【作者】 张才茂；

【导师】 伍津津；

【作者基本信息】 第三军医大学 ， 皮肤病与性病学， 2010， 硕士

【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。更多还原

【Abstract】 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.更多还原