各向异性拓扑结构和刚度细胞培养基底及其在组织工程中的应用

发布时间：2018-01-08 21:15

本文关键词：各向异性拓扑结构和刚度细胞培养基底及其在组织工程中的应用　出处：《中国科学技术大学》2016年博士论文　论文类型：学位论文

【摘要】：细胞生长微环境的拓扑结构、刚度、应力和应变等物理属性是调控细胞生长、增殖、分化和凋亡等生理和病理过程的重要因子。与生物化学方法相比,物理特性在调控某些细胞行为方面更稳定。在细胞培养基底的物理属性中,微纳尺度的拓扑结构和刚度在细胞取向、迁移和分化行为调控及组织工程方面具有重要的应用。组织工程是应用工程和生命科学的原理再生可供研究或移植的生物组织替代物的方法。目前体外培养并可以做临床体内移植的组织主要是皮肤和软骨组织。肌肉和血管组织是除了上述两者外最接近临床应用的组织之一,对这两种组织的研究具有重要的应用前景。占人体总重量40%到45%的骨骼肌,是人体产生自主动作和运动所需要的力的基本来源。造成骨骼肌损伤的原因非常普遍,包括运动造成的肌肉挫伤、事故引起的肌肉撕裂以及绝大多数外科手术造成的肌肉受损。此外,由疾病引起的骨骼肌萎缩以及随年龄增长而造成的骨骼肌老化也使得对骨骼肌组织再生的需求更加迫切。由于大面积的肌肉受损极易产生伤疤,而病变的肌肉缺少可供相关干细胞分化的健康环境,因此在体外进行骨骼肌组织的培养显得尤为重要。血管是最人体最重要的组织之一,动脉粥样硬化等血管病变会严重威胁生命安全,而难以制造微血管网络也使体外再生的人体器官因没有养分供给而快速衰竭。血管在体内外组织及器官中的关键作用,使得制备可供体外血管组织再生的基架至关重要,但血管结构和力学特性上的复杂性也为其基架的加工带来了重重挑战。本文通过微纳加工技术,使用生物兼容材料可控地加工出了具有各向异性的微拓扑结构和刚度的基底,并将其应用于环形和线状骨骼肌和血管组织再生中。本文为体外骨骼肌及血管组织工程的基底和基架的设计与加工提供了新方法,其主要研究内容及成果如下：1.环形肌纤维是尿道括约肌的重要组成部分。本文通过设计结合了拓扑结构和刚度对细胞取向调控作用的基底,在超细胞尺度同时实现了小鼠成肌细胞的环形图形化及细胞相对于环形图形的高度平行排列,并建立了群体细胞在超细胞尺度曲线约束下的有限元模型,分析了引导细胞沿着图形边界方向取向的机理。实验结果表明,超细胞尺度环形约束的宽度和内外边界的平行度对细胞的取向具有显著影响：更窄而内外边界平行的环形约束更有利于细胞沿着环形约束的边界方向取向。然而,环形约束的曲率对细胞的取向并无影响。理论分析表明,细胞与细胞间的作用力可以通过胞间连接传输,细胞的取向与胞间作用力的最大主应力的方向一致,而最大主应力的方向与环形约束的边界平行。当细胞高度汇合时,处于环形约束边界附近的细胞就可以通过胞间连接将环形约束的边界信息传递给处于环形约束中部区域的细胞,细胞在胞间作用力的驱动下,最终沿着胞间最大主应力即环形约束边界的方向取向。高度取向的细胞有利于细胞融合进而分化为肌管,本文的结论为再生环形肌管提供了有力的支撑。另外本文的基底也可作为底层,为在上层再次进行成肌细胞培养提供具有类似体内肌细胞微环境的刚度的条件,从而促进环形肌管再生,为治疗尿道括约肌疾病提供药物筛选及体内移植的可能。2.大面积低成本的骨骼肌肉组织工程具有广阔的应用前景。各向异性的拓扑结构及刚度在引导成肌细胞取向以使其分化为肌管的过程中均有重要作用。本文综合考虑了成本和尺度,提出了基于光敏水凝胶的二次曝光聚合方法,无模具地在水凝胶表面加工出了拓扑结构和相应的空间分布的刚度。二者的叠加作用加速了肌肉组织的再生。实验结果表明,本文使用的基底可以高效地引导成肌细胞取向并形成方向一致的肌管。本文的加工方法具有成本低、加工范围大、可应用于其他光敏水凝胶的特点,因此在骨骼肌组织工程中具有潜在的应用价值。3.动脉和静脉血管包含轴向排列的内皮细胞和周向排列的平滑肌细胞。本文采用软光刻、二次曝光及自卷曲的方法,加工出了内外表面具有垂直约束直径可控的水凝胶管道。管道内壁为轴向沟槽结构,外壁为周向的环形凸起结构和刚度条纹。这两种约束均可使细胞产生接触引导,从而沿着约束的方向取向。不同于在刚性管道外表面喷绕纳米纤维的电纺丝法,本文率先使用生物材料制备出了具备双层垂直约束的管道结构。本文所加工的基架及所提出的方法的可应用于其他水凝胶材料的特点,使他们在体外血管组织再生及体内血管移植方面具有一定的应用前景。
[Abstract]:Topological structure, cell growth micro environment stiffness, stress and strain of physical properties such as growth, proliferation and regulation of cell differentiation and apoptosis, an important factor in many physiological and pathological processes. Compared with chemical and biological methods, physical properties are more stable in regulation of some cell behavior in cell culture. Physical properties of substrate, topology the structure of micro nano scale and stiffness in cell orientation, plays an important role in migration and differentiation regulation and tissue engineering. Tissue engineering is tissue substitute method applying the principle of engineering and life sciences for the study of transplantation or regeneration. At present and in vitro can do clinical transplantation in vivo tissue is mainly the skin and cartilage. Muscle and vascular tissue is one of the two most close to the clinical application of the tissue in the study of the two kinds of organization has important application prospects. For human body The total weight of 40% to 45% of the skeletal muscle, is the basic source of the human body to produce independent action and movement. The force required to cause skeletal muscle injury is very common, including the movement caused by muscle contusion, the accident caused by muscle tear and most surgical muscle damage. In addition, the skeletal muscle atrophy caused by disease along with the age growth caused by the aging of skeletal muscle regeneration of skeletal muscle tissue also makes more urgent needs. Because of the large area of muscle damage prone to scar, and lesions of the muscle lack for stem cell differentiation in a healthy environment, so the cultivation of skeletal muscle tissue in vitro is particularly important. One of the vessels the most important human tissues, atherosclerosis and other vascular diseases will be a serious threat to life safety, and difficult to manufacture micro vascular network also makes human organs for in vitro regeneration No nutrient supply and rapid failure. The key role of vascular tissue in vivo and in organs, the preparation can be vital donor vascular tissue regeneration base, but the complexity of the structure and mechanical properties of the blood vessels but also for the frame processing brings many challenges. This paper through the micro nano processing technology, the use of bio compatible materials controllable machining of micro topology is anisotropic and the stiffness of the substrate, and its application in circular and linear skeletal muscle and vascular tissue regeneration. This paper provides a new method for in vitro skeletal muscle and vascular tissue engineering base and base frame design and processing, the main research contents and results are as follows 1.: circular muscle fiber is an important part of the urethral sphincter. The design combined with the topology and stiffness of the basal cell orientation regulation, in the super cell scale at the same time The mouse into a ring pattern and cell muscle cells relative to the ring pattern height are arranged in parallel, and the establishment of a finite element in the super cell scale curve under the constraint of model groups of cells, analysis the mechanism of cell orientation along the guide direction of the graph boundary. The experimental results show that the super cell measure ring width and outer boundary constraints the parallel degree of orientation has significant effect on cell: a narrow and parallel annular boundary constraints is more conducive to the direction of the annular cells along the boundary constraint orientation. However, there is no constraint on the effect of curvature ring cell orientation. The theoretical analysis shows that the interaction between cells and can be connected by intercellular transmission, and the maximum principal orientation cell cell interactions stress in the same direction, and the direction of the maximum principal stress and ring constraint boundary parallel. When the cell height at the confluence, Near ring boundary cells would pass through the intercellular connection ring constraint boundary information to the central region of the cell in ring constraint, driving force in the intercellular cell, finally the maximum principal stress along the intercellular ring boundary direction. A high degree of orientation of the cell to cell fusion and differentiation for myotubes, the conclusions of this paper provide a strong support for the regeneration of the basal ring myotubes. In addition can also be used as the bottom is in the upper again myoblast culture provides stiffness conditions with similar body muscle cell microenvironment, thereby promoting circular myotube regeneration, and has broad application prospects for the treatment of urethral sphincter disease provide drug screening and in vivo transplantation may.2. large area low cost of skeletal muscle tissue engineering. The topological structure of anisotropy and stiffness in guided fine muscle In order to make the differentiation of cell orientation plays an important role in the process. Myotube considering cost and scale, put forward two exposure polymerization method based on photosensitive hydrogels, no mold on the surface of the hydrogel processing distribution topology and the corresponding spatial stiffness. The two superposition accelerates the regeneration of muscle organization. The experimental results show that the substrate can effectively guide the myoblast orientation and formed myotube direction. With the processing of this method is low cost, large processing range, the characteristics can be applied to other photosensitive hydrogels, therefore has the potential application value of.3. arterial and venous vascular endothelial cells and contains axial week the arrangement of vertical arrangement of smooth muscle cells in skeletal muscle tissue engineering. In this paper, using soft lithography, two exposure method and self crimping, processing the inner and outer surface with a vertical Hydrogel pipeline constraint controllable diameter pipeline wall. The axial groove structure, annular convex structure outer wall circumferential stiffness and stripes. The two constraints can make the cell contact guidance, and along the constrained direction. Different Electrospinning in rigid pipe surface spray around the nanofibers, we first use biological materials were prepared with double pipe structure. The vertical constraint frame processing and the proposed method can be applied to other characteristics of hydrogel materials, so they graft in vascular tissue regeneration in vitro and in vivo blood vessel has a certain application prospect.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q813

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