再生絲素纖維的制備及其在人工韌帶中的應(yīng)用
發(fā)布時(shí)間：2019-02-19 21:10
【摘要】：前交叉韌帶(Anterior cruciate ligament,ACL)是膝關(guān)節(jié)內(nèi)的核心結(jié)構(gòu),對(duì)膝關(guān)節(jié)的穩(wěn)定性起著重要作用。目前我國因運(yùn)動(dòng)和交通事故等造成ACL損傷的現(xiàn)象明顯增多,其損傷斷裂會(huì)導(dǎo)致膝關(guān)節(jié)穩(wěn)定性差等問題的出現(xiàn),嚴(yán)重?fù)p傷還會(huì)影響膝關(guān)節(jié)功能,因此我國對(duì)ACL修復(fù)材料市場需求量急劇增加。絲素蛋白(SF)因其良好的可塑性、生物相容性,可以克服早期移植物的免疫排斥反應(yīng)而成為制備生物醫(yī)用材料的理想素材。發(fā)揮再生SF纖維可降解性以及天然SF纖維優(yōu)良力學(xué)性質(zhì)的優(yōu)勢,本文擬制備以SF為原料的可降解型再生SF纖維復(fù)合韌帶。采用氯化鈣(Ca Cl2)-甲酸(FA)溶解體系在常溫下破壞原纖內(nèi)的結(jié)晶結(jié)構(gòu)分纖溶解蠶絲,利用濕法紡絲、靜電紡絲技術(shù)制備再生SF纖維,探討不同溶解參數(shù)對(duì)溶液溶解性的影響以及溶液性質(zhì)對(duì)再生加工過程、纖維形貌及力學(xué)性能的影響。在此研究基礎(chǔ)上,通過體外降解和細(xì)胞培養(yǎng)實(shí)驗(yàn)驗(yàn)證再生SF纖維的降解性及生物相容性。該溶解體系下溶液中的原纖結(jié)構(gòu)為制備高性能再生纖維提供保障,故選用再生SF長絲、靜電紡再生SF納米纖維以及天然SF纖維為原料構(gòu)建再生SF纖維復(fù)合韌帶,為開拓以SF為基材的韌帶修復(fù)材料提供新思路。傳統(tǒng)溶解方法(Ca Cl2-C2H5OH-H2O)極大地破壞了蠶絲的原纖結(jié)構(gòu),甚至是分子結(jié)構(gòu),嚴(yán)重影響了再生SF材料的性能,同時(shí)對(duì)中性鹽濃度、溶解溫度以及溶解時(shí)間的要求更高。本文采用Ca Cl2-FA溶解體系通過強(qiáng)極性離子產(chǎn)生的水化作用使纖維溶脹、體積增大,削弱了分子間氫鍵作用,可以常溫條件下分纖溶解蠶絲并破壞原纖內(nèi)的β-折疊結(jié)晶結(jié)構(gòu)達(dá)到溶解蠶絲的目的。在SF溶液及干燥膜中都能清晰觀察到原纖結(jié)構(gòu),溶液中的原纖尺寸隨Ca Cl2濃度、溶解溫度的增加呈現(xiàn)減小的趨勢。該溶解方法能獲得粘度更高的溶液,SF在溶液中的溶解度會(huì)隨著Ca Cl2濃度、溶解溫度的增加而增加。此外,該溶解法制備的SF膜材料為水不溶性物質(zhì),去離子水浸泡可以誘導(dǎo)其由無規(guī)卷曲向β-折疊結(jié)構(gòu)轉(zhuǎn)變。溶液中的原纖結(jié)構(gòu)在濕法紡絲過程中經(jīng)過凝固浴并受到剪切作用重組成纖維,以水為凝固浴、質(zhì)量分?jǐn)?shù)為15.0 wt.%的SF溶液濕法紡絲,4倍牽伸后可獲得直徑為10.8±1.4μm、斷裂應(yīng)力達(dá)358.4±43.2 MPa、斷裂伸長率達(dá)24.8±4.2%的SF長絲,力學(xué)性能有明顯提高。此外,水浴凝固具有對(duì)環(huán)境無污染、低成本、易操作等優(yōu)點(diǎn)。將Ca Cl2-FA溶解體系獲得的SF溶液干燥成膜后溶解于甲酸得到仍保留原纖結(jié)構(gòu)的SF溶液進(jìn)行靜電紡絲,采用自制滾筒收集裝置獲得取向納米纖維,并與細(xì)胞外基質(zhì)(ECM)具有相似的尺寸結(jié)構(gòu)。討論不同Ca Cl2濃度制備得到的紡絲液、不同SF濃度對(duì)成纖性的影響,分析滾筒轉(zhuǎn)速和拉伸對(duì)纖維取向性的影響。結(jié)果表明SF質(zhì)量分?jǐn)?shù)為6.0 wt.%、Ca Cl2質(zhì)量分?jǐn)?shù)為5.0 wt.%、滾筒轉(zhuǎn)速為1000 rpm獲得直徑為221±20nm的纖維,1倍牽伸后纖維的斷裂應(yīng)力和斷裂伸長率分別達(dá)到了18.6±3.8 MPa和15.1±2.5%,纖維局部取向度明顯改善,力學(xué)性能較Ca Cl2-C2H5OH-H2O溶解方法的纖維有較大幅度提高。再生SF纖維在PBS溶液中降解相對(duì)緩慢,降解60天,SF納米纖維重量損失15%,纖維間部分出現(xiàn)粘連現(xiàn)象;SF長絲重量損失8.9%,降解只發(fā)生在纖維表面、出現(xiàn)部分裂痕。而蛋白酶XIV溶液對(duì)材料的降解能力明顯增強(qiáng),降解48h時(shí),SF納米纖維幾乎完全降解,SF長絲重量損失61.5%,纖維主體分解。取向排列的SF納米纖維較亂序納米纖維可以在短時(shí)間內(nèi)明顯促進(jìn)細(xì)胞的大量生長,SF長絲和天然SF纖維也利于細(xì)胞生長粘附,具有良好的生物相容性。基于高性能再生SF纖維的制備,采用立式錠子編織機(jī),以SF長絲為軸紗,天然SF纖維編織物、靜電紡SF納米纖維包裹的形式構(gòu)建再生SF纖維復(fù)合韌帶,結(jié)構(gòu)穩(wěn)定,斷裂強(qiáng)力達(dá)2581.7±23.7 N,滿足人體ACL的要求。不同SF纖維在酶溶液中呈現(xiàn)出降解差異,再生SF纖維的快速降解為細(xì)胞再生提供有利的生長空間,降解速度慢、性質(zhì)穩(wěn)定的天然SF纖維支架保證了韌帶材料的力學(xué)要求,實(shí)現(xiàn)了不同材料降解速度差異化的設(shè)計(jì)要求。本文采用Ca Cl2-FA溶解體系獲得的再生SF纖維表現(xiàn)出良好的性能,開發(fā)設(shè)計(jì)出以SF纖維為原料、既保證力學(xué)要求又充分發(fā)揮其良好生物相容性和可降解性的人工韌帶,為制備韌帶修復(fù)材料提供一定的實(shí)驗(yàn)依據(jù)。
[Abstract]:Anterior cruciate ligament (ACL) is the core structure in the knee joint and plays an important role in the stability of the knee joint. At present, the phenomenon of ACL injury caused by sports and traffic accidents in our country is obviously increased, and the damage and fracture can cause the problems of poor stability of the knee joint and the like, and the serious injury can also affect the function of the knee joint, so the demand of the ACL repair material market is rapidly increased. the silk fibroin (SF) has good plasticity and biocompatibility, and can overcome the immune rejection reaction of the early graft and become the ideal material for preparing the biological medical material. It is proposed to prepare the degradable regenerated SF-fiber composite ligament with SF as the raw material to play the advantage of the degradability of the regenerated SF fibers and the excellent mechanical properties of the natural SF fibers. a calcium chloride (Ca 2)-formic acid (FA) dissolving system is adopted to destroy the crystalline structure of the original fiber at normal temperature to dissolve the silk, and the regenerated SF fiber is prepared by wet spinning and electrostatic spinning technology, The effects of different dissolution parameters on the solubility of the solution and the effect of the solution properties on the regeneration process, the morphology and the mechanical properties of the solution were discussed. On the basis of this study, the degradation and biocompatibility of the regenerated SF fibers were verified by in vitro degradation and cell culture experiments. the original fiber structure in the solution under the dissolution system is used for preparing the high-performance regenerated fiber, so that the regenerated SF filament, the electrostatic spinning regenerated SF nano-fiber and the natural SF fiber are used as the raw materials to construct the regenerated SF-fiber composite ligament, in order to provide a new thought for the development of the ligament repair material with the SF as the base material. The traditional dissolution method (Ca 2-C2H5OH-H2O) greatly destroys the original fiber structure of the silk, and even the molecular structure, and the performance of the regenerated SF material is seriously affected, and meanwhile, the requirement of the neutral salt concentration, the dissolution temperature and the dissolution time is higher. In this paper, the dissolution system of Ca 2-FA is used to make the fiber swell, the volume is increased, the intermolecular hydrogen bond is weakened by the hydration of the strong polar ion, and the silk can be dissolved in the fiber at normal temperature and the silk-folded crystal structure in the original fiber can be destroyed to achieve the purpose of dissolving the silk. The original fiber structure can be clearly observed in the SF solution and the dry film, and the size of the original fiber in the solution changes with the concentration of Ca 2 and the increase of the dissolution temperature. The dissolution method can obtain a solution with higher viscosity, and the solubility of the SF in the solution increases with the increase of the concentration of Ca 2 and the dissolution temperature. In addition, the SF film material prepared by the dissolution method is a water-insoluble substance, and the deionized water is soaked to induce the SF film material to be changed from the random curl to the crotch-folding structure. the original fiber structure in the solution is subjected to a coagulation bath in a wet spinning process and is subjected to shearing action to form a fiber, and the water is a coagulating bath, and the mass fraction is 15. 0wt. The mechanical properties of SF-filament with a diameter of 10. 8-1.4. m, a fracture stress of 358. 4-43. 2 MPa and an elongation of break of 24. 8-4. 2% can be obtained after 4-fold drawing, and the mechanical properties of the SF-filament are obviously improved. in addition, the water bath solidification has the advantages of no pollution to the environment, low cost, easy operation and the like. The SF solution obtained by the Ca 2-FA dissolving system is dried to form a film, then dissolved in formic acid to obtain the SF solution which still retains the original fiber structure to carry out electrostatic spinning, and the self-made roller collecting device is adopted to obtain the oriented nano-fiber and has a similar size structure as the extracellular matrix (ECM). The influence of different SF concentration on the fiber orientation was discussed, and the effect of roller speed and tensile on the fiber orientation was analyzed. The results show that the SF mass fraction is 6.0 wt.%, the mass fraction of Ca 2 is 5.0 wt. The fiber with the diameter of 221-20nm was obtained at the speed of 1000 rpm. The fracture stress and the elongation at break of the fiber were 18. 6-3. 8 MPa and 15. 1-2.5%, respectively. The local orientation of the fiber was obviously improved, and the mechanical properties of the fiber with higher mechanical properties were greatly improved than that of the Ca-2-C2H5OH-H2O dissolving method. The degradation of the regenerated SF fibers in the PBS solution was relatively slow, the degradation was 60 days, the weight loss of the SF nano-fiber was 15%, the fiber-to-fiber part had a blocking phenomenon, the weight loss of the SF filament was 8. 9%, and the degradation only occurred on the surface of the fiber, and a partial crack appeared. and the degradation ability of the protease XIV solution to the material is obviously enhanced, and when the degradation 48h, the SF nano-fiber is almost completely degraded, the weight loss of the SF filament is 61.5%, and the fiber body is decomposed. The SF nano-fiber with the orientation arrangement can obviously promote the large-volume growth of the cells in a short time, and the SF filaments and the natural SF fibers are also beneficial to cell growth and adhesion, and have good biocompatibility. based on the preparation of high-performance regenerated SF fiber, a vertical spindle knitting machine is adopted, the SF filament is a shaft yarn, a natural SF fiber braid and an electrostatic spinning SF nano-fiber are wrapped, the regenerated SF fiber composite ligament is constructed, the structure is stable, the breaking strength is up to 2581.7 to 237.N, and the requirements of the human body ACL are met. the different SF fibers exhibit a degradation difference in the enzyme solution, the rapid degradation of the regenerated SF fiber provides an advantageous growth space for cell regeneration, the degradation speed is slow, and the stable natural SF fiber support ensures the mechanical requirements of the ligament material, and the design requirements of differential speed differentiation of different materials are realized. In this paper, the regenerated SF fibers obtained by the Ca 2-FA dissolving system show good performance, and the development of artificial ligament with SF fiber as the raw material, which not only guarantees the mechanical requirements but also gives full play to its good biocompatibility and degradability, provides some experimental basis for the preparation of the ligament repair material.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R318.08
本文編號(hào)：2426877
[Abstract]:Anterior cruciate ligament (ACL) is the core structure in the knee joint and plays an important role in the stability of the knee joint. At present, the phenomenon of ACL injury caused by sports and traffic accidents in our country is obviously increased, and the damage and fracture can cause the problems of poor stability of the knee joint and the like, and the serious injury can also affect the function of the knee joint, so the demand of the ACL repair material market is rapidly increased. the silk fibroin (SF) has good plasticity and biocompatibility, and can overcome the immune rejection reaction of the early graft and become the ideal material for preparing the biological medical material. It is proposed to prepare the degradable regenerated SF-fiber composite ligament with SF as the raw material to play the advantage of the degradability of the regenerated SF fibers and the excellent mechanical properties of the natural SF fibers. a calcium chloride (Ca 2)-formic acid (FA) dissolving system is adopted to destroy the crystalline structure of the original fiber at normal temperature to dissolve the silk, and the regenerated SF fiber is prepared by wet spinning and electrostatic spinning technology, The effects of different dissolution parameters on the solubility of the solution and the effect of the solution properties on the regeneration process, the morphology and the mechanical properties of the solution were discussed. On the basis of this study, the degradation and biocompatibility of the regenerated SF fibers were verified by in vitro degradation and cell culture experiments. the original fiber structure in the solution under the dissolution system is used for preparing the high-performance regenerated fiber, so that the regenerated SF filament, the electrostatic spinning regenerated SF nano-fiber and the natural SF fiber are used as the raw materials to construct the regenerated SF-fiber composite ligament, in order to provide a new thought for the development of the ligament repair material with the SF as the base material. The traditional dissolution method (Ca 2-C2H5OH-H2O) greatly destroys the original fiber structure of the silk, and even the molecular structure, and the performance of the regenerated SF material is seriously affected, and meanwhile, the requirement of the neutral salt concentration, the dissolution temperature and the dissolution time is higher. In this paper, the dissolution system of Ca 2-FA is used to make the fiber swell, the volume is increased, the intermolecular hydrogen bond is weakened by the hydration of the strong polar ion, and the silk can be dissolved in the fiber at normal temperature and the silk-folded crystal structure in the original fiber can be destroyed to achieve the purpose of dissolving the silk. The original fiber structure can be clearly observed in the SF solution and the dry film, and the size of the original fiber in the solution changes with the concentration of Ca 2 and the increase of the dissolution temperature. The dissolution method can obtain a solution with higher viscosity, and the solubility of the SF in the solution increases with the increase of the concentration of Ca 2 and the dissolution temperature. In addition, the SF film material prepared by the dissolution method is a water-insoluble substance, and the deionized water is soaked to induce the SF film material to be changed from the random curl to the crotch-folding structure. the original fiber structure in the solution is subjected to a coagulation bath in a wet spinning process and is subjected to shearing action to form a fiber, and the water is a coagulating bath, and the mass fraction is 15. 0wt. The mechanical properties of SF-filament with a diameter of 10. 8-1.4. m, a fracture stress of 358. 4-43. 2 MPa and an elongation of break of 24. 8-4. 2% can be obtained after 4-fold drawing, and the mechanical properties of the SF-filament are obviously improved. in addition, the water bath solidification has the advantages of no pollution to the environment, low cost, easy operation and the like. The SF solution obtained by the Ca 2-FA dissolving system is dried to form a film, then dissolved in formic acid to obtain the SF solution which still retains the original fiber structure to carry out electrostatic spinning, and the self-made roller collecting device is adopted to obtain the oriented nano-fiber and has a similar size structure as the extracellular matrix (ECM). The influence of different SF concentration on the fiber orientation was discussed, and the effect of roller speed and tensile on the fiber orientation was analyzed. The results show that the SF mass fraction is 6.0 wt.%, the mass fraction of Ca 2 is 5.0 wt. The fiber with the diameter of 221-20nm was obtained at the speed of 1000 rpm. The fracture stress and the elongation at break of the fiber were 18. 6-3. 8 MPa and 15. 1-2.5%, respectively. The local orientation of the fiber was obviously improved, and the mechanical properties of the fiber with higher mechanical properties were greatly improved than that of the Ca-2-C2H5OH-H2O dissolving method. The degradation of the regenerated SF fibers in the PBS solution was relatively slow, the degradation was 60 days, the weight loss of the SF nano-fiber was 15%, the fiber-to-fiber part had a blocking phenomenon, the weight loss of the SF filament was 8. 9%, and the degradation only occurred on the surface of the fiber, and a partial crack appeared. and the degradation ability of the protease XIV solution to the material is obviously enhanced, and when the degradation 48h, the SF nano-fiber is almost completely degraded, the weight loss of the SF filament is 61.5%, and the fiber body is decomposed. The SF nano-fiber with the orientation arrangement can obviously promote the large-volume growth of the cells in a short time, and the SF filaments and the natural SF fibers are also beneficial to cell growth and adhesion, and have good biocompatibility. based on the preparation of high-performance regenerated SF fiber, a vertical spindle knitting machine is adopted, the SF filament is a shaft yarn, a natural SF fiber braid and an electrostatic spinning SF nano-fiber are wrapped, the regenerated SF fiber composite ligament is constructed, the structure is stable, the breaking strength is up to 2581.7 to 237.N, and the requirements of the human body ACL are met. the different SF fibers exhibit a degradation difference in the enzyme solution, the rapid degradation of the regenerated SF fiber provides an advantageous growth space for cell regeneration, the degradation speed is slow, and the stable natural SF fiber support ensures the mechanical requirements of the ligament material, and the design requirements of differential speed differentiation of different materials are realized. In this paper, the regenerated SF fibers obtained by the Ca 2-FA dissolving system show good performance, and the development of artificial ligament with SF fiber as the raw material, which not only guarantees the mechanical requirements but also gives full play to its good biocompatibility and degradability, provides some experimental basis for the preparation of the ligament repair material.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R318.08
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