【作者】 刘君毅；

【导师】 莫秀梅；

【作者基本信息】 东华大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 组织工程学是一门将细胞生物学和材料学相结合的新兴学科。组织工程学通过将种子细胞种植于仿生支架材料上,并在体外培养增殖,在形成新的组织后植入受损部位,从而达到修复和重建原人体组织结构和功能的目的。因此,组织工程学的关键,同时也是具有挑战性的一步就是如何制备具有仿生天然细胞外基质的工程支架。首先,这种组织工程支架要在结构上模拟天然细胞外基质的三维多孔结构；其次,该支架要具有良好的生物安全性和相容性；最后,这种支架要从功能上模拟天然细胞外基质,即能够促进细胞的生长和诱导组织的形成。静电纺丝技术是现阶段一种制备数十到数百纳米纤维的有效方法。由于生物高分子具有良好的生物相容性,近年来国内外都对生物高分子的静电纺丝做了大量的研究。静电纺生物高分子在组织工程支架、组织修复等方面具有独特的优势。本实验通过静电纺丝的方法制备SF-PLGA纳米纤维膜,并通过SEM、FTIR、XRD、接触角、力学性能测试分析该纳米纤维的各项物理、化学性能。实验发现该共混纤维的直径随着PLGA在共混体系中的比例的增加而增大；在共混比例相同时,直径随着溶液总质量体积比的递增而逐渐增大。随着丝素蛋白的含量的增加,力学性能下降,但亲水性有所提高。为了提高静电纺SF-PLGA纳米纤维的耐水性和稳定性,选择甲醇蒸气对SF-PLGA共混纳米纤维进行后处理,并对处理后的SF-PLGA纳米纤维的性能进行了研究,实验发现经过处理后的共混纳米纤维,直径较处理前有所增大。甲醇蒸气处理后的纤维接触角和力学性能都较处理前有所减弱,但纯SF经过处理后,应力变大。通过这些测试,我们对SF-PLGA共混纳米纤维处理前后的物理、化学性质有了基本了解,为后续的细胞实验和动物实验奠定了基础。通过在共混SF-PLGA纳米纤维膜上种植雪旺细胞,用MTT法检测细胞的黏附与增殖情况,在体外评价材料的生物相容性。通过SEM来检测细胞在纤维膜上的生长形态。黏附实验表明细胞在材料上能很好的黏附。增殖实验表明细胞在各种比例的纳米纤维材料上的增殖情况都要好于对照组(玻片),随着时间的增长,细胞的量明显增多。SEM结果显示细胞在纤维膜上能保持良好的细胞形态。SF-PLGA共混纳米纤维膜能为细胞提供良好的生长环境,未表现出细胞毒性。对SF-PLGA共混纳米纤维膜在模拟体内环境37℃PBS缓冲液(7.4±0.1)条件下的体外降解性能进行了探讨。通过对降解不同时间后纳米纤维膜的失重、降解液的pH值变化及纳米纤维膜的形貌观察等一系列的分析和表征,表明了纯的SF静电纺纳米纤维膜基本上不降解,SF-PLGA(2:8)共混纳米纤维膜降解的最快,在SF-PLGA共混纳米纤维膜的降解中发现SF的加入,使PLGA的降解速率加快。采用高速旋转滚筒接收装置制备了取向SF-PLGA共混纳米纤维并研究了其与雪旺细胞的相互作用。实验结果表明纤维的取向情况和滚筒的旋转速率有着密切的关系,随着旋转速率的增加,纤维的取向情况越好。对雪旺细胞在取向、无规的静电纺纤维膜上的增殖进行了研究,增殖实验的结果表明雪旺细胞在SF-PLGA取向和无规静电纺纳米纤维上的增殖均高于取向和无规的PLGA静电纺纳米纤维上的增殖,且细胞在取向SF-PLGA共混纳米纤维上的增殖均高于无规SF-PLGA纳米纤维上的增殖。说明取向SF-PLGA纳米纤维的拓扑结构能引导细胞沿着纤维的方向生长。以SD大鼠作为动物模型,取向SF-PLGA (2:8)纳米纤维制成的神经导管用于桥接大鼠坐骨神经10mm缺损。PLGA纳米纤维制成的神经导管与自体神经作为对照组。术后12周从大体观察、组织学以及图像分析等指标检测比较各组坐骨神经再生的情况。所有的结果均表明：取向SF-PLGA纳米纤维神经导管组再生神经纤维的质量和数量均优于取向PLGA纳米纤维神经导管组。与取向PLGA纳米纤维神经导管相比,SF-PLGA纳米纤维神经导管更能促进周围神经的再生。但是,与自体神经相比较还是略差一点。更多还原

【Abstract】 Tissue engineering (TE) is one of the new subjects which combined Biology and Material Science. The basic concept of TE is aimed to reestablish human tissue and organs by planting the TE scaffolds with functional cells incorporated into the damaged position. Therefore, the key point and the most challenged technique of TE are to fabricate the tissue engineering scaffolds which could mimic the natural extracellular matrix (ECM).Electrospinning is one of the effective methods to produce nanofibers with diameter ranging from several nanometers to hundreds of nanometers. Recently, electrospinning of biopolymers attracts more attentions owing to their good biocompatibility. The electrospun nanofibers have many potential applications in tissue engineering.In this study, the PLGA/SF blended nanofibrous membranes with different weight ratios were fabricated using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) as a solvent via electrospinng. The nanofibrous morphologies were observed by SEM, the results showed that the diameters of resuting nanofibers were affected by solution concentrations and the blend ratio. The water contact angles increased by decreasing the contents of SF. ATR-FTIR and XRD analyses demonstrated no obvious chemical bond reaction between SF and PLGA.The tensile strength and elongation at break of nanofibrous membrances increased with increasing content of PLGA. The unique aspect of the post-processing protocol in this study involves the use of methanol as the post-processing agent to enhance the water resisting property and stability. The nanofibrous morphologies were observed by SEM, the results showed that the nanofibers still maintained an excellent morphology after treated by methanol vapor. XRD analyses demonstrated that the crystallinity occured significant changes. Water contact angles of SF-PLGA decreased after treated, and also decreased while increasing the contents of SF. The tensile strength and elongation at break of post-processed nanofibrous membrances decreased compared with untreated mats except the pure SF mats. The tensile strength of the treated SF sample was increased, but the elongation at break was decreased. The blended nanofibers are potential for their application in tissue engineering scaffolds and to develop functional biomaterials.Schwann cells (SCs) were seeded onto nanofiber mats for adherence and proliferation studies. The cells had a good adherence on nanofibrous mats with SF-PLGA at different ratios, and the amount of the cells’proliferation was more than that of control group on the first day after seeding. SEM images showed that cells maintained good shape on nanofibrous mats. These results demonstrated that treated SF-PLGA nanofibrous scaffolds showed no cytotoxicity toward cell growth with good biocompatibility in vitro.In vitro degradation of SF-PLGA nanofibrous scaffolds were carried out in phosphate buffered saline (7.4±0.1) at 37℃for 6 months. Through a series of analysis and characterization(including loss weight, pH changes of PBS solutions) to fiberous scaffolds after degradation for different time, the results showed that the pure SF nanofibrous scaffolds were not degradable in PBS, the degradation rate of the SF-PLGA(2:8) fibrous scaffolds were faster than others. The adding of SF accelerated the degradation rate of PLGA.Aligned nanofibrous scaffolds of SF-PLGA(2:8) were fabricated by electrospinning technique under optimum condition. The degree of orientation increased with the increase in the rotating drum rates from 500 to 4000rpm. MTT results showed Schwann cells(SCs) had greater proliferation on random/aligner SF-PLGA nanofibrous scaffolds than that on random/aligned PLGA fibrous scaffolds. And SCs had greater proliferation on aligned SF-PLGA nanofibrous scaffolds than random SF-PLGA nanofibrous scaffolds. The alignment of the SF-PLGA nanofibers could control cell orientation and strengthen the interaction between the cell body and the fibers in the longitudinal directon of the fibers.Adult male Sprague-Dawley (SD) rats were used for animal models. The aligned SF-PLGA(2:8) nanofibrous nerve guidance conduit (NGC) was used for bridge implantation across a 10-mm long sciatic nerve defect in rats, PLGA nanofibrous nerve guidance conduit and nerve autografts were used control. The outcome of regenerated nerve at 12 weeks was evaluated by a combination of histological and electron microscopy study.更多还原