Nanostructured biomaterials: investigating hierarchical calcite crystal formation

众所周知，生物体能够产生复杂的矿化结构，这些结构用于各种各样的功能，例如导航、支撑和保护身体的柔软部位等。这种矿化组织的形成需要复杂的生物机制来控制矿化的所有步骤，从控制离子到矿化位点的运输到控制晶体成核、生长和形态，以及微米和宏观矿化组织的形成。迄今为止，还没有研究能够解决在生物矿化过程中所有这些因素是如何整合的。因此，缺少的是对细胞如何控制发生在不同阶段和长度尺度上的过程的基本理解，从离子运输到矿化位点到晶体成核，生长和组装成分层多晶矿化组织的晶体。从长远来看，这个问题将为生物学家和化学家提供新的工具来设计和合成具有生物医学、工业和技术应用潜力的先进材料。该项目的目的是了解细胞如何控制矿化组织的形成，从纳米尺度上形成的第一个晶体，到由高阶结构的晶体组成的成熟矿物。为此，我们将研究由单细胞海藻Emiliana huxleyi和cocolithus pelagicus产生的方解石制成的球石鳞片的形成。这些单细胞提供了独特的模型，在这些模型中，矿化的所有阶段，发生在所有长度尺度上——从矿物形成的开始到成熟的矿物——可以在一个单一的系统中以时间分辨的方式进行研究，以阐明它们如何促进生物矿物的形成和整体特性。为了实现这一目标，我们将结合共聚焦显微镜，冷冻扫描电子显微镜和冷冻透射电子显微镜。这些技术将提供矿物形成各个阶段的详细结构和形态信息。我们将重点关注以下几个方面：1-了解CaCO3运输到球粒囊泡的机制，球粒囊泡是矿化位点。我们将结合活细胞成像和低温扫描电镜，可视化钙离子在矿物形成过程中向矿化部位的运输；阐明晶体成核、生长和形态的控制机制。我们将讨论定向成核是如何由有机模板控制的，以及在球粒形成过程中晶体形态是如何形成的。这一目标将通过低温透射电子显微镜（cryoTEM）来实现，以表征原位球石形成的不同阶段；3-了解最初沉积矿物的性质。我们假设这种矿物最初是作为一种非结晶前体沉积的，后来结晶成方解石。我们打算描述这一初始矿物阶段，并了解其在球粒岩结构形成中的作用。这一目标将通过结合低温透射电镜和拉曼光谱来表征最初沉积的矿物相，并跟踪结晶度的发展。综上所述，这项研究将是第一次在单一系统中可视化生物矿物形成的所有阶段。它将揭示生物体如何控制和调整复杂矿化结构在所有长度尺度上的大小、形态和结构的前所未有的信息。从长远来看，这些结果将为合成具有可控尺寸，结构和材料性能的新型材料提供新的策略，并用于多种工业和技术应用。

Living organisms are well known to produce complex mineralised structures that are used for a wide variety of functions, such as navigation, support and protection of soft parts of the body, among others. The formation of such mineralised tissues requires an intricate biological machinery that controls all steps involved in mineralization, from controlling the transport of ions to the mineralisation site to controlling crystal nucleation, growth and morphology and the formation of micron- and macro-sized mineralized tissues. To date, no study could address how all these factors are integrated during the biomineralisation process. As such, what is missing is a fundamental understanding on how cells control processes occurring at different stages and lengths scales, going from the transport of ions to the mineralisation site to crystal nucleation, growth and assembly of the crystals into a hierarchical multi-crystalline mineralised tissue. This issue in the long term, will provide biologists and chemists with new tools to design and synthesise advanced materials with potential applications for biomedical, industrial and technological applications.The aim of this project is to understand how cells control the formation of mineralized tissue, from the first crystals that are formed at the nano-scale, to the mature mineral that is composed of crystals organised in a higher-order structure. For this, we will investigate the formation of coccolith scales made of calcite that are produced by the unicellular marine algae Emiliana huxleyi and Coccolithus pelagicus. These unicellular provide unique models in which all stages in mineralisation, occurring at all length scales - from the onset of mineral formation to the mature mineral - can be investigated in a single system and in a time-resolved manner to elucidate how they contribute to the formation and overall properties of the biomineral.To achieve this aim, we will combine confocal microscopy, cryo-scanning electron microscopy and cryo-transmission electron microscopy. These techniques will provide detailed structural and morphological information on all stages of mineral formation. We will focus on the following aspects:1- Understand the mechanisms of transport of CaCO3 to the coccolith vesicle, which is the mineralisation site. We will combine live-cell imaging and cryo-scanning electron microscopy, to visualise the transport of calcium ions to the mineralization site during mineral formation;2- Elucidate the mechanisms of control over crystal nucleation, growth and morphology. We will address how oriented nucleation is controlled by an organic template, and how crystal morphology is shaped during coccolith formation. This objective will be achieved using cryo-transmission electron microscopy (cryoTEM) to characterize the different stages of coccolithogenesis in situ;3- Understand the nature of the initially deposited mineral. We hypothesise that the mineral is initially deposited as a non-crystalline precursor which later crystallises into calcite. We intend to characterise this initial mineral phase and understand its role in the formation of coccolith structures. This objective will be achieved combining cryoTEM with Raman spectroscopy to characterise the initially deposited mineral phase, and follow the development of crystallinity.Taken together, this study will be the first time in which all stages in biomineral formation will be visualized in a single system. It will reveal unprecedented information on how living organisms control and tailor the size, morphology and construction of complex mineralized structures at all length scales. In the long term, these results will lead to new strategies for the synthesis of novel materials with controllable size, structure and material properties for several industrial and technological applications.