生物材料表面与微环境构建对细胞行为的调控及在生物医学工程应用

发布时间：2018-05-30 22:18

本文选题：生物材料 + 干细胞　；参考：《山东大学》2016年博士论文

【摘要】：生物材料是一类目的在于和生物系统发生相互作用,来评价、治疗、增强或替代某种组织、器官或者人体功能的材料。理解和认识生物材料-细胞界面以及优化和设计生物材料的结构和性质,将对构建细胞外微环境以及生物医学工程的具体实施具有重要的指导意义。本文针对目前广泛关注的不同类型的微环境中材料学信号对细胞的作用进行了研究,其中包括,材料表面电荷微环境、纳米颗粒微环境、生物因子微环境与仿生细胞外基质微环境,目的是揭示材料表面性质和细胞外微环境和细胞行为的相互作用,为生物材料设计和制备奠定理论和实验基础。本文的研究主要从以下几个方面展开：1.材料表面电荷对干细胞行为的影响：生物材料的表面物理性质和生物化学性质对细胞的贴壁、迁移、增殖和分化等行为有很大的影响。但是在调查单个物理或化学因素与细胞的相互作用时,很多研究并不能在单一变量的体系下进行。在单因素变量的二维模型中研究材料表面性质对细胞行为的影响可以更清楚的认识和理解细胞对材料表面性质的响应。生物材料表面电荷是影响细胞行为的重要因素,但目前关于干细胞对表面电荷的响应尤其是表面电荷对干细胞分化的影响的研究很少。因此,本论文首次利用不同切割方向的铌酸锂单晶薄片构建化学成分相同、表面粗糙度相同的但具有不同电荷表面的单变量简单模型,并用其作为二维培养基底来研究材料表面不同电荷状态对间充质干细胞的贴壁、增殖以及成骨分化的影响。论文证实了相比于负电和中性表面,带正电荷的表面可以促进细胞贴壁铺展以及在体外诱导条件下增强大鼠间充质干细胞向成骨分化。2.细胞外微环境中纳米晶体对干细胞的影响：干细胞在生物医学尤其是再生领域的应用仍然面临着重大挑战,这包括发展先进技术来理解和控制微环境中的各种信号,以及新的手段来追踪和引导植入的干细胞。纳米材料在分子水平上和细胞特异性的作用是理解和控制干细胞的行为的重要工具。比如基于纳米颗粒的载药技术可以靶向释放细胞所需的活性因子,静电纺丝技术合成的纳米纤维支架可以引导细胞的生长及组织形成,以及使用量子点和上转化等纳米材料进行细胞成像和追踪。随着这些大量功能性的纳米材料在生物医学工程上的应用,干细胞对这些人为引入到微环境中的纳米颗粒的响应需要确认和研究。本论文利用溶剂热的方法制备了铌酸锂纳米晶体,利用NIR飞秒激光器作为激发光源在双光子显微镜下观察大鼠间充质干细胞对微环境中铌酸锂纳米晶体的主动内化吞噬,以及利用PCR和免疫染色等生物学手段表征铌酸锂纳米晶体对干细胞分化的影响。论文证实了干细胞对微环境中纳米晶体的主动响应和分化调控。3.通过生长因子的缓释构建组织修复生物微环境：控制细胞行为的细胞外组成不仅包括如上所述的细胞外基质中不可溶的成分,还包括了激素和生长因子等可溶性的分子。所以除了利用生物材料本身表面和结构性质指导细胞行为,生物材料结合可溶性的生长因子来控制细胞分化和功能也是一个构建细胞外微环境的重要手段。目前,外科手术修复肌腱等结缔组织仍旧是临床上一个挑战,缺损处组织修复后很难再达到原来的强度水平。为了在修复前期给缺损处提供初始的力学支持,人们尝试通过增强缝合线的强度以及改进缝合线的抓握方法。虽然这些方法能够一定程度上帮助组织修复,但是这些方法并不能为组织修复创造合适的微环境,也不能调节修复的生物过程。因此,本论文首次提出基于多孔缝合线的生长因子缓释体系来构建组织修复的微环境。研究使用一种简单有效的溶胀-冷冻-干燥方法来制备具有多孔结构鞘层的缝合线而不降低其原有的机械强度。由于多孔结构的存在,缝合线内部的空间就可以被利用起来以实现更高的负载。另外,多孔结构结合使用纤维蛋白交联网络能减缓后续的释放过程,研究实现了PDGF在体外生理环境中长达11天的持续释放。而且释放的PDGF保持了原有的生物活性,能够促进人类骨髓间充质干细胞的增殖。这种基于多孔缝合线的生长因子缓释体系为构建组织修复微环境提供了新的策略。4.利用天然细胞外基质仿生构建细胞外微环境的探索：细胞外基质(ECM)是一个结构和功能都很复杂的混合物,在细胞的行为和表型上起着非常重要的作用,因此人们努力尝试希望能得到一个人造的ECM等价物,来给细胞提供一个模拟天然的微环境。将组织的细胞成分脱除得到的脱细胞基质已经越来越多的被用于组织再生和器官移植等再生医学领域。但是由于天然的细胞外基质主要成分是胶原,支架材料的机械强度较低并且降解速率较快,这制约了其在组织工程尤其是骨组织工程上的应用。因此,本论文将使用优化的脱细胞技术制备猪脱细胞真皮基质(PADM),并使用碳化二亚胺作为交联剂强化PADM,以此尝试模拟细胞外基质微环境。得到的PADM支架保留了胶原天然的多孔结构,并有效保留了胶原和GAG等有效成分,去除了核酸和SDS等免疫反应源；评价了碳化二亚胺作为零长度交联剂对PADM支架的交联作用,及交联对支架强度和降解速率的影响；体外评估了前成骨细胞在交联后PADM支架的生物活性。交联后的PADM的力学强度和降解速率都可以通过交联程度控制,作为仿生支架模拟细胞外微环境也可以很好地支持细胞向支架内部迁移。因此,本研究为使用天然细胞外基质来模拟细胞外基质微环境的探索提出了一种可能性。综上所述,本论文主要致力于研究生物材料表面对细胞行为的调控以及探索细胞外微环境的体外构建,这些研究和探索将进一步促进生物材料在生物医学工程上的应用。
[Abstract]:Biomaterials are a class of materials that aim to interact with biological systems to evaluate, treat, enhance or replace some tissues, organs, or human functions. Understanding and understanding the biomaterial cell interface and optimizing and designing the structure and properties of biological materials will be for the construction of the extracellular microenvironment and biomedical engineering. In this paper, the effect of material signal on the cell is studied in different types of microenvironment, including the surface charge micro environment, nano particle microenvironment, biological factor microenvironment and biomimetic extracellular matrix microenvironment. The purpose is to reveal the surface properties of the material. The interaction between the extracellular microenvironment and cell behavior will lay a theoretical and experimental basis for the design and preparation of biomaterials. This study mainly starts from the following aspects: 1. the effect of surface charge on the behavior of stem cells: the surface physical properties and biochemical properties of biomaterials on cell adhesion, migration, and proliferation. But in investigating the interaction of single physical or chemical factors with cells, many studies can not be carried out in a single variable system. In the two-dimensional model of single factor variables, the study of the effect of material surface properties on cell behavior can be more clearly understood and understood. Response of surface properties. Surface charge of biomaterials is an important factor affecting cell behavior, but there are few studies on the effect of stem cells on surface charge, especially surface charge, on the differentiation of stem cells. Therefore, the first use of different cutting direction lithium niobate single crystal slices to construct the same chemical composition and surface roughness in this paper. A simple single variable model with the same degree but with a different charge surface, and using it as a two-dimensional culture substrate to study the effect of different charge states on the adhesion, proliferation and osteogenesis of mesenchymal stem cells on the surface of the material. And the effect of nanocrystalline on stem cells in the extracellular microenvironment of osteogenic differentiation.2. in vitro induced by rat mesenchymal stem cells: the application of stem cells in biomedicine, especially in the field of regeneration, still faces major challenges, including the development of advanced technology to understand and control the various signals in microenvironment, and the new technology. The means to track and guide implanted stem cells. The role of nanomaterials at molecular level and cell specificity is an important tool for understanding and controlling the behavior of stem cells. For example, nanoparticles based drug loading techniques can target the active factors needed to release the cells, and the nanofiber scaffolds synthesized by the electrospun spinning technology can be guided. Cell growth and tissue formation, and the use of nanomaterials, such as quantum dots and upper conversion, to carry out cell imaging and tracking. With the application of these large functional nanomaterials in biomedical engineering, the response of the stem cells to nano particles introduced into the microenvironment should be confirmed and studied. This paper uses solvent heat. Lithium niobate nanocrystals were prepared by using a NIR femtosecond laser as an excitation source to observe the active endogenous phagocytosis of the rat mesenchymal stem cells to the lithium niobate nanocrystals in microenvironment under two photon microscopy, and the effects of PCR and immunologic staining on the differentiation of the stem cells by using PCR and immunologic staining. The active response and differentiation of nanocrystals in the microenvironment are regulated by stem cells to repair the microenvironment of.3. through the sustained release of growth factors. The extracellular components that control cell behavior include not only the insoluble components in the extracellular matrix as described above, but also the soluble fractions such as hormones and growth factors. So in addition to using the surface and structural properties of biological materials to guide cell behavior, biological materials combine soluble growth factors to control cell differentiation and function is an important means to construct extracellular microenvironment. At present, surgical repair of connective tissue such as tendons remains a clinical challenge, and the defect is repaired in tissue. It is difficult to reach the original strength level. In order to provide initial mechanical support to the defect in the early period of repair, people try to enhance the strength of the suture line and improve the grip of the suture. Although these methods can help the tissue repair to a certain extent, these methods do not create suitable for tissue repair. Microenvironment can not regulate the biological process of repair. Therefore, this paper first proposed a sustained release system of growth factor based on porous suture to construct microenvironment for tissue repair. A simple and effective method of swelling freeze drying was used to prepare suture with porous structure sheath without reducing its original mechanical strength. Due to the existence of the porous structure, the space inside the suture can be used to achieve higher load. In addition, the porous structure combined with fibrin crosslinking network can slow down the subsequent release process. The study realized the sustained release of PDGF for up to 11 days in the physiological environment in vitro. And the release of PDGF maintained the original life. The activity of substance can promote the proliferation of human bone marrow mesenchymal stem cells. This growth factor sustained-release system based on porous suture provides a new strategy for the construction of microenvironment for tissue repair. The extracellular matrix (ECM) of the extracellular matrix (.4.) is a complex structure and function. The mixture plays a very important role in the behavior and phenotype of cells, so people try to try to get a person's ECM equivalent to provide a simulated natural microenvironment. The more the cell components removed from the tissue, the more the cells are used for tissue regeneration and organ transplantation. But because the natural extracellular matrix is mainly collagen, the mechanical strength of the scaffold material is low and the degradation rate is fast, which restricts its application in tissue engineering especially bone tissue engineering. Therefore, this paper will use the optimized acellular technique to prepare the porcine acellular dermal matrix (PADM), and use it in this paper. As a crosslinker, carbonized two imide was used as a crosslinker to simulate the microenvironment of extracellular matrix. The PADM scaffold retained the natural porous structure of collagen, retained effective components such as collagen and GAG, removed the immune response sources such as nucleic acid and SDS, and evaluated the crosslinking of PADM as a zero length cross-linking agent as a zero length cross-linking agent. The effect of interaction and cross-linking on the strength and degradation rate of the scaffold; in vitro evaluation of the bioactivity of the PADM scaffold after cross linking of osteoblasts. The mechanical strength and degradation rate of the crosslinked PADM can be controlled by the degree of cross-linking. As a biomimetic scaffold, the extracellular microenvironment can also be well supported by the migration of the cell to the stent. Therefore, this study provides a possibility for the use of natural extracellular matrix to simulate the microenvironment of extracellular matrix. In summary, this paper focuses on the study of the regulation of cell behavior on the surface of biomaterials and the exploration of the external microenvironment in vitro. These research and exploration will further promote biological materials. Biomedical engineering applications.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08

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