【作者】 陈登龙；

【导师】 李敏；

【作者基本信息】 福建师范大学 ， 高分子化学与物理， 2009， 博士

【摘要】 蜘蛛丝蛋白作为高性能的生物材料如人造肌腱、人工韧带、人工器官、组织修复等组织工程新材料,具有十分诱人的应用前景。国外对蜘蛛丝蛋白作为组织工程材料的研究报道较少,国内以蜘蛛丝蛋白为材料制备组织工程材料除本课题组外也未见相关报道。应用现代生物技术和高分子物理化学手段,将具有特定细胞粘附信号识别功能的RGD三肽的基因重组蛛丝蛋白pNSR16(分子量约为60kD)分别与生物相容性的高分子材料聚乙烯醇(PVA)、聚己内酯(PCL)复合,充分发挥蛋白质和高分子材料两方面的优异性能,可以得到一类综合性能优良的组织工程新材料。为此,本研究采用冷冻干燥/粒子沥滤法和静电纺丝法制备了两类4种重组蛛丝蛋白复合支架材料(pNSR16/PVA和pNSR16/PCL的多孔支架材料和纳米纤维支架材料),对重组蛛丝蛋白复合支架材料结构、理化性能进行分析,并对其生物学性能进行研究。红外光谱研究表明:甲酸使pNSR16分子构象进一步向无规卷曲方向转变,饱和LiBr溶液则使其构象向β-链方向转变;加热和变性都能促使pNSR16分子构象向β-链方向转变;PVA和壳聚糖的加入,能明显促进pNSR16分子构象由无规卷曲向β-链转化。pNSR16/PCL支架材料的红外光谱显示:pNSR16和PCL可能只是以物理形式相结合。力学性能测定表明:PVA可以改善pNSR16支架材料的力学性能;静电纺丝可以提高复合支架材料的力学性能。冷冻干燥/粒子沥滤法制备pNSR16/PVA多孔支架材料的最佳制作配比和工艺是:采用粒径150-250μm的NaCl为致孔剂,pNSR16:PVA=100:4(w/w),NaCl与pNSR16/PVA溶液按1:1(w/v)比例混合后倒膜,经-80℃冷冻干燥时间2小时10分钟后,再用65-75%乙醇变性后洗涤除盐。得到的pNSR16/PVA多孔支架材料孔隙均匀,孔径在100-200μm之间,符合细胞生长的要求;且支架材料整体均匀,PVA与pNSR16相容性较好,未发生相分离现象。静电纺丝法制备粗细均匀的pNSR16/PVA复合纳米纤维的电纺过程参数是:电纺温度45℃,纺丝液浓度15%、电纺电压80kV、固化距离20cm、挤出速度5ml/h。扫描电镜(SEM)结果表明:pNSR16/PVA复合纳米纤维的直径随着纺丝液浓度、纺丝电压、固化距离以及挤出速度的增大均呈现增大的趋势;乙醇处理会引起复合纳米纤维间产生明显的粘连现象。在pNSR16/PCL浓度为30%,NaCl的粒径为150-250μm,NaCl相对于pNSR16/PCL溶液体积的重量为1:1时,可以制得孔径达100μm以上的pNSR16/PCL多孔支架,pNSR16/PCL多孔支架的孔径已基本满足常规细胞培养的要求,但pNSR16/PCL多孔支架的孔结构分布不是很均匀,孔的结构也不是完全相通的。电纺过程参数控制在纺丝液浓度30%、电纺电压80kV、固化距离20cm、挤出速度5ml/h的条件下,可以获得粗细均匀的pNSR16/PCL复合纳米纤维。当pNSR16与PCL的质量比为5:100、纺丝液浓度为30%时,pNSR16/PCL复合纳米纤维直径随着纺丝电压的增大呈现减小的趋势,当电纺电压达90kV时,纤维直径分布不均匀,纤维间有部分粘连;复合纳米纤维直径随着固化距离增大也呈现减小的趋势,复合纳米纤维直径随着挤出速度增大呈现增大的趋势,同时纤维变得不均匀。pNSR16/PVA多孔支架在PBS磷酸盐缓冲液和含有弹性蛋白酶、胰凝乳蛋白酶、胰蛋白酶的PBS磷酸盐缓冲液中的降解行为初步证实了pNSR16/PVA多孔支架具有生物可降解性,说明可以通过改变pNSR16与PVA的混合比例来调节支架的降解速度。pNSR16/PVA支架材料浸提液的细胞毒性反应为1级、符合生物材料的合格标准;鼠成纤维细胞NIH-3T3和人类结肠腺癌细胞Caco-2与pNSR16/PVA多孔支架材料的体外联合培养结果表明:pNSR16/PVA多孔支架具有良好的细胞相容性,有利于细胞的粘附和生长。pNSR16/PVA复合纳米纤维的孔隙率、吸湿性、渗出液在纤维间和纤维内的分布比值、溶涨率分别为84.85%、4.381g/g、6.140g/g,pNSR16/PCL复合纳米纤维的相应指标为86.47%、4.794g/g、14.58g/g,均比多孔支架有明显的增加,表明静电纺丝技术可以有效提高pNSR16/PVA支架作为生物敷料的相关性能指标。pNSR16/PCL复合支架材料的毒性等级在1级以下、符合生物材料的合格标准;pNSR16/PCL复合支架材料的毒性小于纯PCL支架材料,说明pNSR16能增强PCL支架的某些生物学性能。pNSR16/PCL复合支架材料与NIH-3T3细胞的复合培养结果表明:pNSR16能提高PCL的细胞相容性,同时静电纺丝法制成的支架材料在诱导细胞定向生长方面优于冷冻干燥/粒子沥滤法制成的多孔支架材料。更多还原

【Abstract】 As a biomaterial of excellent properties, the spider silk protein has sparked prospect in biomedical applications such as tissue engineer tendon and ligament. However, there are relatively few studies except for some reports from abroad and those from our laboratory.With technologies of biology and physical chemistry, recombinant spider silk which contains peptides RGD could be well composited with synthetic polymers such as PVA and PCL. The porosity scaffolds with pNSR16/PVA and pNSR16/PCL could be produced respectively with Freeze-drying/Particle-leaching Method. Besides, nanofibers with the same materials could be prepared by electrospining. The morphology and structure of blend scaffolds were examined by scanning electron microscopy(SEM) and Fourier transform infrared spectroscopy(FTIR). The biological properties were also detected here.The results of FTIR show the comformation of pNSR16 would transit to random coil after dissolving in formic acid. However, it would transit toβ-sheet in groups with LiBr-dealing or alcohol-dealing. The same effect would be achieved by heating. In the groups where recombinant spider silk mixed with synthetic polymers, it was found that the comformation of pNSR16 could be transited toβ-sheet from random coil after blending with PVA or Chitosan.The results of mechanic characterization show that the mechanic property of pNSR16 scaffolds can be improved when blended with PVA or electrospinning.The optimal protocol of freeze-drying/particle-leaching method for preparing pNSR16/PVA porosity scaffolds is:(1) using NaCl particulates(150-250μm) as pore-forming material and blending with pNSR16/PVA (100/4, w/w) at ratio 1/1.(2) freeze drying at -80℃for 130 min, then leaching the salt with alcohol(65-75%).The scaffolds prepared according to the protocol are highly porous. They are oflarger pore size, ranging from 100μm to 200μm. The well distributed, interconnected and open pore wall structure is critical for cell cultivation.An optimal electrospinning condition was obtained in producing uniform cylindrical nanofibers. It was as follows: temperature 45℃, concentration 15%, voltage 80 kv, distance 20 cm, solution flow rate 5 ml/h. The results show that the fiber diameter tends to increase with the concentration of electrospun solution, the nozzle-to-ground distance, the applied voltage and the solution flow rate. The fibers will adhere together after being dealt with alcohol.According to the same protocol of freeze-drying/particle-leaching method mentioned above, the pNSR16/PCL scaffolds with pore size larger than 100μm can be prepared by using pNSR16/PCL acid solution with concentration 30% and NaCl with particle diameter 150-250μm. Although the pores of scaffolds do not well distributed, the pNSR16/PCL scoffolds are suitable for cell cultivation.For electrospinning of pNSR16/PCL acid solution, an optimal condition was obtained in producing uniform cylindrical nanofibers. It was as follows: temperature 45℃, concentration 30%, voltage 80 kv, distance 20 cm, solution flow rate 5 ml/h. The results show that the fiber diameter tends to decrease with the applied voltage and the nozzle-to-ground distance when the concentration is 30%, the ratio of pNSR16/PCL is 5/100. As solution flow rate increases, the average fiber diameter increases steadily. However, the fibers will adhere together and the diameter is uneven when the voltage or solution flow rate is too high.The degradation behavior of pNSR16/PVA scaffolds in PBS buffer, PBS buffer with elastase or chymotase or trypase was analyzed. It was found that, by adapting the ratio of pNSR16/PVA, the degrading rate was in control. Cyto-compatibility of the porous scaffolds of pNSR16/PVA was investigated by cytotoxicity determination of the extract of the scaffolds. According to the evaluation criterion in ISO10993, cytotoxicity of the extract of the pNSR16/PCL scaffolds is of rank I, so the scaffolds are qualified. Preparation of Cell-scaffold constructs with Caco-2 cells and NIH-3T3 cells indicates that pNSR16/PVA scaffolds have good cyto-compatibility.The porosity of pNSR16/PVA electrospun mats is 84.85%, the absorption capacities 4.381g/g, the ratio of amount osmo-solution between interfibrous and intrafibrous 6.140g/g, while those of pNSR16/PCL electrospun mats are 86.47%, 4.794g/g and 14.58g/g. All the above parameters are higher than those of cast film prepared with same compositions. Cyto-compatibility of the porous complex scaffold of pNSR16/PCL was investigated by cytotoxicity determination of the extract of the scaffolds and preparation of cell-scaffold constructs. According to the evaluation criterion in ISO10993, cytotoxicity of the extract of the pNSR16/PCL scaffolds is of rank I, so the scaffolds are qualified. Results show that the pNSR16/PCL scaffolds promote the adhesion and propagation of NIH 3T3, namely, the scaffolds have good cyto-compatibility. Compared with the porosity scaffolds preparing by freeze-drying/particle-leaching method, electrospun membranes could induce the cell to adhere and proliferate.更多还原