基于PILP途径的CaCO3矿物模板转化介导磷酸钙对胶原内原纤维的仿生矿化研究

Up to now, intensive increasing effort has been exerted to biomimetic mineralization of collagen in vitro. However, the major problem which has not been essentially solved yet still lays in the "biomimetic issue". That is to say, the reconstructed collagen fibrils mineralized from most of conventional mineralizing-systems in vitro greatly differ from the bio-mineralized fibrils of bone tissue, because intrafibrillar mineralization does not occur simply by crystallizing collagen in vitro using supersaturated solutions of hydroxyapatite, which generally nucleated on the surface of collagen fibrils. Whereas the most fundamental level of bone tissue is result of intrafibrillar mineralization, where the nanocrystals of hydroxyapatite are formed within the interstices of the collagen fibrils as well as between fibrils, and the crystals are physically aligned with the long axis of the collagen fibril. Therefore, the lesson of intrafibrillar mineralization must be learned from Nature to achieve the simulation of bone tissue at the most basic level of structure. In recent years, some studies proposed that biominerals may not be synthesized through simple nucleation events, but might be a result of a precursor mechanism which is called the polymer-induced liquid precursor(PILP) process. This PILP process may play a fundamental role in biomineralized tissues. The liquid phase mineral precursor induced from PILP process can infiltrate the collagen fibrils via capillary action created by the gaps and grooves of the collagen matrix, and thus the intrafibrillar mineralization can be resulted in. Based on PILP process, intrafibrillar mineralization has been implemented for collagen sponge with CaCO3 and calcium phosphate (CaP), respectively. However, the inducing process of CaP liquid precursor is comparatively time-consuming, as the PILP process of CaP is much slower than that of CaCO3. Moreover, the mineralizing solution must be refreshed several times to compensate the used Ca2+.Therefore, in order to really achieve intrafibrillar mineralization of collagen and to overcome existing limitations, this project proposes the strategy that the primary intrafibrillar mineralization of collagen can be implemented via CaCO3 PILP process, then the CaP intrafibrillar mineralization of collagen fibrils can be finally achieved through the phase transformation of CaCO3 mineral to CaP in phosphate ion ambience. The initially formed CaCO3 minerals act as templates for the generation of CaP. Additionally, the process of collagen fibrillogenesis is designed to combine with the mineralizing process, which also simulates the naturally biomineralizing procedures. This project can be expected to build up a novel biomimetic mineralization in vitro method for collagen fibrils, and to supply a kind of biomimetic composite scaffolds for bone tissue engineering. Furthermore, this work will lay an important basis for the development of biomimetic bone-repairing materials.

目前，胶原纤维的体外仿生矿化尚未完全解决的主要问题仍然是矿化的"仿生"问题，即没有真正实现原纤维内部的均质矿化（内原纤维矿化），而大部分只是在原纤维表面的非均质矿化。磷酸钙和CaCO3的"聚合物诱导的液态前体"（PILP）过程都能够实现胶原的内原纤维矿化，但由于磷酸钙前体液的诱导难度大而导致矿化的周期长。因此，本申请项目在胶原分子自组装成原纤维的过程中，利用PILP过程诱导生成CaCO3液态前体相而实现CaCO3对胶原内原纤维预仿生矿化，基于CaCO3与磷酸钙之间的热力学稳定性差异，再于磷酸根离子环境中将CaCO3矿物转化为磷酸钙，从而借助于CaCO3矿物的模板化作用，实现磷酸钙对胶原的内原纤维仿生矿化，达到对骨组织多级结构的最低级别的仿生。项目基于PILP途径，通过CaCO3模板转化磷酸钙的思路，将为胶原基质提供一种体外仿生矿化的新方法，并为仿生骨修复材料的构建奠定重要的基础。

由于创伤、病变和老年龄化等原因导致人体骨组织缺损的现象非常普遍,临床对于骨修复和骨替代材料有着很大的需求。骨修复和骨替代材料经过第一代和第二代的发展，目前以能主动诱导和激发组织再生为特征的第三代生物材料为主导性的发展趋势。这类材料要求具有与天然组织相似的组成、结构，能够具有医学诱导再生功能。因此，仿生矿化成为骨组织工程材料领域的研究重点和热点。生物矿化胶原原纤维的结构和矿化特点，是对胶原原纤维实施体外仿生矿化即骨分级结构中最低级别仿生的基础，是从根本上真正实现骨修复替代材料仿生性的依据。PILP模型系统能够对生物矿物某些特征的形成提供机理性的信息。在受到珊瑚矿物转化为羟基磷灰石矿物研究的启示下，本项目在PILP过程的基础上提出这样的仿生矿化设计路线：对同时实现近程有序和远程有序的胶原原纤维，通过PILP过程生成液态的CaCO3前体相，实现CaCO3对胶原内原纤维的预仿生矿化，再促使CaCO3矿物转化为磷酸钙，通过CaCO3矿物的介导和模板化作用实现钙磷矿物对胶原的内原纤维仿生矿化。同时，通过探索Ca2+和酸性矿物蛋白的内源化而提高矿化胶原基质支架的可赋形性。.本项目的主要研究内容和已取得的重要结果在于：CaCO3的PILP过程对胶原的内原纤维预仿生矿化；通过制备胶原液晶水凝胶，结合温度、pH和离子强度的协同控制，同时实现胶原水凝胶支架的近程有序和远程有序，并实现了对这种高度取向的胶原原纤维水凝胶进行CaCO3预矿化，并由此介导实现磷酸钙矿物的矿化；CaCO3矿化液对胶原-海藻酸复合水凝胶的PILP途径预矿化的研究，探索在PILP途径下Ca2+和酸性矿物蛋白的内源化改进；预矿化的胶原原纤维上CaCO3矿物模板向磷酸钙转化过程的研究；CaCO3转化介导的磷酸钙仿生矿化胶原原纤维材料的生物学评价。.本项目的研究工作为进一步真正实现胶原纤维的体外仿生矿化提供了一种新的思路和方法，也为自然骨中胶原纤维的矿化机理提供了一些探索性研究；为骨修复和骨替代仿生材料的开发奠定了较为重要的研究基础。