Periodic Reporting for period 4 - 3D-BioMat (Deciphering biomineralization mechanisms through 3D explorations of mesoscale crystalline structure in calcareous biomaterials )
Reporting period: 2021-09-01 to 2022-08-31
Biocrystallization involves complex biochemical processes where living organisms control the crystalline form of their organo-mineral components. Only considering calcium carbonate polymorphs, a variety of millimetric structures and shapes are observed. These controlled biocrystallization pathways result from the synthesis of otherwise unstable crystalline forms. Mollusc shells, one of the most striking representatives, exhibit different crystalline polymorphs forming various architectures, perfectly illustrated by the famous Pinctada margaritifera pearl oyster.
This microscale architectural diversity contrasts with the quasi-systematic occurrence, in calcareous biominerals, of crystalline spheroid units (granules) of apparent diameter in the 50-500 nm range, coated by a visco-elastic (organic) cortex. This observation does not provide morphological evidence for a crystalline organization. However, the generic characteristic of the granular structure justifies the need of studying the hierarchical arrangement of these 'building-blocks'. The complete understanding of the biomineralization mechanisms could be proven by our capability to fully reproduce the observed structures.
Several bio-crystallization scenarios, able at reproducing some of the biomineral crystalline features, have been proposed. Among them, the polymer-induced liquid-precursor (PILP) process, which could explain most of the observed or inferred crystalline mesoscale properties (crystalline coherence iso-oriented crystalline domains, granular structure with absence of porosity) is gaining more importance. However, a clear demonstration of the relevancy of the PILP process for biomineralization is still lacking.
The question of the biological control on the mineral growth is highly intriguing as well and must be considered simultaneously. It has been recognized that the crystallization process occurs extra cellularly under the control of organic molecules. This active process requires cellular and molecular machinery, producing a leading organic matrix (OM), incorporated within the biomineralized structure itself. Understanding how the OM, and more specifically the proteins, control the mineralization requires identifying the organic components and characterizing their functions. Recently IFREMER has shown that the association between high throughput genomic and proteomic methods enables a real leap forward: 78 matrix proteins have been identified in the pearl oyster. However, their role is speculative. The PILP-based crystallization has been demonstrated for mineralizing systems containing acidic polyelectrolytes. The timely combination of genomic-proteomic results with PILP-based biomimetic crystallization should now provide decisive advances. The ultimate evidences still rely on our capability to analyze the biomimetic crystals at the relevant length scale, at which the granules appear, i. e., the mesoscale.
In this context, the recent breakthrough achieved by the PI and her group, should bring a relevant solution to this problem. In 2011, the PI’s group has proposed a new x-ray crystal-dedicated microscopy, 3D x-ray Bragg ptychography. This approach goes beyond the existing microscopy methods: It gives access to the 3D crystalline properties (crystalline coherence, tilts and strain fields) of a complex, nanostructured and extended crystal, which none of the existing methods is able to provide. In order to fully exploit the specificities of Bragg ptychography for the biomineralization problem, the major limits encountered in the former version have to be pushed back.
Hence, 3D-BioMat aims at deciphering the calcareous biomineralization mechanisms. Using a high-throughput crystalline nano-resolved microscopy, we expect to reveal sets of structural signatures related to the biomineralization pathways. A relevant biomineralization model should be proposed, demonstrating mesoscale structural properties in full agreement with the ones of the biominerals, a decisive proof that can only be brought with 3D Bragg ptychography.
This microscale architectural diversity contrasts with the quasi-systematic occurrence, in calcareous biominerals, of crystalline spheroid units (granules) of apparent diameter in the 50-500 nm range, coated by a visco-elastic (organic) cortex. This observation does not provide morphological evidence for a crystalline organization. However, the generic characteristic of the granular structure justifies the need of studying the hierarchical arrangement of these 'building-blocks'. The complete understanding of the biomineralization mechanisms could be proven by our capability to fully reproduce the observed structures.
Several bio-crystallization scenarios, able at reproducing some of the biomineral crystalline features, have been proposed. Among them, the polymer-induced liquid-precursor (PILP) process, which could explain most of the observed or inferred crystalline mesoscale properties (crystalline coherence iso-oriented crystalline domains, granular structure with absence of porosity) is gaining more importance. However, a clear demonstration of the relevancy of the PILP process for biomineralization is still lacking.
The question of the biological control on the mineral growth is highly intriguing as well and must be considered simultaneously. It has been recognized that the crystallization process occurs extra cellularly under the control of organic molecules. This active process requires cellular and molecular machinery, producing a leading organic matrix (OM), incorporated within the biomineralized structure itself. Understanding how the OM, and more specifically the proteins, control the mineralization requires identifying the organic components and characterizing their functions. Recently IFREMER has shown that the association between high throughput genomic and proteomic methods enables a real leap forward: 78 matrix proteins have been identified in the pearl oyster. However, their role is speculative. The PILP-based crystallization has been demonstrated for mineralizing systems containing acidic polyelectrolytes. The timely combination of genomic-proteomic results with PILP-based biomimetic crystallization should now provide decisive advances. The ultimate evidences still rely on our capability to analyze the biomimetic crystals at the relevant length scale, at which the granules appear, i. e., the mesoscale.
In this context, the recent breakthrough achieved by the PI and her group, should bring a relevant solution to this problem. In 2011, the PI’s group has proposed a new x-ray crystal-dedicated microscopy, 3D x-ray Bragg ptychography. This approach goes beyond the existing microscopy methods: It gives access to the 3D crystalline properties (crystalline coherence, tilts and strain fields) of a complex, nanostructured and extended crystal, which none of the existing methods is able to provide. In order to fully exploit the specificities of Bragg ptychography for the biomineralization problem, the major limits encountered in the former version have to be pushed back.
Hence, 3D-BioMat aims at deciphering the calcareous biomineralization mechanisms. Using a high-throughput crystalline nano-resolved microscopy, we expect to reveal sets of structural signatures related to the biomineralization pathways. A relevant biomineralization model should be proposed, demonstrating mesoscale structural properties in full agreement with the ones of the biominerals, a decisive proof that can only be brought with 3D Bragg ptychography.
3D-BioMat aims at reaching three main objectives:
(1) - Delivering a high throughput 3D x-ray crystalline microscopy,
(2) - Providing the generics of the mesoscale structure from a large panel of biominerals
(3) - Proposing a biomineralization model, including the physico-chemistry and biology
Regarding (1), several major improvements have been brought to our 3D Bragg ptychography approach. A strong speed-up of the acquisition process could be demonstrated, corresponding to a factor of about 10 and an increased field of view, by a factor of about 10. The possibility to retrieve the sample image from a data-set strongly corrupted by positioning errors is now addressed. A full data set can be measured in less than 20 min.
Regarding (2), the structural details of the crystalline properties of a calcite prism from a Pinctada m. shell has been published [Mastropietro2017]. A unique set of samples has been produced by IFREMER and their structures have been characterized in details using a large range of analytical methods. Coherent Raman microscopy has been conducted on Pinctada m. shells and Pinna nobilis shell resulting in the description of the growth cycle of mollusc shells (Duboisset2021).
Regarding (3), a robust crystallisation process could be established, based on the existence of an amorphous precursor. The liquid/liquid phase separation was confirmed in the PILP process. This crystallisation process was used to synthesize a series of synthetic crystals using synthetic molecules, which present excellent agreement with the structure observed in the biogenic samples. In order to go further in the determination of the biomineralization involved-proteins, we used sub-optimally raised (pH x T) shells, expecting that those environmental conditions forced the animal to modify its proteins expression to sustain the increased water acidity and temperature. Proteomics analyses were performed and confronted to the structural analysis performed on these animals, to correlate crystallisation perturbations and increased gene expressions, in link with proteins production. All experiments have been performed and we are finalizing the analysis.
Finally, a series of in vivo experimental strategies were designed and performed with coherent Raman and x-ray Nanodiffraction.
(1) - Delivering a high throughput 3D x-ray crystalline microscopy,
(2) - Providing the generics of the mesoscale structure from a large panel of biominerals
(3) - Proposing a biomineralization model, including the physico-chemistry and biology
Regarding (1), several major improvements have been brought to our 3D Bragg ptychography approach. A strong speed-up of the acquisition process could be demonstrated, corresponding to a factor of about 10 and an increased field of view, by a factor of about 10. The possibility to retrieve the sample image from a data-set strongly corrupted by positioning errors is now addressed. A full data set can be measured in less than 20 min.
Regarding (2), the structural details of the crystalline properties of a calcite prism from a Pinctada m. shell has been published [Mastropietro2017]. A unique set of samples has been produced by IFREMER and their structures have been characterized in details using a large range of analytical methods. Coherent Raman microscopy has been conducted on Pinctada m. shells and Pinna nobilis shell resulting in the description of the growth cycle of mollusc shells (Duboisset2021).
Regarding (3), a robust crystallisation process could be established, based on the existence of an amorphous precursor. The liquid/liquid phase separation was confirmed in the PILP process. This crystallisation process was used to synthesize a series of synthetic crystals using synthetic molecules, which present excellent agreement with the structure observed in the biogenic samples. In order to go further in the determination of the biomineralization involved-proteins, we used sub-optimally raised (pH x T) shells, expecting that those environmental conditions forced the animal to modify its proteins expression to sustain the increased water acidity and temperature. Proteomics analyses were performed and confronted to the structural analysis performed on these animals, to correlate crystallisation perturbations and increased gene expressions, in link with proteins production. All experiments have been performed and we are finalizing the analysis.
Finally, a series of in vivo experimental strategies were designed and performed with coherent Raman and x-ray Nanodiffraction.
Several opportunities, not originally planned in 3D-BioMat, have been made possible since the beginning of the project:
(1) Development of a novel 3D highly-resolved x-ray microscopy for the investigation of polycrystalline material, in collaboration with the group of Prof. Poulsen (DTU). Two articles have been published.
(2) Evidence of the presence of amorphous calcium carbonate in the early stage of the prismatic layer, observed with x-ray pair-distribution function approach using a synchrotron source.An article has been published in PNAS.
(3) Improvement of the Coherent Raman microscope for the in vivo follow-up of the crystallisation process in shells. An in vivo experiment has been included in the project. Interesting results have been obtained and published.
(4) In vivo experiments were also done at synchrotron with x-ray beam to follow the onset of crystallisation (02/22). The results have been analyzed. They show the existence of transient states after the crystallisation, a fully original result. An manuscript has been submitted (11/22).
(1) Development of a novel 3D highly-resolved x-ray microscopy for the investigation of polycrystalline material, in collaboration with the group of Prof. Poulsen (DTU). Two articles have been published.
(2) Evidence of the presence of amorphous calcium carbonate in the early stage of the prismatic layer, observed with x-ray pair-distribution function approach using a synchrotron source.An article has been published in PNAS.
(3) Improvement of the Coherent Raman microscope for the in vivo follow-up of the crystallisation process in shells. An in vivo experiment has been included in the project. Interesting results have been obtained and published.
(4) In vivo experiments were also done at synchrotron with x-ray beam to follow the onset of crystallisation (02/22). The results have been analyzed. They show the existence of transient states after the crystallisation, a fully original result. An manuscript has been submitted (11/22).