【作者】 程成；

【导师】 邵正中；

【作者基本信息】 复旦大学 ， 高分子化学与物理， 2008， 博士

【摘要】 生物矿化机理的研究以及以此为基础的材料的仿生制备是目前生物学家和材料学家们所非常关注的问题。经过近半个世纪的世界各地科研工作者的辛勤工作,人们终于对生物体中完美而精妙的矿物质合成有了初步的了解,对参与其中的大分子的认识也有了大概的轮廓,对这些高性能材料的模拟常识和设计具有新型功能性的聚合物/陶瓷复合物成为了研究热点。动物的丝蛋白,与骨组织中的胶原蛋白一样,都是为人们所关注的重要的结构性蛋白质,虽然它并没有直接被自然界安排在生物矿物的构架之中,但贝类珍珠质中与之极其相似的蛋白质成分的存在使得丝蛋白成为体外模拟生物矿化模板的重要选择,而蚕丝中丝蛋白规整的取向排列结构又使人们考虑到其代替胶原蛋白合成骨替代材料的可能性。同时,由于丝蛋白在生物医药方面的应用研究非常广泛,因此,考察丝蛋白材料在特定溶液环境中的矿化诱导能力是具有指导意义的,如何调控材料的结构,能使得作为软组织工程材料的丝蛋白尽可能的避免诱导矿化,而作为硬组织替代材料的丝蛋白尽可能的促进矿化,是这方面研究中要解决的重要问题。在本论文的研究中,我们选用来源广泛的蚕丝蛋白及其再生溶液和材料,以溶液调控的方法为基础,结合蛋白质的结构特性以及在外界环境影响下的自组装性能,对碳酸钙、磷酸钙盐的溶液结晶过程进行了调控,并在此基础上进行了以丝蛋白材料为有机组分,碳酸钙、二氧化硅等作为无机组分的生物杂化材料的制备和性能研究。另外,我们也对蚕丝以及取向丝蛋白膜和取向壳聚糖膜中的有序结构对矿物沉积的影响做出了深入的讨论。首先,为了制备和研究二氧化硅/丝蛋白复合材料,我们尝试将水溶性的二氧化硅水分散体系即硅溶胶与高浓度丝蛋白水溶液进行不同配比的混合,经过自然条件下缓慢干燥成型并探究二氧化硅纳米粒子与蚕丝蛋白连续材料之间的作用以及两者共存对复合材料性能的影响。研究发现:对于由浇铸成型的再生丝蛋白/硅溶胶复合材料,纳米级的二氧化硅聚集体能够稳定均匀地分布在作为连续相的丝蛋白中。丝蛋白的β-折叠结构以及二氧化硅微粒的阻碍,使得复合材料中分子链段间的相对运动减弱,其动态力学性能得以改善。在对碳酸钙和磷酸钙盐的研究中,我们借鉴了贝类生物珍珠质中类丝蛋白的存在状态和介入方式,将蚕丝蛋白以无定形的溶液状态介入矿物质的结晶过程。采用再生丝蛋白作为可溶性添加物,加入碳酸钙饱和溶液体系,获得了新型的具有特殊形貌和晶型特征的丝蛋白/碳酸钙复合微粒,通过对外界条件的调控,能够分别获得两种具有不同形貌特征的尺寸均匀的微粒产物,微粒表面和切片的扫描电镜结果显示其中长径比约为2:1,长径约为2微米的米粒状微粒具有空心结构,晶型为方解石,而直径约为1微米的球状微粒则是实心结构,为方解石与球文石的混合体。两种微粒中都含有约15%丝蛋白成分,而且丝蛋白分散在碳酸钙微晶之间,介入了微晶的聚集。同时,两种杂化颗粒在母液中的熟化研究表明丝蛋白对文石的生成具有一定程度上的诱导作用。我们通过对溶液初始pH值,结晶温度,丝蛋白浓度以及丝蛋白分子量等影响因素的调控,成功实现了对两种杂化颗粒形成的控制。根据对实验结果的分析,提出了米粒状和球状碳酸钙/丝蛋白杂化微粒的形成机理假设。我们认为:钙离子与丝蛋白分子中带负电的集团之间的作用力导致无定型碳酸钙粒子与丝蛋白的吸附,而此时丝蛋白分子链在溶液中的状态将成为最终产物形成的决定性因素。当丝蛋白在外界条件影响下在溶液中形成具有部分方向性的自组装体时,能够调控碳酸钙的聚集从而形成具有空心结构并且各向异性的米粒状杂化微粒;而当丝蛋白在溶液中的状态是较为舒展的单分子链时,则产生各向同性的球状杂化微粒。另外,对再生丝蛋白对磷酸钙盐结晶的调控作用进行了初步探索,获得了丝蛋白能够加速和促进透钙磷石晶型转变的初步结论。在丝蛋白溶液调控矿化的基础上,我们使用蚕丝以及再生丝蛋白膜作为矿物质结晶的固体基质,对碳酸钙在基质表面的结晶生长行为进行了深入的讨论,其主旨在于探讨固体基质中分子链的取向性对矿物质生长方向的引导和模板作用。在含有丝蛋白溶液的碳酸钙过饱和溶液中,通过浸泡处理的方法,在脱胶丝表面获得了具有明显取向性,平行排列的碳酸钙结晶聚集体。通过对矿化时间和溶液pH环境的调控,能够成功的控制矿物质的沉积速度和沉积量,并得到完整碳酸钙包覆的蚕丝样品。为探讨蚕丝表面有序结构与矿物质生长方向之间的联系,我们选用溴化锂溶液对脱胶丝进行处理破坏其表面结构,之后得到的碳酸钙结晶层没有明显的取向性。不仅如此,取向排列的碳酸钙晶体呈较为罕见的文石晶型,而未完全取向排列的碳酸钙晶体则呈方解石和文石的混合晶型。我们认为:溶液中游离的蛋白质分子束与丝表面的有序部分,由于结构相似性和β-折叠结构的诱导成核作用,具有很强的吸附倾向。而这些吸附在丝表面的游离分子,则充当了碳酸钙粒子与丝基质之间的媒介,从而使得蚕丝中有序结构的模板效应得以体现,引导碳酸钙的取向沉积。预先单轴取向的再生丝蛋白膜表面,在丝蛋白溶液存在情况下,同样生成取向排列的碳酸钙结晶,从而对上述机理提供了有力的支持。另外,我们利用丝蛋白对碳酸钙结晶的调控作用而加强丝蛋白与矿物质之间的结合力,制备了具有规整形貌的丝蛋白/碳酸钙复合多孔支架。经过对复合多孔材料进行了形貌,晶型和组成表征,对碳酸钙的不同形貌与晶型的形成原因进行了深入的讨论并对制得的材料做了初步的力学性能探索研究。最后,我们对碳酸钙在壳聚糖/丝蛋白仿生体系中结晶机制的初步探索。其主要目的在于尝试以壳聚糖/丝蛋白体系模拟贝壳珍珠质中几丁质/类丝蛋白体系,着重考察壳聚糖有序结构对碳酸钙结晶的影响以及丝蛋白作为调控剂在体系中起到的媒介作用。实验发现,丝蛋白能够调控具有规整形貌的碳酸钙层在壳聚糖表面的生长。而具有取向结构的壳聚糖膜能够诱导碳酸钙(104)晶面的选择性吸附。更多还原

【Abstract】 Biological organisms regularly produce bimolecular-inorganic hybrid materials such as bone,teeth,diatoms and shells,which all tend to have much superior mechanical properties over synthetic material hybrids.During the past few decades, Studies on biomineralization,as well as the organic/inorganic composite materials based on the principles of biomineralization,have attracted considerable interest of researchers.Natural animal silks,mainly from silkworms,have been in practically used by man for centuries mainly because of its unique luster,fineness,and mechanical properties.In recent years,extensive studies have been carried on the application of silk fibroin（SF） for non-textile uses.Although not chosen by the nature to involve into the biominerals as collagen,silk fibroin has surprisingly comparability with the silk-like protein in the organic template of nacre.That is why silk fibroin is considered as potential template in biomineralization.In addition,the biomedical application of silk as well as the regenerated silk fibroin materials made it important to control the mineral-regulating ability of silk fibroin.In the present work,we examined the morphology and crystallographic polymorph formation process of calcium carbonate and calcium phosphate in the presence of dissolved silk fibroin;note that the conformation of regenerated silk fibroin（RSF） can be controlled via the solvent environment.Based on the interaction of SF and minerals,SF/silica composite material and SF/calcium carbonate hybrid scaffold were prepared and characterized.Moreover,template effect of degummed silk and uniaxially deformed SF film and chitosan form on the deposition of calcium carbonate was deeply investigated.At first,In order to probe if there is interaction between silk protein and silica, and further more,whether the interaction can improve the mechanical properties of composite materials,the structure and property of the silk fibroin/silica composite material were studied.The results revealed the good compatibility of silk fibroin and silica in the composite system.Comparing to the pure silk fibroin material,the composite material showed better dynamic mechanical property in the range of 15℃to 55℃.Due to both the hydrogen bond and electrostatic interaction between SiO₂ and silk fibroin,the silica particles’ interfusion among the chains of protein had a motive-impeding influence on the protein chains.This influence together with the stableβ-sheet conformation of silk fibroin matrices led the avoidance of modulus dissipation of the material.Mollusk shell is one of the best studied of all calcium carbonate biominerals.Its silk-like binder-matrix protein plays pivotal role during the formation of aragonite crystals in the nacre sheets.In the second part of our work,we provide novel experimental insights into the interaction of mineral and protein using a model system of reconstituted Bombyx mori silk fibroin solutions as templates for the crystallization of calcium carbonate（CaCO₃）.We observed that inherent（self-assembling） aggregation process of silk fibroin molecules affected both of the morphology and crystallographic polymorph of CaCO₃ aggregates.This combination fostered the growth of a novel,rice-grain shaped protein/mineral hybrid with hollow structure with an aragonite polymorph formed after ripening.These observations suggest new hypotheses about the role of silk-like protein in the natural biomineralization process, but it also may serve to shed light on the formation process of those ersatz hybrids regulated by artificially selected structural proteins.Based on the SF solution-mediate mineralization,we investigated the crystallization of calcium carbonate on degummed silks and RSF films which were used as the solid substrate.By dipping in the supersaturated solution of calcium carbonate containing silk fibroin,clusters of parallel arranged calcium carbonatecrystals with obvious orientation were obtained on the surface of silk fibre after degumming.Silk samples completely covered by calcium carbonate deposition were prepared,and the amount and speed of the deposition can be controlled by the time of mineralization and the pH value of the solution.Influence of the oriented structures of the silk surface on the tropism of the mineral growth was studied by treating the silk fibre after degumming with lithium bromide solution to destroy the surface structure.No obvious orientation was found in the obtained calcium carbonate crystals on the treated silks.The results inspired the strong interaction between the oriented silk surface and the dissociative fibroin chains in the solution,which arose from the similar structure and the nucleation induced byβ-sheet structure.Afterwards, oriented deposition of calcium carbonate was obtained while calcium carbonate particles absorbed on the dissociative fibroin moulding by the oriented silk substrate. This mechanism can also be strongly supported by the similar oriented calcium carbonate crystals obtained on the surface of uniaxial deformed regenerated silk fibroin film in present of RSF solution.Moreover,silk fibroin/calcium carbonate composite scaffold was synthesized assisted by the strong combination between silk fibroin and mineral.After the characterization of the morphology,polymorph and component of the porous composites,the mechanisms of the different appearance and crystallization of the calcium carbonate were further discussed and some of the mechanical properties of the products we obtained were investigated.At last,we did some pilot research about the mechanism of crystallization of the calcium carbonate in chitosan/silk fibroin bionic system.Our work mainly focused on the influence of oriented chitosan structure on the crystallization of calcium carbonate and the effects of silk fibroin as intermediate in the system.We found that the growth of calcium carbonate layer with regular structure could be controlled by silk fibroin and the selective adsorption of calcium carbonate（104） crystal plane could be induced by oriented chitosan film.更多还原