天然复合水凝胶的构筑及促血管化研究

发布时间：2018-03-01 06:02

本文关键词： 血管化水凝胶 1-磷酸鞘氨醇羧甲基壳聚糖醛基化透明质酸　出处：《东华大学》2017年硕士论文　论文类型：学位论文

【摘要】：大段骨的缺损修复一直是临床治疗的难题。近年来,骨组织工程制备的仿生骨支架能够从结构和功能上模仿天然骨,为大段骨缺损的修复带来了希望。骨再生是一个复杂过程,包括成骨细胞的长入和增殖、营养物质和氧气的输送等,这些过程都需要血管参与,因此骨移植支架的前期血管化具有重要意义。目前,骨组织工程中常用的有效促血管化的方法有:血管诱导因子引入、成骨细胞和血管内皮细胞与支架材料共培养、相关基因的引入以及显微外科体外重建等。其中,在支架材料内负载促血管化因子是一种简单而有效的方法。本课题以介孔二氧化硅纳米颗粒(MSNs)作为血管化功能性药物1-磷酸鞘氨醇(S1P)的微载体,再将其与透明质酸/壳聚糖基天然高分子制备的水凝胶复合,构建可用于骨支架前期血管化的有效控释载体。首先,利用不同浓度羧甲基化壳聚糖(4,6和8 wt%)与醛基化透明质酸通过席夫碱反应制备三种纯水凝胶样品,通过对纯水凝胶样品的物化性能表征和生物相容性研究筛选出一种可作为后续研究的高强度水凝胶。然后,制备聚电解质藻酸钠(ALG)和壳聚糖(CHI)表面修饰的MSNs,作为药物的微载体,通过聚电解质层修饰提升粒子的生物相容性和增强粒子对药物的装载性能。进一步制备了复合微载体的纳米复合水凝胶,研究纳米粒子的加入量对水凝胶理化性能的影响,对体外药物释放行为以及体外降解行为进行了研究。研究了细胞在纳米复合水凝胶表面和内部的生长情况,并评价了载药纳米复合水凝胶对内皮细胞的募集和体内促血管化的能力。实验结果表明,纯水凝胶的力学性能和交联度随着羧甲基化壳聚糖浓度的增加而增强,但细胞增殖却相应降低。因此,综合考虑水凝胶的力学性能和细胞增殖情况,选取6 wt%羧甲基化壳聚糖用于后续研究。力学性能表征结果显示,纳米粒子的加入明显增强纳米复合水凝胶的力学性能(11.06-36.83 kPa),有效提升了天然水凝胶的力学性能。细胞实验结果表明,纳米复合水凝胶更利于细胞的增殖,少量的牛血清蛋白(BSA)的引入能够改善细胞的铺展。Transwell细胞迁移实验结果显示,载有S1P的纳米复合水凝胶表现对内皮细胞募集的作用。鸡胚尿囊绒毛膜实验和小鼠皮下包埋实验结果进一步表明,载有S1P的纳米复合水凝胶能够有效促进周围组织血管再生。而且,当载药纳米复合水凝胶与大孔支架结合,在小鼠体内植入3周后,大孔支架浅层观察到新生血管的长入。因此,制备的载有S1P的纳米复合水凝胶药物控释载体在骨组织工程血管化领域具有潜在的应用前景。
[Abstract]:In recent years, biomimetic bone scaffolds prepared by bone tissue engineering can mimic natural bone in structure and function, which brings hope to repair large segment bone defect. Bone regeneration is a complex process. These processes, including the growth and proliferation of osteoblasts, the transport of nutrients and oxygen, all require the participation of blood vessels, so the pre-vascularization of bone graft scaffolds is of great significance. The effective vascularization methods used in bone tissue engineering include the introduction of angiogenic factors, co-culture of osteoblasts and vascular endothelial cells with scaffold materials, the introduction of related genes and the reconstruction of microsurgery in vitro. It is a simple and effective method to load vascularization factor into scaffold materials. In this study, mesoporous silica nanoparticles (MSNs) were used as microcarriers of vascularized functional drug, sphingosine 1-phosphate (S1P). Then it was combined with hydrogel prepared by hyaluronic acid / chitosan based natural polymer to construct an effective controlled release carrier for early vascularization of bone scaffold. Three kinds of pure hydrogels were prepared by the reaction of carboxymethylated chitosan with aldohyaluronic acid at different concentrations for 6 and 8 wts through Schiff base reaction. A kind of high strength hydrogel which can be used as a follow-up study was selected by studying the physicochemical properties and biocompatibility of pure hydrogel samples. Then, the surface modified MSNs of polyelectrolyte sodium alginate (ALG) and chitosan (CHI) were prepared and used as microcarriers of the drug. Through the modification of polyelectrolyte layer to improve the biocompatibility of particles and enhance the loading properties of the particles, the nanocomposite hydrogels with composite microcarriers were further prepared, and the effects of the amount of nanoparticles on the physical and chemical properties of hydrogels were studied. The drug release behavior and degradation behavior in vitro were studied. The growth of cells on the surface and inside of nano-composite hydrogels was studied. The ability of drug loaded nano-composite hydrogels to recruit endothelial cells and promote vascularization in vivo was evaluated. The results showed that the mechanical properties and crosslinking degree of pure hydrogels increased with the increase of carboxymethylated chitosan concentration. However, cell proliferation was decreased. Therefore, considering the mechanical properties of hydrogel and cell proliferation, 6 wt% carboxymethylated chitosan was selected for further study. The addition of nano-particles significantly enhanced the mechanical properties of nano-composite hydrogels 11.06-36.83 KPA, and effectively enhanced the mechanical properties of natural hydrogels. The results of cell experiments showed that nano-composite hydrogels were more conducive to cell proliferation. The introduction of a small amount of bovine serum protein (BSA) can improve the proliferation of cells. Transwell cell migration results show that, The effect of S1P loaded nano-composite hydrogel on endothelial cell recruitment. Chicken embryo chorionic chorioallantoic test and mouse subcutaneous embedding test further showed that, Nano-composite hydrogels containing S1P could effectively promote vascular regeneration in peripheral tissues. Moreover, when drug-loaded nano-composite hydrogels were combined with macroporous scaffolds, they were implanted in mice for 3 weeks. The growth of neovascularization was observed in the superficial layer of the macroporous scaffold. Therefore, the S1P loaded nano-composite hydrogel drug controlled release carrier has a potential application prospect in the field of bone tissue engineering vascularization.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08;R68

【引证文献】