RGD-重组蛛丝蛋白复合支架材料的研制
|
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。
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.
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。
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.
引文
[1]Pollok J K,Vacanti J P.Tissue engineering[J].Semin Pediatr Surg,1996,5:191-196.
[2]Sittinger M.Tissue engineering:Artificial tissue replacement containing vital components[J].Laryngorbinootologie,1995,74:695-699.
[3]Langer R,Vacanti J P.Tissue engineering[J].Science,1993,260:920-926.
[4]李世普.生物医用材料导论[M].武汉工业大学出版社,2000.
[5]房乾.RGD-重组蛛丝蛋白和柞蚕丝素蛋白作为组织工程支架材料的研究[D].福建师范大学硕士学位论文.2008
[6]崔福斋,杜昶.骨组织工程[J].世界医疗器械,1999,5:5-54.
[7]Dietmar WH.Scaffolds in tissue engineering bone and cartilage[J].Biomaterials,2000,21:2529-2543.
[8]胡蕴玉.把握契机加快我国组织工程学的应用研究[J].中华骨科杂志,2000,20(9):517-518.
[9]王慧敏.组织工程复相(羟基磷灰石+β-磷酸三钙)多孔陶瓷支架材料的制备研究.昆明理工大学硕士学位论文[D].2005.
[10]方丽茹,翁文剑,等.骨组织工程支架及生物材料研究[J].生物医学工程杂志,2003,20(1):148-152
[11]马朋高,游秀东,姚康德等.骨组织工程及可吸收高分子支架的研究进展[J].化学通报,2001,7:407-410.
[12]Rount A M.Contempotery Biomaterials[J].Chem.Eng.News.1999,3:51-59.
[13]L.G,Griffith,G Naughton.Tissue engineering-current challenges and expanding opportunities[J].Science,2002,295:1009-1016.
[14]J A Hubbell.Bioactive biomaterials[J].Current Opinion in Biotechnology,1999,10:123-129.
[15]J A Hubbell.Tissue engineering[J].Chem.Eng.News,1995,73:42-54.
[16]N D Winblade,H Schmokel,M Baumann et al.Sterically blocking adhesion of cells to biological surfaces with a surface-active copolymer containing poly(ethylene glycol) and phenylboronic acid[J].Biomaterial Mater,2002,59(4):618-631.
[17]胡平,张璐,方壮熙等.电纺丝及其在生物医用材料中的应用.纺织科学研究.2004(2):26-32.
[18]Flemming RG,Muwhy CJ,Abrams GA,et al.Effects of synthetic micro-and nano-structured surfaces on cell behavior[J].Biomaterials,1999(20):573-588.
[19]Chu Benjamin,Hsiao Benjamin S,Hadjiargyrou,et al.Cell storage and delivery.USP:20030054035.
[20]Li Wan Ju,Laurencin,Caterson,et al.Electrospun nanofibers structure:A novel scafold for tisue engineering[J].Journal of Biome Mater,2002,4:613-621.
[21]F.Yanga,R.Muruganb,S.Wangc,S.Ramakrishnaa.Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering[J].Biomaterials,2005,26:2603-2610.
[22]Matthews Jamil A,Boland Eugene D,Wnek Gary E.Eleetrospinning of collagen Nanofibers[J].Biomacromolecules,2002,2:232-238.
[23]Huang L,Apkarian RP,Chaik of EL.High-resolution analysis of engineredtypel collagen nanofibers by electronmicroscopy[J].Scanning,2001,6:372-375.
[24]T.A.Telemeco,C.Ayres,G.L.Bowlin,et al.Regulation of cellular infiltration into tissue engineering scaffolds composed of submicron diameter fibrils produced by electrospinning[J].Acta Biomaterialia,2005,1:377-385.
[25]Mengyan Lia,Mark J.Mondrinosa,Milind R.Gandhia,et al.Electrospun protein fibers as matrices for tissue engineering[J].Biomaterials,2005,26:5999-6008.
[26]H Yoshimoto,Y M Shin,H Terai.A biodegradable nanofiber scaffold by electrospinning and potential for bone tissue engineering[J].Biomateriais,2002,12:2077-2082.
[27]JinHyoung Joon,Park Jaehyung,Cebe Peggy.Engineered films of Bombyxmoil silk-withpoly (ethylene oxide)[J].MaterialsResearch Society Symposium-Proceeding,2003,735:135-140.
[28]Lakshmi S.Nair,Subhabrata Bhattacharyya,Jared D.Bender,et al.Fabrication and Optimization of Methylphenoxy Substituted Polyphosphazene Nanofibers for Biomedical Applications[J].Biomacromolecules,2004,5:2212-2220.
[29]Melvin Schindlera,Ijaz Ahmedb,Jabeen Kamalb,et al.A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture[J].Biomaterials,2005,26:5624-5631.
[30]Zuwei Ma,Masaya K(o|¨)tak,Thomas Yong,et al.Surface engineering of electrospun polyethylene terephthalate(PET) nanofibers towards development of a new material for blood vessel engineering[J].Biomaterials,2005,26:2527-2536.
[31]Stefania A.Riboldia,Maurilio Sampaolesib,Peter Neuenschwanderc,et al.Electrospun degradable polyesterurethane membranes:potential scaffolds for skeletal muscle tissue engineering[J]. Biomaterials,2005,26:4606-4615.
[32]Hench L.,Polak.J M.Third-generation biomedical materials[J].Science,2002;295:1014-1017.
[33]黄建坤.李敏.丝蛋白纤维生物材料与组织工程.中国修复重建外科杂志[J].2004,18(2):127-130
[34]Altman GH.,Diaz F.,Jakuba C.,et al.Silk-based biomaterials[J].Biomaterials,2003,24:401-416.
[35]Altman G H.,Horan,Rebecca L,Lu,Helen H,et.al.Silk matrix for tissue engineered anterior cruciate ligaments[J].Biomaterials,2002,23:4131-4141.
[36]Greenwald D,Shumway S,Albear P.,et al.Mechanical comparison of 10 suture materials before and after in vivo incubation[J].Surg Res 1994,56:372-377.
[37]Mingzhong Li,Masayo Ogiso,Norihiko Minour.Enzymatic degradation behavior of porous silk broin sheets[J].Biomaterials,2003,23:357-365.
[38]Kaiyong Cai,Kangde Yao,Songbai Lin,et al.Poly(d、l-lactic acid) surfaces modied by silk fibroin:effects on the culture of osteoblast in vitro[J].Biomaterials,2002,23:1153-1160.
[39]Kaiyong Cai,Kangde Yao,Yuanlu Cui,et al.Influence of different surface modification treatments on poly(d,l-lactic acid) with silk fi.broin and their effects on the culture of osteoblast in vitro[J].Biomaterials,2002,23:1603-1611.
[40]Minoura N,Aiba S,Ootoh Y.et al.Attachmentand growth of cultured broblast cells on silk protein matrices[J].Biomed Mater Res,1995,29:1215-1221.
[41]Inouye K.,Kurokawa M.,Nishikawa S.,et al.Use of Bombyx mori silk.broin as a substratum for cultivation of animal cells[J].Biochem Biophys Meth,1998,37:159-164.
[42]Sofia S.,McCart hy MB,C.rronowicz G,et al.Functionalizedsilk-based biomaterials for bone formation[J].Biorned MaterRes 2001,54:139-148.
[43]Chiarini A.,Petrini P.,Sabrina Bozzini,et al.Silk fibroin/poly(carbonate)-urethane as a substrate for ce11 growth:in vitro interactions with human cells.Biomaterials[J].2003,24:789-799.
[44]Jinwei Gu,Xinlin Yang,Hesun Zhu.Surface sulfonation of silk fibroin film by plasma treatment and in vitro antithrombogenicity study[J].Materials Science and Engineering.2002,20:199-202.
[45]Cheryl Wong Po Foe,Kaplan D L.Genetic engineering of fibrous proteins:spider dragline silk and collagen[J].Advanced Drug,2002,54:1131-1143.
[46]Vollrath F.,Barth P.,et al.Local tolerance to spider silks and protein polymers in vivo[J].2002,16(4):229-234.
[47]Minoura N.et al.Physico-chemical properties of silk fibroin membrane as biomaterials[J]. Materials,1990,11:430-434.
[48]Muller,W.S.,Samuelson,L.A.,Fossey,S.A.et al.Formation and properties of silk thin films[J].Science and Biotechnology,1994,544:342-352.
[49]李明忠,卢神,吴徵宇等,多孔丝素膜的制备及其形态结构[J].纺织学报,2000,21(5):268-271.
[50]Li MZ,Tao W,Kuga S,et al.Controlling molecular conformation of regenerated wild silk fibroin by aqueous ethanol treatment[J].Polymers for Advanced Technologies,2003,14:694-698.
[51]Kweon H.Y,Park H.Y..Structural and conformational changes of regenerated Antheraea pemyi silk fibroin films treated with methanol solution[J].Appy.Polym.Sci.1999,73:2887-2894.
[52]G.Freddi,P.Monti,M.Nagura et al.Structure and molecular conformation of tussali silk fibroin films:effect of heat treatment[J].Polymaterial.Science,(Polym.Phys),1997,35:841-847.
[53]Li MZ,Tao W,Lu SZ,et al.Comphant film of regenerated Antheraea pernyi silkfibroin by chemical crosslinking[J],International Journal of Biological Macromolecules,2003,32:159-163.
[54]G.Freddi,M.Tsukada,S.Beretta,Structure and physical properties of silk fibroin/polyaccrylamide blend films[J].Appl.Polym.Sci.1999,71:1563-1571.
[55]Yang G,Zhang L.,Cao X.D..Structure and mieroporous formation of cellulose/silk fibroin blend membranes PartⅡ effect of post-treatment by alkali[J].Journal of membrane Science,2002,210:379-387.
[56]Yang G,Zhang L.Regenerated cellulose microporous membranes by mixing cellulose cuoxam with a water soluble polymer[J].Membrane Science,1996,114:149-155.
[57]Jin HJ,Park J,Vailuzzi R,et al.Biomaterials film of Bombyx Mori Silk Fibroin with poly(ethylene oxide)[J].Biomacromolicules,2004,5(3):711-717.
[58]Chen X,Knight DP,Shao Z,et al.Conformation transition in silk protein films monitored by time-resolved Fourier transform infrared spectroscopy:effect of potassium ions on Nephila spidroins films[J].Biochemistry,2002,41(50):14944-14950.
[59]黄君霆.蜘蛛丝研究的动向[J].丝绸,1999,9:48-49.
[60]郑震.蜘蛛丝蛋白的生物制造技术及纤维加工[J].国外纺织技术,2000,12:2-4.
[61]叶金兴.蛛丝制作纺织材料的生物技术[J].现代纺织技术,2000,4:57-58.
[62]黄智华.蛛丝蛋白工程菌高密度发酵、表达产物纯化与湿法纺丝.福建师范大学硕士学位论文[D].2004.
[63]Lewis R V,Hinman M,Kothakota S,et al.Expression and purification of a spider silk protein:A new strategy for production repetitive proteins.Protein[J].Expression and Purification,1996,7:400-406.
[64]Liivak O,Blye A,Shah N,et al.A Mierofabricated wet-spinning apparatus to spin fibers of silk[J].Macromoleeules,1998,31:2947-2951.
[65]Seidel A,Liivak O,Jelinski L W.Artificial spinning of spider silk[J].Maeromoleeules,1998,31:6733-6736.
[66]Areidiaeono S,M.Mello C,Butler M,et al.Aqueous Processing and Fiber Spinning of Recombinant Spider Silks[J].Macromolecules,2002,35:1262-1266.
[67]Lazaris A,Arcidiacono S,Huang Y,et al.Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells[J].Scienee,2002,295:472-476.
[68]Shao Z,Vollrath F,Yang Y,et al.Strueture and Behavior of Regenerated Spider Silk[J].Macromolecules,2003,36:1157-1161.
[69]盛家镛,潘志娟,陈宇岳等.蜘蛛丝的化学组成与结构初探[J].丝绸,2000,(4):8-12.
[70]赵昀,陆长德.用蛋白质工程改进蚕丝性能的研究[D].中国科学院上海生物化学研究所,博士学位论文,2000.
[71]Seidel A,Liivak O,Jelinski L W.Artificial spinning of spider silk[J].Maeromolecules,1998,31:6733-6736.
[72]Liivak O,Blye A,Shah N,et al.A mierofabrieated wet-spinning apparatus to spin fibers of silk proteins[J].Strueture-property correlations.Macromolecules,1998,1:2947-2951.
[73]Fahnestock.Recombinanfly produced spider silk[N].United States Patent,July 31,2001.
[74]Lock.Process for spinning polypeptide fibers[N].United States Patent,December 15,1992.
[75]Steven Arcidiaeono,Charlene M.Miehelle Bufler,et al.Aqueous Processing and Fiber Spinning of Recombinant Spider Silks[J].Macromolecules,2002,35:1262-1266.
[76]Anthoula Lazaris,Steven Arcidiacono,Yue Huaug,et al.Spider silk fibers spun from soluble recombinant silk produced in mammalian cells[J].Scinece,2002,295:472-476.
[77]Zarkoob.Synthetically spun silk nanofibers and a process for making the same[N].United States Patent,August 29,2000.
[78]Weisshart K.Silk bioteelmology Reviews in Molecular[J].Bioteehnology,2000,74:65-66.
[79]Wong Po,Foo C,Keplan D L.Genetie engineering of fibrous proteins:sprider dragline silk and collagen[J].Advaced Drug,2002,54(8):1131-1143.
[80]Miao Y,Zhang Y,Nakagaki K,et al.Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system[J].Appl Microbiol Biotechnol,2005,13:1-8.
[81]Scheller J,Henggeler D,Viviani A,et al.Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation[J].Transgenic Res,2004,13(1):51-57.
[82]Falmestock SR,Yao Z,Bedzyk LA.Microbial production of spider silk proteins[J].Biotechnol.2000,74(2):105-109.
[83]Fukushima Y.Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk[J].Biopolymers,1998,45(4):269-279.
[84]Anthoula Lazaris,Steven Arcidiacono,Yue Huang,et al.Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells[J].Science,2002,295:472-476.
[85]Fukushima Y.Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk[J].Biopolymers,1998,45(4):269-279.
[86]张前军,祖旭宇,梁宋平等.拖丝蛋白全基因亚克隆文库的构建和部分序列的测定[J].南华大学学报(医学版).2004,(2):13-16.
[87]章文贤,李敏,涂桂云等.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.2003,20:15-17.
[88]章文贤,李敏,涂桂云等.重组拖丝蛋白基因在大肠杆菌中表达条件的优化[J].福建师范大学学报.2003,(2):62-64.
[89]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,(3):331-334.
[90]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[J].2001(11):7-9.
[91]黄全生,刘霞,王义琴等.基因枪轰击法将蜘蛛丝蛋白基因(ADF3)转入海岛棉的研究[J].新疆农业科学.2004,(4):59-61.
[92]黄建坤.RGD-蛛丝蛋白生物合成与丝膜制备[D].福建师范大学硕士论文.2004.
[93]牛恒尧.Araneusdiadematu拖丝蛋白ADF3的原核表达及棉花纤维特异表达载体的构建[D].中国科学院遗传学研究所硕士论文.2001.
[94]陈新,黄郁芳,邵正中等.蜘蛛吐丝过程中钾的作用[J].高等学校化学学报,2004(6):181-184.
[95]Lazaris.A,.Arcidiacono.S,Huang,Y.et al.Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells[J].Science,2002,295:472-476.
[96]Dicko C.,Vollrath F.,Kenney JM.Silk protein refolding is controlled by changing pH[J].Biomacromolecules,2004,(5):704-710.
[97]Dicko C.,Kernney JM.,Knight D.et al.Transition to a beta-sheet-rich structure in spidroin in vitro:the effects of pH and cations[J].Biochemistry,2004,43:14080-14087.
[98]Terry A.E.,KnighL D.P.,Porter,D.et al.pH Induced Changes in the Rheology of Silk Fibroin Solution from the Middle Division of Bombyx mori Silkworm[J].Biomacromolecules 2004,(5):768-772.
[99]彭显能,陈新,武培怡等.二维相关红外光谱研究再生蚕丝蛋白膜的构象与温度之间的关系[J].化学学报,2004,21:2127-2130.
[100]Knight,D.P.,Vollrath,F.Changes in element composition in the secretory pathway for dragline silk in the golden orb web spider,Nephila edulis[J].Naturwissenschaften,2001,88:179-182.
[101]Tanaka,T.,M agoshi,L,M agoshi,Y.,et al.Spherulites of Tussah Silk Fibroin:Structure,thermal properties and growth rates[T].Journal of Thermal Analysis and Calorimetry.2001,64:645-650.
[102]Ochi,A,Hossain,K.S,Magoshi,J,et al.gheology and dynamic light scattering of silk flbroin solution extracted from the middle division of Bombyx mori silkwonn[J].Biomacromolecules 2002,3(6):1187-1196.
[103]K.Gellynck,P.Verdonk,K.F.Almqvist,et al.Chondrocyte Growth in Porous Spider Silk 3D-Scaffolds[J].European Cells and Materials.2005,10(2):45-48.
[104]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.
[105]李敏,黄建坤,涂桂云.RGD-蜘蛛拖丝蛋白聚合物的生物合成与纯化[J].中国生物工程杂志,2004,21(6):1006-1010.
[106]李敏,涂桂云,黄智华,黄曦.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.
[107]阮超然,黄晶星,魏梅红,李敏.高分子量RGD-蛛丝蛋白重组体构建、高密度发酵及纯化[J].生物工程学报,2007,23(5):858-861.
[108]K.Gellynck,P.Verdonk,K.F.Almqvist,E.Van Nimmenl,T.Gheysens,Mertens,L.Van Langenhove,P.Kiekens,A.Verbruggen.European Cells and Materials.2005,10(Supp1.2)
[109]XING Ben-gang,LIANG Hong.Joumal of Guangxi Normal University[J].Natural Science Edition,1997(3):46-49.
[110]LI Min,HUANG Jian-kun,TU Gui-yunet al.Biologieal Synthesis and Purification of Spider Dragline Silk Protein Polymers Containing RGD Three Peptide[J].Journal of Biomedical Engineering.2004,(6):1006-1010.
[111]LI Min,CHEN Deng-long,TU G-ui-yun.[P]:CN 200510076750.8,2005.
[112]QIAN Guo-di,YAO Yu-liang.Journal of Suzhou Institute of silk textile technology[J].1983,(4):26-30.
[113]Suzhou Institute of silk textile technology[J].Silk-manufacturing Chemistry.China textile&Apparel Press,1996:62.
[114]CHEN Xin,ZHOU Li,SHAO Zheng-zhong,et al.Conformation transition of silk protein membranes monitored by time-resolved FT-IR spectroscopy-Conformation transition behavior of regenerated silk fibroin membranes in alcohol solution at high eoncentrations[J].Acta Chimica Sinica,2003,(4):625-629.
[115]LI M Z,WU Z Y,ZHANG C S,et al.Study on Porous Silk Fibroin Materials.Ⅱ.Preparation And Characteristics of Spongy Porous Silk Fibroin Materials.Journal of Applied Polymer[J]Science,2001,79:2192-2199.
[116]Nazarov R,Jin H J,Kaplan D L.Porous 3-D Scaffolds from Regenerated Silk Fibroin[J].Biomacromolecules,2004,(5):718-726.
[117]姚康德,尹玉姬编著.组织工程相关生物材料[M].北京:化学工业出版社,2003.
[1118]师昌绪主编.材料大词典[M].北京.化学工业出版社,1994.
[119]沈家骢.纳米生物医用材料[J].中国医学科学院学报,2006,(4):472-474.
[120]陈登龙,李敏,房乾.静电纺丝技术在纳米结构高分子支架中的应用[J].中国修复重建外科杂志,2007,21(4):411-415.
[121]Chu Bejamin,Benjamin S,Fang Dufei,et al.Biodegradable and/or bioabsorbable fibrous articles and methods for using the articles in medical applications[P].PCT:WO 02/092 339,2002.5.15.
[122]Christopher J Buehko,Louic Chen,Yu Shen,et al.Processing andmierostructural characterization of porous biocompatible protein polymerthinfilms[J].Polymer,1999,40:7397-7407.
[123]Smith Daniel,Reneker Darrell,Kataphinan Woraphon.Electrcspun skin masks and USeS thereof [P].PCT/US:00/27775,2000.10.6.
[124]Zheng Ming Huang,Y Z Zhang,M Komki,et al.A review on polymer nanofibers by electrospinning and their application in nanocomposites[J].Composites Science and Technology,2003,63:2223-2253.
[125]刘华,房乾,陈登龙等.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.
[126]陈登龙,房乾,涂桂云等.重组蛛丝蛋白pNSR16/聚乙烯醇复合材料的研究.功能材料[J].2007,38(7):1194-1196.
[127]陈登龙,房乾,姚清华等.傅里叶红外光谱研究重组蛛丝蛋白分子构象的影响因素[J].高分子材料科学与工程.2009,25(1):104-106.
[128]李敏,陈登龙,吴钦缘.静电纺丝仪.[P]:CN 200610125960.6,2006.
[129]陈登龙,房乾,涂桂云等.重组蛛丝蛋白pNSR16/聚乙烯醇复合材料的研究[J].功能材料.2007,38(7):1194-1196.
[130]佘厚德.羟基磷灰石及组织工程用聚己内酯复合支架的制备和研究[D].福建师范大学硕士论文,2007.
[131]Boontheekul T,Mooney DJ.Protein-based signaling systems in tissue engineering[J].Curr Opin Biotech,2003,14:559-565.
[132]HERSEL U,DAHMEN C,KESSLER H.RGD modified pol-ymers:biomaterials for stimulated cell adhesion and beyond[J].Biomaterials,2003,24(10):4385-4415.
[133]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):210-212.
[134]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):24-27.
[135]Iiohner U,Hutmacher DW,See Y,et al.Individually CAD-CAM technique designed,hioresoibahle 3-dimensional polycaprolactone framework for experimental reconstruction of craniofacial defects in the pig[J].Mund Kiefer Gesiclitschir,2002,6(3):162-167.
[136]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.
[137]李国义,梁传余,郑艳等.气管软骨组织工程管状泡沫支架的研制[J].中华医学网络杂志2004,1(2):43-45.
[138]Ung-Jin Kim,Jaehyung Park,et al.Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin[J].Biomaterials.2005,26:2775-2785.
[139 Vassilis Karageorgiou,David Kaplan.Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials[J].2005,26:5474-5491.
[140]Chu Bejamin,Benjamin S,Fang Dufei,et al.Biodegradable and / or bioabsorbablc fibrous articles and methods for using the articles in medical applications[P].PCT:WO 02/092 339,2002.5.15.
[141]Christopher J Buehko,Louie Chen,Yu Shen,et al.Processing andmierostructural characterization of porous biocompatible protein polymerthinfilms[J].Polymer,1999,40:7397-7407.
[142]Smith Daniel,Reneker Darrell,Kataphinan Woraphon.Electrcspun skin masks and USeS thereof [P].PCT / US:00/27 775,2000.10.6.
[143]Zheng Ming Huang,Y Z Zhang,M Komki,et al.A review on polymer nanofibers by electrospinning and their application in nanocomposites[J].Composites Science and Technology,2003,63:2223-2253.
[144]E.J.Chon,T.T.Phan,I.J.Lim,et al.Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution[J].Acta Biomaterialia,2007,3:321-330.
[145]B.Linnemann,R.Ali,T.Grie 等.聚己内酯(PCL)纳米纤维的静电纺丝[J].国际纺织导报,2006,(4):22-24.
[146]赵洁,全大萍,廖凯荣等.组织工程中生物可降解高分子的功能化及生物学修饰[J].高分子通报,2005,6:70-75.
[147]Ulrich Hersel,Claudia Dahrnen,Horst Kessler.RGD modified polymers:biomaterials for stimulated cell adhesion and beyond[J].Biomaterials.2003,24:4385-4415.
[148]Stephane Biltresse,Mireille Attolini,Jacqueline Marchand-Brynaert.Cell adhesive PET membranes by surface grafting of RGD peptidomimetics[J].Biornaterials.2005,26:4576-458
[2]Sittinger M.Tissue engineering:Artificial tissue replacement containing vital components[J].Laryngorbinootologie,1995,74:695-699.
[3]Langer R,Vacanti J P.Tissue engineering[J].Science,1993,260:920-926.
[4]李世普.生物医用材料导论[M].武汉工业大学出版社,2000.
[5]房乾.RGD-重组蛛丝蛋白和柞蚕丝素蛋白作为组织工程支架材料的研究[D].福建师范大学硕士学位论文.2008
[6]崔福斋,杜昶.骨组织工程[J].世界医疗器械,1999,5:5-54.
[7]Dietmar WH.Scaffolds in tissue engineering bone and cartilage[J].Biomaterials,2000,21:2529-2543.
[8]胡蕴玉.把握契机加快我国组织工程学的应用研究[J].中华骨科杂志,2000,20(9):517-518.
[9]王慧敏.组织工程复相(羟基磷灰石+β-磷酸三钙)多孔陶瓷支架材料的制备研究.昆明理工大学硕士学位论文[D].2005.
[10]方丽茹,翁文剑,等.骨组织工程支架及生物材料研究[J].生物医学工程杂志,2003,20(1):148-152
[11]马朋高,游秀东,姚康德等.骨组织工程及可吸收高分子支架的研究进展[J].化学通报,2001,7:407-410.
[12]Rount A M.Contempotery Biomaterials[J].Chem.Eng.News.1999,3:51-59.
[13]L.G,Griffith,G Naughton.Tissue engineering-current challenges and expanding opportunities[J].Science,2002,295:1009-1016.
[14]J A Hubbell.Bioactive biomaterials[J].Current Opinion in Biotechnology,1999,10:123-129.
[15]J A Hubbell.Tissue engineering[J].Chem.Eng.News,1995,73:42-54.
[16]N D Winblade,H Schmokel,M Baumann et al.Sterically blocking adhesion of cells to biological surfaces with a surface-active copolymer containing poly(ethylene glycol) and phenylboronic acid[J].Biomaterial Mater,2002,59(4):618-631.
[17]胡平,张璐,方壮熙等.电纺丝及其在生物医用材料中的应用.纺织科学研究.2004(2):26-32.
[18]Flemming RG,Muwhy CJ,Abrams GA,et al.Effects of synthetic micro-and nano-structured surfaces on cell behavior[J].Biomaterials,1999(20):573-588.
[19]Chu Benjamin,Hsiao Benjamin S,Hadjiargyrou,et al.Cell storage and delivery.USP:20030054035.
[20]Li Wan Ju,Laurencin,Caterson,et al.Electrospun nanofibers structure:A novel scafold for tisue engineering[J].Journal of Biome Mater,2002,4:613-621.
[21]F.Yanga,R.Muruganb,S.Wangc,S.Ramakrishnaa.Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering[J].Biomaterials,2005,26:2603-2610.
[22]Matthews Jamil A,Boland Eugene D,Wnek Gary E.Eleetrospinning of collagen Nanofibers[J].Biomacromolecules,2002,2:232-238.
[23]Huang L,Apkarian RP,Chaik of EL.High-resolution analysis of engineredtypel collagen nanofibers by electronmicroscopy[J].Scanning,2001,6:372-375.
[24]T.A.Telemeco,C.Ayres,G.L.Bowlin,et al.Regulation of cellular infiltration into tissue engineering scaffolds composed of submicron diameter fibrils produced by electrospinning[J].Acta Biomaterialia,2005,1:377-385.
[25]Mengyan Lia,Mark J.Mondrinosa,Milind R.Gandhia,et al.Electrospun protein fibers as matrices for tissue engineering[J].Biomaterials,2005,26:5999-6008.
[26]H Yoshimoto,Y M Shin,H Terai.A biodegradable nanofiber scaffold by electrospinning and potential for bone tissue engineering[J].Biomateriais,2002,12:2077-2082.
[27]JinHyoung Joon,Park Jaehyung,Cebe Peggy.Engineered films of Bombyxmoil silk-withpoly (ethylene oxide)[J].MaterialsResearch Society Symposium-Proceeding,2003,735:135-140.
[28]Lakshmi S.Nair,Subhabrata Bhattacharyya,Jared D.Bender,et al.Fabrication and Optimization of Methylphenoxy Substituted Polyphosphazene Nanofibers for Biomedical Applications[J].Biomacromolecules,2004,5:2212-2220.
[29]Melvin Schindlera,Ijaz Ahmedb,Jabeen Kamalb,et al.A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture[J].Biomaterials,2005,26:5624-5631.
[30]Zuwei Ma,Masaya K(o|¨)tak,Thomas Yong,et al.Surface engineering of electrospun polyethylene terephthalate(PET) nanofibers towards development of a new material for blood vessel engineering[J].Biomaterials,2005,26:2527-2536.
[31]Stefania A.Riboldia,Maurilio Sampaolesib,Peter Neuenschwanderc,et al.Electrospun degradable polyesterurethane membranes:potential scaffolds for skeletal muscle tissue engineering[J]. Biomaterials,2005,26:4606-4615.
[32]Hench L.,Polak.J M.Third-generation biomedical materials[J].Science,2002;295:1014-1017.
[33]黄建坤.李敏.丝蛋白纤维生物材料与组织工程.中国修复重建外科杂志[J].2004,18(2):127-130
[34]Altman GH.,Diaz F.,Jakuba C.,et al.Silk-based biomaterials[J].Biomaterials,2003,24:401-416.
[35]Altman G H.,Horan,Rebecca L,Lu,Helen H,et.al.Silk matrix for tissue engineered anterior cruciate ligaments[J].Biomaterials,2002,23:4131-4141.
[36]Greenwald D,Shumway S,Albear P.,et al.Mechanical comparison of 10 suture materials before and after in vivo incubation[J].Surg Res 1994,56:372-377.
[37]Mingzhong Li,Masayo Ogiso,Norihiko Minour.Enzymatic degradation behavior of porous silk broin sheets[J].Biomaterials,2003,23:357-365.
[38]Kaiyong Cai,Kangde Yao,Songbai Lin,et al.Poly(d、l-lactic acid) surfaces modied by silk fibroin:effects on the culture of osteoblast in vitro[J].Biomaterials,2002,23:1153-1160.
[39]Kaiyong Cai,Kangde Yao,Yuanlu Cui,et al.Influence of different surface modification treatments on poly(d,l-lactic acid) with silk fi.broin and their effects on the culture of osteoblast in vitro[J].Biomaterials,2002,23:1603-1611.
[40]Minoura N,Aiba S,Ootoh Y.et al.Attachmentand growth of cultured broblast cells on silk protein matrices[J].Biomed Mater Res,1995,29:1215-1221.
[41]Inouye K.,Kurokawa M.,Nishikawa S.,et al.Use of Bombyx mori silk.broin as a substratum for cultivation of animal cells[J].Biochem Biophys Meth,1998,37:159-164.
[42]Sofia S.,McCart hy MB,C.rronowicz G,et al.Functionalizedsilk-based biomaterials for bone formation[J].Biorned MaterRes 2001,54:139-148.
[43]Chiarini A.,Petrini P.,Sabrina Bozzini,et al.Silk fibroin/poly(carbonate)-urethane as a substrate for ce11 growth:in vitro interactions with human cells.Biomaterials[J].2003,24:789-799.
[44]Jinwei Gu,Xinlin Yang,Hesun Zhu.Surface sulfonation of silk fibroin film by plasma treatment and in vitro antithrombogenicity study[J].Materials Science and Engineering.2002,20:199-202.
[45]Cheryl Wong Po Foe,Kaplan D L.Genetic engineering of fibrous proteins:spider dragline silk and collagen[J].Advanced Drug,2002,54:1131-1143.
[46]Vollrath F.,Barth P.,et al.Local tolerance to spider silks and protein polymers in vivo[J].2002,16(4):229-234.
[47]Minoura N.et al.Physico-chemical properties of silk fibroin membrane as biomaterials[J]. Materials,1990,11:430-434.
[48]Muller,W.S.,Samuelson,L.A.,Fossey,S.A.et al.Formation and properties of silk thin films[J].Science and Biotechnology,1994,544:342-352.
[49]李明忠,卢神,吴徵宇等,多孔丝素膜的制备及其形态结构[J].纺织学报,2000,21(5):268-271.
[50]Li MZ,Tao W,Kuga S,et al.Controlling molecular conformation of regenerated wild silk fibroin by aqueous ethanol treatment[J].Polymers for Advanced Technologies,2003,14:694-698.
[51]Kweon H.Y,Park H.Y..Structural and conformational changes of regenerated Antheraea pemyi silk fibroin films treated with methanol solution[J].Appy.Polym.Sci.1999,73:2887-2894.
[52]G.Freddi,P.Monti,M.Nagura et al.Structure and molecular conformation of tussali silk fibroin films:effect of heat treatment[J].Polymaterial.Science,(Polym.Phys),1997,35:841-847.
[53]Li MZ,Tao W,Lu SZ,et al.Comphant film of regenerated Antheraea pernyi silkfibroin by chemical crosslinking[J],International Journal of Biological Macromolecules,2003,32:159-163.
[54]G.Freddi,M.Tsukada,S.Beretta,Structure and physical properties of silk fibroin/polyaccrylamide blend films[J].Appl.Polym.Sci.1999,71:1563-1571.
[55]Yang G,Zhang L.,Cao X.D..Structure and mieroporous formation of cellulose/silk fibroin blend membranes PartⅡ effect of post-treatment by alkali[J].Journal of membrane Science,2002,210:379-387.
[56]Yang G,Zhang L.Regenerated cellulose microporous membranes by mixing cellulose cuoxam with a water soluble polymer[J].Membrane Science,1996,114:149-155.
[57]Jin HJ,Park J,Vailuzzi R,et al.Biomaterials film of Bombyx Mori Silk Fibroin with poly(ethylene oxide)[J].Biomacromolicules,2004,5(3):711-717.
[58]Chen X,Knight DP,Shao Z,et al.Conformation transition in silk protein films monitored by time-resolved Fourier transform infrared spectroscopy:effect of potassium ions on Nephila spidroins films[J].Biochemistry,2002,41(50):14944-14950.
[59]黄君霆.蜘蛛丝研究的动向[J].丝绸,1999,9:48-49.
[60]郑震.蜘蛛丝蛋白的生物制造技术及纤维加工[J].国外纺织技术,2000,12:2-4.
[61]叶金兴.蛛丝制作纺织材料的生物技术[J].现代纺织技术,2000,4:57-58.
[62]黄智华.蛛丝蛋白工程菌高密度发酵、表达产物纯化与湿法纺丝.福建师范大学硕士学位论文[D].2004.
[63]Lewis R V,Hinman M,Kothakota S,et al.Expression and purification of a spider silk protein:A new strategy for production repetitive proteins.Protein[J].Expression and Purification,1996,7:400-406.
[64]Liivak O,Blye A,Shah N,et al.A Mierofabricated wet-spinning apparatus to spin fibers of silk[J].Macromoleeules,1998,31:2947-2951.
[65]Seidel A,Liivak O,Jelinski L W.Artificial spinning of spider silk[J].Maeromoleeules,1998,31:6733-6736.
[66]Areidiaeono S,M.Mello C,Butler M,et al.Aqueous Processing and Fiber Spinning of Recombinant Spider Silks[J].Macromolecules,2002,35:1262-1266.
[67]Lazaris A,Arcidiacono S,Huang Y,et al.Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells[J].Scienee,2002,295:472-476.
[68]Shao Z,Vollrath F,Yang Y,et al.Strueture and Behavior of Regenerated Spider Silk[J].Macromolecules,2003,36:1157-1161.
[69]盛家镛,潘志娟,陈宇岳等.蜘蛛丝的化学组成与结构初探[J].丝绸,2000,(4):8-12.
[70]赵昀,陆长德.用蛋白质工程改进蚕丝性能的研究[D].中国科学院上海生物化学研究所,博士学位论文,2000.
[71]Seidel A,Liivak O,Jelinski L W.Artificial spinning of spider silk[J].Maeromolecules,1998,31:6733-6736.
[72]Liivak O,Blye A,Shah N,et al.A mierofabrieated wet-spinning apparatus to spin fibers of silk proteins[J].Strueture-property correlations.Macromolecules,1998,1:2947-2951.
[73]Fahnestock.Recombinanfly produced spider silk[N].United States Patent,July 31,2001.
[74]Lock.Process for spinning polypeptide fibers[N].United States Patent,December 15,1992.
[75]Steven Arcidiaeono,Charlene M.Miehelle Bufler,et al.Aqueous Processing and Fiber Spinning of Recombinant Spider Silks[J].Macromolecules,2002,35:1262-1266.
[76]Anthoula Lazaris,Steven Arcidiacono,Yue Huaug,et al.Spider silk fibers spun from soluble recombinant silk produced in mammalian cells[J].Scinece,2002,295:472-476.
[77]Zarkoob.Synthetically spun silk nanofibers and a process for making the same[N].United States Patent,August 29,2000.
[78]Weisshart K.Silk bioteelmology Reviews in Molecular[J].Bioteehnology,2000,74:65-66.
[79]Wong Po,Foo C,Keplan D L.Genetie engineering of fibrous proteins:sprider dragline silk and collagen[J].Advaced Drug,2002,54(8):1131-1143.
[80]Miao Y,Zhang Y,Nakagaki K,et al.Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system[J].Appl Microbiol Biotechnol,2005,13:1-8.
[81]Scheller J,Henggeler D,Viviani A,et al.Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation[J].Transgenic Res,2004,13(1):51-57.
[82]Falmestock SR,Yao Z,Bedzyk LA.Microbial production of spider silk proteins[J].Biotechnol.2000,74(2):105-109.
[83]Fukushima Y.Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk[J].Biopolymers,1998,45(4):269-279.
[84]Anthoula Lazaris,Steven Arcidiacono,Yue Huang,et al.Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells[J].Science,2002,295:472-476.
[85]Fukushima Y.Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk[J].Biopolymers,1998,45(4):269-279.
[86]张前军,祖旭宇,梁宋平等.拖丝蛋白全基因亚克隆文库的构建和部分序列的测定[J].南华大学学报(医学版).2004,(2):13-16.
[87]章文贤,李敏,涂桂云等.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.2003,20:15-17.
[88]章文贤,李敏,涂桂云等.重组拖丝蛋白基因在大肠杆菌中表达条件的优化[J].福建师范大学学报.2003,(2):62-64.
[89]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,(3):331-334.
[90]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[J].2001(11):7-9.
[91]黄全生,刘霞,王义琴等.基因枪轰击法将蜘蛛丝蛋白基因(ADF3)转入海岛棉的研究[J].新疆农业科学.2004,(4):59-61.
[92]黄建坤.RGD-蛛丝蛋白生物合成与丝膜制备[D].福建师范大学硕士论文.2004.
[93]牛恒尧.Araneusdiadematu拖丝蛋白ADF3的原核表达及棉花纤维特异表达载体的构建[D].中国科学院遗传学研究所硕士论文.2001.
[94]陈新,黄郁芳,邵正中等.蜘蛛吐丝过程中钾的作用[J].高等学校化学学报,2004(6):181-184.
[95]Lazaris.A,.Arcidiacono.S,Huang,Y.et al.Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells[J].Science,2002,295:472-476.
[96]Dicko C.,Vollrath F.,Kenney JM.Silk protein refolding is controlled by changing pH[J].Biomacromolecules,2004,(5):704-710.
[97]Dicko C.,Kernney JM.,Knight D.et al.Transition to a beta-sheet-rich structure in spidroin in vitro:the effects of pH and cations[J].Biochemistry,2004,43:14080-14087.
[98]Terry A.E.,KnighL D.P.,Porter,D.et al.pH Induced Changes in the Rheology of Silk Fibroin Solution from the Middle Division of Bombyx mori Silkworm[J].Biomacromolecules 2004,(5):768-772.
[99]彭显能,陈新,武培怡等.二维相关红外光谱研究再生蚕丝蛋白膜的构象与温度之间的关系[J].化学学报,2004,21:2127-2130.
[100]Knight,D.P.,Vollrath,F.Changes in element composition in the secretory pathway for dragline silk in the golden orb web spider,Nephila edulis[J].Naturwissenschaften,2001,88:179-182.
[101]Tanaka,T.,M agoshi,L,M agoshi,Y.,et al.Spherulites of Tussah Silk Fibroin:Structure,thermal properties and growth rates[T].Journal of Thermal Analysis and Calorimetry.2001,64:645-650.
[102]Ochi,A,Hossain,K.S,Magoshi,J,et al.gheology and dynamic light scattering of silk flbroin solution extracted from the middle division of Bombyx mori silkwonn[J].Biomacromolecules 2002,3(6):1187-1196.
[103]K.Gellynck,P.Verdonk,K.F.Almqvist,et al.Chondrocyte Growth in Porous Spider Silk 3D-Scaffolds[J].European Cells and Materials.2005,10(2):45-48.
[104]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.
[105]李敏,黄建坤,涂桂云.RGD-蜘蛛拖丝蛋白聚合物的生物合成与纯化[J].中国生物工程杂志,2004,21(6):1006-1010.
[106]李敏,涂桂云,黄智华,黄曦.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.
[107]阮超然,黄晶星,魏梅红,李敏.高分子量RGD-蛛丝蛋白重组体构建、高密度发酵及纯化[J].生物工程学报,2007,23(5):858-861.
[108]K.Gellynck,P.Verdonk,K.F.Almqvist,E.Van Nimmenl,T.Gheysens,Mertens,L.Van Langenhove,P.Kiekens,A.Verbruggen.European Cells and Materials.2005,10(Supp1.2)
[109]XING Ben-gang,LIANG Hong.Joumal of Guangxi Normal University[J].Natural Science Edition,1997(3):46-49.
[110]LI Min,HUANG Jian-kun,TU Gui-yunet al.Biologieal Synthesis and Purification of Spider Dragline Silk Protein Polymers Containing RGD Three Peptide[J].Journal of Biomedical Engineering.2004,(6):1006-1010.
[111]LI Min,CHEN Deng-long,TU G-ui-yun.[P]:CN 200510076750.8,2005.
[112]QIAN Guo-di,YAO Yu-liang.Journal of Suzhou Institute of silk textile technology[J].1983,(4):26-30.
[113]Suzhou Institute of silk textile technology[J].Silk-manufacturing Chemistry.China textile&Apparel Press,1996:62.
[114]CHEN Xin,ZHOU Li,SHAO Zheng-zhong,et al.Conformation transition of silk protein membranes monitored by time-resolved FT-IR spectroscopy-Conformation transition behavior of regenerated silk fibroin membranes in alcohol solution at high eoncentrations[J].Acta Chimica Sinica,2003,(4):625-629.
[115]LI M Z,WU Z Y,ZHANG C S,et al.Study on Porous Silk Fibroin Materials.Ⅱ.Preparation And Characteristics of Spongy Porous Silk Fibroin Materials.Journal of Applied Polymer[J]Science,2001,79:2192-2199.
[116]Nazarov R,Jin H J,Kaplan D L.Porous 3-D Scaffolds from Regenerated Silk Fibroin[J].Biomacromolecules,2004,(5):718-726.
[117]姚康德,尹玉姬编著.组织工程相关生物材料[M].北京:化学工业出版社,2003.
[1118]师昌绪主编.材料大词典[M].北京.化学工业出版社,1994.
[119]沈家骢.纳米生物医用材料[J].中国医学科学院学报,2006,(4):472-474.
[120]陈登龙,李敏,房乾.静电纺丝技术在纳米结构高分子支架中的应用[J].中国修复重建外科杂志,2007,21(4):411-415.
[121]Chu Bejamin,Benjamin S,Fang Dufei,et al.Biodegradable and/or bioabsorbable fibrous articles and methods for using the articles in medical applications[P].PCT:WO 02/092 339,2002.5.15.
[122]Christopher J Buehko,Louic Chen,Yu Shen,et al.Processing andmierostructural characterization of porous biocompatible protein polymerthinfilms[J].Polymer,1999,40:7397-7407.
[123]Smith Daniel,Reneker Darrell,Kataphinan Woraphon.Electrcspun skin masks and USeS thereof [P].PCT/US:00/27775,2000.10.6.
[124]Zheng Ming Huang,Y Z Zhang,M Komki,et al.A review on polymer nanofibers by electrospinning and their application in nanocomposites[J].Composites Science and Technology,2003,63:2223-2253.
[125]刘华,房乾,陈登龙等.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.
[126]陈登龙,房乾,涂桂云等.重组蛛丝蛋白pNSR16/聚乙烯醇复合材料的研究.功能材料[J].2007,38(7):1194-1196.
[127]陈登龙,房乾,姚清华等.傅里叶红外光谱研究重组蛛丝蛋白分子构象的影响因素[J].高分子材料科学与工程.2009,25(1):104-106.
[128]李敏,陈登龙,吴钦缘.静电纺丝仪.[P]:CN 200610125960.6,2006.
[129]陈登龙,房乾,涂桂云等.重组蛛丝蛋白pNSR16/聚乙烯醇复合材料的研究[J].功能材料.2007,38(7):1194-1196.
[130]佘厚德.羟基磷灰石及组织工程用聚己内酯复合支架的制备和研究[D].福建师范大学硕士论文,2007.
[131]Boontheekul T,Mooney DJ.Protein-based signaling systems in tissue engineering[J].Curr Opin Biotech,2003,14:559-565.
[132]HERSEL U,DAHMEN C,KESSLER H.RGD modified pol-ymers:biomaterials for stimulated cell adhesion and beyond[J].Biomaterials,2003,24(10):4385-4415.
[133]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):210-212.
[134]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):24-27.
[135]Iiohner U,Hutmacher DW,See Y,et al.Individually CAD-CAM technique designed,hioresoibahle 3-dimensional polycaprolactone framework for experimental reconstruction of craniofacial defects in the pig[J].Mund Kiefer Gesiclitschir,2002,6(3):162-167.
[136]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.
[137]李国义,梁传余,郑艳等.气管软骨组织工程管状泡沫支架的研制[J].中华医学网络杂志2004,1(2):43-45.
[138]Ung-Jin Kim,Jaehyung Park,et al.Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin[J].Biomaterials.2005,26:2775-2785.
[139 Vassilis Karageorgiou,David Kaplan.Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials[J].2005,26:5474-5491.
[140]Chu Bejamin,Benjamin S,Fang Dufei,et al.Biodegradable and / or bioabsorbablc fibrous articles and methods for using the articles in medical applications[P].PCT:WO 02/092 339,2002.5.15.
[141]Christopher J Buehko,Louie Chen,Yu Shen,et al.Processing andmierostructural characterization of porous biocompatible protein polymerthinfilms[J].Polymer,1999,40:7397-7407.
[142]Smith Daniel,Reneker Darrell,Kataphinan Woraphon.Electrcspun skin masks and USeS thereof [P].PCT / US:00/27 775,2000.10.6.
[143]Zheng Ming Huang,Y Z Zhang,M Komki,et al.A review on polymer nanofibers by electrospinning and their application in nanocomposites[J].Composites Science and Technology,2003,63:2223-2253.
[144]E.J.Chon,T.T.Phan,I.J.Lim,et al.Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution[J].Acta Biomaterialia,2007,3:321-330.
[145]B.Linnemann,R.Ali,T.Grie 等.聚己内酯(PCL)纳米纤维的静电纺丝[J].国际纺织导报,2006,(4):22-24.
[146]赵洁,全大萍,廖凯荣等.组织工程中生物可降解高分子的功能化及生物学修饰[J].高分子通报,2005,6:70-75.
[147]Ulrich Hersel,Claudia Dahrnen,Horst Kessler.RGD modified polymers:biomaterials for stimulated cell adhesion and beyond[J].Biomaterials.2003,24:4385-4415.
[148]Stephane Biltresse,Mireille Attolini,Jacqueline Marchand-Brynaert.Cell adhesive PET membranes by surface grafting of RGD peptidomimetics[J].Biornaterials.2005,26:4576-458