RGD-重组蛛丝蛋白/聚己内酯复合支架材料的研究
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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。
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.
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。
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.
引文
[1]Antonios G Mikos,Johnnas,T emenoff.Formation of highly porous biodegradable scaffolds for tissue engineering[J].Electronic Journal of Biotechnology,2000,2:114-119.
[2]姚康德,王向辉,侯信.组织工程相关生物材料[J].天津理工学院学报,2000(4):1-5.
[3]Rount AM.Contempotery Biomaterials[J].Chem.Eng.News.1999,77(3):51-59.
[4]Hutmacher DW,Schantz JT,Zein I,er al.Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling[J].J Biomed Mater Res,2001,55(2):203-216.
[5]Lannutti J,Reneker D,Ma T,et al.Electrospinning for tissue engineering scaffolds.Mat Sci Eng C,2007,27:504-509.
[6]Nam Y S,Yoon I J,Park T G.A novel fabrication method of macroporous biodegradable polymer scaffold using gas foaming salt as a porogen additive[J].Journal of Biomedical materials Research,2006,53:1-7.
[7]Antonios G.Mikes,Johnna S.et al.Formation of highly porous biodegradable scaffolds for tissue engineering[J].Electronic Journal of Biotechnology,2000,3(2):114-119.
[8]Mikos AG,Thorsen AJ,Czerwonka LA,et al.Preparation and characterization of poly(L-lactic acid) foams[J].Polymer,1994,35:1068-1077.
[9]王彦平.生物可降解性组织工程支架材料的制备[J].甘肃科技,2003,19(12):30-31.
[10]Leong K F,Cheah C M.Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs Chua[J].Biomaterials,2003,24:2363-2378.
[11]Xiong Z,Yan Y N,Zhang R J,et al.Fabrication of porous scaffolds for bone tisue engineering via low temperature deposition[J].Scripta Materialia,2002,46:771-776.
[12]Whang K,Thomas C H,& Healy K E.A novel method to fabricate bioabsorble scaffolds[J].Polymer,1995,36:837-842.
[13]Zhang R Y,& Ma P X.Porous poly(L-lactic acid)/apatitec omposites created by Biomimetic process[J].Journal of Biomedical Materials Research,1999,45:285-293.
[14]Nam YS,& Park TG.Biodegradable polymeric microcellular foams by modified Thermally induced phase separation method[J].Biomaterials,1999,20:1783-1790.
[15]杨晓芳,奚廷斐.生物材料生物相容性评价研究进展[J].生物医学工程杂志,2001,21(1):123-128.
[16]冯凌云,陈晓明.生物陶瓷材料的生物学性能评价[J].武汉工业大学学报,1996,18(2):124.
[17]李宝玉.生物医学材料[M].北京:化学工业出版社,2003:265-302.
[18]奚廷斐.医疗器械生物学评价[J].中国医疗器械信息,1999,5(3):4.
[19]黄曦,张秋轩,周志华.重组蛛丝蛋白支架材料的细胞相容性.The 5th Asian International Symposium on BiometriCs,福建厦门大学,2006年11月15-18日.
[20]Marletta G,Ciapetti G,Satriano C,et al.The effect of irradiation modification and RGD sequence adsorption on the response of human osteoblasts to polyeaprolaetone.[J].Biomaterials.2005,26(23):4793-804.
[21]K.C.迪伊,D.A.普莱奥,R.比齐奥斯.组织-生物材料相互作用导论.北京:化学工业出版社,2005:38-41.
[22]HERSEL U,DAHMEN C,KESSLER H.RGD modified pol-ymers:biomaterials for stimulated cell adhesion and beyond[J].Biomaterials,2003,24(10):4385-4415.
[23]杨国利.RGD序列与种植体骨整合[J].国际口腔医学杂志,2007,34(3).
[24]高虹.植物细胞中的RGD摸体及受体[J].植物细胞学杂志,2006,28:833-838.
[25]卢玲,王迎军.生物材料细胞粘附肽仿生修饰的研究进展[J].材料导报,2005,19(1):11-20.
[26]Rachael H Schmedlen,Kristyn S Masters,Jennifer L West.Photocrosslinkable polyvinyl alcohol hydrogels that can be modified with cell adhesion peptides for use in tissue engineering[J].Biomaterials,2002,23:4325.
[27]Yong Doo Park,Nicola Tirelli,Jeffrey A Hubbell.Photopolymerized hyaturonie acid-based hydrogels and interpenetrating networks[J].Biomaterials,2003,24:893.
[28]Syed S Lateef,Samuel Boateng,Thomas J Hartman.et al.GRGDSP peptid-bound silicone membranes withstand mechanical flexing in vitro and display enhanced fibroblast adhesion[J].Biomaterials,2002,23:3159.
[29]Ferris DM,Moodie GD,Dimond PM,et al.RGD-coatedtitaniurn implants stimulate increased bone formation in vivo[J].Biomaterials,1999,20:23-29.
[30]Li H,Ke Y,Hu Y.Polymer nanofibers prepared by template melt extrusion[J].J Appl Polym Sci,2006,99(3):1018-1023.
[31]Yang Z,Xu B.Supramolecular hydrogels based on biofunctional nanofibers of self-assembled small molecules[J].J Mater Chem,2007,17(23):2385-2393.
[32]Ellison C J,Phatak A,Giles D W,et al.Melt blown nanofibers:Fiber diameter distributions and onset of fiber breakup[J].Polymer,2007,48(20):3306-3316.
[33]Teo W E,Ramakrishna S.A review on electrospinning design and nanofibre assemblies[J].Nanotechnology,2006,17(14):89-106.
[34]Gu B K,Shin M K,Sohn K W,et al.Direct fabrication of twisted nanofibers by electrospinning[J].Appl Phys Lett,2007,90(26):263-272.
[35]Panda P K,Ramakrishna S.Electrospinning of alumina nanofibers using different precursors[J].J Mater Sci,2007,42(6):2189-2193.
[36]Kim G,Kim W.Formation of oriented nanofibers using electrospinning[J].Appl Phys Lett,2006,88(23):233-241.
[37]吴大诚,杜仲良,高绪珊编.纳米纤维[M].北京:化学工业出版社,2003:44-56.
[38]K.J.Pawlowski,H.L.Belvin,D.L.Raney et al.Electrospinning of a micro-air vehicle wing shin[J].Polymer,2003,(44):1309-1314.
[39]Greiner A,wendorff J H.Electrospinning:A fascinating method for the preparation of ultrathin fibers[J].Angew Chem Int Edit,2007,46(30):5670-5703.
[40]Tan S H,Inai R,Kotaki M,et al.Systematic parameter study for ultra-fine fiber fabrication via electrospinning[J].Polymer,2005,46(16):6128-6134.
[41]Ashammakhi N,Ndreu A,Piras AM,et al.Biodegradable nanomats produced by electrospinning:Expanding multifunctionality and potential for tissue engineering[J].J Nanosci Nanotechnol,2007,7:862.
[42]Chiu J B,Liu C,Hsiao B S,et al.Functionalization of poly(L-lactide) nanofibrous scaffolds with bioactive collagen molecules[J].J Biomed Mater Res Part A,2007,83(4):1117-1127.
[43]Venugopal J,Low S,Choon A T,et al.Interaction of cells and nanofiber scaffolds in tissue engineering[J].J Biomed Mater Res Part B:Appl Biomater,2008,84(1):34-48.
[44]Matthews J A,Wnek G E,Simpson D C,et al.Electrospinning of Collagen Nanofibers[J].Biomacromolecules,2002,3(2):232-238.
[45]Li W,Laurencin C T,Caterson E J,et al.Electrospun nanofibrous su'ucture:A novel scafold for tissue engineering[J].J Biome Mater Bes,2002,60(4):613-621.
[46]Yoshimota H,Shin Y M,Vacanti J P,et al.A biodegradable nanofiber scafold by electrospinning and its potential for bone tisue engineerlng[J].Biomateriais,2003,24:2077-2082.
[47]T.B Bini,Shujun Gao,Shu Wang,et al.Poly(1-lactide-co-glycolide) biodegradable microfibers and electrospun nanofibers for nerve tissue engineering:an in vitro study[J].J Mater Sci,2006,41:6453-6459.
[48]MN Giraud,D Keller,D Balazs,et al.Selection of tuning PCL nanofiber non-wovens for muscle tissue engineering[J].European Cells and Materials,2007,14(3):68.
[49]Min BM,You Y,Kim JM,et al.Formation of nanostructured poly(lactic-co-glycolic acid)/chitin matrix and its cellular response to normal human keratinocytes and fibroblasts[J].Carbohydr Polym,2004,57:285-292.
[50]何创龙,黄争鸣,张彦中等.静电纺丝法制备组织工程纳/微米纤维支架[J].自然科学进展,2005,15(10):1175-1182.
[51]Lewis,R.Unraveling the weave of spider silk[J].Bioscience,1996,46:636-638
[52]Weisshart K.Silk biotechnology[J].Reviews in Molecular Bioteehnology,2000,74:65-66.
[53]Wong Po Foo C,Kaplan D L.Genefic engineering of fibrous proteins:spider dragline silk and collagen[J].Adv Drug Deliv Rev,2002,54(8):1131-1143.
[54]Kubik S.High-Performance Fibers from Spider Silk[J].Angew.Chem.Int.Ed,2002,41(15):2721-2723.
[55]Scheller J,Guhrs K H,Grosse F.et al.Production of spider silk protein in tobacco and potato[J].Nat Bioteehnol,2001,19(6):573-577.
[56]Scheller J,Henggeler D,Viviant A,et al.purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation[J].Transgenic Research,2004,13(1):51-57.
[57]Barr L A,Fahnestock S R,Yang J.Production and purification of recombinant DP1B silk- like protein in plants[J].Mol Breeding,2004(13):345-356.
[58]Yang J,Barr L A,Fahnestock S R,et al.High yield recombinant silk- like protein production in transgenic plants through protein targeting[J].Transgenic Research,2005(14):313-324.
[59]庞志其.21世纪信息技术生态纺织品国际研讨会论文集[C].北京:北京科学技术协会,2003:234-236.
[60]牛恒尧,王义琴,萨其拉等.在大肠杆菌中表达蜘蛛拖丝融合蛋白[J].高技术通讯,2001,11:5-7.
[61]田保中,汪生鹏,王建南等.类蜘蛛丝丝素蛋白SPF198在毕赤酵母中的分泌表达[J].蚕业科学,2006,20:276-279.
[62]Miao Y,Zhang Y,Nakagaki K.Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bae-to-Bac/BmNPV baculovirus expression system[J].Appl Microbiol Biotechnol,2005,9(13):1-8.
[63]李敏,章文贤,黄智华等.蜘蛛拖丝蛋白基因的构建及在大肠杆菌中的表达[J],生物工程学报,2002,18(3):331-334.
[64]章文贤,李敏等.组织工程新材料蜘蛛拖丝蛋白的重组培养[J].中国临床康复,2003,20(7):2782-2784.
[65]李敏,黄建坤等.RGD-蜘蛛拖丝蛋白聚合物的生物合成与纯化[J].生物医学工程学杂志,2004,21(6):1006-1010.
[66]阮超然,黄晶星,魏梅红,李敏.高分子量RGD-蛛丝蛋白重组体的构建、高密度发酵及纯化[J].生物工程学报,2007,23(5):858-861.
[67]李敏,涂桂云,黄智华等.RGD蛛丝蛋白基因工程菌高密度发酵条件的研究[J].生物医学工程学杂志,2005,22(6):1206-1209.
[68]Allmeling C.,Jokuszies A,Reimers K,et al.Use of spider silk fibres as an innovative material in a biocompatible artificial nerve conduit.[J].Cell.Mol.Med.2006,10(3):770-777.
[69]Vepari,C.and Kaplan,D.L.Silk as a biomaterial[J].Prog.Polym.Sei.2007,32:991-1007.
[70]Arcidiacono,S.et al.(2002) Aqueous processing and fiber spinning of recombinant spider silks[J].Macromolecules,35:1262-1266.
[71]Huemmerich,D.et al.Processing and modification of films made from recombinant spider silk proteins[J].Appl.Phys.A,2006,82:219-222.
[72]Junghans,F.et al.Preparation and mechanical properties of layers made of recombinant spider silk proteins and silk from silk worm.Appl.Phys.A,2006,82:253-260.
[73]Gellynck,K.et al.Chondrocyte growth in porous spider silk 3Dscaffolds[J].Eur.Cell.Mater.,2005,10(2):45.
[74]Hermanson,K.D.et al.Engineered microcapsules fabricated from reconstituted spider silk[J].Adv.Mater,2007,19:1810-1815.
[75]刘华,房乾,陈登龙,李敏.重组蛛丝蛋白支架材料在大鼠体内的组织相容性[J].中华生物医学工程杂志,2007,13(4):223-226.
[76]Bini,E.et al.RGD-functionalized bioengineered spider dragline silk biomaterial[J].Biomacromolecules,2006,7:3139-3145.
[77]Wong Po Foo,C.et al.Novel nanocomposites from spider silksilica fusion(chimeric)proteins[J].Proc.Natl.Acad.Sci.U.S.A.2006,103:9428-9433.
[78]Huang,J.et al.The effect of genetically engineered spider silkdentin matrix protein 1chimeric protein on hydroxyapatite nucleation[J].Biomaterials,2007,28:2358-2367.
[79]Hutmacher DW,Schantz JT,Tamai TK,et al.Evaluation of ultra-thin poly (epsilon-caprolactone) films for tissue-engineered skin[J].Tissue Eng,2001,7:441-455.
[80]Berrban M,Zahidi A.Preparation of poly-ε-capro|actone nanocapsules by nanoprecipitation and their evaluation using benzyl benzoate and G-C-MS[J].J Pharm Clin,1999,18:3132-3146.
[81]Pitt CG.Polycaprolactone and its copolymers.In Larger Ran Chas in M Eds.Biodegradable polymers as drug delivery systems[J].New York:Marcel Dekker,1990,70.
[82]宋存先,王彭延,孙洪范等.PCL在体内的降解、吸收和排泄[J].生物医学工程学杂志,2000,17:25-28.
[83]陈建海,黄春霞,陈志良.PCL材料的生物相容性与毒理学研究[J].生物医学工程学杂志,2000,17:380-382.
[84]艾合麦提·玉素甫,陈统一,陈中伟.可降解聚己内酯材料的体外生物学特征:安全性评价[J].中国临床康复,2004,8:5704-5705.
[85]贾骏,段嫄嫄,周建学等.PCL电纺纤维的制备及生物相容性研究[J].中国美容医学,2007,16:603-606.
[86]Rutledge B,Huyette D,Day D,et al.Treatment of osteomyelitis with local antibiotics delivered via bioabsorbable polymer[J].Clin Orthop,2003,411:280-287.
[87]Shenoy DB,D'Souza RJ,Tiwari SB,et al.Potential application of polymeric microsphere suspension as subcutaneous depot for insulin[J].Drug Dev Ind Pharm,2003,29:555-563.
[88]黄争鸣,杨爱昭.将药源包覆到PCL超细纤维的芯部[J].高分子学报,2006,1:48-52.
[89]Luong-Van E,Grφndahl L,Nurcombe V,et al.In vitro biocompatibility and bioactivity of microencapsulated heparan sulfate[J].Biomaterials,2007,28:2127-2136.
[90]Kim HW,Lee EJ,Jun IK,et al.Degradation and drug release of phosphate glass/polycaprolactone biological composites for hard-tissue regeneration[J].Biomed Mater Res B Appl Biomater,2005,75:34-41.
[91]Takahiro Miyai,Atsuo Ito,Gaku Tamazawa,et al.Antibiotic-loaded poly-ε-caprolactone and porous β-tricalcium phosphate composite for treating osteomyelitis[J].Biomaterials,2007,9:350-358.
[92]Forrest ML,Ya(?)ez JA,Remsberg CM,et al.Paclitaxel Prodrugs with Sustained Release and High Solubility in Poly(ethylene glycol)-b-poly(epsilon-caprolactone)Micelle Nanocarriers:Pharmacokinetic Disposition,Tolerability,and Cytotoxicity[J].Pharm Res,2008,25:194-206.
[93]Hutmacher DW.Mechanical properties and cell cultural response of polycaprolaetone scaffolds designed and fabricated via fused deposition modeling[J].Biomed Mater Res,2001,55:203-216.
[94]Swieszkowski W,Tuan BH,Kurzydlowski KJ,Hutmacher DW,et al.Repair and regeneration of osteochondral defects in the articular joints[J].Biomol Eng,2007,24:489-495.
[95]Bunaprasert T,Thongmarongsri N,Thanakit V,Tissue engineering of cartilage with porous polycarprolactone-alginate scaffold:the first report of tissue engineering in Thailand[J].Med Assoc Thai,2006,9:108-114.
[96]Kee Woei NG,B.Eng,Dietmar W.,Hutmacher,et al.Evaluation of Ultra-Thin Poly(ε-Caprolactone) Films for Tissue-Engineered Skin[J].Tissue Engineering,2001,7:441-454.
[97]Ng KW,Achuth HN,Moochhala S,et al.In vivo evaluation of an ultra-thin polycaprolactone film as a wound dressing[J].Biomater Sci Polym Ed,2007,18:925-938.
[98]Chong EJ,Phan TT,Lim IJ,Zhang YZ,Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution[J].Acta Biomater.2007,3:321-330.
[99]Duan Y,Wang Z,Yan W,et al.Preparation of collagen-coated electrospun nanofibers by remote plasma treatment and their biological properties[J].Biomater Sci Polym Ed,2007,18:1153-1164.
[100]Pankajakshan D,Krishnan V K,Krishnan LK.Vascular tissue generation in response to signaling molecules integrated with a novel poly(epsilon-caprolactone)-fibrin hybrid scaffold[J].Tissue Eng Regen Med.2007,1:389-397.
[101]Jones DS,Djokic J,McCoy CP,et al.Poly(epsilon-caprolactone) and poly(epsilon-caprolactone)-polyvinylpyrrolidone-iodine blends as ureteral biomaterials:characterisation of mechanical and surface properties,degradation and resistance to encrustation in vitro[J].Biomaterials.2002,23:4449-4458.
[102]Ferreira P,Coelho JF,Gil MH.Development of a new photocrosslinkable biodegradable bioadhesive[J].Int J Pharm,2007,12:107-116.
[103]李靖扬,艾合麦提·玉素甫,陈平波等.PCL膜对同种异体肌腱移植后粘连预防作用的实验研究[J].新疆医科大学学报,2005,28:973-975.
[104]Molamma P.Prabhakaran,J.Venugopal,Tan Ter Chyan,et al.Electrospun Biocomposite Nanofibrous Scaffolds for Neural Tissue Engineering[J].Tissue Engineering:Part A,2008,14(11):1-11.
[105]Ilaria Amato,Gabriela Ciapetti,Stefania Pagani,et al.Expression of cell adhesion receptors in human osteoblasts cultured on biofunctionalized poly-(ε-caprolactone)surfaces[J].Biomaterials,2007,28:3668-3678.
[106]A.Karakecili,C.Satriano,M.Gumusderelioglu,et al.Relationship between the fibroblastic behaviour and surface properties of RGD-immobilized PCL membranes[J].J Mater Sci:Mater Med,2007,18:317-319.
[107]Chung TW,Yang MG,Liu DZ,et al.Enhancing growth human endothelial cells on Arg-Gly-Asp(RGD) embedded poly(epsilon-caprolactone)(PCL) surface with nanometer scale of surface disturbance[J].J Biomed Mater Res A,2005,72(2):213-219.
[108]Matthias Gabriel,Geerten P.Van Nieuw Amerongen,Victor W.M.Van Hinsbergh,et al.Direct grafting of RGD-motif-containing peptide on the surface of polycaprolactone films[J].J.Biomater.Sci.Polymer Edn,2006,179(5):567-577.
[109]Ji Suk Choi,Kam W.Leong,Hyuk Sang Yoo.In vivo wound healing of diabetic ulcers using electrospun nanofibers immobilized with human epidermal growth factor (EGF)[J].Biomaterials,2008,29:587-596.
[110]Houde She,Xiufeng Xiao,Rongfang Liu.Preparation and characterization of polycaprolactone-chitosan composites for tissue engineering applications[J].J Mater Sci,2007,42:8113-8119.
[111]K.Gellynck,P.Verdonk,K.F.Almqvist,et al.Chondrocyte Growth in Porous Spider Silk 3D-Scaffolds[J].European Cells and Materials,2005,10(Suppl.2).
[112]Scheller J,Henggeler D,Viviani A,Conrad U.Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation[J].Transgenic Res.2004,13(1):51-57.
[113]Vollrath F.,Barth P.,et al,Local tolerance to spider silks and protein polymers In Vivo[J].2002,16(4):229-234.
[114]刘培生,马晓明.多孔材料检测方法[M].北京:冶金工业出版社,2006:107
[115]刘培生.多孔材料引论脚[M].北京:清华大学出版社,2004:30.
[116]陈登龙,房乾,涂桂云,黄曦,李敏.pNSR16/聚乙烯醇复合材料的研究[J].功能材料,2007,38(7):1194-1196.
[117]蒋青锋,陈建海,陈志良等.控缓释制剂载体材料聚己内酯的制备及表征[J].华西药学杂志,2004,19(4):260-263.
[118]Nam YS,Park TG.Porous biodegradable polymeric scaffolds prepared by thermally induced phase separation[J].J Biomed Mater Res,1999,47:8-17.
[119]石桂欣,王身国,贝建中.聚乳酸与聚乳酸-羟基乙酸多孔细胞支架的制备及孔隙的表征[J].功能高分子学报,2001,14(1):7-11.
[120]Woodward S C,Brewer P S,Moatamed F.The intraeellular degradation of poly-(ε-caprolactone)[J].J Biomed Mater Res,1985,19:437-444.
[121]Sabita S,Stephen D G.Screening of vitro cytotoxicity by the adhesive test[J].Biomaterials,1990,11(3):133-136.
[122]田嘉军,陈敦春,王定国.医疗器械生物学试验细胞增殖度法影响因素的探讨[J].北京生物医学工程,2003,22(1):55-56.
[123]赵晓伟,林建华,王梓壬.自制磷酸钙骨水泥的生物相容性和安全性[J].福建医科大学学报,2002,36(4):388-392.
[124]Hussain RF,Nouri AM,Oliver RT.A new aproach for measurement of eytotoxieity using colometric assay[J].J Biomed Mater Res,1982,160:89-96.
[125]吕晓迎,Kappert HF.牙科材料细胞毒性评定的新方法(MTT试验)[J].中华口腔医学杂志,1995,30:377-379.
[126]汤顺清,毛萱,石海涛等.组织工程用β-磷酸三钙/聚乳酸支架材料性能评价[J].暨南大学学报(自然科学版),2002,23(5):67-70.
[2]姚康德,王向辉,侯信.组织工程相关生物材料[J].天津理工学院学报,2000(4):1-5.
[3]Rount AM.Contempotery Biomaterials[J].Chem.Eng.News.1999,77(3):51-59.
[4]Hutmacher DW,Schantz JT,Zein I,er al.Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling[J].J Biomed Mater Res,2001,55(2):203-216.
[5]Lannutti J,Reneker D,Ma T,et al.Electrospinning for tissue engineering scaffolds.Mat Sci Eng C,2007,27:504-509.
[6]Nam Y S,Yoon I J,Park T G.A novel fabrication method of macroporous biodegradable polymer scaffold using gas foaming salt as a porogen additive[J].Journal of Biomedical materials Research,2006,53:1-7.
[7]Antonios G.Mikes,Johnna S.et al.Formation of highly porous biodegradable scaffolds for tissue engineering[J].Electronic Journal of Biotechnology,2000,3(2):114-119.
[8]Mikos AG,Thorsen AJ,Czerwonka LA,et al.Preparation and characterization of poly(L-lactic acid) foams[J].Polymer,1994,35:1068-1077.
[9]王彦平.生物可降解性组织工程支架材料的制备[J].甘肃科技,2003,19(12):30-31.
[10]Leong K F,Cheah C M.Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs Chua[J].Biomaterials,2003,24:2363-2378.
[11]Xiong Z,Yan Y N,Zhang R J,et al.Fabrication of porous scaffolds for bone tisue engineering via low temperature deposition[J].Scripta Materialia,2002,46:771-776.
[12]Whang K,Thomas C H,& Healy K E.A novel method to fabricate bioabsorble scaffolds[J].Polymer,1995,36:837-842.
[13]Zhang R Y,& Ma P X.Porous poly(L-lactic acid)/apatitec omposites created by Biomimetic process[J].Journal of Biomedical Materials Research,1999,45:285-293.
[14]Nam YS,& Park TG.Biodegradable polymeric microcellular foams by modified Thermally induced phase separation method[J].Biomaterials,1999,20:1783-1790.
[15]杨晓芳,奚廷斐.生物材料生物相容性评价研究进展[J].生物医学工程杂志,2001,21(1):123-128.
[16]冯凌云,陈晓明.生物陶瓷材料的生物学性能评价[J].武汉工业大学学报,1996,18(2):124.
[17]李宝玉.生物医学材料[M].北京:化学工业出版社,2003:265-302.
[18]奚廷斐.医疗器械生物学评价[J].中国医疗器械信息,1999,5(3):4.
[19]黄曦,张秋轩,周志华.重组蛛丝蛋白支架材料的细胞相容性.The 5th Asian International Symposium on BiometriCs,福建厦门大学,2006年11月15-18日.
[20]Marletta G,Ciapetti G,Satriano C,et al.The effect of irradiation modification and RGD sequence adsorption on the response of human osteoblasts to polyeaprolaetone.[J].Biomaterials.2005,26(23):4793-804.
[21]K.C.迪伊,D.A.普莱奥,R.比齐奥斯.组织-生物材料相互作用导论.北京:化学工业出版社,2005:38-41.
[22]HERSEL U,DAHMEN C,KESSLER H.RGD modified pol-ymers:biomaterials for stimulated cell adhesion and beyond[J].Biomaterials,2003,24(10):4385-4415.
[23]杨国利.RGD序列与种植体骨整合[J].国际口腔医学杂志,2007,34(3).
[24]高虹.植物细胞中的RGD摸体及受体[J].植物细胞学杂志,2006,28:833-838.
[25]卢玲,王迎军.生物材料细胞粘附肽仿生修饰的研究进展[J].材料导报,2005,19(1):11-20.
[26]Rachael H Schmedlen,Kristyn S Masters,Jennifer L West.Photocrosslinkable polyvinyl alcohol hydrogels that can be modified with cell adhesion peptides for use in tissue engineering[J].Biomaterials,2002,23:4325.
[27]Yong Doo Park,Nicola Tirelli,Jeffrey A Hubbell.Photopolymerized hyaturonie acid-based hydrogels and interpenetrating networks[J].Biomaterials,2003,24:893.
[28]Syed S Lateef,Samuel Boateng,Thomas J Hartman.et al.GRGDSP peptid-bound silicone membranes withstand mechanical flexing in vitro and display enhanced fibroblast adhesion[J].Biomaterials,2002,23:3159.
[29]Ferris DM,Moodie GD,Dimond PM,et al.RGD-coatedtitaniurn implants stimulate increased bone formation in vivo[J].Biomaterials,1999,20:23-29.
[30]Li H,Ke Y,Hu Y.Polymer nanofibers prepared by template melt extrusion[J].J Appl Polym Sci,2006,99(3):1018-1023.
[31]Yang Z,Xu B.Supramolecular hydrogels based on biofunctional nanofibers of self-assembled small molecules[J].J Mater Chem,2007,17(23):2385-2393.
[32]Ellison C J,Phatak A,Giles D W,et al.Melt blown nanofibers:Fiber diameter distributions and onset of fiber breakup[J].Polymer,2007,48(20):3306-3316.
[33]Teo W E,Ramakrishna S.A review on electrospinning design and nanofibre assemblies[J].Nanotechnology,2006,17(14):89-106.
[34]Gu B K,Shin M K,Sohn K W,et al.Direct fabrication of twisted nanofibers by electrospinning[J].Appl Phys Lett,2007,90(26):263-272.
[35]Panda P K,Ramakrishna S.Electrospinning of alumina nanofibers using different precursors[J].J Mater Sci,2007,42(6):2189-2193.
[36]Kim G,Kim W.Formation of oriented nanofibers using electrospinning[J].Appl Phys Lett,2006,88(23):233-241.
[37]吴大诚,杜仲良,高绪珊编.纳米纤维[M].北京:化学工业出版社,2003:44-56.
[38]K.J.Pawlowski,H.L.Belvin,D.L.Raney et al.Electrospinning of a micro-air vehicle wing shin[J].Polymer,2003,(44):1309-1314.
[39]Greiner A,wendorff J H.Electrospinning:A fascinating method for the preparation of ultrathin fibers[J].Angew Chem Int Edit,2007,46(30):5670-5703.
[40]Tan S H,Inai R,Kotaki M,et al.Systematic parameter study for ultra-fine fiber fabrication via electrospinning[J].Polymer,2005,46(16):6128-6134.
[41]Ashammakhi N,Ndreu A,Piras AM,et al.Biodegradable nanomats produced by electrospinning:Expanding multifunctionality and potential for tissue engineering[J].J Nanosci Nanotechnol,2007,7:862.
[42]Chiu J B,Liu C,Hsiao B S,et al.Functionalization of poly(L-lactide) nanofibrous scaffolds with bioactive collagen molecules[J].J Biomed Mater Res Part A,2007,83(4):1117-1127.
[43]Venugopal J,Low S,Choon A T,et al.Interaction of cells and nanofiber scaffolds in tissue engineering[J].J Biomed Mater Res Part B:Appl Biomater,2008,84(1):34-48.
[44]Matthews J A,Wnek G E,Simpson D C,et al.Electrospinning of Collagen Nanofibers[J].Biomacromolecules,2002,3(2):232-238.
[45]Li W,Laurencin C T,Caterson E J,et al.Electrospun nanofibrous su'ucture:A novel scafold for tissue engineering[J].J Biome Mater Bes,2002,60(4):613-621.
[46]Yoshimota H,Shin Y M,Vacanti J P,et al.A biodegradable nanofiber scafold by electrospinning and its potential for bone tisue engineerlng[J].Biomateriais,2003,24:2077-2082.
[47]T.B Bini,Shujun Gao,Shu Wang,et al.Poly(1-lactide-co-glycolide) biodegradable microfibers and electrospun nanofibers for nerve tissue engineering:an in vitro study[J].J Mater Sci,2006,41:6453-6459.
[48]MN Giraud,D Keller,D Balazs,et al.Selection of tuning PCL nanofiber non-wovens for muscle tissue engineering[J].European Cells and Materials,2007,14(3):68.
[49]Min BM,You Y,Kim JM,et al.Formation of nanostructured poly(lactic-co-glycolic acid)/chitin matrix and its cellular response to normal human keratinocytes and fibroblasts[J].Carbohydr Polym,2004,57:285-292.
[50]何创龙,黄争鸣,张彦中等.静电纺丝法制备组织工程纳/微米纤维支架[J].自然科学进展,2005,15(10):1175-1182.
[51]Lewis,R.Unraveling the weave of spider silk[J].Bioscience,1996,46:636-638
[52]Weisshart K.Silk biotechnology[J].Reviews in Molecular Bioteehnology,2000,74:65-66.
[53]Wong Po Foo C,Kaplan D L.Genefic engineering of fibrous proteins:spider dragline silk and collagen[J].Adv Drug Deliv Rev,2002,54(8):1131-1143.
[54]Kubik S.High-Performance Fibers from Spider Silk[J].Angew.Chem.Int.Ed,2002,41(15):2721-2723.
[55]Scheller J,Guhrs K H,Grosse F.et al.Production of spider silk protein in tobacco and potato[J].Nat Bioteehnol,2001,19(6):573-577.
[56]Scheller J,Henggeler D,Viviant A,et al.purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation[J].Transgenic Research,2004,13(1):51-57.
[57]Barr L A,Fahnestock S R,Yang J.Production and purification of recombinant DP1B silk- like protein in plants[J].Mol Breeding,2004(13):345-356.
[58]Yang J,Barr L A,Fahnestock S R,et al.High yield recombinant silk- like protein production in transgenic plants through protein targeting[J].Transgenic Research,2005(14):313-324.
[59]庞志其.21世纪信息技术生态纺织品国际研讨会论文集[C].北京:北京科学技术协会,2003:234-236.
[60]牛恒尧,王义琴,萨其拉等.在大肠杆菌中表达蜘蛛拖丝融合蛋白[J].高技术通讯,2001,11:5-7.
[61]田保中,汪生鹏,王建南等.类蜘蛛丝丝素蛋白SPF198在毕赤酵母中的分泌表达[J].蚕业科学,2006,20:276-279.
[62]Miao Y,Zhang Y,Nakagaki K.Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bae-to-Bac/BmNPV baculovirus expression system[J].Appl Microbiol Biotechnol,2005,9(13):1-8.
[63]李敏,章文贤,黄智华等.蜘蛛拖丝蛋白基因的构建及在大肠杆菌中的表达[J],生物工程学报,2002,18(3):331-334.
[64]章文贤,李敏等.组织工程新材料蜘蛛拖丝蛋白的重组培养[J].中国临床康复,2003,20(7):2782-2784.
[65]李敏,黄建坤等.RGD-蜘蛛拖丝蛋白聚合物的生物合成与纯化[J].生物医学工程学杂志,2004,21(6):1006-1010.
[66]阮超然,黄晶星,魏梅红,李敏.高分子量RGD-蛛丝蛋白重组体的构建、高密度发酵及纯化[J].生物工程学报,2007,23(5):858-861.
[67]李敏,涂桂云,黄智华等.RGD蛛丝蛋白基因工程菌高密度发酵条件的研究[J].生物医学工程学杂志,2005,22(6):1206-1209.
[68]Allmeling C.,Jokuszies A,Reimers K,et al.Use of spider silk fibres as an innovative material in a biocompatible artificial nerve conduit.[J].Cell.Mol.Med.2006,10(3):770-777.
[69]Vepari,C.and Kaplan,D.L.Silk as a biomaterial[J].Prog.Polym.Sei.2007,32:991-1007.
[70]Arcidiacono,S.et al.(2002) Aqueous processing and fiber spinning of recombinant spider silks[J].Macromolecules,35:1262-1266.
[71]Huemmerich,D.et al.Processing and modification of films made from recombinant spider silk proteins[J].Appl.Phys.A,2006,82:219-222.
[72]Junghans,F.et al.Preparation and mechanical properties of layers made of recombinant spider silk proteins and silk from silk worm.Appl.Phys.A,2006,82:253-260.
[73]Gellynck,K.et al.Chondrocyte growth in porous spider silk 3Dscaffolds[J].Eur.Cell.Mater.,2005,10(2):45.
[74]Hermanson,K.D.et al.Engineered microcapsules fabricated from reconstituted spider silk[J].Adv.Mater,2007,19:1810-1815.
[75]刘华,房乾,陈登龙,李敏.重组蛛丝蛋白支架材料在大鼠体内的组织相容性[J].中华生物医学工程杂志,2007,13(4):223-226.
[76]Bini,E.et al.RGD-functionalized bioengineered spider dragline silk biomaterial[J].Biomacromolecules,2006,7:3139-3145.
[77]Wong Po Foo,C.et al.Novel nanocomposites from spider silksilica fusion(chimeric)proteins[J].Proc.Natl.Acad.Sci.U.S.A.2006,103:9428-9433.
[78]Huang,J.et al.The effect of genetically engineered spider silkdentin matrix protein 1chimeric protein on hydroxyapatite nucleation[J].Biomaterials,2007,28:2358-2367.
[79]Hutmacher DW,Schantz JT,Tamai TK,et al.Evaluation of ultra-thin poly (epsilon-caprolactone) films for tissue-engineered skin[J].Tissue Eng,2001,7:441-455.
[80]Berrban M,Zahidi A.Preparation of poly-ε-capro|actone nanocapsules by nanoprecipitation and their evaluation using benzyl benzoate and G-C-MS[J].J Pharm Clin,1999,18:3132-3146.
[81]Pitt CG.Polycaprolactone and its copolymers.In Larger Ran Chas in M Eds.Biodegradable polymers as drug delivery systems[J].New York:Marcel Dekker,1990,70.
[82]宋存先,王彭延,孙洪范等.PCL在体内的降解、吸收和排泄[J].生物医学工程学杂志,2000,17:25-28.
[83]陈建海,黄春霞,陈志良.PCL材料的生物相容性与毒理学研究[J].生物医学工程学杂志,2000,17:380-382.
[84]艾合麦提·玉素甫,陈统一,陈中伟.可降解聚己内酯材料的体外生物学特征:安全性评价[J].中国临床康复,2004,8:5704-5705.
[85]贾骏,段嫄嫄,周建学等.PCL电纺纤维的制备及生物相容性研究[J].中国美容医学,2007,16:603-606.
[86]Rutledge B,Huyette D,Day D,et al.Treatment of osteomyelitis with local antibiotics delivered via bioabsorbable polymer[J].Clin Orthop,2003,411:280-287.
[87]Shenoy DB,D'Souza RJ,Tiwari SB,et al.Potential application of polymeric microsphere suspension as subcutaneous depot for insulin[J].Drug Dev Ind Pharm,2003,29:555-563.
[88]黄争鸣,杨爱昭.将药源包覆到PCL超细纤维的芯部[J].高分子学报,2006,1:48-52.
[89]Luong-Van E,Grφndahl L,Nurcombe V,et al.In vitro biocompatibility and bioactivity of microencapsulated heparan sulfate[J].Biomaterials,2007,28:2127-2136.
[90]Kim HW,Lee EJ,Jun IK,et al.Degradation and drug release of phosphate glass/polycaprolactone biological composites for hard-tissue regeneration[J].Biomed Mater Res B Appl Biomater,2005,75:34-41.
[91]Takahiro Miyai,Atsuo Ito,Gaku Tamazawa,et al.Antibiotic-loaded poly-ε-caprolactone and porous β-tricalcium phosphate composite for treating osteomyelitis[J].Biomaterials,2007,9:350-358.
[92]Forrest ML,Ya(?)ez JA,Remsberg CM,et al.Paclitaxel Prodrugs with Sustained Release and High Solubility in Poly(ethylene glycol)-b-poly(epsilon-caprolactone)Micelle Nanocarriers:Pharmacokinetic Disposition,Tolerability,and Cytotoxicity[J].Pharm Res,2008,25:194-206.
[93]Hutmacher DW.Mechanical properties and cell cultural response of polycaprolaetone scaffolds designed and fabricated via fused deposition modeling[J].Biomed Mater Res,2001,55:203-216.
[94]Swieszkowski W,Tuan BH,Kurzydlowski KJ,Hutmacher DW,et al.Repair and regeneration of osteochondral defects in the articular joints[J].Biomol Eng,2007,24:489-495.
[95]Bunaprasert T,Thongmarongsri N,Thanakit V,Tissue engineering of cartilage with porous polycarprolactone-alginate scaffold:the first report of tissue engineering in Thailand[J].Med Assoc Thai,2006,9:108-114.
[96]Kee Woei NG,B.Eng,Dietmar W.,Hutmacher,et al.Evaluation of Ultra-Thin Poly(ε-Caprolactone) Films for Tissue-Engineered Skin[J].Tissue Engineering,2001,7:441-454.
[97]Ng KW,Achuth HN,Moochhala S,et al.In vivo evaluation of an ultra-thin polycaprolactone film as a wound dressing[J].Biomater Sci Polym Ed,2007,18:925-938.
[98]Chong EJ,Phan TT,Lim IJ,Zhang YZ,Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution[J].Acta Biomater.2007,3:321-330.
[99]Duan Y,Wang Z,Yan W,et al.Preparation of collagen-coated electrospun nanofibers by remote plasma treatment and their biological properties[J].Biomater Sci Polym Ed,2007,18:1153-1164.
[100]Pankajakshan D,Krishnan V K,Krishnan LK.Vascular tissue generation in response to signaling molecules integrated with a novel poly(epsilon-caprolactone)-fibrin hybrid scaffold[J].Tissue Eng Regen Med.2007,1:389-397.
[101]Jones DS,Djokic J,McCoy CP,et al.Poly(epsilon-caprolactone) and poly(epsilon-caprolactone)-polyvinylpyrrolidone-iodine blends as ureteral biomaterials:characterisation of mechanical and surface properties,degradation and resistance to encrustation in vitro[J].Biomaterials.2002,23:4449-4458.
[102]Ferreira P,Coelho JF,Gil MH.Development of a new photocrosslinkable biodegradable bioadhesive[J].Int J Pharm,2007,12:107-116.
[103]李靖扬,艾合麦提·玉素甫,陈平波等.PCL膜对同种异体肌腱移植后粘连预防作用的实验研究[J].新疆医科大学学报,2005,28:973-975.
[104]Molamma P.Prabhakaran,J.Venugopal,Tan Ter Chyan,et al.Electrospun Biocomposite Nanofibrous Scaffolds for Neural Tissue Engineering[J].Tissue Engineering:Part A,2008,14(11):1-11.
[105]Ilaria Amato,Gabriela Ciapetti,Stefania Pagani,et al.Expression of cell adhesion receptors in human osteoblasts cultured on biofunctionalized poly-(ε-caprolactone)surfaces[J].Biomaterials,2007,28:3668-3678.
[106]A.Karakecili,C.Satriano,M.Gumusderelioglu,et al.Relationship between the fibroblastic behaviour and surface properties of RGD-immobilized PCL membranes[J].J Mater Sci:Mater Med,2007,18:317-319.
[107]Chung TW,Yang MG,Liu DZ,et al.Enhancing growth human endothelial cells on Arg-Gly-Asp(RGD) embedded poly(epsilon-caprolactone)(PCL) surface with nanometer scale of surface disturbance[J].J Biomed Mater Res A,2005,72(2):213-219.
[108]Matthias Gabriel,Geerten P.Van Nieuw Amerongen,Victor W.M.Van Hinsbergh,et al.Direct grafting of RGD-motif-containing peptide on the surface of polycaprolactone films[J].J.Biomater.Sci.Polymer Edn,2006,179(5):567-577.
[109]Ji Suk Choi,Kam W.Leong,Hyuk Sang Yoo.In vivo wound healing of diabetic ulcers using electrospun nanofibers immobilized with human epidermal growth factor (EGF)[J].Biomaterials,2008,29:587-596.
[110]Houde She,Xiufeng Xiao,Rongfang Liu.Preparation and characterization of polycaprolactone-chitosan composites for tissue engineering applications[J].J Mater Sci,2007,42:8113-8119.
[111]K.Gellynck,P.Verdonk,K.F.Almqvist,et al.Chondrocyte Growth in Porous Spider Silk 3D-Scaffolds[J].European Cells and Materials,2005,10(Suppl.2).
[112]Scheller J,Henggeler D,Viviani A,Conrad U.Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation[J].Transgenic Res.2004,13(1):51-57.
[113]Vollrath F.,Barth P.,et al,Local tolerance to spider silks and protein polymers In Vivo[J].2002,16(4):229-234.
[114]刘培生,马晓明.多孔材料检测方法[M].北京:冶金工业出版社,2006:107
[115]刘培生.多孔材料引论脚[M].北京:清华大学出版社,2004:30.
[116]陈登龙,房乾,涂桂云,黄曦,李敏.pNSR16/聚乙烯醇复合材料的研究[J].功能材料,2007,38(7):1194-1196.
[117]蒋青锋,陈建海,陈志良等.控缓释制剂载体材料聚己内酯的制备及表征[J].华西药学杂志,2004,19(4):260-263.
[118]Nam YS,Park TG.Porous biodegradable polymeric scaffolds prepared by thermally induced phase separation[J].J Biomed Mater Res,1999,47:8-17.
[119]石桂欣,王身国,贝建中.聚乳酸与聚乳酸-羟基乙酸多孔细胞支架的制备及孔隙的表征[J].功能高分子学报,2001,14(1):7-11.
[120]Woodward S C,Brewer P S,Moatamed F.The intraeellular degradation of poly-(ε-caprolactone)[J].J Biomed Mater Res,1985,19:437-444.
[121]Sabita S,Stephen D G.Screening of vitro cytotoxicity by the adhesive test[J].Biomaterials,1990,11(3):133-136.
[122]田嘉军,陈敦春,王定国.医疗器械生物学试验细胞增殖度法影响因素的探讨[J].北京生物医学工程,2003,22(1):55-56.
[123]赵晓伟,林建华,王梓壬.自制磷酸钙骨水泥的生物相容性和安全性[J].福建医科大学学报,2002,36(4):388-392.
[124]Hussain RF,Nouri AM,Oliver RT.A new aproach for measurement of eytotoxieity using colometric assay[J].J Biomed Mater Res,1982,160:89-96.
[125]吕晓迎,Kappert HF.牙科材料细胞毒性评定的新方法(MTT试验)[J].中华口腔医学杂志,1995,30:377-379.
[126]汤顺清,毛萱,石海涛等.组织工程用β-磷酸三钙/聚乳酸支架材料性能评价[J].暨南大学学报(自然科学版),2002,23(5):67-70.