聚噻吩基导电水凝胶研制及其在心肌组织工程中的应用研究

发布时间：2018-03-20 09:11

本文选题：导电水凝胶　切入点：多孔支架　出处：《天津大学》2016年博士论文　论文类型：学位论文

【摘要】：本文通过“一步法”策略制备了两种导电水凝胶(Conductive hydrogel,CH),由优良导电性能的聚噻吩衍生物和具有良好生物相容性的天然高分子组成。综合评价了CH溶胀性能、力学强度、电学性能及蛋白吸附特性。以棕色脂肪干细胞(BADSCs)为模型,系统研究了CH的组成对BADSCs粘附、增殖及向心肌细胞分化的调控作用及相关分子机制。聚噻吩基双网络导电水凝胶研制及性能评价:为了克服传统两步法制备导电水凝胶时组分不均匀的缺点,以聚3-噻吩乙酸(PTAA)为导电刚性网络,光交联明胶(MAAG)为柔性网络,在二甲基亚砜(DMSO)溶液中通过羰基二咪唑(CDI)交联PTAA的羧基,同时采用光交联的方法交联MAAG,两种反应同时进行且互不干涉,形成均一的双网络导电水凝胶(HEDN)。采用高分辨氢谱核磁共振(1HNMR)和傅里叶红外光谱(FTIR)追踪HEDN的形成,并系统研究了HEDN组成对其溶胀性能、力学强度和导电性能的调控作用。结果发现HEDN的理化性能可以通过调节PTAA网络和MAAG网络的比例实现调控。双网络水凝胶的压缩力学强度在22.7 kPa到493.1 kPa之间,电导率约10-4 S·cm-1,与天然心肌组织的电导率相似。在此基础上,将BADSCs种植在HEDN表面,在施加电刺激的作用下,通过原位荧光染色、免疫组织化学、免疫荧光、Western-Blotting等手段详细研究了材料性能对BADSCs粘附、增殖以及向心肌细胞(MCs)分化的调控作用。结果发现HEDN可支持BADSCs的粘附与增殖,同时其行为与HEDN水凝胶的组成存在一定的相关性,例如:当PTAA/MAAG质量比例为1:1时,BADSCs表现出最好的增殖能力。更为重要的是HEDN水凝胶可调控BADSCs向心肌细胞分化的能力,PTAA网络的引入可明显增强心肌特异性蛋白(Cardiac troponin T和α-actinin)的表达,同时促进细胞间通讯。此外,电刺激可明显增强BADSCs向心肌细胞分化的现象。聚噻吩基粒子增强型多孔支架研制及工程化心肌组织体外构建:为了增强心肌组织工程支架材料的导电性和易加工性能,以聚乙烯二氧噻吩(PEDOT)海藻酸钠为基材,通过一步法原位聚合、化学交联方式在水相制备了聚乙烯二氧噻吩/海藻酸钠水凝胶(PAEH),冷冻干燥后获得多孔支架材料(PAMCS)。通过透射电镜和扫描电镜研究了支架中材料的组合方式和支架的形貌结构,同时系统研究了材料组成和结构对PAMCS溶胀特性、降解性能、力学强度、电学性能和对蛋白的吸附能力的影响。结果表明,PAEH的储能模量超过100 kPa,导电率大于10-2 S·cm-1,同时可通过调整PEDOT与海藻酸的比例控制其蛋白吸附能力。在此基础上,以BADSCs为种子细胞,成功构建了基于PAMCS支架的工程化心肌组织,系统的研究了BADSCs在PAMCS中的生物学行为,结果表明,相比单纯海藻酸钠支架,PEDOT的引入明显增强支架的生物相容性,HE以及激光共聚焦结果表明BADSCs在PAMCS中分布均匀,生长良好。且PEDOT的引入可增强BADSCs向心肌细胞分化的能力,提高其cTnT和α-actinin蛋白的表达及Connexin 43的表达。
[Abstract]:This article through the "one-step" strategy has prepared two kinds of conductive hydrogel (Conductive hydrogel, CH), by the excellent conductive properties of polythiophene derivatives and natural polymers with good biocompatibility. The comprehensive evaluation of CH swelling properties, mechanical strength, electrical properties and protein adsorption. Brown adipose derived stem cells (BADSCs as a model system) on the CH of the composition of BADSCs adhesion, regulation of proliferation and differentiation into cardiomyocyte cells and the related molecular mechanism. Evaluation of polythiophene based conductive hydrogel preparation and properties of double network: in order to overcome the traditional two step preparation of conductive hydrogel component is not uniform, with poly (thiophene acetic acid 3- PTAA) for conducting rigid optical network (MAAG) is a flexible crosslinked gelatin network in two dimethyl sulfoxide (DMSO) solution by two carbonyl imidazole (CDI) cross-linked PTAA carboxyl, adopting the crosslinking of MA light AG, two kinds of reaction simultaneously without mutual interference, double network form a uniform conductive hydrogel (HEDN). Using high resolution nuclear magnetic resonance spectroscopy (1HNMR) and Fourier transform infrared spectroscopy (FTIR) to track the formation of HEDN, and study the HEDN composition on the swelling properties, mechanical strength and regulation of conductive properties the results showed that the regulation by adjusting the PTAA network and the MAAG ratio of the physical and chemical properties of HEDN can be compressed. The mechanical strength of double network hydrogel at 22.7 kPa to 493.1 kPa, the conductivity of about 10-4 S cm-1, and the natural cardiac tissue conductivity. On this basis, the BADSCs grown on the surface of HEDN and in the stimulation effect, by in situ fluorescence staining, immunohistochemistry, immunofluorescence, Western-Blotting and other means of a detailed study of the material properties on the BADSCs adhesion, proliferation and differentiation into cardiomyocytes (MCs) as the control . results showed that the proliferation and adhesion of HEDN can support BADSCs, while the composition of its behavior and HEDN hydrogel has certain relevance, for example: when the mass ratio of PTAA/MAAG to 1:1, BADSCs showed the best proliferation ability. What is more important is the HEDN hydrogel could regulate BADSCs differentiation into cardiomyocytes, the introduction of PTAA network could enhance the cardiac specific protein (Cardiac, troponin T and alpha -actinin) expression, while promoting intercellular communication. In addition, electrical stimulation can significantly enhance the cardiomyogenic differentiation of BADSCs phenomenon. Construction of polythiophene based particle enhanced development and tissue engineering scaffold in vitro: in order to enhance the myocardial tissue scaffold the conductivity and processability of polyethylene, two oxygen thiophene (PEDOT) sodium alginate as base material, through a one-step in situ polymerization, chemical crosslinking methods were prepared in aqueous polyethylene system Two oxygen thiophene / sodium alginate hydrogel (PAEH), obtained after freeze drying of porous scaffolds (PAMCS). The morphology by transmission electron microscope and scanning electron microscope study of the combination and stent materials, at the same time, the system of material composition and structure on PAMCS degradation properties, swelling properties, mechanical strength, electrical properties and effects the protein adsorption capacity. The results showed that the storage modulus of PAEH more than 100 kPa, the conductivity is greater than 10-2 S cm-1, at the same time can be controlled by adjusting the PEDOT protein and the ratio of alginate adsorption capacity. On this basis, using BADSCs as seed cells, successfully constructed myocardial tissue engineering based on the PAMCS scaffold. Systematic study on the biological behavior of BADSCs, in PAMCS the results show that compared with pure sodium alginate stent, the introduction of PEDOT significantly enhanced the compatibility of scaffold organisms, HE and confocal laser scanning results It indicates that BADSCs is well distributed and well distributed in PAMCS, and the introduction of PEDOT can enhance the ability of BADSCs to differentiate into cardiomyocytes, increase the expression of cTnT and -actinin and the expression of Connexin 43.

【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;TQ427.26

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