絲素蛋白質(zhì)電凝膠的制備及其性能研究
發(fā)布時(shí)間：2019-03-01 18:46
【摘要】：蠶絲絲素蛋白質(zhì)水凝膠具有良好的生物相容性、生物降解性、保水性和孔隙連通性等,已被研究用于軟組織工程修復(fù)、藥物緩釋、基因工程等領(lǐng)域,為當(dāng)前生物醫(yī)用材料和再生醫(yī)學(xué)領(lǐng)域的研究熱點(diǎn)。作為軟組織修復(fù)材料,絲素蛋白質(zhì)水凝膠常通過(guò)交聯(lián)、接枝或機(jī)械作用等方法制備,但存在耗時(shí)較長(zhǎng)、凝膠機(jī)械性能差、引入有機(jī)溶劑、形貌難以調(diào)控等缺點(diǎn),難以滿足臨床應(yīng)用的要求。因此,迫切需要建立一種新型的絲素水凝膠制備方法,構(gòu)筑性能良好且又綠色溫和的絲素水凝膠。針對(duì)這一問(wèn)題,本研究著眼于絲素蛋白質(zhì)大分子的理化性能特性,采用綠色、溫和環(huán)保的靜電場(chǎng)組裝技術(shù),通過(guò)調(diào)節(jié)絲素水溶液的pH值和外加低壓電場(chǎng),快速有效地制備性能優(yōu)異的絲素水凝膠,為軟組織修復(fù)工程提供一種新型的絲素支架。在深入理解絲素大分子的理化特性和目標(biāo)材料性能的基礎(chǔ)上,本研究設(shè)想通過(guò)pH值(pI附近)和低壓電場(chǎng)電泳的協(xié)同作用,調(diào)控絲素蛋白質(zhì)大分子的組裝行為,降低或抵消絲素大分子之間和之內(nèi)的相互排斥作用,強(qiáng)化絲素大分子之間的親疏水相互作用和相互糾纏、抱合的程度,獲得穩(wěn)定的二級(jí)結(jié)構(gòu)(β-sheets);同時(shí),改變部分絲素大分子的排列方式,獲得機(jī)械性能優(yōu)異、穩(wěn)定性好的絲素電凝膠。通過(guò)改變穩(wěn)定直流電壓、絲素蛋白質(zhì)溶液濃度和pH獲得絲素蛋白質(zhì)電凝膠制備優(yōu)化條件:穩(wěn)定直流電壓24 V,絲素溶液濃度為5wt%,pH值為4。通過(guò)吸水率、壓縮強(qiáng)度等機(jī)械性能的測(cè)試來(lái)評(píng)價(jià)絲素蛋白質(zhì)電凝膠;采用掃描電鏡(SEM)觀察絲素電凝膠的形貌;傅立葉紅外光譜(FTIR)、X射線衍射(XRD)、拉曼光譜(Raman)等表征了凝膠的聚集態(tài)結(jié)構(gòu)和二級(jí)結(jié)構(gòu);通過(guò)體外模型藥物的裝載和釋放實(shí)驗(yàn)探究了凝膠的藥物緩釋性能;最后通過(guò)體外蛋白酶XIV的降解實(shí)驗(yàn)和對(duì)大鼠腎小管上皮細(xì)胞相容性的實(shí)驗(yàn),評(píng)價(jià)了絲素蛋白質(zhì)電凝膠的生物降解行為和細(xì)胞相容性。研究結(jié)果表明,采用調(diào)節(jié)pH值和低壓電場(chǎng)協(xié)同作用制備的絲蛋白質(zhì)電凝膠(SF pH-e-gel)較自然形成的絲蛋白凝膠(SF gel)和調(diào)節(jié)pH值制備的絲蛋白凝膠(SF pH-gel)具有更顯著的機(jī)械性能。壓縮強(qiáng)度約為7.90MPa,壓縮模量約為70.0MPa,遠(yuǎn)高于SF gel的0.25MPa和1.0MPa,并且其含水率可達(dá)約390%。其次,SEM結(jié)果表明,SF pH-e-gel材料孔呈規(guī)整的圓形、孔徑較小且孔壁較厚,與傳統(tǒng)絲素凝膠的層狀或片狀孔結(jié)構(gòu)不同。FTIR、XRD、Raman等表征結(jié)果表明,SF pH-e-gel內(nèi)β-sheet結(jié)構(gòu)的含量較低,而α-helix的含量較其他兩種凝膠高,silk I型結(jié)晶的含量明顯高于另外兩凝膠。再次,以羅丹明B作為模型藥物的緩釋實(shí)驗(yàn)結(jié)果表明,當(dāng)絲素溶液的藥物初始濃度為10μg/mL時(shí),SF pH-e-gel的藥物包覆率可達(dá)87.5%;在120h內(nèi),藥物的釋放量約為60%。而在蛋白酶XIV作用下,SF pH-e-gel體外降解28d,降解量約為80%,表明SF pH-e-gel支架具備良好的可生物降解性。最后,選擇大鼠腎小管上皮細(xì)胞作為模型細(xì)胞,接種于SF pH-e-gel支架上,通過(guò)不同時(shí)間點(diǎn)細(xì)胞形態(tài)和活力測(cè)試,結(jié)果表明凝膠支架能支持上皮細(xì)胞的黏附、分化和增殖,具有良好的細(xì)胞相容性。通過(guò)本文的研究,建立了一種綠色環(huán)保、快速有效制備絲素蛋白質(zhì)凝膠的新方法,所制備凝膠具備良好的機(jī)械性能、優(yōu)異的生物相容性、藥物緩釋性和生物降解性;闡明了SF pH-e-gel的形成機(jī)理。其主要形成機(jī)制:通過(guò)調(diào)控絲素蛋白質(zhì)的pH值,降低或消除絲素大分子之間或分子內(nèi)的排斥力,使分子鏈相互靠近;在靜電場(chǎng)的作用下,絲素大分子被牽拉、伸直,在熱力學(xué)作用下,進(jìn)一步相互糾纏,形成較多的α-helix結(jié)構(gòu);同時(shí),在絲素大分子相互靠近、分子鏈熱運(yùn)動(dòng)和親疏水性相互作用的共同影響下,形成穩(wěn)定的β-sheet結(jié)構(gòu),并與上述的α-helix結(jié)構(gòu)一起組裝成結(jié)晶含量較高、不溶于水的固體凝膠。主要以α-helix結(jié)構(gòu)形成的silk I型結(jié)晶,使凝膠彈性模量降低;以β-sheet結(jié)構(gòu)形成的silk II型結(jié)晶,起到提高凝膠強(qiáng)度和模量的作用。本研究制備的絲素蛋白質(zhì)電凝膠改善了傳統(tǒng)絲素蛋白質(zhì)凝膠機(jī)械性能差的問(wèn)題,表現(xiàn)出良好的生物相容性和生物降解性,為組織工程中軟組織的修復(fù)提供了一種新型的生物材料。
[Abstract]:Silk fibroin hydrogel has good biocompatibility, biodegradability, water retention and pore connectivity. It has been used in the fields of soft tissue engineering repair, drug slow release, gene engineering and so on. As a soft tissue repair material, the silk fibroin hydrogel is prepared by a method such as cross-linking, grafting or mechanical action, but has the disadvantages of long time consumption, poor gel mechanical property, introduction of organic solvent, difficult regulation of appearance and the like, and is difficult to meet the requirements of clinical application. Therefore, it is urgent to establish a new method for preparing silk fibroin hydrogel, which is a good and green silk fibroin hydrogel. aiming at the problem, the research focuses on the physical and chemical properties of the silk fibroin macromolecules, adopts a green, mild and environment-friendly electrostatic field assembly technology, and rapidly and effectively prepares the silk fibroin hydrogel with excellent performance by adjusting the pH value of the silk fibroin aqueous solution and the applied low-voltage electric field, And provides a novel silk fibroin bracket for soft tissue repair engineering. On the basis of in-depth understanding of the physical and chemical properties of the silk fibroin and the properties of the target material, this study envisages the co-action of the pH value (near pI) and the low-voltage electric field electrophoresis to control the assembly behavior of the silk fibroin macromolecules. the mutual exclusion between the silk fibroin macromolecules is reduced or eliminated, the interaction of the hydrophilic and hydrophobic water between the silk fibroin macromolecules and the entanglement of the silk fibroin macromolecules are enhanced, the degree of the binding is improved, and the stable secondary structure is obtained; and meanwhile, the arrangement mode of the partial silk fibroin macromolecules is changed, And the silk fibroin electrogel with excellent mechanical performance and good stability is obtained. The optimized conditions were obtained by changing the stable DC voltage, the concentration of the fibroin solution and the pH. The optimized conditions were as follows: the stable DC voltage is 24 V, the concentration of the silk fibroin solution is 5% by weight, and the pH value is 4. The electrogel of silk fibroin was evaluated by the tests of water absorption, compressive strength and other mechanical properties. The morphology of the silk fibroin gel was observed by scanning electron microscope (SEM), Fourier transform infrared (FTIR), X-ray diffraction (XRD), The aggregation state structure and the secondary structure of the gel are characterized by Raman and the like; the drug sustained-release performance of the gel is explored through the loading and release experiments of the in-vitro model drug; and finally, the experiment of the degradation experiment of the in vitro protease XIV and the compatibility of the renal tubular epithelial cells of the rat is carried out, The biodegradability and cell compatibility of silk fibroin electrogel were evaluated. The results show that the SF gel and SF-gel prepared by the synergistic effect of pH and low-voltage electric field have more significant mechanical properties than that of the naturally-formed silk fibroin gel (SF gel) and the adjusted pH value. The compressive strength is about 7.90 MPa, the compression modulus is about 70.0 MPa, much higher than that of the SF gel of 0.25 MPa and 1.0 MPa, and the water content can reach about 390%. The results showed that the pore size of SF-e-gel material was regular, the pore diameter was small and the pore wall was thick, and it was different from the lamellar or sheet-like pore structure of the traditional silk fibroin gel. The results of FTIR, XRD and Raman show that the content of the S-sheet in the SF-e-gel is low, and the content of the HCO3-help is higher than that of the other two gels, and the content of the sikI-type crystals is higher than that of the other two gels. The results of the sustained-release experiment with rhodamine B as a model drug show that the drug-coating rate of SF-e-gel can reach 87.5% when the initial concentration of the drug in the solution is 10. m u.g/ mL, and the release amount of the drug is about 60% in 120 h. Under the action of protease XIV, the degradation of SF-e-gel in vitro was about 80%, indicating that the SF-e-gel scaffold had good biodegradability. In the end, the rat renal tubular epithelial cells were selected as model cells, seeded on the SF pH-e-gel scaffold, and the cell morphology and viability were tested by different time points. The results showed that the gel scaffold can support the adhesion, differentiation and proliferation of the epithelial cells, and has good cell compatibility. In this paper, a new method of green environmental protection and rapid and effective preparation of silk fibroin gel is established. The prepared gel has good mechanical properties, excellent biocompatibility, drug release and biodegradability, and the formation mechanism of SF pH-e-gel is clarified. The main forming mechanism is that by regulating the pH value of the silk fibroin protein, the repulsive force between the silk fibroin macromolecules or the molecules is reduced or eliminated, so that the molecular chains are close to each other; under the action of the electrostatic field, the silk fibroin macromolecules are pulled and straightened, and the silk fibroin macromolecules are further entangled with each other under the action of thermodynamics; And meanwhile, under the mutual influence of the mutual proximity of the silk fibroin macromolecules, the thermal movement of the molecular chain and the hydrophobic interaction, a stable l-sheet structure is formed, and is assembled into a solid gel with higher crystal content and is insoluble in water together with the carbon-help structure described above. Silk type I crystals, which are mainly formed by the structure of the Si-helix structure, are used to reduce the elastic modulus of the gel, and the silk type II crystal formed by the structure of the Si-sheet structure plays a role in improving the strength and the modulus of the gel. The silk fibroin electrogel prepared by the method improves the problem of poor mechanical property of the traditional silk fibroin gel, has good biocompatibility and biodegradability, and provides a novel biological material for the repair of soft tissue in the tissue engineering.
【學(xué)位授予單位】：武漢紡織大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ427.26
本文編號(hào)：2432700
[Abstract]:Silk fibroin hydrogel has good biocompatibility, biodegradability, water retention and pore connectivity. It has been used in the fields of soft tissue engineering repair, drug slow release, gene engineering and so on. As a soft tissue repair material, the silk fibroin hydrogel is prepared by a method such as cross-linking, grafting or mechanical action, but has the disadvantages of long time consumption, poor gel mechanical property, introduction of organic solvent, difficult regulation of appearance and the like, and is difficult to meet the requirements of clinical application. Therefore, it is urgent to establish a new method for preparing silk fibroin hydrogel, which is a good and green silk fibroin hydrogel. aiming at the problem, the research focuses on the physical and chemical properties of the silk fibroin macromolecules, adopts a green, mild and environment-friendly electrostatic field assembly technology, and rapidly and effectively prepares the silk fibroin hydrogel with excellent performance by adjusting the pH value of the silk fibroin aqueous solution and the applied low-voltage electric field, And provides a novel silk fibroin bracket for soft tissue repair engineering. On the basis of in-depth understanding of the physical and chemical properties of the silk fibroin and the properties of the target material, this study envisages the co-action of the pH value (near pI) and the low-voltage electric field electrophoresis to control the assembly behavior of the silk fibroin macromolecules. the mutual exclusion between the silk fibroin macromolecules is reduced or eliminated, the interaction of the hydrophilic and hydrophobic water between the silk fibroin macromolecules and the entanglement of the silk fibroin macromolecules are enhanced, the degree of the binding is improved, and the stable secondary structure is obtained; and meanwhile, the arrangement mode of the partial silk fibroin macromolecules is changed, And the silk fibroin electrogel with excellent mechanical performance and good stability is obtained. The optimized conditions were obtained by changing the stable DC voltage, the concentration of the fibroin solution and the pH. The optimized conditions were as follows: the stable DC voltage is 24 V, the concentration of the silk fibroin solution is 5% by weight, and the pH value is 4. The electrogel of silk fibroin was evaluated by the tests of water absorption, compressive strength and other mechanical properties. The morphology of the silk fibroin gel was observed by scanning electron microscope (SEM), Fourier transform infrared (FTIR), X-ray diffraction (XRD), The aggregation state structure and the secondary structure of the gel are characterized by Raman and the like; the drug sustained-release performance of the gel is explored through the loading and release experiments of the in-vitro model drug; and finally, the experiment of the degradation experiment of the in vitro protease XIV and the compatibility of the renal tubular epithelial cells of the rat is carried out, The biodegradability and cell compatibility of silk fibroin electrogel were evaluated. The results show that the SF gel and SF-gel prepared by the synergistic effect of pH and low-voltage electric field have more significant mechanical properties than that of the naturally-formed silk fibroin gel (SF gel) and the adjusted pH value. The compressive strength is about 7.90 MPa, the compression modulus is about 70.0 MPa, much higher than that of the SF gel of 0.25 MPa and 1.0 MPa, and the water content can reach about 390%. The results showed that the pore size of SF-e-gel material was regular, the pore diameter was small and the pore wall was thick, and it was different from the lamellar or sheet-like pore structure of the traditional silk fibroin gel. The results of FTIR, XRD and Raman show that the content of the S-sheet in the SF-e-gel is low, and the content of the HCO3-help is higher than that of the other two gels, and the content of the sikI-type crystals is higher than that of the other two gels. The results of the sustained-release experiment with rhodamine B as a model drug show that the drug-coating rate of SF-e-gel can reach 87.5% when the initial concentration of the drug in the solution is 10. m u.g/ mL, and the release amount of the drug is about 60% in 120 h. Under the action of protease XIV, the degradation of SF-e-gel in vitro was about 80%, indicating that the SF-e-gel scaffold had good biodegradability. In the end, the rat renal tubular epithelial cells were selected as model cells, seeded on the SF pH-e-gel scaffold, and the cell morphology and viability were tested by different time points. The results showed that the gel scaffold can support the adhesion, differentiation and proliferation of the epithelial cells, and has good cell compatibility. In this paper, a new method of green environmental protection and rapid and effective preparation of silk fibroin gel is established. The prepared gel has good mechanical properties, excellent biocompatibility, drug release and biodegradability, and the formation mechanism of SF pH-e-gel is clarified. The main forming mechanism is that by regulating the pH value of the silk fibroin protein, the repulsive force between the silk fibroin macromolecules or the molecules is reduced or eliminated, so that the molecular chains are close to each other; under the action of the electrostatic field, the silk fibroin macromolecules are pulled and straightened, and the silk fibroin macromolecules are further entangled with each other under the action of thermodynamics; And meanwhile, under the mutual influence of the mutual proximity of the silk fibroin macromolecules, the thermal movement of the molecular chain and the hydrophobic interaction, a stable l-sheet structure is formed, and is assembled into a solid gel with higher crystal content and is insoluble in water together with the carbon-help structure described above. Silk type I crystals, which are mainly formed by the structure of the Si-helix structure, are used to reduce the elastic modulus of the gel, and the silk type II crystal formed by the structure of the Si-sheet structure plays a role in improving the strength and the modulus of the gel. The silk fibroin electrogel prepared by the method improves the problem of poor mechanical property of the traditional silk fibroin gel, has good biocompatibility and biodegradability, and provides a novel biological material for the repair of soft tissue in the tissue engineering.
【學(xué)位授予單位】：武漢紡織大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ427.26
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