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1 Journal of Clinical Rehabilitative Tissue Engineering Research January 1, 2008 Vol.12, No.1 /**** Cell biocompatibility of electrospun chitosan/collagen composite nanofibers Wang Pei-wei 1, Chen Zong-gang 1, Wang Hong-sheng 1, He Chuang-long 1, Mo Xiu-mei 1,2 Abstract AIM: Electrospunning is a process that the charged polymer solution or melt jet efflux in the electrostatic field to prepare the polymer nanofibers. This study aimed to prepare electrospun chitosan/collagen composite nanofibers and evaluate their cell biocompatibility. METHODS: The experiment was carried out in the Laboratory of Chemistry, Chemical Biotechnology at Donghua University between November 2006 and October Taking hexafluoroisopropanol/trifluoroacetic acid as solvent, the composite nanofibers were prepared by electrospunning according to the mass ratio of chitosan/collagen as 100:0, 80:20, 50:50, 20:80 and 0:100.The composite nanofibers were inoculated with porcine iliac artery endothelial cells in vitro. The cell adhesion and proliferation as well as cell morphologies were evaluated by MTT method and HE stain, respectively. RESULTS: The porcine iliac artery endothelial cells adhered tightly on surface of electrospun chitosan/collagen composite nanofibers, exhibited plump and fusiform shape, as well as good growth. MTT results showed that nanofibers could obviously enhance the adhesion and proliferation of endothelial cells on the surface of materials, and the cells had the best spreading and attachment on the composite nanofibers with a ratio of 20:80, then 50:50. CONCLUSION: The electrospun chitosan/collagen composite nanofibers hold great potential for tissue engineering application due to their good cell biocompatibility. Wang PW, Chen ZG, Wang HS, He CL, Mo XM.Cell biocompatibility of electrospun chitosan/collagen composite nanofibers.zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(1):5-9() [ / / // MTT /MTT /./[J] (1):5-9 [ 1 Laboratory of Biotechnology, 2 State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Wang Pei-wei, Studying for master's degree, Laboratory of Biotechnology, wangpeiwei@yahoo. cn Correspondence to: Mo Xiu-mei, Laboratory of Biotechnology; State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, xmm@dhu.edu.cn >><< Supported by: the National Natural Science Foundation of, No *; Shanghai Pujiang Program in 2005, No. 05PJ14013*; Major Fundamental Research Program of Shanghai, No. 05DJ14006*; Programme of Introducing Talents of Discipline to Universities (111 Project), No. b07024* Received: Accepted: ISSN CN /R CODEN: ZLKHAH 5

2 wangpeiwei@ yahoo.cn 1, xmm@dhu.edu.cn * PJ14013* DJ14006* ( 111 b07024)* :R :A : (2008) ( /N Y) (ECM) Formhals [1-3] 1934 [4-5] (GAGs) [6-7] [8-9] / [10-12] / (PIECs) M10685% 0.8~ Fluorochem Ltd. 25% DMEM2.5 g/l MTTSigma- MK3 S-2700 / [13] / G 2.5 ml 20 kv12 cm 0.8 ml/h/ 25% 12 h h PBS4 hpbs3100 L DMEM 37 SEM Image J PIECs0.1 DMEM CO 2 24 h 2.5 g/l 24 / 6 P.O.Box 1200,Shenyang kf @sina.com

3 ./ g/l10 min 2 min2 min 5 min 10 min() s30 s0.7 2 MTT L L 1 24 MTTPIECs/ 200 L 0.1DMEM 200 L CO h123 d PBS3400 L MTT40 L5 g/l4 h 400 L10 min492 nm A SEM 1SEM / 2.2 / / 2 / PIECs/ Figure 1 1 achitosancollagen =1000 bchitosancollagen =8020 cchitosancollagen =5050 dchitosancollagen =2080 echitosancollagen =0100 Morphology of chitosan/collagen composite nanofibers in different mass ratios by scanning electron microscope5 000 SEM / ISSN CN /R CODEN: ZLKHAH 7

4 achitosancollagen =1000 cchitosancollagen =5050 echitosancollagen =0100 bchitosancollagen =8020 dchitosancollagen =2080 ftissue culture plate Figure 2 Cell morphology on the chitosan/collagen composite nanofibers / / hmtt / 2080 A/ TCP Figure 3 Adhesion of cells to the chitosan/collagen composite t(culture)/h nanofibers 3 / 8 Absorbance d / A 3 d2 d1 d A/ 2080A 4 P < TCP t(culture)/d Figure 4 Cell proliferation on the chitosan/collagen composite nanofibers 4 / [14] MTT [15-17] [18] Absorbance P.O.Box 1200,Shenyang kf @sina.com

5 ./ / / PIECsPIECs dmtt PIECs/ / 1 Formhals A. Process and apparatus for preparing artificial thread [P].US. Patent, Formhals A. Method and apparatus for spinning [P].U.S. Patent, Formhals A. Method and apparatus for spinning [P].U.S. Patent, Ma ZKotaki MInai Ret a1.potential of nanofiber matrix as tissue-engineering scaffolds. Tissue Engineering 2005;1 1(1-2): Qian YF,Mo XM,Ke QF,et al.zhongguo Zuzhi Gongcheng Yanjiu Yu Linchuang Kangfu 2007; 11(22): ,,,. [J]., 2007, 11(22): Duan B, Dong CH, Yuan XY,et al. Electrospinning of chitosan solutions in acetic acid with poly(ethylene oxide). J Biomater Sci Polym Edn 2004;15(6): Bhattarai N, Edmondson D, Veiseh O, et al. Electrospun chitosan-based nanofibers and their cellular compatibility. Biomaterials 2005;26(31): Matthews JA, Wnek GE, Simpson DG,et al. Electrospinning of collagen nanofibers. Biomacromolecules 2002;3(2): Boccafoschi F, Habermehl J, Vesentini S, et al. Biological performances of collagen-based scaffolds for vascular tissue engineering. Biomaterials 2005;26(35): Shanmugasundaram N, Ravichandran P, Reddy PN, et al. Collagen-chitosan polymeric scaffolds for the in vitro culture of human epidermoid carcinoma cells. Biomaterials 2001;22(14): Chen JP, Chang GY,Chen JK. Electrospun collagen/chitosan nanofibrous membrane as wound dressing. Colloids and Surfaces A: Physicochemical and Engineering Aspects (2007), doi: /j.colsurfa Sionkowska A, Wisniewski M, Skopinska J, et al. Molecular interactions in collagen and chitosan blends. Biomaterials 2004;25(5): Chen Z, Mo XM, Qing FL. Electrospinning of collagen-chitosan complex. Materials Letters 2007; 61(16): Merzkirch C, Davies N, Zilla P. Engineering of vascular ingrowth matrices: are protein domains an alternative to peptides? Anat Rec 2001;263(4): Wang ZH,Xiao YZ.Gaofenzi Cailiao Kexue Yu Gongcheng 2005;21(5):19-22,. [J].,2005,21(5): Wu CG,Fang NT,Pan LF. Zhongguo Zuzhi Gongcheng Yanjiu Yu Linchuang Kangfu 2007;1(26): ,,. [J]., 2007, 11(26): Li JG,Cui CX,Qi YM,et al. Zhongguo Zuzhi Gongcheng Yanjiu Yu Linchuang Kangfu 2007; 11(35): ,,,. // [J]., 2007, 11(35): Ma AJ,Yang QF,Liang W,et al.cailiao Kexue Yu Gongcheng Xuebao 2005;23(5): ,,,.[J].,2005,23(5): ISSN CN /R kf @sina.com kf @sina.com kf @sina.com kf @sina.com ISSN CN /R CODEN: ZLKHAH 9

助剂改善其止血效果 1 实验 1.1 原料和试剂家蚕蛹经过提取蛹油蛋白质后剩余的残渣 ( 主要成分为蛹皮 ), 烘干除杂粉碎后待用 ; 壳聚糖 ( 成都市科龙化工试剂厂 ), 脱乙酰

助剂改善其止血效果 1 实验 1.1 原料和试剂家蚕蛹经过提取蛹油蛋白质后剩余的残渣 ( 主要成分为蛹皮 ), 烘干除杂粉碎后待用 ; 壳聚糖 ( 成都市科龙化工试剂厂 ), 脱乙酰研究与技术蚕蛹壳聚糖复合止血材料的制备及凝血性能初探陈超, 单其艳, 杨铭, 蓝广芊, 吴大洋 ( 西南大学纺织服装学院, 重庆 400716) 摘要 : 探讨以蚕蛹壳聚糖 (CS) 和明胶 (GEL) 为主要原料制备复合止血材料的工艺及凝血效果首先