Nanostructured biomaterials: investigating hierarchical calcite crystal formation

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.

The formation of mineralised tissues such as bone, teeth and shells requires an intricate cellular machinery that regulates the following factors: a) the transport and deposition of ions to the mineralisation site; b) the nucleation of crystals on an organic substrate and the timing of nucleation; c) crystal growth along specific directions to shape its morphology; and d) how individual crystals come together to form a hierarchical structure. The goal of this research is to elucidate how all these factors are controlled during biomineralisation, leading to the formation of a complex mineralised tissue. For this, I will study coccolith biomineralisation in the unicellular marine algae Emiliana huxleyi and Coccolithus pelagicus as model systems. We will focus on the following aspects:
1- Understand the mechanisms of transport of CaCO3 to the mineralisation site. We will combine live-cell imaging by confocal microscopy and cryo-scanning electron microscopy, to visualise the transport of calcium-containing granules to the mineralization site;
2- We will elucidate how oriented nucleation is controlled by an organic template, and how crystal morphology is shaped during coccolith formation. We will use cryo-transmission electron microscopy (cryoTEM) to characterize the different stages of coccolithogenesis in situ;
3- We will study whether calcite is preceded by an amorphous calcium carbonate precursor and the role of this phase in the formation of coccolith structures. We will combine cryoTEM with Raman spectroscopy to characterise the initially deposited mineral phase, and follow the development of crystallinity.
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.

In the short term, the beneficiaries from this proposed research will be scientists from other research fields and institutes. This study will represent the first time in which the whole process of mineral formation by organisms will be visualised in a single system, from the transport of mineral, to the onset of crystal nucleation, crystal growth and formation of a mineralised tissue. Thus, it will reveal unprecedented information on how living organisms control and tailor the size, morphology and construction of complex mineralised structures at all length scales. Thus, this research will have a strong impact in the fields of biomineralisation, crystal growth, biomaterials and biomimetic synthetic chemistry, significantly stimulating research in those areas.
In addition, the astonishing material properties of biominerals often combine strength, toughness, sophistication, miniaturization, resistance and adaptability as the result of the integration between the organic and inorganic phases and their hierarchical assembly into complex structures. We anticipate that project outputs will provide new tools to further expand our understanding on the formation and properties of functional biomaterials, and to design synthetic routes to control and tailor the size, shape, complexity and function of crystals and biomaterials. In the long term, this understanding will lead to new routes for the synthesis of specialized hybrid organic-inorganic materials such as tissue engineering scaffolds, re-enforced polymers, sensors, catalyst supports and opto-electronic devices. The feasibility of this approach has already been demonstrated in the fields of nanoelectronics, semiconductors, nanowires, silicification and biomedic engineering, where materials with controllable sizes, composition, morphology, crystallinity and hierarchical organization have been produced.
A further impact this research will have is in developing environmentally-friendly methods to the fabrication of advanced, functional materials. The formation of biominerals in Nature demonstrates that such advanced hybrid materials can be synthesised in aqueous media, under ambient conditions and using renewable feedstocks exclusively. These conditions are in stark contrast with the currently used industrial synthetic methods, which place a high demand on natural resources and have a high ecological footprint. It is our aim, with this project, to develop biomimetic routes using water-based production methods effective at ambient temperatures and provide an alternative to oil-based polymers for the synthesis of materials, addressing current health, industrial, technological, energetic and ecological-related challenges.
Finally, ocean acidification due to an increase in CO2 emissions is one of the challenges our society currently faces. Coccolithophores play an important role in the global carbon cycle and in controlling the alkalinity, carbonate chemistry and CO2 cycle in the oceans. Several studies have already shown that elevated CO2 levels affect calcification by coccolithophores, however the mechanisms are not understood. Importantly, the question of how coccolithophores will respond to future high CO2 conditions will have significant implications to for the ocean's carbon cycle and for global climate. Therefore, elucidating biomineralisation mechanisms will help scientists understand the effect of ocean acidification on calcification and will allow the use of these organisms as proxies to gauge the environmental impact of increased CO2 emissions and content in the oceans.
Overall, this research will have a broad impact in a number of different areas, spanning biology, chemistry, materials chemistry, the biomedical, technological and industrial sectors, and the environmental sciences. It will significantly help the UK to maintain a leading position in scientific research and in the industrial sector.