基于微流控乳液模板的有序多孔材料的制备研究

发布时间：2018-06-04 20:05

本文选题：组织工程 + 微流控技术　；参考：《东南大学》2017年硕士论文

【摘要】：20世纪80年代,组织工程学被提出,通过科学家的努力,利用组织工程技术在裸鼠上成功形成具有皮肤覆盖的人耳廓形态软骨,这意味着组织工程从基础迈向临床应用的广阔前景。组织工程被定义为一门以细胞生物学和材料科学相结合,进行体外或体内构建组织或器官的新兴学科。组织工程的基本原理是,从病人体内分离取得种子细胞,种植在具有良好生物相容性并且在体内可逐步降解吸收的组织工程多孔支架上形成细胞-支架复合物,细胞在支架上增殖、分化,然后将此复合物植入同一病人组织病损部位,在体内继续增殖并分泌细胞外基质,形成新的与自身功能和形态相适应的组织或器官,从而达到修复病损组织或器官的目的。组织工程的核心是建立细胞与生物支架结构,支架对培养细胞的黏附、分化、增殖、组织形成起着重要作用。好的组织工程支架需要具有三维多孔结构、良好的生物相容性、可生物降解性、一定的生物力学性能。传统的支架制备技术主要有:纤维编织技术、成孔剂析出法、气体发泡技术、热致相分离等,制备支架的方法多种多样,但各有优缺点,很难同时满足理想组织工程支架的几个特点。后来,一种新的制备方式被提出,科学家们发展出以尺寸均一的固体微球作为模板制备得到孔洞大小均一、孔隙率高的组织工程支架。这种方法能同时满足对支架的性能要求,但步骤仍较为繁琐。在本论文中,我们提出利用微流控技术对微球模板法进行改进,一步式构建满足组织工程要求的有序多孔三维支架。微流控技术是一门在微电子、微制作、生物工程和纳米技术等基础上发展而来的全新的交叉学科,通过利用流体的流动剪切力与表面张力之间的相互作用,在微尺度通道内将连续流体分割成离散的尺寸在微米级别的液滴。微流控方法方便简单,可实时调控支架孔洞大小,调控范围在几十微米至几百微米之间,适用于不同组织培养,获得支架连通性好,大小均一,可满足组织工程的多种需求。本论文具体开展工作如下:(一)微流控芯片的构建:不同微流控芯片的设计可以实现单重乳液、双重乳液、多重乳液等不同乳液的形成。根据后期所需乳液模板的液滴为单重乳液,设计相应的微流控芯片,流体通道分为内相入口端、外相入口端、收集端,为了更好的观察内外相液滴剪切情况,在剪切处加入一定长度方管。通过微流控芯片的设计,探讨剪切端管道尺寸对液滴尺寸的影响、单分散性、稳定性的影响因素,最终获得稳定的乳液液滴。(二)乳液液滴模板的制备:在微流控技术和微流控芯片构建的基础上,为了实现有序多孔支架良好的生物相容性和可生物降解性,提出三种乳液液滴的制备体系,尝试利用海藻酸钠或PEG-DA或丝素蛋白溶液作为外相溶液,无毒性的食用油或是硅油作为内相溶液,形成乳液液滴。探索合适的表面活性剂,使液滴不会破裂,液滴间不会相互融合,以形成排列规整的液滴模板。通过对流体流速的调控,可探讨内外相流速对液滴尺寸、液滴形成间距的影响。(三)有序多孔材料的制备:研究确定三种体系下能稳定形成乳液液滴模板后,对材料进行固化。PEG-DA具有光敏性,通过紫外光照射,可使PEG-DA聚合成凝胶。在海藻酸钠中加入钙离子,通过钙离子置换钠离子形成海藻酸钙分子,使得海藻酸钠凝胶化。丝素蛋白经过冷冻干燥处理后可形成海绵状结构。利用酒精将支架材料中的乳液液滴模板洗去,经过冷冻干燥,获得具有有序孔洞结构的支架材料。(四)有序多孔材料的生物应用探究:作为组织工程的核心,支架材料对培养细胞的黏附、分化、增殖起着重要作用。在获得的PEG-DA多孔支架上接种HepG2细胞,探究其在细胞培养上的应用。
[Abstract]:In 1980s, tissue engineering was proposed that, through the efforts of scientists, tissue engineering techniques have been used to successfully form human ear shaped cartilage with skin covering on nude mice. This means the broad prospect of tissue engineering from foundation to clinical application. Tissue engineering is defined as a combination of cell biology and material science. The basic principle of tissue engineering is to isolate and obtain seed cells from the patient, and to form a cell scaffold complex on a porous scaffold that has good biocompatibility and can gradually degrade and absorb in the body. The cells proliferate, differentiate, and then grow on the scaffold. The complex is implanted in the lesion site of the same patient and continues to proliferate and secrete the extracellular matrix in the body to form a new tissue or organ that adapts to its own functions and forms, so as to achieve the purpose of repairing the damaged tissues or organs. The core of the tissue engineering is to establish the structure of the cell and biological scaffold, and the adhesion of the scaffold to the cultured cells. Differentiation, proliferation, and tissue formation play an important role. Good tissue engineering scaffolds need three-dimensional porous structure, good biocompatibility, biodegradability, and certain biomechanical properties. Traditional scaffolding techniques include fiber braiding technology, pore forming agent precipitation, gas foaming technology, thermally induced phase separation and so on. There are a variety of methods, but each has its advantages and disadvantages. It is difficult to meet the characteristics of the ideal tissue engineering scaffold at the same time. Later, a new preparation method has been proposed that scientists have developed a tissue engineering scaffold with uniform size and high porosity with homogeneous solid microspheres as a template. This method can meet the requirements at the same time. The performance of the scaffolds is required, but the steps are still tedious. In this paper, we propose to use microfluidic technology to improve the microsphere template method and one step to build an ordered porous three-dimensional scaffold that meets the requirements of the tissue engineering. Microfluidic technology is developed on the basis of microelectronics, microfabrication, biological engineering and nanotechnology. By using the interaction between the flow shear force and the surface tension of the fluid, the new cross section divides the continuous fluid into the micrometer droplets in the microscale channel. The microfluidic method is convenient and simple, and can adjust the size of the hole in real time. The control range is between several hundred microns and hundreds of microns. In this paper, the design of microfluidic chips: the design of different microfluidic chips can realize the formation of different emulsions, such as single emulsion, double emulsion, multiple emulsion, etc., according to the emulsion template needed in the later period. The flow channel is divided into the internal phase entrance, the external phase entrance and the collection end. In order to better observe the shear condition of the internal and external liquid droplets, the length of the length square tube is added to the shearing place. The effect of the pipe size on the droplet size is discussed by the design of the microfluidic chip, and the single dispersion and stability are discussed. (two) preparation of emulsion droplet templates: on the basis of microfluidic technology and microfluidic chip construction, in order to achieve good biocompatibility and biodegradability of ordered porous scaffolds, three kinds of emulsion droplets were prepared to try to use sodium alginate or PEG-DA or silk fibroin. As an external solution, the protein solution is an external solution, nontoxic edible oil or silicone oil is used as an internal solution to form an emulsion droplet. To explore the appropriate surface active agent, the droplets will not break down, and the droplets will not be fused to form a regular droplet template. The droplet size and droplet can be discussed by the flow velocity regulation. The influence of formation spacing. (three) preparation of ordered porous materials: the study determines that after the three systems can stabilize the emulsion droplet template, the.PEG-DA is photosensitive to the material, and the PEG-DA can be polymerized into gelatin through UV irradiation. Calcium ions are added to sodium alginate and calcium ions are replaced by sodium ions to form calcium alginate. It makes sodium alginate gelatinization. Silk fibroin can form a sponge like structure after freezing drying. Using alcohol to remove the emulsion drop template in the scaffold material and freeze drying to obtain the scaffold materials with ordered pore structure. (four) biological application of ordered porous materials: the core of tissue engineering, scaffold Material plays an important role in the adhesion, differentiation and proliferation of cultured cells. HepG2 cells are inoculated on the obtained PEG-DA porous scaffold to explore its application in cell culture.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08

【参考文献】