本文關(guān)鍵詞：低晶不溶性絲蛋白支架的制備及體外細(xì)胞實(shí)驗(yàn)研究　出處：《蘇州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 絲蛋白 多孔支架 二級(jí)結(jié)構(gòu) 力學(xué)性能 軟組織

【摘要】：蠶絲因具有良好的細(xì)胞相容性、優(yōu)良的機(jī)械性能和可生物降解性而成為一種普適性的生物材料,在組織工程領(lǐng)域引起了廣泛的關(guān)注。然而不同組織的修復(fù)對(duì)支架的微觀形貌、力學(xué)性能及降解性能等都有著不同的要求,調(diào)控支架的結(jié)構(gòu)和性能使其與人體組織相匹配成為關(guān)鍵問題。新鮮絲蛋白溶液可采用冷凍干燥法制備多孔材料,但其成孔性一般較差,不利于細(xì)胞的生長、增殖及遷移。本文先對(duì)新鮮絲蛋白溶液進(jìn)行預(yù)處理,即緩慢濃縮再稀釋獲得納米纖維絲蛋白溶液,然后用該溶液制備絲蛋白支架,觀察絲蛋白材料處理前后微觀形貌的變化。實(shí)驗(yàn)結(jié)果表明,緩慢濃縮處理可以誘導(dǎo)絲蛋白溶液中納米球向納米纖維轉(zhuǎn)變,明顯提高支架的成孔性從而可獲得具有良好孔結(jié)構(gòu)的絲蛋白材料。在此基礎(chǔ)上,向緩慢濃縮處理的絲蛋白溶液中添加不同含量的甘油制備結(jié)構(gòu)可調(diào)的支架,并對(duì)支架的二級(jí)結(jié)構(gòu)、穩(wěn)定性、降解性能等進(jìn)行研究。實(shí)驗(yàn)結(jié)果表明甘油可以改變絲蛋白的二級(jí)結(jié)構(gòu),使絲蛋白由無規(guī)結(jié)構(gòu)向silk I和silk II結(jié)構(gòu)轉(zhuǎn)變,提高支架的穩(wěn)定性,甘油含量對(duì)力學(xué)性能有著一定的調(diào)控作用,從而通過添加甘油及改變其含量實(shí)現(xiàn)了對(duì)支架結(jié)構(gòu)和性能的調(diào)控。另一方面,基于絲蛋白分子自組裝原理,在不添加任何有機(jī)試劑的條件下,通過改變分子運(yùn)動(dòng)性進(jìn)一步實(shí)現(xiàn)對(duì)絲蛋白支架結(jié)構(gòu)和性能的調(diào)控。將絲蛋白納米纖維溶液與新鮮絲蛋白溶液按絲蛋白干重比1:15進(jìn)行混合,通過改變混合溶液的冷凍-解凍次數(shù)或?qū)ζ溥M(jìn)行50oC溫度處理來調(diào)節(jié)絲蛋白在溶液中的組裝形態(tài),隨后采用冷凍干燥法制備不同的絲蛋白多孔支架。結(jié)果表明,隨著冷凍-解凍次數(shù)的增加,支架中silk II含量增加,壓縮模量增大,而溫度處理則有利于支架中silk I結(jié)構(gòu)增加,壓縮模量相應(yīng)降低。因此,反復(fù)冷凍-解凍和溫度處理也可以實(shí)現(xiàn)對(duì)二級(jí)結(jié)構(gòu)和力學(xué)性能的調(diào)控,獲得性能不同的支架。最后,將SD大鼠骨髓間充質(zhì)干細(xì)胞(r BMSCs)在支架上進(jìn)行體外細(xì)胞培養(yǎng),研究不同性能的絲蛋白多孔支架對(duì)r BMSCs增殖和分化的影響。結(jié)果表明細(xì)胞在所有支架上增殖情況良好,且實(shí)驗(yàn)組增殖情況明顯優(yōu)于對(duì)照組。DNA含量結(jié)果進(jìn)一步證明了支架良好的細(xì)胞相容性,且支架模量越低,細(xì)胞增殖情況越好。隨后選取不同力學(xué)性能的支架復(fù)合r BMSCs進(jìn)行體外培養(yǎng),研究力學(xué)性能變化對(duì)干細(xì)胞分化的影響,結(jié)果表明細(xì)胞在模量在8~17k Pa的絲蛋白支架上優(yōu)先向成肌細(xì)胞分化,而在壓縮模量在1~7k Pa的支架上向內(nèi)皮細(xì)胞分化。綜上所述,本論文通過溫和的方法調(diào)控絲蛋白的納米結(jié)構(gòu)、親水作用及分子運(yùn)動(dòng)性,從而制備微觀結(jié)構(gòu)、二級(jí)結(jié)構(gòu)、力學(xué)性能可控的絲蛋白多孔支架,以實(shí)現(xiàn)不同的功能,提高了絲蛋白在不同組織修復(fù)領(lǐng)域的適用性。
[Abstract]:Because silk has a good biocompatibility, excellent mechanical properties and biodegradability and become a kind of universal biological material, has attracted wide attention in the field of tissue engineering. However, the morphology of different tissues of stent repair, mechanical properties and degradation properties have different requirements, structure the performance of the control and support of human tissue matching becomes a key issue. The fresh silk fibroin solution can be prepared by freeze drying method of porous materials, but the hole is generally poor, is not conducive to cell growth, proliferation and migration. The article focuses on the fresh silk fibroin solution pretreatment, slowly concentrated and then diluted to obtain nano fiber protein solution, preparation of silk fibroin protein and then the solution was observed, the morphology of silk protein materials before and after processing. The experimental results show that the slow concentration could induce silk protein Change of nanoparticles solution to nano fiber, improve stent porosity can be obtained with good pore structure of silk protein materials. On this basis, adding different content to the silk fibroin solution slowly in glycerol concentration treatment preparation support structure can be adjusted, and the two level structure, the stability of support study on degradation performance, etc. The experimental results show that glycerol can change the two level structure of silk protein, the silk protein changed from random structure to silk I and silk II structure, improve the stability of the stent, the glycerol content plays a role in control of certain mechanical properties, and by adding glycerol and change its content to achieve the regulation on the structure and properties of the scaffold. On the other hand, based on the principle of molecular self-assembly of silk protein, without adding any organic reagent conditions, by changing the molecular motion of the further realization of the silk scaffold structure Regulation and performance. The silk fibroin nanofiber solution and fresh silk protein fibroin solution according to dry weight ratio of 1:15 were mixed by changing the mixed solution of freezing thawing cycles or the 50oC temperature to adjust the assembly structure of silk protein in solution, followed by freeze drying method of silk fibroin porous scaffold preparation different. The results show that with the increase of freezing thawing cycles, increase the silk content of II stent, compression modulus increases, while the temperature is conducive to the increase of silk in I frame structure, compression modulus decreased. Therefore, repeated freezing and thawing temperature treatment can achieve the control of the two level structure and mechanical properties the performance of different support. Finally, the SD of rat bone marrow mesenchymal stem cells (R BMSCs) were cultured on the scaffold, study the different properties of silk fibroin porous scaffold on the proliferation of BMSCs and R The effect of differentiation. The results showed that the cell proliferation in all scaffolds, and cell growth in experimental group was significantly better than the control group.DNA content results prove that support good cell compatibility, and support the lower modulus, the proliferation of R BMSCs composite scaffold is better. Then choosing different mechanical properties in vitro, influence the study on mechanics behavior of the differentiation of stem cells. The results show that the silk scaffold modulus in 8~17k Pa cells in preference to differentiation into muscle cells, and the compression modulus in support of Pa 1~7k points to the endothelial cells. In summary, the gentle method in this paper through the nano structure regulation of silk protein, and hydrophilic interaction molecular motion, thus preparing the micro structure, two level structure of silk fibroin porous scaffold, controllable mechanical properties, in order to achieve different functions, improve the silk protein in different tissue repair The applicability of the complex domain.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R318.08

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本文編號(hào)：1390881