组织工程真皮动态三维应变培养装置和抗张强度检测装置的研制
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摘要
如何促进和加速伤口的愈合,减少疤痕形成,是人们历来关注的重要课题,尤其是大面积烧伤时自体皮源不足的情况如何及时覆盖创面,是临床一个急需解决的重要问题,皮肤组织工程为解决这一问题带来了新的希望,组织工程皮肤为伤口的愈合和修复提供了一种好的可选方案,在临床上有着巨大需求。现有的组织工程皮肤如已商品化的Apligraft等双层胶原凝胶类产品已具有真表皮双层结构,但均缺乏完整的基底膜,真表皮结合力不强,易于分离,弹性差,易脆,力学性能还未达到令人满意的程度,难以满足移植手术的操作要求,这已成为皮肤组织工程急需解决的关键技术问题。
力学生物学(mechanobiology)是诞生于上世纪90年代的一门新兴交叉学科,为提高组织工程产品力学性能提供了新手段。研究内容主要有力学刺激信号、细胞对力学刺激信号的感受及转导机制和细胞受力后的反应三部分内容。众多研究表明,流体剪切力、静水压、负压吸引、拉伸应变及压缩应变等机械外力对细胞的增殖、分化、基因表达及蛋白合成有显著的影响。而组织工程皮肤可以作为力学生物学研究的良好模型,可研究机械外力对其物理特性以及细胞生物学功能的影响。不同组织细胞对应变大小的承受力有巨大的差异,受到的机械力作用方式也各不相同,研究的关键技术是研制适合不同组织的施加机械力的装置,并探索合适的应变大小、频率及加载时间。力学生物学研究的主要模式是根据所研究的细胞在体所受外力作用形式及应变大小来设计相应的施加机械力装置,利用装置对所研究细胞施加机械力,研究细胞对力学信号感受、转导机制及受力后的反应,从而在细胞及分子水平探索外力对机体的影响。因此本研究从研制施加机械力培养装置和检测力学性能两个方面展开,为组织工程皮肤力学生物学进一步研究提供条件。
目的:
1.研制能够稳定、有效地模拟皮肤在体受力情况的组织工程真皮动态三维应变培养装置。
2.验证建立的组织工程真皮动态三维应变培养装置的有效性。
3.为了测定动态三维应变培养后组织工程皮肤的物理特性,对本实验室与重庆大学联合研制的高敏度小张力膜状生物材料力学检测装置进行改进和完善。
方法:
1.设计制作采用周期性流动培养基驱动的组织工程真皮动态三维应变培养装置。
2.采用本实验室常规方法制作的复方壳多糖组织工程真皮,分别观察常规静态培养及动态三维应变培养下细胞分布、增殖、细胞外基质合成情况。
3.将本实验室研制的高敏度小张力膜状生物材料力学性能检测装置的加载系统改为气体加载系统、夹具升级为夹持精确、可靠且可视的夹持系统,更换为高精度传感器,进行准确度及稳定性测试。
结果:
1.研制的周期性流动培养基驱动的组织工程真皮动态三维应变培养装置能满足组织工程真皮动态三维应变培养要求。
2.初步结果显示动态三维应变培养后组织工程真皮中细胞分布均匀、有序,与受力方向一致;细胞增殖更快,I型胶原合成增多,证明该装置能够模拟皮肤在体力学刺激条件,为下一步研究组织工程皮肤力学生物学提供了重要条件。
3.改进后的高敏度小张力膜状生物材料力学性能检测装置外形小巧、夹持可靠、试件变化可视、操作方便、快捷,且仪器具有良好的准确度及稳定性,为组织工程产品力学性能检测提供了有效检测手段。
结论:
成功研制了组织工程真皮动态三维应变培养装置和抗张强度检测装置,可应用于组织工程皮肤力学生物学的进一步研究,为下一步的研究铺平了道路。
Background:
It is an important issue that how to promote and accelerate wound healing in order to reduce scar formation, while people have always been concerned about, especially when there is insufficient donor skin in extensive burns, how to cover wound in time is an urgent clinical problem which need to be addressed. Skin tissue engineering has brought new hope to cope with this challenge, tissue-engineered skin provides a good option for wound healing and repair and has a huge clinical demand. Current tissue-engineered skin products such as Apligraft and other commercial collagen gel tissue-engineered skin have epidermis and dermis structure, but lack of complete basement membrane, the binding of epidermis and dermis is not strong, their flexibility is poor, and fragile,have not yet reached satisfactory mechanical properties for the transplant operation.These drawback has become an key technical problem that need to be solved urgently for tissue-engineered skin.
Mechanobiology provides a new way to improve the mechanical properties of tissue engineering. It is a new interdisciplinary subject that born in 90’s of the last century. The contents mainly include mechanical stimulation signal, cells feeling the mechanical stimulation signal, force transduction mechanisms and cellular response to mechanical stimulation. A lot of studies have shown that fluid shear stress, hydrostatic pressure, negative pressure, tensile strain, compression strain and other mechanical external forces have significant impact on cell proliferation and differentiation, gene expression and protein synthesis.
The reaction of different tissue cells to mechanical strain showed huge different amplitude, and the type of mechanical force is different to every kind tissue. The key technology of mechanobiology research is to develop the device to impose mechanical force for different organizations and cells, and to explore appropriate strain amplitude, * Supported by National High Technology Research and Development Program of China(863 Program) (2006AA02A121) frequency and processing time. The main mode of mechanobiology research is to develop the sutiable device which based on the mechanical force type and strain amplitude in vivo, then impose mechanical force to cells in vitro with the devices, to study how the cells feel mechanical signal, the mechanical force transduction mechanism and the cellular response to mechanical force, thus to research the influence of mechanical force to body on cellular and molecular level.
Objectives:
1. To establish a stable and effective dynamic three-dimensional stain culture equipment for culturing tissue-engineered dermis which can simulate the mechanical force effect to the skin in vivo.
2. To confirm the effectiveness of the established dynamic three-dimensional strain culture equipment for tissue-engineered dermis.
3. To improve the high-sensitivity mechanical properties testing device for small tension membranaceous biomaterials manufactured by our laboratory and Chongqing University.
Methods:
1. Designed and made dynamic three-dimensional strain culture equipment for tissue-engineered dermis that drived by the cyclical media flow.
2. Observed the cell distribution and viability, extracellular matrix synthesis and mechanical properties of composite chitosan tissue-engineered dermis under our laboratory conventional methods and dynamic three-dimensional culture.
3. Loading system was replaced by airy pressure loading system; the clamp was upgraded to clamping accurately, reliable and visual clamping system; the sensor was replaced by a high-precision sensor, tested accuracy and stability.
Results:
1. Developed dynamic three-dimensional strain culture equipment for tissue- engineered dermis which was drived by cyclical media flow.
2. Tissue-engineered dermis cells distributed evenly, orderly, and the cell distributed along with mechanical force direction after been imposed dynamic three-dimensional strain; cell proliferation was faster, the extracellular matrix synthesis was higher which mechanical strain was performanced.
3. Improved the original instrument into a small, delicate, reliable clamp, test piece change visible, convenient and fast test device, which was very accurate and stable.
Conclusion:
A dynamic three-dimensional strain culture equipment for tissue-engineered dermis and a tensile strength testing equipment are developed successfully, which can be used to the research of tissue-engineered skin mechanobiology in future.
力学生物学(mechanobiology)是诞生于上世纪90年代的一门新兴交叉学科,为提高组织工程产品力学性能提供了新手段。研究内容主要有力学刺激信号、细胞对力学刺激信号的感受及转导机制和细胞受力后的反应三部分内容。众多研究表明,流体剪切力、静水压、负压吸引、拉伸应变及压缩应变等机械外力对细胞的增殖、分化、基因表达及蛋白合成有显著的影响。而组织工程皮肤可以作为力学生物学研究的良好模型,可研究机械外力对其物理特性以及细胞生物学功能的影响。不同组织细胞对应变大小的承受力有巨大的差异,受到的机械力作用方式也各不相同,研究的关键技术是研制适合不同组织的施加机械力的装置,并探索合适的应变大小、频率及加载时间。力学生物学研究的主要模式是根据所研究的细胞在体所受外力作用形式及应变大小来设计相应的施加机械力装置,利用装置对所研究细胞施加机械力,研究细胞对力学信号感受、转导机制及受力后的反应,从而在细胞及分子水平探索外力对机体的影响。因此本研究从研制施加机械力培养装置和检测力学性能两个方面展开,为组织工程皮肤力学生物学进一步研究提供条件。
目的:
1.研制能够稳定、有效地模拟皮肤在体受力情况的组织工程真皮动态三维应变培养装置。
2.验证建立的组织工程真皮动态三维应变培养装置的有效性。
3.为了测定动态三维应变培养后组织工程皮肤的物理特性,对本实验室与重庆大学联合研制的高敏度小张力膜状生物材料力学检测装置进行改进和完善。
方法:
1.设计制作采用周期性流动培养基驱动的组织工程真皮动态三维应变培养装置。
2.采用本实验室常规方法制作的复方壳多糖组织工程真皮,分别观察常规静态培养及动态三维应变培养下细胞分布、增殖、细胞外基质合成情况。
3.将本实验室研制的高敏度小张力膜状生物材料力学性能检测装置的加载系统改为气体加载系统、夹具升级为夹持精确、可靠且可视的夹持系统,更换为高精度传感器,进行准确度及稳定性测试。
结果:
1.研制的周期性流动培养基驱动的组织工程真皮动态三维应变培养装置能满足组织工程真皮动态三维应变培养要求。
2.初步结果显示动态三维应变培养后组织工程真皮中细胞分布均匀、有序,与受力方向一致;细胞增殖更快,I型胶原合成增多,证明该装置能够模拟皮肤在体力学刺激条件,为下一步研究组织工程皮肤力学生物学提供了重要条件。
3.改进后的高敏度小张力膜状生物材料力学性能检测装置外形小巧、夹持可靠、试件变化可视、操作方便、快捷,且仪器具有良好的准确度及稳定性,为组织工程产品力学性能检测提供了有效检测手段。
结论:
成功研制了组织工程真皮动态三维应变培养装置和抗张强度检测装置,可应用于组织工程皮肤力学生物学的进一步研究,为下一步的研究铺平了道路。
Background:
It is an important issue that how to promote and accelerate wound healing in order to reduce scar formation, while people have always been concerned about, especially when there is insufficient donor skin in extensive burns, how to cover wound in time is an urgent clinical problem which need to be addressed. Skin tissue engineering has brought new hope to cope with this challenge, tissue-engineered skin provides a good option for wound healing and repair and has a huge clinical demand. Current tissue-engineered skin products such as Apligraft and other commercial collagen gel tissue-engineered skin have epidermis and dermis structure, but lack of complete basement membrane, the binding of epidermis and dermis is not strong, their flexibility is poor, and fragile,have not yet reached satisfactory mechanical properties for the transplant operation.These drawback has become an key technical problem that need to be solved urgently for tissue-engineered skin.
Mechanobiology provides a new way to improve the mechanical properties of tissue engineering. It is a new interdisciplinary subject that born in 90’s of the last century. The contents mainly include mechanical stimulation signal, cells feeling the mechanical stimulation signal, force transduction mechanisms and cellular response to mechanical stimulation. A lot of studies have shown that fluid shear stress, hydrostatic pressure, negative pressure, tensile strain, compression strain and other mechanical external forces have significant impact on cell proliferation and differentiation, gene expression and protein synthesis.
The reaction of different tissue cells to mechanical strain showed huge different amplitude, and the type of mechanical force is different to every kind tissue. The key technology of mechanobiology research is to develop the device to impose mechanical force for different organizations and cells, and to explore appropriate strain amplitude, * Supported by National High Technology Research and Development Program of China(863 Program) (2006AA02A121) frequency and processing time. The main mode of mechanobiology research is to develop the sutiable device which based on the mechanical force type and strain amplitude in vivo, then impose mechanical force to cells in vitro with the devices, to study how the cells feel mechanical signal, the mechanical force transduction mechanism and the cellular response to mechanical force, thus to research the influence of mechanical force to body on cellular and molecular level.
Objectives:
1. To establish a stable and effective dynamic three-dimensional stain culture equipment for culturing tissue-engineered dermis which can simulate the mechanical force effect to the skin in vivo.
2. To confirm the effectiveness of the established dynamic three-dimensional strain culture equipment for tissue-engineered dermis.
3. To improve the high-sensitivity mechanical properties testing device for small tension membranaceous biomaterials manufactured by our laboratory and Chongqing University.
Methods:
1. Designed and made dynamic three-dimensional strain culture equipment for tissue-engineered dermis that drived by the cyclical media flow.
2. Observed the cell distribution and viability, extracellular matrix synthesis and mechanical properties of composite chitosan tissue-engineered dermis under our laboratory conventional methods and dynamic three-dimensional culture.
3. Loading system was replaced by airy pressure loading system; the clamp was upgraded to clamping accurately, reliable and visual clamping system; the sensor was replaced by a high-precision sensor, tested accuracy and stability.
Results:
1. Developed dynamic three-dimensional strain culture equipment for tissue- engineered dermis which was drived by cyclical media flow.
2. Tissue-engineered dermis cells distributed evenly, orderly, and the cell distributed along with mechanical force direction after been imposed dynamic three-dimensional strain; cell proliferation was faster, the extracellular matrix synthesis was higher which mechanical strain was performanced.
3. Improved the original instrument into a small, delicate, reliable clamp, test piece change visible, convenient and fast test device, which was very accurate and stable.
Conclusion:
A dynamic three-dimensional strain culture equipment for tissue-engineered dermis and a tensile strength testing equipment are developed successfully, which can be used to the research of tissue-engineered skin mechanobiology in future.
引文
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1. Suki B, Bates JH. Extracellular matrix mechanics in lung parenchymal diseases[J]. Respir Physiol Neurobiol, 2008, 163(1-3): 33-43.
2. Hahn C, Schwartz MA. The role of cellular adaptation to mechanical forces in atherosclerosis[J]. Arterioscler Thromb Vasc Biol, 2008, 28(12): 2101-2107.
3. Suresh S, Spatz J, Mills JP, et al. Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria[J]. Acta Biomater, 2005, 1(1): 15-30.
4. Block JA, Shakoor N. The biomechanics of osteoarthritis: implications for therapy[J]. Curr Rheumatol Rep, 2009, 11(1): 15-22.
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15. Puso MA, Weiss JA. Finite element implementation of anisotropic quasi-linear viscoelasticity using a discrete spectrum approximation[J]. J Biomech Eng, 1998, 120(1): 62-70.
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19. Billiar KL, Sacks MS. Biaxial mechanical properties of the native and glutaraldehyde- treated aortic valve cusp: Part II--A structural constitutive model[J]. J Biomech Eng, 2000, 122(4): 327-335.
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45. Garvin J, Qi J, Maloney M, et al. Novel system for engineering bioartificial tendons and application of mechanical load[J]. Tissue Eng, 2003, 9(5): 967-979.
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49.汤顺波,贾乃文.悬膜结构塑性失稳破坏计算[J].空间结构, 2001, 7(4): 24-29.
1. Suki B, Bates JH. Extracellular matrix mechanics in lung parenchymal diseases[J]. Respir Physiol Neurobiol, 2008, 163(1-3): 33-43.
2. Hahn C, Schwartz MA. The role of cellular adaptation to mechanical forces in atherosclerosis[J]. Arterioscler Thromb Vasc Biol, 2008, 28(12): 2101-2107.
3. Suresh S, Spatz J, Mills JP, et al. Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria[J]. Acta Biomater, 2005, 1(1): 15-30.
4. Block JA, Shakoor N. The biomechanics of osteoarthritis: implications for therapy[J]. Curr Rheumatol Rep, 2009, 11(1): 15-22.
5.冯元桢.生物力学[M].北京:科学出版社,1983:160-l64.
6. Flynn DM, Peura GD, Grigg P, et al. A finite element based method to determine the properties of planar soft tissue[J]. J Biomech Eng, 1998, 120(2): 202-210.
7. Mow VC, Kuei SC, Lai WM, et al. Biphasic creep and stress relaxation of articular cartilage in compression? Theory and experiments[J]. J Biomech Eng, 1980, 102(1): 73-84.
8. Mow VC, Holmes MH, Lai WM. Fluid transport and mechanical properties of articular cartilage: a review[J]. J Biomech, 1984, 17(5): 377-394.
9. Mow VC, Gibbs MC, Lai WM, et al. Biphasic indentation of articular cartilage--II. A numerical algorithm and an experimental study[J]. J Biomech, 1989, 22(8-9): 853-861.
10. Oomens CW, van Campen DH, Grootenboer HJ. A mixture approach to the mechanics of skin[J]. J Biomech, 1987, 20(9): 877-885.
11.黄立独,汪勤悫.皮肤层蠕变分析的混合解法[J].应用数学和力学, 1994, 15(12): 1075-1082.
12. Bischoff JE, Arruda EM, Grosh K. Finite element modeling of human skin using an isotropic, nonlinear elastic constitutive model[J]. J Biomech, 2000, 33(6): 645-652.
13. Humphrey JD, Strumpf RK, Yin FC. A constitutive theory for biomembranes: application to epicardial mechanics[J]. J Biomech Eng, 1992, 114(4): 461-466.
14. Humphrey JD, Strumpf RK, Yin FC. Determination of a constitutive relation forpassive myocardium: I. A new functional form[J]. J Biomech Eng, 1990, 112(3): 333-339.
15. Puso MA, Weiss JA. Finite element implementation of anisotropic quasi-linear viscoelasticity using a discrete spectrum approximation[J]. J Biomech Eng, 1998, 120(1): 62-70.
16. Pereira JM, Mansour JM, Davis BR. Dynamic measurement of the viscoelastic properties of skin[J]. J Biomech, 1991, 24(2): 157-162.
17. Bischoff JE,Arruda EM. Grosh K. Finite element simulations of orthotropic hyperelasticity[J]. Finite Elements in Analysis and Design, 2002, 38(10): 983-998.
18. Bischoff JE, Arruda EM, Grosh K. Orthotropic hyperelasticity in terms of an arbitrary molecular chain model[J]. Journal of Applied Mechanics, Transactions of the ASME, 2002, 69(2): 198-201.
19. Billiar KL, Sacks MS. Biaxial mechanical properties of the native and glutaraldehyde- treated aortic valve cusp: Part II--A structural constitutive model[J]. J Biomech Eng, 2000, 122(4): 327-335.
20.刘策励,成海平.人体股前区皮肤生物力学特性的初步研究[J].生物医学工程学杂志, 1997, 14(4): 380-382.
21. Kvistedal YA, Nielsen PM. Investigating stress-strain properties of in-vivo human skin using multiaxial loading experiments and finite element modeling[J]. Conf Proc IEEE Eng Med Biol Soc, 2004, 7: 5096-5099.
22. Schock RB, Brunski JB, Cochran GVB.In vitro experiments on pressure sore biomechanics: stresses and strains in indented tissues[C]. ASME 1982 Advances in Bioengineering. New York, 1982: 88-91.
23. Lanir Y, Dikstein S, Hartzshtark A, et al. In-vivo indentation of human skin[J]. J Biomech Eng, 1990, 112(1): 63-69.
24.黄立独,汪勤悫,麦福达.皮肤层在钢印挤压作用下的生物力学研究[J].中国生物医学工程学报, 1997,16(1) : 1-7.
25. Zheng YP, Huang DT, Mak AFT. Experimental studies of indentor misalignment for indentation test on soft tissues[C]. Proceedings of the 19th Annual International Conference of the IEEE. Chicago, 1997 (5): 2250-2253.
26. Zheng YP, Mak AFT. Extraction of effective Young’s modulus of skin andsubcutaneous tissues from manual indentation data[C]. Proceedings of the 19th Annual International Conference of the IEEE. Chicago, 1997(5): 2246-2249.
27. Scalari G, Eisinberg A, Menciassi A, et al. Micro-instrumentation for non-invasive measurement of mechanical properties of tissues[C]. 1st Annual International of Microtechnologies in Medicine and Biology. Lyon, France,2000:199-202.
28. Oka H, Irie T, Hao SY, et a1. Portable system for measuring biomechanical properties[C]. Proceedings of the 8th Instrumentation and Measurement Technology Conference of the IEEE. Atlanta, 1991: 556-561.
29. Pan LH, Zan L, Foster FS. In vivo high frequency ultrasound assessment of skin elasticity[C]. Proceedings of Ultrasonics Symposium of the IEEE, 1997:1087-1091.
30. Brienza DM, Chung KC, Brubaker CE, et al. A system for the analysis of seat support surfaces using surface shape control and simultaneous measurement of applied pressures[J]. IEEE Trans Rehabil Eng, 1996, 4(2): 103-113.
31. Wang J, Brienza DM, Yuan YW, et a1. Biomechanical analysis of buttock soft tissue using computer-aided seating system[J]. Engineering in Medicine and Biology Society, 1998, 5(29): 2757-2759.
32. Gennisson JL, Baldeweck T, Tanter M, et al. Assessment of elastic parameters of human skin using dynamic elastography[J]. IEEE Trans Ultrason Ferroelectr Freq Control, 2004, 51(8): 980-989.
33.范志宏,关文祥,金一涛,等.快速扩张后皮肤生物力学特性的实验研究[J].中华整形烧伤外科杂志, 1994(1).34-37.
34.章晶,胡华新,等.扩张皮肤移植后的生物力学特性[J].中国生物医学工程学报, 2002, 21(2): 144-148.
35.曾衍钧,杨坚,许传青,等.不同扩张方案下离体皮肤的生物力学特性比较[J].中国生物医学工程学报, 2003, 22(6): 559-563.
36.霍然,王洋,李尚滨,等.皮肤软组织重复扩张的生物力学[J].中华医学美学美容杂志, 2005, 11(1): 39-41.
37. Banes AJ, Tsuzaki M, Hu P, et al. PDGF-BB, IGF-I and mechanical load stimulate DNA synthesis in avian tendon fibroblasts in vitro[J]. J Biomech, 1995, 28(12): 1505-1513.
38. Lee AA, Delhaas T, Waldman LK, et al. An equibiaxial strain system for culturedcells[J]. Am J Physiol, 1996, 271(4 Pt 1): C1400-C1408.
39. Wang JH, Thampatty BP, Lin JS, et al. Mechanoregulation of gene expression in fibroblasts[J]. Gene, 2007, 391(1-2): 1-15.
40. Yost MJ, Simpson D, Wrona K, et al. Design and construction of a uniaxial cell stretcher[J]. Am J Physiol Heart Circ Physiol, 2000, 279(6): H3124-H3130.
41. Wang JH, Jia F, Yang G, et al. Cyclic mechanical stretching of human tendon fibroblasts increases the production of prostaglandin E2 and levels of cyclooxygenase expression: a novel in vitro model study[J]. Connect Tissue Res, 2003, 44(3-4): 128-133.
42. Wang JH, Yang G, Li Z. Controlling cell responses to cyclic mechanical stretching[J]. Ann Biomed Eng, 2005, 33(3): 337-342.
43. Yang G, Crawford RC, Wang JH. Proliferation and collagen production of human patellar tendon fibroblasts in response to cyclic uniaxial stretching in serum-free conditions[J]. J Biomech, 2004, 37(10): 1543-1550.
44. Yang G, Im HJ, Wang JH. Repetitive mechanical stretching modulates IL-1beta induced COX-2, MMP-1 expression, and PGE2 production in human patellar tendon fibroblasts[J]. Gene, 2005, 363: 166-172.
45. Garvin J, Qi J, Maloney M, et al. Novel system for engineering bioartificial tendons and application of mechanical load[J]. Tissue Eng, 2003, 9(5): 967-979.