發(fā)布時(shí)間：2018-05-16 21:49

  本文選題：蠶絲-PLGA支架 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《浙江省醫(yī)學(xué)科學(xué)院》2015年碩士論文

【摘要】：前交叉韌帶是膝關(guān)節(jié)內(nèi)重要穩(wěn)定結(jié)構(gòu),損傷后缺乏自我愈合能力,通常需要利用移植物關(guān)節(jié)鏡下重建前交叉韌帶。各種移植物都有其局限性,利用組織工程方法構(gòu)建有望克服目前移植物的問題。理想的組織工程支架應(yīng)具有良好的生物安全性、組織相容性,能為韌帶/肌腱早期活動(dòng)提供可靠的力學(xué)強(qiáng)度,為種子細(xì)胞的生長(zhǎng)繁殖和生理功能發(fā)揮提供空間。本實(shí)驗(yàn)通過(guò)蠶絲-PLGA支架的細(xì)胞毒性、生物安全性,力學(xué)強(qiáng)度細(xì)胞相容性,支架降解液與細(xì)胞增殖的研究,得出蠶絲-PLGA支架是一種較理想的組織工程支架。第一部分：蠶絲-PLGA支架的細(xì)胞毒性及生物安全性目的：對(duì)蠶絲-PLGA混合編織支架進(jìn)行體外細(xì)胞毒性實(shí)驗(yàn)和動(dòng)物生物安全性評(píng)價(jià),探討蠶絲-PLGA混合編織支架材料的細(xì)胞毒性和生物安全性,為該材料臨床應(yīng)用提供理論依據(jù)。方法：制備蠶絲-PLGA細(xì)絲混合編織支架,并制備該支架材料的浸提液,通過(guò)MTT法檢測(cè)25%、50%、100%濃度的蠶絲-PLGA混合編織支架材料浸提液對(duì)兔骨髓間充質(zhì)干細(xì)胞(MSCs)的細(xì)胞毒性作用；采用蠶絲-PLGA細(xì)絲混合編織支架浸提液進(jìn)行動(dòng)物實(shí)驗(yàn)研究：急性全身毒性試驗(yàn)、皮內(nèi)刺激試驗(yàn)、溶血試驗(yàn)、熱原試驗(yàn),并根據(jù)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行生物安全性的分析、評(píng)價(jià)。結(jié)果：在細(xì)胞毒性中,MSCs于不同濃度實(shí)驗(yàn)組及陰性對(duì)照組中生長(zhǎng)均好,細(xì)胞呈長(zhǎng)梭形等形態(tài),細(xì)胞形態(tài)飽滿有光澤,MTT法結(jié)果表明各時(shí)間點(diǎn)各濃度實(shí)驗(yàn)組與陰性對(duì)照組之間的A值差異不顯著(P0.05),各時(shí)間點(diǎn)各濃度浸提液對(duì)MSCs細(xì)胞的相對(duì)增殖率均在93%以上,毒性分級(jí)為0級(jí)或1級(jí),無(wú)細(xì)胞毒性。同時(shí)支架材料無(wú)急性全身毒性作用,無(wú)皮內(nèi)刺激反應(yīng),不具溶血作用,符合生物材料溶血試驗(yàn)的要求。熱原試驗(yàn)表明,3只實(shí)驗(yàn)兔體溫升高數(shù)分別為0.4、0.2、0.3℃,均低于0.6℃,總值小于1.4℃,不具熱原反應(yīng)。結(jié)論：蠶絲-PLGA混合編織支架材料細(xì)胞相容性良好,無(wú)細(xì)胞毒性,符合材料毒性的安全標(biāo)準(zhǔn),而且具有良好的生物相容性,是一種較有潛力的組織工程韌帶/肌腱的支架材料。第二部分：蠶絲-PLGA支架的力學(xué)強(qiáng)度和生物相容性目的：通過(guò)對(duì)蠶絲-PLGA支架的力學(xué)性能及其與骨髓間充質(zhì)干細(xì)胞體外共培養(yǎng)的細(xì)胞相容性實(shí)驗(yàn),探討使用該材料構(gòu)建組織工程韌帶的可行性。方法：通過(guò)捻擰編織法和緯編針織法制備蠶絲-PLGA共聚物細(xì)絲混合支架,并以纖維連接蛋白作表面修飾,檢測(cè)支架材料的力學(xué)性能。將制備的兔骨髓間充質(zhì)干細(xì)胞種植在蠶絲-PLGA共聚物細(xì)絲混合支架上進(jìn)行體外共培養(yǎng),觀察細(xì)胞與支架復(fù)合生長(zhǎng)、基質(zhì)形成,以及細(xì)胞與支架結(jié)合的情況。結(jié)果：蠶絲-PLGA混合編織支架呈乳白色,質(zhì)地均勻,韌性強(qiáng),為螺旋上升的繩索狀,直徑為2.3mm。支架材料的最大負(fù)荷、拉伸強(qiáng)度、斷點(diǎn)伸長(zhǎng)率、彈性模量分別為(315.06 ±30.77)N、(75.83±7.46)MPa、(61.39±7.26)％、(213.58±23.45)MPa。掃描電鏡觀察顯示,骨髓間充質(zhì)干細(xì)胞貼附于支架表面生長(zhǎng),增殖良好,細(xì)胞大多呈梭形,伸出偽足匍匐于材料的表面,形態(tài)較佳,伸展良好,呈立體狀生長(zhǎng),并分泌基質(zhì)。緯編針織法制備的蠶絲-PLGA復(fù)合網(wǎng)狀支架外觀呈乳白色,質(zhì)地均勻,韌性強(qiáng),支架寬約為5mm。支架的最大載荷、抗拉強(qiáng)度、斷點(diǎn)伸長(zhǎng)率、彈性模量分別為(118.32± 11.21)N、(52.90±5.03)MPa、49.79%±5.16%、(177.25±19.18)MPa。支架-MSCs復(fù)合物體外培養(yǎng)2天的掃描電鏡觀察顯示：MSCs細(xì)胞附于支架表面生長(zhǎng),黏附在支架上,呈梭形,形態(tài)較佳,呈立體狀生長(zhǎng),增殖良好,并分泌基質(zhì)。結(jié)論：表明蠶絲-PLGA共聚物繩狀和網(wǎng)狀支架都具有良好的機(jī)械性能及細(xì)胞相容性,是一種較有潛力的組織工程韌帶的支架材料。第三部分：蠶絲-PLGA支架降解液與骨髓間充質(zhì)干細(xì)胞的增殖目的：觀察蠶絲-PLGA細(xì)絲混合編織支架體外長(zhǎng)期降解過(guò)程中降解液對(duì)兔骨髓間充質(zhì)干細(xì)胞增殖活性的影響。方法：將蠶絲-PLGA細(xì)絲混合編織支架材料置于完全培養(yǎng)基中體外降解14周,每周換液1次,測(cè)定各周支架降解液的pH值。將兔骨髓間充質(zhì)干細(xì)胞分組培養(yǎng),實(shí)驗(yàn)組加入各周支架降解液和新鮮完全培養(yǎng)基各100μL,陰性對(duì)照組加入完全培養(yǎng)基200μL,培養(yǎng)4d.MTT法檢測(cè)細(xì)胞增殖、生長(zhǎng)情況。結(jié)果：①支架降解液pH值的變化：前3周下降緩慢,從7.00降到6.89；第4周起下降較快,6-11周較低,在5.16-5.67之間；12-14周呈上升趨勢(shì),回升到6.95。②骨髓間充質(zhì)干細(xì)胞形態(tài)：實(shí)驗(yàn)組及陰性對(duì)照組細(xì)胞增殖生長(zhǎng)及形態(tài)狀況基本相似。降解7-10周支架降解液對(duì)細(xì)胞的生長(zhǎng)有抑制作用,細(xì)胞數(shù)量相對(duì)較少、較疏,而其余各周支架降解液對(duì)細(xì)胞生長(zhǎng)無(wú)明顯抑制作用。③骨髓間充質(zhì)干細(xì)胞的增殖：1-6周及11-14周的支架降解液對(duì)細(xì)胞增殖無(wú)顯著影響,細(xì)胞相對(duì)增殖率均在92.1%以上,毒性分級(jí)為0或1級(jí)；7-10周的支架降解液雖對(duì)細(xì)胞增殖有抑制作用,但細(xì)胞相對(duì)增殖率為82.5%-87.9%,毒性分級(jí)為1級(jí),為合格。結(jié)論：表明蠶絲-PLGA共聚物混合編織支架降解液具有良好的細(xì)胞相容性。
[Abstract]:Anterior cruciate ligament (ACL) is an important stable structure in the knee joint, and it lacks self healing ability after injury. It is usually necessary to reconstruct the anterior cruciate ligament using graft arthroscopy. All kinds of grafts have its limitations. The tissue engineering scaffold should have good biological safety. Integrity and histocompatibility can provide a reliable mechanical strength for early ligament / tendon activity and provide space for the growth, reproduction and physiological function of seed cells. In this experiment, the cytotoxicity, biosafety, mechanical strength cell compatibility of the silk -PLGA scaffold, the study of the scaffold degradation liquid and cell proliferation, and the -PLGA branch of silkworm silk were obtained in this experiment. The frame is an ideal scaffold for tissue engineering. Part 1: cytotoxicity and biological safety of silk -PLGA scaffold: the cytotoxicity test and animal biological safety evaluation of silk -PLGA braided braid in vitro, and the cytotoxicity and biological safety of the silk -PLGA braid scaffold materials are discussed. The clinical application provides the theoretical basis. Methods: the silk -PLGA filaments mixed braiding stents were prepared and the extracts of the scaffold material were prepared. The cytotoxic effects of the extracts of the silk -PLGA mixed woven scaffold material on the rabbit bone marrow mesenchymal stem cells (MSCs) were detected by MTT, and the mixed braiding of the silk -PLGA filaments was used. Animal experimental study of scaffold leaching solution: acute systemic toxicity test, intradermal stimulation test, hemolysis test, pyrogen test, and biological safety analysis according to the experimental data. Results: in cytotoxicity, MSCs grew well in different concentration experimental group and negative control group, cell shape was long shuttle shape, cell shape The MTT method showed that there was no significant difference in A between the experimental group and the negative control group at each time point (P0.05). The relative proliferation rate of the extract solution to MSCs cells at each time point was above 93%, the toxicity was grade 0 or 1, and the scaffold material had no acute systemic toxicity and no intradermal spines. The irritable reaction, which was not hemolytic, accorded with the requirements of the hemolytic test of biomaterials. The thermometer test showed that the temperature rise of the 3 experimental rabbits was 0.4,0.2,0.3 C, lower than 0.6, and less than 1.4, and no thermion reaction. Conclusion: the silk -PLGA braided scaffold has good cytocompatibility and no cytotoxicity, which conforms to the toxicity of the material. Safety standards, with good biocompatibility, is a potential scaffold for tissue engineering ligaments / tendons. The second part: the mechanical strength and biocompatibility of silk -PLGA scaffold: the mechanical properties of silk -PLGA scaffold and the compatibility with bone marrow mesenchymal stem cells in vitro The feasibility of using this material to construct tissue engineering ligaments was explored. Methods: silk -PLGA copolymer filaments mixed scaffold was prepared by twisting and weft knitting method and weft knitting method, and the mechanical properties of the scaffold were detected with fibronectin as surface modification. The prepared rabbit bone marrow mesenchymal stem cells were planted in the silk -PLGA copolymer. The combined growth of cells and scaffolds, the formation of matrix, and the combination of the cells with the scaffold were observed on the mixed scaffold. Results: the silk -PLGA braided braid was milky white, with a uniform texture, strong toughness, a spiral rise rope, and the maximum load, tensile strength and breakpoint elongation of the 2.3mm. scaffold. The modulus of elasticity was (315.06 + 30.77) N, (75.83 + 7.46) MPa, (61.39 + 7.26)% and (213.58 + 23.45) MPa. scanning electron microscopy (213.58 + 23.45). The bone marrow mesenchymal stem cells were attached to the surface of the scaffold, with good proliferation, mostly spindle shaped and protruding on the surface of the material. The silkworm -PLGA composite mesh scaffold prepared by weft knitting method has a milky white appearance, uniform texture and strong toughness, and the width of the scaffold is about the maximum load of 5mm. scaffold, tensile strength, elongation at breakpoint, modulus of elasticity (118.32 + 11.21) N, (52.90 + 5.03) MPa, 49.79% + 5.16%, and (177.25 + 19.18) MPa. scaffold -MSCs compound for 2 days outside. The scanning electron microscopy (SEM) showed that MSCs cells attached to the surface of the scaffold grew on the scaffold, attached to the scaffold and were spindle shaped, with a better shape, good proliferation, and secreted matrix. Conclusion: the silk -PLGA copolymer and reticular scaffold have good mechanical properties and cell compatibility, which is a potential tissue engineering toughening. The third part: the third part: the proliferation of silkworm silk scaffold degradation solution and bone marrow mesenchymal stem cells: the effect of the degradation solution on the proliferation activity of rabbit bone marrow mesenchymal stem cells in the long term degradation process of silk -PLGA filament mixed braid. Method: the mixed silk -PLGA silk braid scaffold material is placed completely The culture medium was degraded for 14 weeks in vitro and changed 1 times a week to determine the pH value of the scaffold degradation solution in each week. The rabbit bone marrow mesenchymal stem cells were grouped into groups, the experimental group was added to the scaffold degradation solution and the fresh complete medium 100 mu L, the negative control group was added to the complete medium 200 mu L, and the cell proliferation and growth were detected by 4d.MTT method. The results were as follows: 1 The changes in the pH value of the scaffold degradation fluid: the decline in the first 3 weeks was slow, from 7 to 6.89, and the decrease was faster in the fourth week, the 6-11 week was lower, and between the 12-14 weeks. The 12-14 weeks showed an upward trend, and rose to the form of 6.95. bone marrow mesenchymal stem cells: the growth growth and morphology of the cells in the experimental group and the negative control group were basically similar. The degradation of the scaffold was reduced for 7-10 weeks. The solution has a inhibition effect on cell growth, and the number of cells is relatively small and sparse, while the rest of the scaffold degradation solution has no obvious inhibitory effect on cell growth for the rest of the week. The proliferation of bone marrow mesenchymal stem cells: 1-6 weeks and 11-14 weeks of scaffold degradation has no significant effect on cell proliferation, the relative proliferation rate of the cells is above 92.1% and the toxicity classification is 0. Or 1 grade; 7-10 weeks of scaffold degradation solution had inhibitory effect on cell proliferation, but the relative proliferation rate of cells was 82.5%-87.9% and the toxicity grade was 1, which was qualified. Conclusion: the results showed that the mixed woven scaffold degradation solution of silk -PLGA copolymer had good cytocompatibility.
【學(xué)位授予單位】：浙江省醫(yī)學(xué)科學(xué)院
【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)：1898559