【作者】 魏梅红；

【导师】 李敏； 陈登龙；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 聚己内酯(PCL)是一种新型脂肪族聚酯,以其良好的机械性能、无毒性、可塑性等优良性能广泛应用于生物材料领域,但材料表面缺乏细胞识别位点,对细胞的亲和力不够,植入体内会引起炎症反应等成为制约其作为理想组织工程材料的主要障碍。蛛丝蛋白纤维作为高性能生物材料具有十分诱人的应用前景。国外对蛛丝蛋白作为生物材料的研究报道较少,国内以蛛丝蛋白为材料制备组织工程生物材料除本课题组外尚未见相关报道。应用现代生物技术和高分子物理化学手段将具有特定细胞黏附信号识别功能的RGD三肽的基因重组蛛丝蛋白pNSR16与PCL共混,充分发挥蛋白质和高分子材料两方面的优异性能。采用冷冻干燥/粒子滤粒法和静电纺丝技术制备pNSR16/PCL复合多孔支架和纳米纤维支架,对复合支架材料结构、理化性能进行分析,并对其降解性能和生物学性能进行研究,得到一类综合性能优良的组织工程新材料。冷冻干燥/粒子滤粒法制备pNSR16/PCL多孔支架材料的最佳制作配比和工艺是:溶液浓度0.3g/mL、致孔剂NaCl用量0.5g、粒径为150-250μm,可以制得孔径达到100μm以上pNSR16/PCL多孔支架,支架孔径能基本满足常规细胞培养的要求;电纺过程参数控制纺丝液浓度30%、电压80kV、固化距离20cm、挤出速度5ml/h条件下可获得粗细均匀的pNSR16/PCL复合纳米纤维。扫描电镜(SEM)观察到添加pNSR16不影响PCL支架材料的表面形貌。傅立叶红外光谱分析表明pNSR16与PCL以物理方式共混,二者之间并未形成新化学键。热性能测试结果表明66.5℃为支架材料的熔点(或软化点),416.7℃对应为热分解温度,添加pNSR16并未影响PCL的热性能。力学性能测试结果表明,多孔支架制作过程中致孔剂的加入提高了支架材料的力学性能,静电纺丝可明显提高支架材料的力学性能。对多孔支架及纳米纤维支架的孔隙率和吸湿性进行测试比较发现,静电纺纳米纤维膜的孔隙率平均达90%,吸湿性达3.0g/g以上,而浇注多孔膜孔隙率平均仅达到81%,吸湿性仅1.5g/g,电纺丝技术可以有效的提高pNSR16/PCL复合膜作为生物敷料的相关性能指标。选择磷酸盐溶液、含猪胰脂肪酶的磷酸盐溶液,通过残余重量百分比及SEM考察pNSR16/PCL复合支架的体外降解行为。结果表明由于蛋白脱落造成孔隙空间大,pNSR16/PCL复合支架材料的降解速度较PCL快。另外,支架材料的降解与材料的大小及表面积有关,纤维膜降解速度较多孔膜快。说明pNSR16/PCL复合支架具有一定的生物可降解性。评价pNSR16/PCL复合支架的细胞相容性,采用MTT的方法考察支架材料的细胞毒性,并通过NIH-3T3细胞与支架复合培养考察其对细胞黏附、生长和分化的影响。MTT实验结果表明,与单纯PCL浸提液相比,pNSR16/PCL复合材料浸提液能较明显促进NIH-3T3细胞的生长和增殖,且效果随着pNSR16比例的增大而越好,根据细胞毒性评定标准,分析pNSR16/PCL复合支架材料毒性等级在1级以下,符合生物材料的要求。SEM和常规HE染色观察发现,pNSR16/PCL复合支架更利于NIH-3T3细胞黏附,细胞在材料表面增殖形成多层结构。细胞在纳米纤维膜上生长呈现一定的方向性,但是基本停留在膜表面生长,并未见伸入膜孔隙内部,而在浇注多孔膜上不仅在表面大量扩增,且逐渐伸入孔隙内部。细胞支架粘附率及增殖曲线测定结果表明,pNSR16/PCL复合支架的细胞粘附率明显高于PCL支架,pNSR16/PCL复合纳米纤维膜高于pNSR16/PCL复合浇注多孔膜。细胞在各组支架上均能正常增殖,在PCL材料上增殖速度较慢,在pNSR16/PCL复合纳米纤维膜上刚开始增殖较快,以后则因为空间限制细胞增殖速率较多孔膜慢。免疫组化实验结果说明NIH-3T3细胞在各组支架上均可正常表达bFGF因子。说明RGD-重组蛛丝蛋白明显改善了PCL材料的细胞相容性,该复合材料具备种子细胞生长、增殖的微环境,初步认为可进一步应用于组织工程各领域。以医用胶原为对照,将各组支架材料植入SD大鼠肌肉,观察机体的炎症反应和材料在体内的变化。pNSR16/PCL复合支架材料以及医用胶原在术后30d根据评定标准,均呈Ⅰ级-Ⅱ级反应,评价为合格,材料局部炎性反应消退,炎症细胞基本消失,肌纤维结构排列有序,纤维囊壁趋向变薄,有大量新生血管出现。组织相容性优劣顺序为医用胶原＞pNSR16/PCL复合支架材料＞PCL支架材料。结果表明pNSR16/PCL复合支架材料具有良好的生物相容性和生物可降解性,与组织无排斥作用,能减轻炎症反应,初步认为其作为组织工程支架材料具有一定的可行性。更多还原

【Abstract】 Polycaprolactone (PCL) is a kind of aliphatic polyester with widely applications in biological materials. However, lack of cell-binding signals on the surface and causing inflammatory reaction after implantation have become the major obstacle to be an ideal tissue engineering material. As a kind of biomaterials with high performance, spider protein fibrin has a good prospect of application. Reports on spider protein fibrin as biomaterials abroad have been rare. No reports about making biomaterias from spider protein inland have been seen besides us.Genetic recombination spider protein(pNSR16) with Arg-Gly-Asp(RGD) tripeptide which can provide cell-binding signals was blended with PCL using modern techniques of biological and physical chemistry of polymers. pNSR16/PCLcomposite porous scaffolds were prepared by using freeze-drying/partical leaching method and pNSR16/PCLcomposite nanofibre scaffolds by electrospinning. A series of properties of the composite scaffolds such as structure, physico-chemical properties, biodegradation and biocompatibility were characterized, so as to make a new biomaterial with superior performance both from protein and polymer material.The optimum preparation technology of freeze-drying/partical leaching method was:solution concentration 0.3g/mL,quantity of porogen NaCl 0.5g and particle diameter 150-250um. In this condition,pNSR16/PCLcomposite porous scaffolds with bore diameter greater than 100μcan be made, such a bore diameter can meet the requirement of routine cell culturing. pNSR16/PCLcomposite nanofibres with diameter evenly disposed were made while solution concentration was controlled at 0.3g/mL,applied voltage80kV, dnozzle-to-ground distance 20cm and extrusion speed 5ml/h.The morphologies of scaffolds were evaluated using SEM, result showed that introduction of pNSR16 into PCL didn’t affect the appearance of PCL scaffolds. FTIR analysis indicated that pNSR16 blended with PCL physically,no through chemical bond.TG/DSC tests revealed that the melting point of scaffolds was 66.5℃, decomposition point was 416.7℃, introduction of pNSR16 didn’t change thermal property of PCL. Introduction of porogen can improve mechanical properties of porous scaffolds, scaffolds made by electrospinning had better maechanical properties. Porosity and hygroscopicity of scaffolds as wound dressings were also characterized, results showed that porosity and hygroscopicity of porous scaffolds were 81% and 1.5 g/g respectively,while that of nanofibre scaffolds were 90% and 3.0 g/g respectively, indicating electrospinning can improve performance of scaffolds as wound dressings.The vitro degradation behaviour of pNSR16/PCL composite scaffolds were investigated by the phosphate solution and the phosphate solution containing with porcine lipase. Results showed that the loss weight of the samples increased with the protein-content increasing, attributing to protein breaking off the scaffolds so as to make more space. Furthermore, nanofibre scaffolds had a more thorough degradation than porous scaffolds because degradation of materials have a relation to its size and surface areas.The in vitro biocompatibility of pNSR16/PCL composite scaffolds in this study using NIH-3T3 mouse fibroblasts. Different cellular aspects were analyzed in order to know the cell viability during cell culture on pNSR16/PCL composite scaffolds: scaffolds cytotoxicity,adhesion, proliferation, morphology, and secret ion of bFGF. MTT assay results showed that comparing to that of PCLscaffolds, toxic rank of pNSR16/PCL composite scaffolds was below 1 level, satisfying the requirements of biomaterials. SEM and HE results indicated that comparing with PCL scaffolds, pNSR16/PCL composite scaffolds with RGD had a better cell adhesion and nanofibre scaffolds higher than porous scaffolds.. Cells on the scaffolds can form multi-layered cells on the surface. Cells had a more orientated growth on nanofibre scaffolds,but only on the surface,no into the pore. Cells on all scaffolds growed normally, excepting slower on PCL scaffolds. Immunohistochemistry results indicated that cells on the scaffolds can normally proliferate and secret bFGF. All results provided evidences of good adhesion, growth, viability, morphology and normal physiologic function of cells on pNSR16/PCL composite scaffolds. Therefore, it can be provisionally concluded that pNSR16/PCL composite scaffold is a suitable and biocompatible material to be further used in the areas of tissue engineering.Histocompatibility of scaffolds were evaluated by implanting into SD mouse in vivo and examined at different periods,comparing with collagen(medical grade). Results indicated that after implanting 30d, inflammatory response of pNSR16/PCL composite scaffolds and collagen both subsided, according to evaluation standards, its I-IIgrade,indicating qualification. Order of histocompatibility was:collagen(medical grade) >pNSR16/PCL composite scaffolds >PCL scaffolds. It can be provisionally concluded that pNSR16/PCL composite scaffold had favourable biocompatibility and biodegradation which made it possible to be tissue engineering materials.更多还原