界面仿生沉积修饰衍生骨支架的制备及生物学性能的研究

发布时间：2018-08-31 18:21

【摘要】：目的:骨移植是临床上最常用的颌面部骨缺损修复方法。异种骨(如牛、羊、猪等哺乳动物)的松质骨内部存在着与人体骨结构、成份极为相似的无机骨支架,且来源广泛,是一种良好的、能够制备成骨移植材料的天然原料。异种骨可以通过不同的方法脱去抗原,制备获得仅具备无机成份的生物衍生骨支架,该支架是一种极具发展潜力的骨移植材料。然而,经去抗原处理后的生物衍生骨支架内部的主要成份为羟基磷灰石,其性质稳定、不易降解,无法在植入早期提供充足的成骨原料,从而影响其成骨活性。因此,为了获得更好的成骨活性,需要对支架表面进行修饰。目前,羟基磷灰石表面修饰的方法众多,大多数方法程序较为复杂,实现条件较为苛刻。所以,探索出一种简便、容易实现的生物衍生骨支架表面修饰方法是十分必要的。方法:1.取牦牛松质骨骨块,在甲醇/氯仿混合液、30%过氧化氢溶液中交替反复浸泡,脱去抗原,再经900℃煅烧2小时,制备出去抗原的衍生骨支架,应用SEM、XRD、FTIR对材料的结构、成份进行表征。2.将制得的衍生骨支架材料在37℃的2倍模拟体液中连续浸泡,进行仿生沉积表面修饰,应用SEM、XRD、FTIR分别对浸泡1周、3周、5周后的材料进行结构、成份的表征。3.将MC3T3-E1细胞株进行体外诱导并获得成骨细胞,将其与各组材料进行共培养,观察成骨细胞在材料表面的早期黏附、铺展、增殖行为,研究材料表面成骨细胞碱性磷酸酶的活性,从而分析不同表面修饰后材料的早期体外成骨性能。结果:1.采用化学物理联合法能够完全脱去异种骨中的抗原成份,获得了骨支架材料,其内部呈多孔结构,成份表征为羟基磷灰石。2.衍生骨支架材料经过仿生沉积后,其表面均形成了矿化沉积物,随着浸泡时间延长,沉积物也在逐渐增多,在仿生沉积5周时,沉积物覆盖面积最大,沉积物分布最均匀。XRD、FTIR检测证实了材料表面形成的沉积物是含有CO32-的、结构较为复杂的类骨磷灰石。3.与未进行仿生沉积处理的骨支架材料相比,成骨细胞在仿生沉积修饰后的材料表面粘附和增殖量更大,成骨细胞的碱性磷酸酶活性表达更强,且成骨细胞在材料表面铺展状态良好,伪足向四周充分伸展。结论:经化学物理联合法可以制备出完全脱抗原的骨支架,通过2倍仿生体液沉积后,材料表面可以沉积生成类骨磷灰石,浸泡沉积5周后,衍生骨支架材料能够更好地促进成骨细胞在材料表面早期黏附、铺展和增殖,且细胞的功能状态良好。
[Abstract]:Objective: bone transplantation is the most commonly used method for repairing maxillofacial bone defects in clinic. Xenogeneic bone (* bovine, sheep, pig and other mammals) has inorganic bone scaffold which is very similar to human bone structure and components. It has a wide range of sources and is a good natural raw material for the preparation of bone graft materials. Bio-derived bone scaffolds with inorganic components can be prepared by removing antigens in the same way. This scaffold is a promising bone graft material. However, hydroxyapatite is the main component in the scaffolds after removing antigens, which is stable in nature and difficult to be degraded, and can not provide sufficient formation in the early stage of implantation. In order to obtain better osteogenic activity, it is necessary to modify the surface of the scaffolds. At present, there are many methods to modify the surface of the scaffolds. Most of the methods are complicated and the conditions are harsh. Methods: 1. The antigen-derived bone scaffolds were prepared by soaking the cancellous bone of yak in methanol/chloroform mixture, 30% hydrogen peroxide solution and calcining at 900 C for 2 hours. The structures and compositions of the scaffolds were characterized by SEM, XRD and FTIR. Immersion in simulated body fluid at 7 C for 2 times, surface modification by biomimetic deposition, SEM, XRD and FTIR were used to characterize the structure and composition of the material after immersion for 1 week, 3 weeks and 5 weeks respectively. 3. MC3T3-E1 cell line was induced in vitro and osteoblasts were obtained. The osteoblasts were co-cultured with various groups of materials to observe the early stage of osteoblasts on the surface of the material. Adhesion, spreading and proliferation were studied to study the activity of osteoblast alkaline phosphatase on the surface of the material, and to analyze the early osteogenic properties of the material after different surface modification. Results: 1. The antigenic components of xenogeneic bone were completely removed by chemical-physical combination method, and the bone scaffold material was obtained. Its internal structure was porous and its composition was characterized as hydroxyl. Matrix apatite. 2. After biomimetic deposition, mineralized sediments were formed on the surface of the derived bone scaffolds. With the extension of immersion time, the sediments increased gradually. At 5 weeks of biomimetic deposition, the sediment coverage was the largest and the sediment distribution was the most uniform. Comparing with bone scaffolds without biomimetic deposition, osteoblasts adhered and proliferated more on the surface of biomimetic deposition modified materials, and the expression of alkaline phosphatase activity of osteoblasts was stronger. The osteoblasts spread well on the surface of materials, and the pseudopodia extends fully around the material. Bone scaffolds can be prepared by chemical and physical methods. After 2 times biomimetic humoral deposition, bone-like apatite can be deposited on the surface of the material. After 5 weeks of immersion and deposition, the derived bone scaffolds can promote the early adhesion, spreading and proliferation of osteoblasts on the surface of the material, and the function of the cells is good.
【学位授予单位】：北华大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08

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