Ⅰ型胶原蛋白-P3/4HB组织工程瓣膜无纺复合支架的体外构建及表征分析
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摘要
第一部分静电纺丝技术构建P3/4HB无纺支架及其对细胞生物学行为的影响
目的:运用静电纺丝技术构建P3/4HB无纺支架,探讨支架拓扑结构对肌成纤维细胞生长的影响。
方法:采用静电纺丝技术制备具有不同纤维直径、孔径的微米和亚微米级P3/4HB无纺支架,接种大鼠肌成纤维细胞,体外培养1周后行扫描电镜观察,沉淀法及MTT法分别检测各支架上的细胞黏附及增殖情况。
结果:大鼠肌成纤维细胞在直径为1.24μm,孔径为38.3μm的微米级电纺支架上8小时的黏附率为50.37%,体外培养7天后细胞增殖数为3.54×105个/ml,与直径为0.58μm的亚微米支架上细胞增殖无明显差异,但后者细胞黏附率仅为43.30%,且限制了细胞在支架上的内向性生长,直径为57.8μm的微米支架细胞增殖数均较前二种支架低。
结论:电纺支架的纤维直径和孔径等拓扑结构对肌成纤维细胞的生长具有重要的调节作用,微米电纺支架是一种适合肌成纤维细胞生长的理想三维多孔支架。
第二部分氧等离子体表面改性P3/4HB无纺支架及其对细胞粘附性的影响
目的:研究氧等离子体表面改性对P3/4HB无纺支架的作用。
方法:氧等离子体改性P3/4HB无纺支架,改性的效能通过检测接触角及X射线光电子能谱分析进行结构表征,接种大鼠肌成纤维细胞,检测细胞黏附率以进行功能表征,并检测改性前后支架最大应力变化。
结果:等离子体改性后的P3/4HB无纺支架表面氧含量增加了12.1%,C-O(C-OH)峰、COOR(COOH)峰分别增加0.2%、6.2%,接触角下降了44°,而最大应力无明显变化,改性后支架细胞黏附率较预处理组为高。
结论:氧等离子体处理P3/4HB支架,可在支架表面引入-OH、-COOH等活性基团,改善材料亲水性,提高细胞黏附力,并为进一步共价接枝生物活性单体创造了条件。
第三部分Ⅰ胶原蛋白化学接枝P3/4HB体外构建组织工程复合支架
目的:构建Ⅰ型胶原蛋白-P3/4HB复合支架,并探讨其对细胞生长的影响。
方法:氧等离子体改性P3/4HB无纺支架,引入活性基团,通过化学接枝技术,将Ⅰ型胶原蛋白接枝在P3/4HB无纺支架表面,采用X射线光电子能谱分析及茚三酮法定性、定量检测接枝效果;体外接种大鼠肌成纤维细胞,MTT法检测复合支架的细胞增殖情况以进行功能表征。
结果:电子能谱分析结果显示,接枝后支架表面出现酰胺基的谱峰;复合支架在磷酸盐缓冲液中浸洗48h后,胶原涂层含量无明显变化;与对照组比较,肌成纤维细胞在复合支架上增殖较多。
结论:通过化学缩合反应,可有效制备Ⅰ型胶原蛋白-P3/4HB复合支架,接枝的胶原涂层具有稳定的生物学效应,可显著促进细胞增殖。
PartⅠFabrication of P3/4HB Nonwoven Scaffolds and Effects on the Behavior of Myofibroblasts
Objective: To fabricate P3/4HB nonwoven scaffolds by electrospinning and investigate the effects of fiber diameter and pore size on myofibroblasts cultured on electrospun scaffolds in vitro.
Methods: P3/4HB nonwoven scaffolds with different fiber diameters and pore sizes were fabricated by electrospinning, and then seeded by myofibroblasts harvested from rats. After 1 week’s cultivation, SEM was performed, cell count and MTT test were used to examine cell attachment and proliferation respectively.
Results: Myofibroblasts were adhered well and proliferated on the microfiber scaffold (1.24μm in diameter) ,whereas reduced cell adhesion on the sub-microfiber scaffold (0.58μm in diameter) and poor cell spreading on microfiber scaffold (2.79μm in diameter,)were observed.
Conclusions: Fiber diameter、pore size have influence on cell adhesion ,proliferation and various other cell behaviors. Microfiber electrospun scaffold is an optimal 3D matrix for myofibroblast.
PartⅡOxygen Plasma Surface Modification of P3/4HB Scaffolds and Effects on the Adhesion of Myofibroblasts
Objective: To investigate the effects of oxygen plasma surface modification on electrospun P3/4HB nonwoven scaffolds.
Methods: Electrospun P3/4HB nonwoven scaffolds were modified by low pressure oxygen plasma and physicochemical characteristics of the modified surfaces were investigated by x-ray photoelectron spectrometer, contact angle measurements together with maximum stress measurements, the cell adhesion capability tests on modified scaffolds and serum preconditioned scaffolds were carried out using myofibroblasts harvested from rats.
Results :XPS show the increase in the oxygen content、(C-OH)group and COOR(COOH)group of P3/4HB scaffolds after oxygen plasma surface modification,water contact angle decreased, myofibroblasts attachment were improved ,whereas the maximum stress unchanged.
Conclusions: Hydrophilic functional groups such as carboxy group and hydroxyl group can be generated on the surface of P3/4HB nonwoven scaffolds by using oxygen plasma surface modification, which implied its tremendous potential use in tissue engineering field.
PartⅢImmobilization of TypeⅠCollagen on P3/4HB Electrospun Scaffolds
Objective: To fabricate and characterize collagen-anchored P3/4HB composite scaffolds.
Methods: Electrospun P3/4HB nonwoven scaffolds were modified by low pressure oxygen plasma treatment, then they were collagen immobilized using the coupling reaction. Physicochemical characteristics of the modified surfaces were investigated by x-ray photoelectron spectrometer (XPS) and the ninhydrin method , the cell proliferation behavior on surface-modified scaffolds were analyzed using myofibroblasts harvested from rats.
Results: XPS spectra confirmed that typeⅠcollagen was immobilized onto the surface of P3/4HB nonwoven scaffolds successfully and the proliferation abilities of myofibroblasts cultured on the modified scaffolds were significantly improved.
Conclusions: It’s an effective method to fabricate collagen-anchored P3/4HB nonwoven scaffolds by immobilizing typeⅠcollagen onto the surface of P3/4HB, which implied its tremendous potential use in tissue engineering field.
目的:运用静电纺丝技术构建P3/4HB无纺支架,探讨支架拓扑结构对肌成纤维细胞生长的影响。
方法:采用静电纺丝技术制备具有不同纤维直径、孔径的微米和亚微米级P3/4HB无纺支架,接种大鼠肌成纤维细胞,体外培养1周后行扫描电镜观察,沉淀法及MTT法分别检测各支架上的细胞黏附及增殖情况。
结果:大鼠肌成纤维细胞在直径为1.24μm,孔径为38.3μm的微米级电纺支架上8小时的黏附率为50.37%,体外培养7天后细胞增殖数为3.54×105个/ml,与直径为0.58μm的亚微米支架上细胞增殖无明显差异,但后者细胞黏附率仅为43.30%,且限制了细胞在支架上的内向性生长,直径为57.8μm的微米支架细胞增殖数均较前二种支架低。
结论:电纺支架的纤维直径和孔径等拓扑结构对肌成纤维细胞的生长具有重要的调节作用,微米电纺支架是一种适合肌成纤维细胞生长的理想三维多孔支架。
第二部分氧等离子体表面改性P3/4HB无纺支架及其对细胞粘附性的影响
目的:研究氧等离子体表面改性对P3/4HB无纺支架的作用。
方法:氧等离子体改性P3/4HB无纺支架,改性的效能通过检测接触角及X射线光电子能谱分析进行结构表征,接种大鼠肌成纤维细胞,检测细胞黏附率以进行功能表征,并检测改性前后支架最大应力变化。
结果:等离子体改性后的P3/4HB无纺支架表面氧含量增加了12.1%,C-O(C-OH)峰、COOR(COOH)峰分别增加0.2%、6.2%,接触角下降了44°,而最大应力无明显变化,改性后支架细胞黏附率较预处理组为高。
结论:氧等离子体处理P3/4HB支架,可在支架表面引入-OH、-COOH等活性基团,改善材料亲水性,提高细胞黏附力,并为进一步共价接枝生物活性单体创造了条件。
第三部分Ⅰ胶原蛋白化学接枝P3/4HB体外构建组织工程复合支架
目的:构建Ⅰ型胶原蛋白-P3/4HB复合支架,并探讨其对细胞生长的影响。
方法:氧等离子体改性P3/4HB无纺支架,引入活性基团,通过化学接枝技术,将Ⅰ型胶原蛋白接枝在P3/4HB无纺支架表面,采用X射线光电子能谱分析及茚三酮法定性、定量检测接枝效果;体外接种大鼠肌成纤维细胞,MTT法检测复合支架的细胞增殖情况以进行功能表征。
结果:电子能谱分析结果显示,接枝后支架表面出现酰胺基的谱峰;复合支架在磷酸盐缓冲液中浸洗48h后,胶原涂层含量无明显变化;与对照组比较,肌成纤维细胞在复合支架上增殖较多。
结论:通过化学缩合反应,可有效制备Ⅰ型胶原蛋白-P3/4HB复合支架,接枝的胶原涂层具有稳定的生物学效应,可显著促进细胞增殖。
PartⅠFabrication of P3/4HB Nonwoven Scaffolds and Effects on the Behavior of Myofibroblasts
Objective: To fabricate P3/4HB nonwoven scaffolds by electrospinning and investigate the effects of fiber diameter and pore size on myofibroblasts cultured on electrospun scaffolds in vitro.
Methods: P3/4HB nonwoven scaffolds with different fiber diameters and pore sizes were fabricated by electrospinning, and then seeded by myofibroblasts harvested from rats. After 1 week’s cultivation, SEM was performed, cell count and MTT test were used to examine cell attachment and proliferation respectively.
Results: Myofibroblasts were adhered well and proliferated on the microfiber scaffold (1.24μm in diameter) ,whereas reduced cell adhesion on the sub-microfiber scaffold (0.58μm in diameter) and poor cell spreading on microfiber scaffold (2.79μm in diameter,)were observed.
Conclusions: Fiber diameter、pore size have influence on cell adhesion ,proliferation and various other cell behaviors. Microfiber electrospun scaffold is an optimal 3D matrix for myofibroblast.
PartⅡOxygen Plasma Surface Modification of P3/4HB Scaffolds and Effects on the Adhesion of Myofibroblasts
Objective: To investigate the effects of oxygen plasma surface modification on electrospun P3/4HB nonwoven scaffolds.
Methods: Electrospun P3/4HB nonwoven scaffolds were modified by low pressure oxygen plasma and physicochemical characteristics of the modified surfaces were investigated by x-ray photoelectron spectrometer, contact angle measurements together with maximum stress measurements, the cell adhesion capability tests on modified scaffolds and serum preconditioned scaffolds were carried out using myofibroblasts harvested from rats.
Results :XPS show the increase in the oxygen content、(C-OH)group and COOR(COOH)group of P3/4HB scaffolds after oxygen plasma surface modification,water contact angle decreased, myofibroblasts attachment were improved ,whereas the maximum stress unchanged.
Conclusions: Hydrophilic functional groups such as carboxy group and hydroxyl group can be generated on the surface of P3/4HB nonwoven scaffolds by using oxygen plasma surface modification, which implied its tremendous potential use in tissue engineering field.
PartⅢImmobilization of TypeⅠCollagen on P3/4HB Electrospun Scaffolds
Objective: To fabricate and characterize collagen-anchored P3/4HB composite scaffolds.
Methods: Electrospun P3/4HB nonwoven scaffolds were modified by low pressure oxygen plasma treatment, then they were collagen immobilized using the coupling reaction. Physicochemical characteristics of the modified surfaces were investigated by x-ray photoelectron spectrometer (XPS) and the ninhydrin method , the cell proliferation behavior on surface-modified scaffolds were analyzed using myofibroblasts harvested from rats.
Results: XPS spectra confirmed that typeⅠcollagen was immobilized onto the surface of P3/4HB nonwoven scaffolds successfully and the proliferation abilities of myofibroblasts cultured on the modified scaffolds were significantly improved.
Conclusions: It’s an effective method to fabricate collagen-anchored P3/4HB nonwoven scaffolds by immobilizing typeⅠcollagen onto the surface of P3/4HB, which implied its tremendous potential use in tissue engineering field.
引文
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13. Sodian, R, et al. Fabrication of a trileaflet heart valve scaffold from a polyhydroxyalkanoate biopolyester for use in tissue engineering. Tissue Eng, 2000. 6(2): p. 183-8.
14.严乐平,吴刚,王迎军,等.聚β-羟基烷酸醋的改性研究进展.化工进展, 2006. 25(11): p. 1266-9.
15.王铁柱,赵强,成国祥,等.聚( 3-羟基丁酸酯)的化学改性研究进展.塑料, 2004. 33(1): p. 48-53.
16. Mitchell, S.A., M.R. Davidson, R.H. Bradley, et al. Improved cellular adhesion to acetone plasma modified polystyrene surfaces. J Colloid Interface Sci, 2005. 281(1): p. 122-9.
17.史嘉玮,董念国,孙宗全,等. RGD肽联合转化生长因子_1对组织工程瓣膜构建的影响.中华医学杂志, 2006. 86(29).
18. Ying, L, et al. Immobilization of galactose ligands on acrylic acid graft-copolymerized poly(ethylene terephthalate) film and its application to hepatocyte culture. Biomacromolecules, 2003. 4(1): p. 157-65.
19. Ro, H.e, et al. Integrins:Verastility,modulation and signaling in cell adhesion. Cell, 1992. 69: p. 15.
20. Masafumi Nakagawa, F.TSH..J.T, et al. Improvement of cell adhesion on poly(L-lactide) by atmospheric plasma treatment. Journal of Biomedical Materials Research Part A, 2006. 77A(1): p. 112-118.
21. Wang, Y, et al. Improvement in hydrophilicity of PHBV films by plasma treatment. J Biomed Mater Res A, 2006. 76(3): p. 589-95.
22. E. Occhiello, M.mgfgh, et al. Oxygen-plasma-treated polypropylene interfaces with air, water, and epoxy resins: Part I. Air and water. Journal of Applied Polymer Science, 1991. 42(2): p. 551-559.
23.任煜,邱夷平,等.低温等离子体对高聚物材料表面改性处理时效性的研究进展.材料导报, 2007. 21(1): p. 56-59.
1. Langer R, V.J. et al. Tissue engineering. Science, 1993: p. 260-920.
2.李世普,等.生物医用材料导论第一版,武汉,武汉工业大学出版社. 2000: p. 302- 303.
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5. Thomas C, F., Abhay Pandit, et al. Living artificial heart valve alternatives: a review. European Cells and Materials, 2003. 6: p. 28-45.
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7. Schoen, F.J., et al. Cardiac valves and valvular pathology Update on function, disease, repair, and replacement. Cardiovascular Pathology, 2005. 14: p. 189-194.
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10. JD, K., et al. Specific regional and directional contractile responses of aortic cusp tissue. J Heart Valve Dis, 2004. 13: p. 798-803.
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22.翟万银,常江,赵强,等组织工程心脏瓣膜研究进展.国外医学生物医学工程分册, 2005. 28(6): p. 340-344.
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24. Steinhoff G, S.U., Karim N, et al. Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduits. Circulation, 2000. 102: p. 1050-55.
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1. Bisson, I, et al. Acrylic acid grafting and collagen immobilization on poly(ethylene terephthalate) surfaces for adherence and growth of human bladder smooth muscle cells. Biomaterials, 2002. 23(15): p. 3149-58.
2. Gupta, B, et al. Plasma-induced graft polymerization of acrylic acid onto poly(ethylene terephthalate) films: characterization and human smooth muscle cell growth on grafted films. Biomaterials, 2002. 23(3): p. 863-71.
3. Tyan, Y.C, et al. Assessment and characterization of degradation effect for the varied degrees of ultra-violet radiation onto the collagen-bonded polypropylene non-woven fabric surfaces. Biomaterials, 2002. 23(1): p. 65-76.
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6. Zuwei Ma, C.G. J.S, et al. Paraffin spheres as porogen to fabricate poly(L-lactic acid) scaffolds with improved cytocompatibility for cartilage tissue engineering. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2003. 67B(1): p. 610-617.
7. Dewez, J.-L, et al. Competitive adsorption of proteins: Key of the relationship between substratum surface properties and adhesion of epithelial cells. Biomaterials, 1999. 20(6): p. 547-559.
8.董念国,史嘉玮,胡平,等.组织工程心脏瓣膜支架的研究现状和发展趋势.中华实验外科杂志, 2007. 24(3): p. 2.
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11. Wilson GJ, Y.H., Klement P, et al. Acellular matrix allograft small caliber vascular prostheses. ASAIO Trans, 1990. 36(3): p. 340-3.
12.王碧,叶勇,程劲,等.胶原蛋白制备生物医学材料的特征及改性方法.化学世界, 2003(11): p. 606-610.
13. Sodian, R, et al. Fabrication of a trileaflet heart valve scaffold from a polyhydroxyalkanoate biopolyester for use in tissue engineering. Tissue Eng, 2000. 6(2): p. 183-8.
14.严乐平,吴刚,王迎军,等.聚β-羟基烷酸醋的改性研究进展.化工进展, 2006. 25(11): p. 1266-9.
15.王铁柱,赵强,成国祥,等.聚( 3-羟基丁酸酯)的化学改性研究进展.塑料, 2004. 33(1): p. 48-53.
16. Mitchell, S.A., M.R. Davidson, R.H. Bradley, et al. Improved cellular adhesion to acetone plasma modified polystyrene surfaces. J Colloid Interface Sci, 2005. 281(1): p. 122-9.
17.史嘉玮,董念国,孙宗全,等. RGD肽联合转化生长因子_1对组织工程瓣膜构建的影响.中华医学杂志, 2006. 86(29).
18. Ying, L, et al. Immobilization of galactose ligands on acrylic acid graft-copolymerized poly(ethylene terephthalate) film and its application to hepatocyte culture. Biomacromolecules, 2003. 4(1): p. 157-65.
19. Ro, H.e, et al. Integrins:Verastility,modulation and signaling in cell adhesion. Cell, 1992. 69: p. 15.
20. Masafumi Nakagawa, F.TSH..J.T, et al. Improvement of cell adhesion on poly(L-lactide) by atmospheric plasma treatment. Journal of Biomedical Materials Research Part A, 2006. 77A(1): p. 112-118.
21. Wang, Y, et al. Improvement in hydrophilicity of PHBV films by plasma treatment. J Biomed Mater Res A, 2006. 76(3): p. 589-95.
22. E. Occhiello, M.mgfgh, et al. Oxygen-plasma-treated polypropylene interfaces with air, water, and epoxy resins: Part I. Air and water. Journal of Applied Polymer Science, 1991. 42(2): p. 551-559.
23.任煜,邱夷平,等.低温等离子体对高聚物材料表面改性处理时效性的研究进展.材料导报, 2007. 21(1): p. 56-59.
1. Langer R, V.J. et al. Tissue engineering. Science, 1993: p. 260-920.
2.李世普,等.生物医用材料导论第一版,武汉,武汉工业大学出版社. 2000: p. 302- 303.
3. Terada S, S.M., Sevy A, et al. Tissue engineering in the twenty-first century. Yonsei Med J, 2000(14): p. 685-691.
4. D.W, H., et al. Scaffolds in Tissue Engineering Bone and Cartilage. Biomaterials, 2000. 21: p. 2529-43.
5. Thomas C, F., Abhay Pandit, et al. Living artificial heart valve alternatives: a review. European Cells and Materials, 2003. 6: p. 28-45.
6. Vesely, I., et al. Heart Valve Tissue Engineering. Circulation Research, 2005. 95: p. 743-755.
7. Schoen, F.J., et al. Cardiac valves and valvular pathology Update on function, disease, repair, and replacement. Cardiovascular Pathology, 2005. 14: p. 189-194.
8. Mendelson, K.M.F.J.S.K., et al. Heart Valve Tissue Engineering: Concepts, Approaches, Progress, and Challenges. Annals of Biomedical Engineering 2006. 34(12): p. 1799-1819.
9. Takkenberg, M.Y.J., et al. Will heart valve tissue engineering change the world? Nature Clinical Practice Cardiovascular Medicine 2004. 2(2): p. 60-61.
10. JD, K., et al. Specific regional and directional contractile responses of aortic cusp tissue. J Heart Valve Dis, 2004. 13: p. 798-803.
11. D?rthe Schmidt, S.P.H., et al. Tissue engineered heart valves based on human cells. SWISS MED WKLY, 2005. 135: p. 618-623.
12. P,Hoerslrup, S.N.S., et al. Heart valve tissue engineering. Transplant Immunology 2004. 12: p. 359-365.
13. Shinoka T, B.C., Tanel RE, et al. Tissue engineering heart valves: valve leaflet replacement study in a lamb model. Ann Thorac Surg, 1995. 60(6): p. 513-516.
14. Shinoka T, M.X., et al.Tissue engineered heart valve autologous valve leaflet replacement study in a lamb model Circulation, 1996. 94: p. 164-168.
15. Kim WG, C.S., Kang MC, et al. Tissue-engineered heart valve leaflets: an animal study. Int J Artif Organs, 2001. 24(9): p. 642-8.
16. Sodian, R., et al.Fabrication of a trileaflet heart valve scaffold from a polyhydroxyalkanoate biopolyester for use in tissue engineering. Tissue Eng, 2000. 6(2): p. 183-8.
17. Ralf Sodian, S.P.H., Jason S Sperling, et al. Tissue engineering of heart valves: in vitro experiences. The Annals of Thoracic Surgery, 2000. 70(1): p. 140-144.
18. Taylor PM, A.S., Dreger SA, Yacoub MH, et al. Human cardiac valve interstitial cells in collagen sponge: a biological three-dimensional matrix for tissue engineering J Heart Valve Dis, 2002. 11(3): p. 298-306.
19. Kofidis T, A.P., Wachsmann B, et al. A novel bioartificial myocardial tissue and its prospective use in cardiac surgery. Eur J Cardiothorac Surg, 2002. 22(2): p. 238-243.
20. Ye Q, Z.G., Benedikt P, et al. Fibrin gel as a three dimensional matrix in cardiovascular tissueengineering. Eur J Cardiothorac Surg 2000. 17(5): p. 587-591.
21. Augustinus Bader, T.S., et al. Tissue engineering of heart valves– human endothelial cell seeding of detergent acellularized porcine valves. Eur J Cardiothorac Surg, 1999. 14: p. 279-284.
22.翟万银,常江,赵强,等组织工程心脏瓣膜研究进展.国外医学生物医学工程分册, 2005. 28(6): p. 340-344.
23. Cebotari S, M.H., Kallenback K, et al. Construction of autologous human heart valves based on an acellular allograft matrix. Circulation, 2002. 106: p. 63-68.
24. Steinhoff G, S.U., Karim N, et al. Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduits. Circulation, 2000. 102: p. 1050-55.
25. Dohmen PM, L.A., Hotz H, et al. Ross operation with a tissueengineered heart valves. Ann Thorac Surg, 2002. 74: p. 1438-1442.
26. Ralf Sodian, C.L., et al. Tissue Engineering of Autologous Human Heart Valves Using Cryopreserved Vascular Umbilical Cord Cells. Ann Thorac Surg 2006. 81: p. 2207-16.
27.顾其胜,侯春林,徐政,等实用生物医用材料学.上海科学技术出版社, 2005.
28. Niels Grabow, K.S., et al. Mechanical and Structural Properties of a Novel Hybrid Heart Valve Scaffold for Tissue Engineering. Artificial Organs 2004. 28(11): p. 971–979
29. Christof Stamm, A.K., et al. Biomatrix/Polymer Composite Material for Heart Valve Tissue Engineering. Ann Thorac Surg, 2004. 78: p. 2084-93.
30.史嘉玮,董念国,孙宗全,等RGD肽联合转化生长因子_1对组织工程瓣膜构建的影响.中华医学杂志, 2006. 86(29).