membrane material. The experiment was divided into four groups: blank control group, simple membrane material group, ADM BMP-2 group, and bFGF loaded membrane material group. 8mm 脳 7mm elliptical full-thickness bone defects were prepared on the left parietal bone of female Wistar rats aged 8 weeks. The above membrane materials were covered on the surface of the defect respectively, and a blank control group was set up. The experimental animals were killed at 1 week, 2 weeks and 8 weeks after operation. After 1 and 2 weeks of operation, the recruitment of mesenchymal stem cells (MSCs) was observed by CD34 / CD90 immunofluorescence double staining. Eight weeks after operation, osteopontin (OPN) expression was detected by immunohistochemistry to detect osteogenic differentiation of stem cells. All the data were expressed as mean 卤standard deviation (mean 卤SD). Using SPSS 17.0 software package, the single factor ANOVA of related data was carried out, 伪 0.05. Results (1) the morphology and structure of the lyophilized membrane material were not changed significantly. The survival rate of the experimental animals after operation was about 90%, and there was no obvious inflammation in the defect. The results of immunofluorescence double staining of CD34 / CD90 showed that ADM bFGF had a strong recruitment effect on stem cells at week 1 and week 2. The results of HE staining showed that the defects of ADM BMP-2 and ADM bFGF in ADM group were significantly higher than those in control group at 8 weeks. The bone healing of all experimental groups with ADM membrane was better than that of ADM BMP-2 group, and the bone defect of ADM bFGF group was almost completely healed. The mean optical density (IOD) of ADM BMP-2 / ADM bFGF group was significantly higher than that of blank control group and ADM group at 8 weeks, but significantly higher than that of blank control group and ADM group. Conclusion: compared with the traditional growth factor BMP-2, bFGF has the function of early recruitment and proliferation of stem cells, while preserving its ability of osteogenic differentiation. The combination of bFGF and GTR membrane material not only plays a role in maintaining the space of barrier membrane. And it can accelerate the healing of bone defect effectively.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781

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