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1 生物工程学报 Chin J Biotech 2009, April 25; 25(4): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 组织工程与细胞培养 - 孙秀娟 1,3, 范代娣 1,2, 朱晨辉 1,2, 马晓轩 2, 骆艳娥 2, 陈岚 3, 郭佳庆 1, , , : 将类人胶原蛋白与透明质酸按不同比例复合, 控制透明质酸的终浓度 (W/V) 分别为 % 0.05% 0.1%, 用京尼平交联, 采用真空冷冻干燥方法构建出血管支架材料 通过扫描电镜 XPS 分析 拉力测试 压力爆破实验 细胞毒性实验 血管支架细胞种植实验及小鼠皮下植入等方法对其表面超微结构 表面元素组成 力学性能 细胞毒性等级 细胞相容性 组织相容性进行了研究 结果表明 : 当透明质酸的含量为 0.05% 时, 类人胶原蛋白 - 透明质酸支架的孔径比较均匀, 孔隙率达 94.38%, 应力为 ( ± 7.9) kpa, 爆破压力为 ( ± 8.2) kpa, 细胞毒性实验合格, 同时具有良好的细胞相容性 组织相容性及降解性能 : 类人胶原蛋白, 透明质酸, 血管支架, 生物相容性 2 Characterization and biocompatibility of human-like collagen-hyaluronic acid scaffold for blood vessel Xiujuan Sun 1,3, Daidi Fan 1,2, Chenhui Zhu 2, Xiaoxuan Ma 2, Yan e Luo 2, Lan Chen 3, and Jiaqing Guo 2 1 Shaanxi Key Laboratory of Degradable Biomaterials, Northwest University, Xi an , China 2 Biomaterials and Fermentation Engineering Center of Shaanxi, Xi an , China 3 Faculty of Life Science, Northwest University, Xi an , China Abstract: Human-like collagen (HLC) was cross-linked with hyaluronic acid by genipin in different ratio. The concentrations of hyaluronic acid in the mixture were 0, 0.01%, 0.05% and 0.1%. The blood vessel tubular grafts were then fabricated by freeze-drying. Microstructure, element composite, mechanical properties, cytotoxicity grade, and biocompatibility of different vascular scaffold groups were studied by scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), tensile test, burst pressure experiment, cytotoxicity experiment, endothelial cells planted in blood vessel scaffolds and hypodermic embedding of mice. The results showed that HLC-HA (0.05%) tubular scaffold exhibited interconnected well-distributed and porous structure and porosity of 94.38%; achieved the desirable mechanical property with stress of (1000.8±7.9) kpa and burst pressure of (1058.6±8.2) kpa, hypocytotoxicity, favourable cytocompatibility, hisocompatibility and disposition of degradation. Keywords: human-like collagen, hyaluronic acid, blood vessel scaffold, biocompatibility Received: October 7, 2008; Accepted: February 9, 2009 Supported by: National Natural Science Foundation of China (No ), Scientific Research Program of Shaanxi Provincial Department of Education(No. 08JK452 ), Scientific Technology Program of Xi an City (No.YF07078 ). Corresponding author: Daidi Fan. Tel: ; fandaidi@nwu.edu.cn 国家自然科学基金项目 (No ), 陕西省教育厅专项科研计划项目 (No. 08JK452 ), 西安市科技计划项目 (No.YF07078 ) 资助

2 592 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4, [1],,, [2], (Hyaluronic acid, HA) (Human-like collagen, HLC),, [3], [4 6], HA,,, [7], [8] ; HA [9],,, [10] [11] ; [12] -,, 1 材料和方法 1.1 材料, 97 kd; (HA) ; ; BHK-21 ; (EVC304) ; 1640 Gibco ; ; MTT(3-(4, )-2, 5- ) (DMSO) Amresco ; Sigma ; 1.2 仪器 ULT V34, REVCO; FREEZONE6, LABCONCO; S-570, ; 5565, INSTRON; XPS PHI-5400, PE; CM1850, LEICA; ESCO-2, ESCO;, REVCO; MULTISKAN MK 3, THERMO;, NIKON;, PTCA, Medtronic 1.3 实验方法 1.3.1, % 0.05% 0.1% 15 min,, 0.625% 5 mm 7 mm 4 o C 3 d, 80 o C 2 h, 1.3.2,, E5100SEM, S kv [13], WD mm, mbar, PHI-5400(PE, USA) X, ml, W 1, W s,,

3 : - 593,, W 2,, W 3, =(W 2 W 3 W s )/ (W 1 W 3 ) mm 6 mm 0.35 mm,, 2 mm/min,, ,,,, MTT ISO, HLC-HA(0.05%) (10% 1640 ) 0.2 g/ml, 37 o C, 3 d, 100%,, 100% 75% 50% 25%,, 4 o C 37 o C 5% CO 2 BHK-21, BHK /ml 96, 24 h,, 100% 75% 50% 25%, 6, 200 μl 48 h, MTT,, (Relative growth rate, RGR) RGR= / 100%, [14] HLC HLC-HA(0.05%), cm, 24, 75% 24 h, 75%, PBS 3, 1 ml 10% o C 24 h,, /ml, ml, 37 o C 0.5% CO 2 3 h, 0.5 ml 37 o C 0.5% CO 2 3 d,,,, , 28~32 g, 5, 6 [15],,, 1 cm,,, 1 cm 0.5 cm (Co 60 ), ,, 10% 24 h,, HE, , T, P< 实验结果 2.1 支架形态 1,, - 2, HLC HLC-HA(0.01%), ; HLC-HA(0.05%),, (60±9) μm, ; HLC HLC-HA(0.01%), HLC-HA(0.1%),, (90±8) μm, (100~300 μm) [2] 2.2 XPS 表面元素分析 71.78% 7.73% 79.64% 0.97% 20.49% 19.38% 3, -

4 594 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4, 90% 4, 90%, HLC, HLC-HA(0.05%) 94.38% 图 1 类人胶原蛋白 - 透明质酸血管支架的外部形态 Fig. 1 Appearance of human-like collagen-hyaluronic acid blood vessel graft. 图 4 各组血管支架的孔隙率 Fig. 4 Porosity of different groups of blood vessel grafts. 图 2 各种血管支架扫描电镜 Fig. 2 SEM micrographs of different blood vessel grafts. (A) HLC. (B) HLC-HA (0.01%). (C) HLC-HA (0.05%). (D) HLC- HA (0.1%). 图 3 不同样品 C1s N1s O1s 峰的 XPS 分析图 Fig. 3 XPS C1s, N1s and O1s core level spectra of different samples. 2.3 材料孔隙率测定结果,, 2.4 血管支架的力学性能测试 1 HLC-HA HLC LSD (P<0.01), 3 HLC 3 (P>0.05), HLC-HA(0.05%) (1000.8±7.9)kPa, HLC (216.7±9.1)kPa;, HLC-HA(0.01%) (P<0.01);, HLC-HA(0.05%) HLC-HA(0.1%)) HLC HLC-HA(0.01%), HLC-HA HLC 5A~5C,,,, 2.5 血管支架压力爆破实验 HLC-HA(0.05%), (1058.6±8.2)kPa HLC-HA HLC, 2.6 血管支架材料细胞毒性的测定 2, 100% 75% 50% 25% HLC-HA(0.05%), 48 h BHK-21 90%,,, 1,

5 : 表 1 类人胶原蛋白 - 透明质酸支架材料的拉伸性能 (n=5) Table 1 Tensile properties for human-like collagen-hyaluronic acid scaffolds (n=5) Samples Name Stress (kpa) Strain (%) Young s modulus (MPa) HLC 216.7± ± ±0.4 Dry samples HLC-HA (0.01%) 815.6± ± ±0.5 HLC-HA (0.05%) ± ± ±0.6 HLC-HA (0.1%) 900.6± ± ±0.9 HLC 110.5± ± ±0.5 Wet samples HLC-HA (0.05%) 190.5± ± ±0.4 HLC-HA (0.01%) 180.4± ± ±0.3 图 5 各组血管支架材料在室温下的应力应变曲线图 Fig. 5 Curve of stress-strain for groups of scafflods were obtained at room temperature. (A) Dry scaffold of HLC-HA(0.1%). (B) Dry scaffold of HLC-HA(0.01%). (C) HLC-HA (0.01%) (in system of normal sodium).

6 596 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4 表 2 血管支架材料细胞毒性的测定结果 (n=6, x ± s) Table 2 Results of cell toxicity were obtained from groups of vessel scaffolds (n=6, x ± s) Groups OD 490 ( x ±s) RGR (%) Toxicity fractionation Blank 0.645± % leaching liquor 0.591± % leaching liquor 0.608± % leaching liquor 0.638± % leaching liquor 0.640± 血管支架细胞种植实验 7 HLC-HA(0.05%)( B D) HLC( A C),, [13], HLC-HA(0.05%) 2.8 皮下植入实验 HLC-HA(0.05%), HE 8, 1,, ( A); 3, ( B); 5,, ( C); 9, ( D) 图 6 各组血管支架压力爆破实验结果 Fig. 6 Results of burst pressure experiment for groups of scaffolds (n=5). 图 8 类人胶原蛋白 - 透明质酸 (0.05%) 皮下植入实验 HE 染色结果 Fig. 8 HE micrographs of human-like collagen-hyaluronic acid scaffolds (0.05%) implanted in mices for one week(a), three weeks (B), five weeks (C) and nine weeks (D). Magnification: A, 80 ; B, 80 ; C, 100 ; D, 讨论 图 7 脐带静脉内皮细胞 (EVC304) 在血管支架内表面的生长情况 : (A) 类人胶原蛋白 ( 光学显微镜 10 ); (B) 类人胶原蛋白 - 透明质酸 (0.05%)( 光学显微镜 10 ); (C) 类人胶原蛋白 ( 扫描电镜 300 ); (D) 类人胶原蛋白 - 透明质酸 (0.05%)( 扫描电镜 1000 ) Fig. 7 Growth of endothelial cells from umbilical core vein in internal surface of blood vessel scaffolds. (A) HLC. (B) HLC-HA (0.05%). (C) HLC (SEM). (D) HLC-HA (0.05%) (SEM). Magnification: A, 10 ; B, 10 ; C, 300 ; D, 1000.,,, 90% 100~300 μm, [16]

7 : , HLC-HA(0.05%),, 90%,,, HLC-HA(0.1%) 60~90 μm,, OH COO,,,, HA [10,17,18],,,,, HLC-HA, (1000.8±7.9) kpa, [17] - (665±5) kpa, [11] (1.08±0.20) MPa, (190.5±2.1) kpa;, HLC-HA(0.01%) (20.6±0.7)%, HLC-HA(0.01%) ; HLC-HA(0.05%) HLC-HA (0.1%) HLC-HA(0.01%) HLC,,,,,,, HLC-HA(0.05%), (2700~5000 mmhg) [19],, MTT [20] HLC-HA(0.05%) 1,, HLC-HA(0.05%), HLC,, HLC-HA(0.05%) [21] 3 ; 5, ; 9,, ; 20,,, 4 结论, 90%, (1000.8±7.9) kpa, (1058.6±8.2) kpa, ;, ;

8 598 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4 - (0.05%),,, REFERENCES [1] American Heart Association Heart and Stroke Statistical Update. Dallas: American Heart Association, [2] Wu YC, Cui L, Cao YL. The research and development of tissue engineering vessel. J Tiss Eng Rec Surg, 2006, 2(5): ,,.., 2006, 2(5): [3] Mi Y, Xi JF, Fan DD, et al. The biocompatibility of human-like collagen. J Northwest Univ (Natl Sci Ed), 2004, 34(1): ,,,.. ( ), 2004, 34(1): [4] Zhai Y, Cui FZ, Wang Y. Formation of nanohydroxylapatite on recombinant human-like collagen fibrils. Curr Appl Phys, 2005, 5: [5] Ma M, Fan DD. Hemostatic effect and histocom-patibility of chitosan-human-like collagen on rabbit liver wound surface. J Fourth Mil Med Univ, 2007, 28 (12): ,. -., 2007, 28(12): [6] Zhai Y, Cui FZ. Recombinant human-like collagen directed growth of hydroxyapatite nanocrystals. J Cryst Growth, 2006, 291: [7] Huang SL, Ling PX. Crosslinking Hyaluronic acid application in medicine. Food Drug, 2004, 10: ,.., 2004, 10: [8] Liu JJ, Zeng CQ. Effect of hyaluronan in angiogenesis of tumor. J Beihua Univ (Natl Sci), 2003, 10(4): ,. ( ), 2003, 10(4): [9] Wang YH, Wang FS, Guo XP. Angiogenesis effects of low molecular weight hyaluronic acid. Chin Pharm J, 2007, 5(42): ,,.., 2007, 5(42): [10] Huang ZB, Gu QS. A neotype crosslinking agent genipen application and development in biomedical engineering. Shanghai J Biomed Eng, 2003, 24(1): ,.., 2003, 24(1): [11] Li L, Chen J, Yu XX. Preparing biological tissues for tissue-engineered vascular scaffold by crosslinking agent genipin. J Clin Rehab Tiss Eng Res, 2007, 11(35): ,,.., 2007, 11(35): [12] Yao FL, Li XQ, Yu X, et al. Crosslinking reaction of chitosan and gelatin with genipin. J Tianjin Univ, 2007, 40(12): ,,.., 2007, 40(12): [13] Suzan TM, Paul J, Ronnie W, et al. Increased angiogenesis and blood vessel maturation in acellular collagen heparin scaffolds containing both FGF2 and VEGF. Biomaterials, 2007, 28: [14] United States Pharmacopoeia Convention. Biological Tests/Biological Reactivity Tests, In Vivo. USP XXII. Rockville, [15] GB/T /ISO Biological evaluation of medical devices-part 1: Evaluation and testing. GB/T /ISO :. [16] Cao YL. Tissue Engineering. Beijing: Science Press, 2007: :, 2007: [17] Lu Q, Zhang SJ, Hu K. Cytocompatibility and blood compatibility of multifunctional, fibroin/collagen/heparin scaffolds. Biomaterials, 2007, 28: [18] Sung HW, Liang IL, Chen CN, et al. Stability of a biological tissue fixed with a naturally occurring crosslink agent (genipin). J Biomed Mater Res, 2001, 55: [19] Joseph D, Michael M, Robert M, et al. A biological hybrid model for collagen-based tissue engineered vascular constructs. Biomaterials, 2003, 24: [20] Wang X, Shi YP, Zhu XT, et al. Evaluation on the cytotoxicity of polymeric material for medical use by MTT-assay. Shandong J Biomed Eng, 2003, 1: , 嬿,,. MTT., 2003, 1: [21] Wang X, Qu L, Dou ZY, et al. Biomaterials in tissue engineering scaffolds. China Biotechnol, 2003, 23(10): ,,,,., 2003, 23(10):