一种组织工程化培养生物反应器的研究
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摘要
本文根据国内外组织工程化培养生物反应器研究现状，利用有限元方法分析了旋转壁式生物反应器内部流场情况和剪应力分布情况，探讨了生物反应器与培养物之间的传质过程以及脉冲压力对传质能力的影响。并在计算机仿真和理论分析的基础上，研制了一种具有内循环的灌流旋转壁式生物反应器，对其性能进行了相关实验和检测。
通过理论分析给出了旋转壁式生物反应器中培养物受到剪应力以及流动轨迹的矢量图。建立了旋转壁式生物反应器内部流场的有限元模型，使用ANSYS有限元分析软件计算了培养物表面剪应力大小和分布，描绘了生物反应器空间流场的速度矢量云图，并与理论分析比较，结果取得了较好的一致性。
通过对生物反应器与培养物之间传质过程的分析，得出了影响传质能力的关键因素，研究表明适当脉冲压力对传质能力有促进作用，并可以用脉冲压力实现高传质能力生物反应器的设计。
基于理论分析和有限元仿真，设计了一台具有内循环的、在线供气供液的并带有脉冲加压装置的旋转壁式生物反应器系统。生物反应器性能测试实验的结果表明：在生物反应器中培养14天后的细胞／羟基磷灰石支架材料以及细胞／松质骨支架材料，其细胞的增殖和形态都明显优于在5％CO_2孵箱中培养的同类材料。
Based on the development of the bioreactor used to the culture of tissue engineering in the nation and the overseas, this thesis analyzed the ulterior fluid field and the shearing stress distribution of the rotating wall bioreactor by using the finite element method, discussed the mass transfer process between the bioreactor and the culture and the infection of the impulse press. Based on the computer simulation and the theoretically analysis, we have designed and manufactured a rotating wall perfusion bioreactor system with interior circulation, and tested its capability with correlative experimentations.
Through the theoretically analysis the vector figures of the shearing stress and the moving track of the culture within the rotating wall bioreactor have been given out. We also built a finite element model about the rotating wall bioreactor interior fluid field, calculated the shearing stress' values and the distribution status of the cultures, described the fluid velocity vector figure of the bioreactor space, and compared with the theoretically analysis results, the research results were primely consistent with them.
We also analyzed the mass transfer process between the bioreactor and the culture, found some key factors which evidently affected the ability of the mass transfer. We proved that proper impulse press could enhance the mass transfer, and we could design a kind of high mass transfer bioreactor by the impulse press.
Based on the theoretically analysis and the finite element simulation, we have designed a rotating wall bioreactor system with interior circle, offering gas and medium on-line and impulse press device. This bioreactor can offer a culture space about 700ml. The bioreactor can culture micro-carrier and cell/tissue scaffold.
The testing results of the bioreactor system indicated that our bioreactor has a better ability to culture the scaffold with cells: hydroxy-phosphorite scaffolds and cancellous bone with cells which cultured our bioreactor system proliferated very markedly; cells cultured in it about 14 days have better modality than cultured in the 5%
Researching of the bioreactor used to the culture of tissue engineering _ Abstract
incubater.
通过理论分析给出了旋转壁式生物反应器中培养物受到剪应力以及流动轨迹的矢量图。建立了旋转壁式生物反应器内部流场的有限元模型，使用ANSYS有限元分析软件计算了培养物表面剪应力大小和分布，描绘了生物反应器空间流场的速度矢量云图，并与理论分析比较，结果取得了较好的一致性。
通过对生物反应器与培养物之间传质过程的分析，得出了影响传质能力的关键因素，研究表明适当脉冲压力对传质能力有促进作用，并可以用脉冲压力实现高传质能力生物反应器的设计。
基于理论分析和有限元仿真，设计了一台具有内循环的、在线供气供液的并带有脉冲加压装置的旋转壁式生物反应器系统。生物反应器性能测试实验的结果表明：在生物反应器中培养14天后的细胞／羟基磷灰石支架材料以及细胞／松质骨支架材料，其细胞的增殖和形态都明显优于在5％CO_2孵箱中培养的同类材料。
Based on the development of the bioreactor used to the culture of tissue engineering in the nation and the overseas, this thesis analyzed the ulterior fluid field and the shearing stress distribution of the rotating wall bioreactor by using the finite element method, discussed the mass transfer process between the bioreactor and the culture and the infection of the impulse press. Based on the computer simulation and the theoretically analysis, we have designed and manufactured a rotating wall perfusion bioreactor system with interior circulation, and tested its capability with correlative experimentations.
Through the theoretically analysis the vector figures of the shearing stress and the moving track of the culture within the rotating wall bioreactor have been given out. We also built a finite element model about the rotating wall bioreactor interior fluid field, calculated the shearing stress' values and the distribution status of the cultures, described the fluid velocity vector figure of the bioreactor space, and compared with the theoretically analysis results, the research results were primely consistent with them.
We also analyzed the mass transfer process between the bioreactor and the culture, found some key factors which evidently affected the ability of the mass transfer. We proved that proper impulse press could enhance the mass transfer, and we could design a kind of high mass transfer bioreactor by the impulse press.
Based on the theoretically analysis and the finite element simulation, we have designed a rotating wall bioreactor system with interior circle, offering gas and medium on-line and impulse press device. This bioreactor can offer a culture space about 700ml. The bioreactor can culture micro-carrier and cell/tissue scaffold.
The testing results of the bioreactor system indicated that our bioreactor has a better ability to culture the scaffold with cells: hydroxy-phosphorite scaffolds and cancellous bone with cells which cultured our bioreactor system proliferated very markedly; cells cultured in it about 14 days have better modality than cultured in the 5%
Researching of the bioreactor used to the culture of tissue engineering _ Abstract
incubater.
引文
[1] Langer R, Vacanti J P. Tissue engineering. Science, 1993, 260: 920～926.
[2] Lysaght M T, Solliven K F, Tsui J H. An economic survey of the tissue engineering industry. Proceed Inter Symp Control RelBioact Mater., Controlled Release Society Ine, Deerfield, 1998, 25: 107～108.
[3] 潘君，王远亮，唐丽灵等．组织工程研究进展．国外医学生物医学工程分册，2000，23(5)：257～261．
[4] Vacanti CA, Cima LG, Ratkowski D, et al. Tissue engineered growth of new cartilage in the shape of a human ear using syntbetic polymers seeded with chondrocytes. Mat Res Sco Symp Proc. 1992, 252: 367～374.
[5] Puelacher WC, Vacanti JP, Kim SW. Fabdcation of nasal implants using haman shape-specific polymer scaffolds seeded with chondrocytes. An Coll Surgeons Surg Forum, 1993, 44: 678～680.
[6] Kim B S, Mooney D J. Development of biocompatabile synthetic extracellular matrices for tissue engineering. Trends in Biotech, 1998, 16: 224～225.
[7] 姚康德，尹玉姬．组织工程相关生物材料．化学工业出版社，2003．
[8] 张元兴，许学书．生物反应器工程．第一版．上海．华东理工大学出版社，2001，266～292
[9] Cherry RS, Elefthedos TP. Physical mechanisms of cell damage in microcarrier cell culture bioreactors. BioteehnoI and Bioeng, 1988, 32: 1001～1014.
[10] Goodwin TJ, Prewett TL, Wolf DA, et al. Reduce shear stress: a major copmonent in the ability of mammalian tissues to form three-dimensional assemblies in stimulated microgravity. J Cell Biochem, 1993, 51: 301～311.
[11] Cynthia M. Begley. The Fluid Dynamic and Shear Environment in the NASA/JSC Rotating-Wall Perfused-Vessel Bioreactor. Biotechnol and Bioeng, 2000, 70: 32～40
[12] Smith PL, Rusk SF, Ellison BE. et al. Invitro stimulation of articular chondrocyte mRNA and extmcellar matrix synthesis by hydrostaticpressure. J. Orthop Res, 1996, 14: 53.
[13] Freed E, Robert L, Ivan M, et al. Tissue engineering if cartilage in space. Medical Sciences, 1997; 94: 13885～13890.
[14] 徐小增等 1996．实用新型细胞培养生物反应器．CN 2236493Y
[15] Kamen A. A, Charaie C, Andre G, et al. Design parameters and performance of a suface baffled helical ribbon impeller bioreactor for the culture of shear sensitive cells. Chemical Engineering Science, 1992, 47(9-11): 2375～2380.
[16] Emilio Molina Grima. Characterizayion of shear rates in airlift bioreactors for animal cellculture. Biotechnology, 1997, 54: 195～210.
[17] 王斯靖，陈因良，潘厚昌．动物细胞培养反应器氧传递率的研究．生物工程学报，1993，9(1)：16～20．
[18] 张元兴，陈因良．动物细胞培养反应器的流体循环和氧传递规律．华东化工学院学报，1989，15(4)：504～508．
[19] 欧阳平凯，陆永明，肖民耕等．气升式生物反应器的混合性能研究．生物工程进展，1992，12(5)：9～13．
[20] Ku K, Kuo MJ, Delente J, et al. Development of a hollow-fiber system for large-scale culture of mammalian cells. Biotechnol and Bioeng, 1981, 23: 79～95.
[21] Karel SF, Libicki SB, Robertson CR. The immobilization of whole cells. Chem Eng Sci, 1985, 40: 1321～1354.
[22] Piret JM, Cooney CL. Model of oxygen transport limitations in hollow fiber bioreactors. Biotechnol and Bioeng, 1991, 37: 80～92.
[23] Efthymiou GS, Shuler ML. Elimination of diffusional limitation in a membrance entrapped cell reactor. Biotechnol and Bioeng, 1999, 62: 183～192.
[24] ERIC M. DARLING, B. S., and KYRIACOS A. ATHANASIOU, Ph. D., P. E. Ariticular Cartilage Bioreactors and Bioprosesses. Tissue engineering, 2003, 9(1): 9～26.
[25] Isabelle Walther. Development of a miniature bioreactor for continuous culture in a space laboratory. Biotechnology, 1994, 38: 21～32
[26] Obradovic B, Rebecca LC, Vunjak-Novakovic G. Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage. Biotechnol and Bioeng, 1999, 63(2): 198～205.
[27] Lisa. E. Freed. Tissue engineering of cartilage in space. Proc. Natl. Acad Sci. USA 1997, 94: 13885～13890
[28] 陶祖莱等．在线供气、供液空心旋转圆盘式细胞三维培养器．2000，专利号
CN2398289Y.
[29] H. Gao. Surface Transformation of Bioactive Glass in Bioreactors Simulating Microgravity Conditions. Part one: Experimental Study. Biotechnol and Bioeng, 2001, 75: 369～378.
[30] S. J. Kleis. A Viscous Pump Bioreactor. Biotechnol and Bioeng, 1990, 36: 771～777.
[31] Muzzio FJ, Unger DR, Liu M, et al. Computational and experimental investigation of flow and particle settling in a roller bottle bioreactor. Biotechnol and Bioeng, 1999, 63(2): 185～196.
[32] 吴望一．流体力学(下册)．北京大学出版社，1983．
[33] 刘涛，杨凤鹏．精通ANSYS．清华大学出版社，2001．
[34] [美]J 金克普利斯．传递过程与单元操作，北京：清华大学出版社，1985．
[35] 丁力，袁修干，赵玉珍．生物皮肤传质的研究．北京航空航天大学学报，2001，27(2)：171～174．
[36] 张元兴，徐学书．生物反应器工程，上海：华东理工大学出版社，2001．
[37] 李琳，丁富新，袁乃驹．微孔膜气体分布器对气液传质的影响．高校化工工程学报，1992，6(2)：147～152．
[38] 黄廷章，刘福海，杨正明．复杂化学流体在多孔介质中的传质．力学学报，2002，34(2)：256～260．
[39] 雷晓晓，吴望一，温功碧等．恶性肿瘤的传质问题(Ⅰ)-流体动力学部分．应用数学和力学，1998，19(11)：947～952．
[40] 雷晓晓，吴望一，温功碧等．恶性肿瘤的传质问题(Ⅱ)-药物传输部分．应用数学和力学，1998，19(12)：1063～1070．
[41] 吴望一，是长春，王露．毛细血管-组织间流体交换的双重介质模型．力学学报 1989，21(6)：649～656．
[42] Shea. L. D., D. Wang, R. T. Franceschs, et al. Engineered bone development from a pre-osteoblast cell line on the three-dimensional scaffolds. Tissue Engineering, 2000, 6(6): 605～617.
[42]．王斯靖等．CellCul-20A反应器的丝网间液体交换速率及氧传递模型．生物工程学报．1993，9(4)：372～378．
[43] 杨汝清．现代机械设计——系统与结构．上海科学技术文献出版社，2000．
[44] Schwarz, Ray P. Gas permeable bioreactor and method of use. 1997, United States Patent: 5702941.
[45] Masako, Geert. The fluid shear stress distribution on the membrane of leukocytes in the microcireulation. 2003, Journal of Biomechanical Engineering, 2003, 125: 628～638.
[46] 朱文箐，金慰芳，张丽丽等．MTT法分析培养成骨细胞的存活和增殖能力。上海医科大学学报，1995，22(4)：254．
[47] Stein GS, Lian JB. Molecular mechanisms mediating proliferation differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev, 1993, 14: 424-442.
[48] Sugihara-seki, M.. Flow around cells adhered to a microvessel wall. Ⅰ. fluid stresses and forces acting on the sells. biorheology, 2000, 37: 341～359.
[49] Sugihara-seki, M.. Flow around cells adhered to a microvessel wall. Ⅱ. comparison to flow around adherent cells in channel flow. biorheology, 2001, 38: 3～13
[50] P Prendergast, D Kelly. The response of cells to mechanical loading: a finite element analysis. Biomech., 2001. 30: 539～548.
[51] Brown, T. D. Techniques for mechanical stimulation of cells in vitro: a review. Biomech., 2000, 33: 3～11.
[52] Hutmacher, D. W. Scaffolds in tissue engineering bone and cartilage. Biomaterials, 2000, 21: 2529～2540.
[53] Hansen, U., Schunke, M., Domm, et al. Combination of reduced oxygen tension and intermittent hydrostatic pressure: a useful tool in articular cartilage tissue engineering. Biomech., 2001, 34: 941～955.
[54] Parkkinen, J., Ikonen, J., Lammi, M., et al. Effects of cyclic hydrostatic pressure on proteoglycan synthesis in cultured chondrocytes and articular cartilage explants. Arch. Biochem. Biophys, 1993, 50: 430～451.
[2] Lysaght M T, Solliven K F, Tsui J H. An economic survey of the tissue engineering industry. Proceed Inter Symp Control RelBioact Mater., Controlled Release Society Ine, Deerfield, 1998, 25: 107～108.
[3] 潘君，王远亮，唐丽灵等．组织工程研究进展．国外医学生物医学工程分册，2000，23(5)：257～261．
[4] Vacanti CA, Cima LG, Ratkowski D, et al. Tissue engineered growth of new cartilage in the shape of a human ear using syntbetic polymers seeded with chondrocytes. Mat Res Sco Symp Proc. 1992, 252: 367～374.
[5] Puelacher WC, Vacanti JP, Kim SW. Fabdcation of nasal implants using haman shape-specific polymer scaffolds seeded with chondrocytes. An Coll Surgeons Surg Forum, 1993, 44: 678～680.
[6] Kim B S, Mooney D J. Development of biocompatabile synthetic extracellular matrices for tissue engineering. Trends in Biotech, 1998, 16: 224～225.
[7] 姚康德，尹玉姬．组织工程相关生物材料．化学工业出版社，2003．
[8] 张元兴，许学书．生物反应器工程．第一版．上海．华东理工大学出版社，2001，266～292
[9] Cherry RS, Elefthedos TP. Physical mechanisms of cell damage in microcarrier cell culture bioreactors. BioteehnoI and Bioeng, 1988, 32: 1001～1014.
[10] Goodwin TJ, Prewett TL, Wolf DA, et al. Reduce shear stress: a major copmonent in the ability of mammalian tissues to form three-dimensional assemblies in stimulated microgravity. J Cell Biochem, 1993, 51: 301～311.
[11] Cynthia M. Begley. The Fluid Dynamic and Shear Environment in the NASA/JSC Rotating-Wall Perfused-Vessel Bioreactor. Biotechnol and Bioeng, 2000, 70: 32～40
[12] Smith PL, Rusk SF, Ellison BE. et al. Invitro stimulation of articular chondrocyte mRNA and extmcellar matrix synthesis by hydrostaticpressure. J. Orthop Res, 1996, 14: 53.
[13] Freed E, Robert L, Ivan M, et al. Tissue engineering if cartilage in space. Medical Sciences, 1997; 94: 13885～13890.
[14] 徐小增等 1996．实用新型细胞培养生物反应器．CN 2236493Y
[15] Kamen A. A, Charaie C, Andre G, et al. Design parameters and performance of a suface baffled helical ribbon impeller bioreactor for the culture of shear sensitive cells. Chemical Engineering Science, 1992, 47(9-11): 2375～2380.
[16] Emilio Molina Grima. Characterizayion of shear rates in airlift bioreactors for animal cellculture. Biotechnology, 1997, 54: 195～210.
[17] 王斯靖，陈因良，潘厚昌．动物细胞培养反应器氧传递率的研究．生物工程学报，1993，9(1)：16～20．
[18] 张元兴，陈因良．动物细胞培养反应器的流体循环和氧传递规律．华东化工学院学报，1989，15(4)：504～508．
[19] 欧阳平凯，陆永明，肖民耕等．气升式生物反应器的混合性能研究．生物工程进展，1992，12(5)：9～13．
[20] Ku K, Kuo MJ, Delente J, et al. Development of a hollow-fiber system for large-scale culture of mammalian cells. Biotechnol and Bioeng, 1981, 23: 79～95.
[21] Karel SF, Libicki SB, Robertson CR. The immobilization of whole cells. Chem Eng Sci, 1985, 40: 1321～1354.
[22] Piret JM, Cooney CL. Model of oxygen transport limitations in hollow fiber bioreactors. Biotechnol and Bioeng, 1991, 37: 80～92.
[23] Efthymiou GS, Shuler ML. Elimination of diffusional limitation in a membrance entrapped cell reactor. Biotechnol and Bioeng, 1999, 62: 183～192.
[24] ERIC M. DARLING, B. S., and KYRIACOS A. ATHANASIOU, Ph. D., P. E. Ariticular Cartilage Bioreactors and Bioprosesses. Tissue engineering, 2003, 9(1): 9～26.
[25] Isabelle Walther. Development of a miniature bioreactor for continuous culture in a space laboratory. Biotechnology, 1994, 38: 21～32
[26] Obradovic B, Rebecca LC, Vunjak-Novakovic G. Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage. Biotechnol and Bioeng, 1999, 63(2): 198～205.
[27] Lisa. E. Freed. Tissue engineering of cartilage in space. Proc. Natl. Acad Sci. USA 1997, 94: 13885～13890
[28] 陶祖莱等．在线供气、供液空心旋转圆盘式细胞三维培养器．2000，专利号
CN2398289Y.
[29] H. Gao. Surface Transformation of Bioactive Glass in Bioreactors Simulating Microgravity Conditions. Part one: Experimental Study. Biotechnol and Bioeng, 2001, 75: 369～378.
[30] S. J. Kleis. A Viscous Pump Bioreactor. Biotechnol and Bioeng, 1990, 36: 771～777.
[31] Muzzio FJ, Unger DR, Liu M, et al. Computational and experimental investigation of flow and particle settling in a roller bottle bioreactor. Biotechnol and Bioeng, 1999, 63(2): 185～196.
[32] 吴望一．流体力学(下册)．北京大学出版社，1983．
[33] 刘涛，杨凤鹏．精通ANSYS．清华大学出版社，2001．
[34] [美]J 金克普利斯．传递过程与单元操作，北京：清华大学出版社，1985．
[35] 丁力，袁修干，赵玉珍．生物皮肤传质的研究．北京航空航天大学学报，2001，27(2)：171～174．
[36] 张元兴，徐学书．生物反应器工程，上海：华东理工大学出版社，2001．
[37] 李琳，丁富新，袁乃驹．微孔膜气体分布器对气液传质的影响．高校化工工程学报，1992，6(2)：147～152．
[38] 黄廷章，刘福海，杨正明．复杂化学流体在多孔介质中的传质．力学学报，2002，34(2)：256～260．
[39] 雷晓晓，吴望一，温功碧等．恶性肿瘤的传质问题(Ⅰ)-流体动力学部分．应用数学和力学，1998，19(11)：947～952．
[40] 雷晓晓，吴望一，温功碧等．恶性肿瘤的传质问题(Ⅱ)-药物传输部分．应用数学和力学，1998，19(12)：1063～1070．
[41] 吴望一，是长春，王露．毛细血管-组织间流体交换的双重介质模型．力学学报 1989，21(6)：649～656．
[42] Shea. L. D., D. Wang, R. T. Franceschs, et al. Engineered bone development from a pre-osteoblast cell line on the three-dimensional scaffolds. Tissue Engineering, 2000, 6(6): 605～617.
[42]．王斯靖等．CellCul-20A反应器的丝网间液体交换速率及氧传递模型．生物工程学报．1993，9(4)：372～378．
[43] 杨汝清．现代机械设计——系统与结构．上海科学技术文献出版社，2000．
[44] Schwarz, Ray P. Gas permeable bioreactor and method of use. 1997, United States Patent: 5702941.
[45] Masako, Geert. The fluid shear stress distribution on the membrane of leukocytes in the microcireulation. 2003, Journal of Biomechanical Engineering, 2003, 125: 628～638.
[46] 朱文箐，金慰芳，张丽丽等．MTT法分析培养成骨细胞的存活和增殖能力。上海医科大学学报，1995，22(4)：254．
[47] Stein GS, Lian JB. Molecular mechanisms mediating proliferation differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev, 1993, 14: 424-442.
[48] Sugihara-seki, M.. Flow around cells adhered to a microvessel wall. Ⅰ. fluid stresses and forces acting on the sells. biorheology, 2000, 37: 341～359.
[49] Sugihara-seki, M.. Flow around cells adhered to a microvessel wall. Ⅱ. comparison to flow around adherent cells in channel flow. biorheology, 2001, 38: 3～13
[50] P Prendergast, D Kelly. The response of cells to mechanical loading: a finite element analysis. Biomech., 2001. 30: 539～548.
[51] Brown, T. D. Techniques for mechanical stimulation of cells in vitro: a review. Biomech., 2000, 33: 3～11.
[52] Hutmacher, D. W. Scaffolds in tissue engineering bone and cartilage. Biomaterials, 2000, 21: 2529～2540.
[53] Hansen, U., Schunke, M., Domm, et al. Combination of reduced oxygen tension and intermittent hydrostatic pressure: a useful tool in articular cartilage tissue engineering. Biomech., 2001, 34: 941～955.
[54] Parkkinen, J., Ikonen, J., Lammi, M., et al. Effects of cyclic hydrostatic pressure on proteoglycan synthesis in cultured chondrocytes and articular cartilage explants. Arch. Biochem. Biophys, 1993, 50: 430～451.