【作者】 明津法；

【导师】 左保齐；

【作者基本信息】 苏州大学 ， 纺织材料与纺织品设计， 2014， 博士

【摘要】 骨作为人体最大的组织器官，承担着生命活动的重要职责，我国每年因交通事故、股骨头坏死等原因导致骨缺损的患者超过百万人，骨组织修复一直是再生医学领域的难题和研究热点之一。临床修复过程中迫切需要具有生物相容性和生物降解性的再生骨修复支架材料。现有研究表明丝素蛋白（SF）具有良好的生物相容性及再生加工性能。由于人体本身是一典型的胶体系统，制备类细胞外基质结构的SF基水凝胶支架用作骨修复材料成为新的研究方向。本文在研究SF的silk I结构及纤维化水凝胶的制备方式基础上，引入海藻酸钠（SA），制备SF/SA纤维水凝胶，研究纤维凝胶的制备条件及理化性能，在此研究基础上，采用仿生矿化原理，探讨纤维凝胶体系对羟基磷灰石（HAp）晶体生长形貌的调控；通过体外细胞培养实验，研究骨髓间充质干细胞在纤维凝胶材料上的生长情况。本文通过改变环境温湿度等影响因素，调控SF溶液的干燥速率，成功制备SF的silk I结晶结构，并建立SF溶液形成silk I结构的干燥速率方程。系统地对silk I结构形成影响因素进行分析，溶液浓度和干燥速率均会影响silk I结构的形成；silk I结构的形成过程中伴随着SF分子通过氢键、亲疏水性及静电作用等形成稳定纳米纤维网状形貌。Silk I结构膜的性能研究结果表明：材料断面有大量的纳米纤维存在，可在相对较低的温度条件（5或-20℃）下长期保持silk I结构；silk I结构膜在干态下表现出良好的力学性能，其断裂应力为38.68±6.21MPa。以酸/盐为溶解体系溶解SF纤维获得再生SF溶液，并采用9.3M溴化锂溶液再溶解，获得SF水溶液，该溶液中SF为纳米线状形貌，且为无规卷曲结构；采用该两步法溶解制备的SF溶液的凝胶过程为自上而下渐进式进行的，凝胶形成过程中SF分子形成纤维网状形貌的同时其结构也由无规卷曲转变成β-sheet；此外，SF纤维凝胶具有良好的压缩力学性能，为类细胞外基质结构水凝胶的制备提供了前提条件。将两步法获得的SF溶液与SA溶液共混，制备SF/SA纤维凝胶。研究结果表明SA添加量增加，会延迟胶凝时间；SF/SA纤维凝胶中SF的二级结构以β-sheet为主，同时，纤维凝胶的力学性能随SA含量的增加，其压缩应力呈现下降趋势。从化学组成和结构仿生的角度出发，以SF/SA纤维凝胶为矿化模板，在纤维凝胶体系中诱导HAp晶体的生长。实验结果表明，通过改变矿化过程中的矿化温度、矿化时间及溶液pH值等影响因素，可以调控获得形貌相近的棒状HAp晶体。同时，运用有机基质诱导成核理论中的静电积累-Ionotropic模型，对纤维凝胶体系中HAp晶体形貌的调控进行了探讨。根据激光共聚焦显微镜、扫描电子显微镜及MTT测试结果可知，人骨髓间充质干细胞（hMSCs）和小鼠骨髓间充质干细胞（BMSCs）在SF基纤维凝胶上有良好的粘附和增殖能力。同时，SF/SA纤维凝胶中SA含量的变化对hMSCs细胞的粘附和增殖影响不明显。通过本文的研究，阐明了SF基纤维水凝胶的制备方法及其形成机理，并发现纤维凝胶体系能够调控棒状HAp晶体的生长，为制备骨组织修复材料提供了一定的实验依据。更多还原

【Abstract】 As the body’s largest tissues and organs, bone has an important responsibility of lifeactivities. At present, the number of patients is more than one million people every year inChina, which are caused by traffic accident, femoral head necrosis and other reasons. Bonetissue repair is one of difficult problem and research topic in the field of regenerativemedicine, what’s more, clinical rehabilitation urgently needs a large number of regeneratedscaffold materials with biocompatibility and biodegradability. Up to now, many literaturesindicate silk fibroin (SF) has good biocompatibility and regeneration processingperformance. At the same time, human body itself is a typical colloid system, how toprepare the similar extracellular matrix structure of SF-based hydrogel scaffold serving asbone repair materials has been a new research field.Based on these researches such as silk I conformation of SF and the preparation of SFnanofiber hydrogels, this paper prepares silk fibroin/sodium alginate (SF/SA) nanofiberhydrogels through simply mixing solution. And then, it researches the preparationconditions and physicochemical properties of SF/SA nanofiber hydrogels. Moreover, usingbiomimetic mineralization principle, this paper also discusses hydroxyapatite (HAp)crystals growth regulating and controlling in nanofiber hydrogels. In addition, through cellculture in vitro, the proliferation of bone marrow stem cells is discussed on the nanofiberhydrogels.Firstly, the drying rate of SF solution is controlled by changing temperature, humidityand other factors, and on this basis, the silk I conformation of SF is successfully prepared,simultaneously, the drying rate equation of silk I conformation is established. At the sametime, this paper systematically analyzes some factors affecting silk I formation. The resultsshow solution concentration and drying rate will affect the conformation of silk I formation.Furthermore, in the silk I formation process, SF molecules are self-assembled into stablenanofiber network through hydrogen bond, hydrophilicity and electrostatic interactions etc.In addition, SF films with silk I conformation are analyzed and the results are as follows: many nanofibers are existed in the cross-section of films; silk I conformation of SF issustained for a long time at relatively low temperature condition such as5℃or-20℃.At the same time, SF film with silk I conformation shows excellent mechanical propertiesand its breaking stress reaching up to38.68±6.21MPa.Secondly, SF fibers are dissolved in acid/salt solution, and then, the regenerated SFmaterials are dissolved in9.3M lithium bromide solution, obtaining SF aqueous solution.This dissolution process is called two-step dissolution method. In SF aqueous solution, SFmolecules are nanofiber structure and its secondary structure is random coil. The gelationprocess of SF solution is top-down, simultaneously, SF molecules are self-assembled intonanofiber networks, and its secondary structure is transferred from random coil to β-sheet.In addition, SF nanofiber hydrogels have excellent compressive strain. The preparationprocess of SF nanofiber hydrogels is provided a method for preparing similar extracellularmatrix structure of hydrogels.SF/SA nanofiber hydrogels are prepared by mixing SF and SA solution with variousproportions, which SF solution is prepared by two-step dissolution method. It is found thatthe content of SA increasing, the gelation time of nanofiber hydrogel delaying. Thesecondary structure of SF is mainly β-sheet in nanofiber hydrogels, at the same time, thecompressive stress of SF/SA nanofiber hydrogels is trending downward with increasing SAcontent in hydrogels. Furthermore, from the perspective of chemical composition andstructural bionics, SF/SA nanofiber hydrogel as a template is used to control HAp crystalgrowth in hydrogel system. The results demonstrate that HAp nanorods crystals are grownthrough changing temperature, mineralization times, pH and other factors in mineralprocess. At the same time, the morphology of HAp crystals controlling by nanofiberhydrogels is discussed by using electrostatic accumulation-Ionotropic model in nucleationtheory induced by organic matrix.Based on confocal scanning laser microscope, scanning electron microscope, andMTT assay, human mesenchymal stem cells (hMSCs) and bone marrow mesenchymalstem cells (BMSCs) have individually good adhesion and proliferation on SF-basednanofiber hydrogels, at the same time, there are no signs of inhibition of SF/SA nanofiberhydrogel to attachment and proliferation of hMSCs under changing the SA content inhydrogels.Through above researches, this paper clarifies the preparation method and physicochemical properties of SF-based nanofiber hydrogels and its formation mechanism.At the same time, HAp crystal growth is controlled in hydrogel systems. The researchideas may provide some experimental results for preparation of bone tissue repairmaterials.更多还原