本文選題：組織工程 + 微流控技術(shù)　； 參考：《東南大學(xué)》2017年碩士論文

【摘要】：20世紀(jì)80年代,組織工程學(xué)被提出,通過科學(xué)家的努力,利用組織工程技術(shù)在裸鼠上成功形成具有皮膚覆蓋的人耳廓形態(tài)軟骨,這意味著組織工程從基礎(chǔ)邁向臨床應(yīng)用的廣闊前景。組織工程被定義為一門以細(xì)胞生物學(xué)和材料科學(xué)相結(jié)合,進(jìn)行體外或體內(nèi)構(gòu)建組織或器官的新興學(xué)科。組織工程的基本原理是,從病人體內(nèi)分離取得種子細(xì)胞,種植在具有良好生物相容性并且在體內(nèi)可逐步降解吸收的組織工程多孔支架上形成細(xì)胞-支架復(fù)合物,細(xì)胞在支架上增殖、分化,然后將此復(fù)合物植入同一病人組織病損部位,在體內(nèi)繼續(xù)增殖并分泌細(xì)胞外基質(zhì),形成新的與自身功能和形態(tài)相適應(yīng)的組織或器官,從而達(dá)到修復(fù)病損組織或器官的目的。組織工程的核心是建立細(xì)胞與生物支架結(jié)構(gòu),支架對(duì)培養(yǎng)細(xì)胞的黏附、分化、增殖、組織形成起著重要作用。好的組織工程支架需要具有三維多孔結(jié)構(gòu)、良好的生物相容性、可生物降解性、一定的生物力學(xué)性能。傳統(tǒng)的支架制備技術(shù)主要有:纖維編織技術(shù)、成孔劑析出法、氣體發(fā)泡技術(shù)、熱致相分離等,制備支架的方法多種多樣,但各有優(yōu)缺點(diǎn),很難同時(shí)滿足理想組織工程支架的幾個(gè)特點(diǎn)。后來,一種新的制備方式被提出,科學(xué)家們發(fā)展出以尺寸均一的固體微球作為模板制備得到孔洞大小均一、孔隙率高的組織工程支架。這種方法能同時(shí)滿足對(duì)支架的性能要求,但步驟仍較為繁瑣。在本論文中,我們提出利用微流控技術(shù)對(duì)微球模板法進(jìn)行改進(jìn),一步式構(gòu)建滿足組織工程要求的有序多孔三維支架。微流控技術(shù)是一門在微電子、微制作、生物工程和納米技術(shù)等基礎(chǔ)上發(fā)展而來的全新的交叉學(xué)科,通過利用流體的流動(dòng)剪切力與表面張力之間的相互作用,在微尺度通道內(nèi)將連續(xù)流體分割成離散的尺寸在微米級(jí)別的液滴。微流控方法方便簡單,可實(shí)時(shí)調(diào)控支架孔洞大小,調(diào)控范圍在幾十微米至幾百微米之間,適用于不同組織培養(yǎng),獲得支架連通性好,大小均一,可滿足組織工程的多種需求。本論文具體開展工作如下:(一)微流控芯片的構(gòu)建:不同微流控芯片的設(shè)計(jì)可以實(shí)現(xiàn)單重乳液、雙重乳液、多重乳液等不同乳液的形成。根據(jù)后期所需乳液模板的液滴為單重乳液,設(shè)計(jì)相應(yīng)的微流控芯片,流體通道分為內(nèi)相入口端、外相入口端、收集端,為了更好的觀察內(nèi)外相液滴剪切情況,在剪切處加入一定長度方管。通過微流控芯片的設(shè)計(jì),探討剪切端管道尺寸對(duì)液滴尺寸的影響、單分散性、穩(wěn)定性的影響因素,最終獲得穩(wěn)定的乳液液滴。(二)乳液液滴模板的制備:在微流控技術(shù)和微流控芯片構(gòu)建的基礎(chǔ)上,為了實(shí)現(xiàn)有序多孔支架良好的生物相容性和可生物降解性,提出三種乳液液滴的制備體系,嘗試?yán)煤Ｔ逅徕c或PEG-DA或絲素蛋白溶液作為外相溶液,無毒性的食用油或是硅油作為內(nèi)相溶液,形成乳液液滴。探索合適的表面活性劑,使液滴不會(huì)破裂,液滴間不會(huì)相互融合,以形成排列規(guī)整的液滴模板。通過對(duì)流體流速的調(diào)控,可探討內(nèi)外相流速對(duì)液滴尺寸、液滴形成間距的影響。(三)有序多孔材料的制備:研究確定三種體系下能穩(wěn)定形成乳液液滴模板后,對(duì)材料進(jìn)行固化。PEG-DA具有光敏性,通過紫外光照射,可使PEG-DA聚合成凝膠。在海藻酸鈉中加入鈣離子,通過鈣離子置換鈉離子形成海藻酸鈣分子,使得海藻酸鈉凝膠化。絲素蛋白經(jīng)過冷凍干燥處理后可形成海綿狀結(jié)構(gòu)。利用酒精將支架材料中的乳液液滴模板洗去,經(jīng)過冷凍干燥,獲得具有有序孔洞結(jié)構(gòu)的支架材料。(四)有序多孔材料的生物應(yīng)用探究:作為組織工程的核心,支架材料對(duì)培養(yǎng)細(xì)胞的黏附、分化、增殖起著重要作用。在獲得的PEG-DA多孔支架上接種HepG2細(xì)胞,探究其在細(xì)胞培養(yǎng)上的應(yīng)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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R318.08

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