# Book of abstract-17th International symposium on Biomineralization, Saint Etienne, France, August 28th to September 1st

Joël Gautron, Marthe Rousseau

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Joël Gautron, Marthe Rousseau. Book of abstract-17th International symposium on Biomineralization, Saint Etienne, France, August 28th to September 1st. 2023. hal-04200775

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Submitted on 8 Sep 2023

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## BOOK OF ABSTRACTS

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### Venue

Faculty of medicine
Jean Monnet University
Saint Etienne, France

### Date

28th August
1st September
2023

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Welcome note of the conference chairs

It is our great pleasure to welcome you to Saint Etienne to join us at the 17th International Symposium on Biomineralization (BIOMIN XVII).

Since 1970, year of the first symposium on biomineralization organized in Germany, the biomineralization community has been making great advances in different aspects of biomineralization, including the hierarchical structures of biominerals and their development, the important genes and functional proteins of biomineralization, the mechanisms of biomineralization in living systems and biomimetic mineralization, the discovery of non-classical crystallization pathways, and the fabrication of advanced functional materials inspired by biomineralization.

The knowledge gained in biomineralization has been applied in hard tissue repair, pharmaceutical, agricultural, energy and environmental sciences, paleontology, and materials-assisted artificial life systems. Biomineralization becomes an important resource for developing new structured materials and solving health-care problems. We believe that we are standing on the historic stage to link the fundamental studies of biomineralization to the challenging global problems.

This biannual conference has been organized in many countries in the world, but only once in a French speaking place, Monaco in 1993. It is time to organize it in France.

Due to the pandemic the BIOMIN XVI edition has been organized virtually by our Chinese colleagues in August 2021.

It is a great pleasure to welcome you to Saint Etienne to have the opportunity to meet face-to-face again

<figure><img src="image_4.png" /><figcaption>Marthe Rouseau</figcaption></figure>

3

<figure><img src="image_5.png" /><figcaption>Joël Gautron</figcaption></figure>
# Organization

**Venue**
Faculty of medicine, Jean Monnet University
10 Rue de la Marandière,
42270 Saint-Priest-en-Jarez
France

**Conference Website** https://www.biomin2023.com/en/page/welcome/

**Conference chairs**

Marthe ROUSSEAU
U1059 INSERM - SAINBIOSE - LBTO
Faculty of medicine
10, rue de la Marandière
42270 Saint-Priest-en-Jarez, France

Joël GAUTRON
INRAE – University of Tours
UMR BOA
37380 Nouzilly, France

**Scientific Committee**

<table><tr><td>France</td><td>A. GUIGNANDON: Université de Saint Etienne<br>C. BLIN: Université de Nice<br>D. EGLIN: Ecole des mines, Saint Etienne<br>F. MARIN: Université de Bourgogne Franche-Comté<br>M.H. LAFAGE-PROUST: Université de Saint Etienne<br>S. RÉHAULT-GODBERT: National research Institute for agriculture, food and environment</td></tr><tr><td>Spain</td><td>A. CHECA: University of Granada</td></tr><tr><td>Germany</td><td>D. GEBAUER: University of Hannover<br>H. COLFEN: University of Konstanz<br>P. FRATZL: Max-Planck institute for colloids and interfaces<br>S. WOLF: Friedrich-Alexander University, institute of glass and ceramics<br>N. KROEGER: Professor at the University of Dresden</td></tr><tr><td>Monaco</td><td>S. TAMBUTTE: Scientific Center of Monaco</td></tr><tr><td>Netherlands</td><td>N. SOMMERDIJK: Radboud Institute for Molecular Life Sciences</td></tr><tr><td>Canada</td><td>M.D. Mc KEE: Mac Gill University<br>M.T. HINCKE: University of Ottawa</td></tr><tr><td>Israël</td><td>S. WEINER: Weizmann institute<br>T. MASS: University of Haïfa, School of marine sciences<br>L. ADDADI: Weizmann Institute of Israël</td></tr><tr><td>China</td><td>Y. MA: Beijing Institute of Technology</td></tr><tr><td>Japan</td><td>M. SUZUKI: University of Tokyo</td></tr><tr><td>USA</td><td>E. BENIASH: Professor in Oral and Craniofacial Sciences, and Bioengineering at the University of Pittsburgh<br>D. JOESTER: Associate Professor on Materials Science and Engineering at the Northwestern University</td></tr></table>

**Local Organizing Committee**

Michel DUCLOS, Tours
Prof David EGLIN, Saint Etienne
Jacky EZAGAL, Tours
Dr Alain GUIGNANDON, Saint Etienne
Prof Marie-Hélène LAFAGE-PROUST, Saint Etienne
Dr Thierry MOREAU, Tours
Simon PALUSSIERRE, Tours
Dr Sophie REHAULT-GODBERT, Tours
Odile SABIDO, Saint Etienne
# The detailed program

## Monday 28 August 2023

**11-00 - 14:00 Registration and poster set-up**

**13:45 - 14:45 Opening Session**

13:45 Opening and welcome
Marthe Rousseau and Joël Gautron cochairs of the conference
XXXXXXX

14:15 Opening keynote Lecture
Steve Weiner (Weizmann Institute of Science, Israël)
Future advances and perspectives in biomineralization research

**14:45 - 18:00 Topic 1 Fundamental of biomineralization**
**(Chairs N. Kroger and T. Mass)**

14:45 Topic 1 Keynote lecture
Stephan Wolf (Friedrich-Alexander-University, Erlangen-Nuremberg, Germany)
Calcareous Mineral Formation — from complex solutes to graded structures

15:15 (T1 O1) A. I. Arns, D. Evans, R. Schiebel, A. Jantschke, S. E. Wolf, G. H. Haug
bridging biomineralisation fundamentals to paleoclimate
reconstructions

15:30 (T1 O2) K. Benzerara, J. Gaëtan, G. Gaschignard, C. Mangin, M. A. Khan, N.
Mehta
The biomineralization of intracellular amorphous carbonates by bacteria

15:45 (T1 O3) H. Ehrlich
Insights into Biosilicification: how does sophisticated biological glass grow

16:00 (T1 O4) S. Milita, T. Zaquin, S. Fermani, D. Montroni, I. Pinkas, L. Barba, G.
Falini, T. Mass
Assembly of the intraskeletal coral organic matrix during calcium carbonate formation

**16:15 - 17:00 Coffee break and posters**

17:00 (T1 O5) C.A. Schmidt, A. Hopanchuk, E. Tambutté, S. Tambutté, P. Gilbert
Amorphous and crystalline precursors to coral skeleton formation in acidifying oceans

17:15 (T1 O6) N. Kroger, C. Heinze, I. Babenko, F. Benjamin
The molecular basis for pore pattern morphogenesis in diatom silica

17:30 (T1 O7) M.L. Lemloh
Diversity and function of biomineralized materials in ciliates

17:45 (T1 O8) J. Tyska, J. Golen, U. Bickmeyer, J. Bijma, K. Godos, Y. Nagai, T. Toyofuku
Organic architecture of chamber biomineralization in main foraminiferal calcifiers

**18:00 - 20:00 Welcome Cocktail**
**8:15 - 12:45 Topic 2 (Session 1) Biomineral structures and crystallization mechanisms (Chairs S. Wolf and A. Checa)**

**8:15** **Topic 2 Keynote lecture**
Helmut Cölfen (University of Konstanz, Germany)
Biomimetic Mineralization to investigate Biomineral structure formation and mineralization mechanisms

**8:45 (T2 O1)** I. Zlotnikov, D. Karpov
The role of lattice distortions in coccoliths morphogenesis

**9:00 (T2 O2)** O. Ben Joseph, L. Aram, D. de Haan, K. Rechav, A. Gal
Intracellular biomineralization of rhombohedral calcite by haploid life phases of coccolithophores

**9:15 (T2 O3)** K. Berent, K. Nalepka, P. Czaja, Ł. Maj, T. Machniewicz, M. Bieda-Niemiec, K. Sztwiertnia, A.G. Checa
Twin-based strengthening mechanism in a crossed-lamellar structure

**9:30 (T2 O4)** J. Huang, H. Liu
Calcification of the egg capsule in the apple snail: the organic matrix and the calcium-rich granules

**9:45 (T2 O5)** W. E. G. Müller, X. Wang
Biomimetic transformations of amorphous calcium phosphate to dental and ossifying crystalline hydroxyapatite

**10:00 (T2 O6)** K. Berent, A.G. Checa
Organization of a plywood biomaterial: the calcitic cross-foliated microstructure of limpets

**10:15 - 11:00 Coffee break and posters**

**11:00 (T2 O7)** F. Nudelman, A.L. Rossi, F. Laidlaw, G. Graziani, G. Falini
Structural and mechanical adaptation of Lingula anatina shells

**11:15 (T2 O8)** N. Oehlsen, J. von Döhren, S. Weiner, J. Ibrahim, L. Stegbauer
Stabilization of Amorphous Calcium Phosphate by Barium: A Novel Approach Inspired by a Marine Worm

**11:30 (T2 O9)** M. de Frutos, A.B. Rodríguez-Navarro, X. Li, A.G. Checa
Monochromated STEM-EELS analysis of the structure and composition of biogenic calcite reveals the biomineral growth pattern

**11:45 (T2 O10)** T. Okumura, M. Suzuki, T. Kogure
Anisotropic lattice parameters change in aragonite induced by Na+ substitution

**12:00 (T2 O11)** V. Sardhalia, C. Do Reis Ferreira, S. Amini, P. Fratzl, M. De Frutos, D. Talbot, A. Vallée, A. Fitch, S. Checchia, N. Nassif, T. Azaïs, M. Albéric
Halochromic ACC crystallization in the presence of naphthoquinones unveils biomineral pigmentation in sea urchins

**12:15 (T2 O12)** C. Grenier, E. Griesshaber, W. Schmahl, B. Bernin, A.G. Checa
Microstructures and biomineralization patterns of gymnolaemate bryozoans (Bryozoa, Gymnolaemata)

**12:30 (T2 O13)** N. Shaked, S. Barinova, I. Pinkas, S. Addadi, S. Weiner, L. Addadi
Calcite and guanine formation by the freshwater green alga *Phacotus lenticularis*
**12:45 - 14:15** Lunch and poster session

**14:15 - 15:30** Topic 5 immunity in biomineralized barriers
(Chairs S. Réhault-Godbert and M. Mc Kee)

**14:15** Topic 5 Keynote lecture
Sophie Réhault-Godbert (INRAE-Université de Tours, France)
Dynamics of the chicken chorioallantoic membrane and the eggshell during chicken embryonic
development: a fine regulation between eggshell decalcification and maintenance of egg defences

**14:45 (T5 O1)** C.R. Ferreira, C. Djediat, X. Lekube, U. Izagirre, N. Garcia-Veslaco; Y. Politi, L. Bertinetti, N. Nassif, M. Albéric
Shedding light on the nature of sea urchin pigment-bearing vesicles

**15:00 (T5 O2)** L. Zorzetto, E. Scoppola, E. Raguin, P.Fratzl, C.M. Bidan
Induced mineralization in E. coli biofilms: the role of extracellular matrix

**15:15 (T5 O3)** P. Działak, M. Słowakiewicz, A. Borkowski
Morphology and size of mineral particles may be affected by bacteriophages

**15:30 - 18:00** Topic 8 biomineralization and environmental changes
(Chair S. Fitzer)

**15:30** Topic 8 Keynote lecture
Susan Fitzer (University of Stirling, UK)
Impacts of climate change on mollusc biomineralisation pathways

**16:00 (T8 O1)** I. Coronado1, J.A. Cruz1,, A. Owczarek, M.Sáenz-Navajas, J.R. Mateos-Carralafuente, P. Cózar, E. Fernández-Martínez, L. Fernández-Díaz, J. Stolarski
Secondary aragonite recrystallization during otoliths diagenesis: natural and experimental evidence

**16:15 - 17:00** Coffee break and posters

**17:00 (T8 O2)** T.Mass, F. Scucchia, P. Zaslansky
Primary coral polyps responses to decreasing seawater pH: observations from cell to the complete
organism

**17:15 (T8 O3)** A. Venn, M. Gilbert, E. Tambutté, S. Tambutté
Investigating the impact of ocean acidification on CaCO3 crystal growth rates in the reef coral
Stylophora pistillata

**17:30 (T8 O4)** D.M. Chevrier, A. Juhin, N. Menguy, R. Bolzoni, P.E.D. Soto-Rodriguez, M. Kojadinovic-Sirinelli, G.A. Paterson, R. Belkhou, W. Williams, F. Skouri-Panet, A. Kosta, H. Le Guenno, E. Pereiro, D. Faivre, K. Benzerara, C.L. Monteil, C.T. Lefevre
Organization and magnetic properties of magnetic ectosymbiotic bacteria optimize a collective
magnetotaxis behavior in a microbial holobiont

**17:45 (T8 O5)** M. Hu
Cellular pH regulatory systems underlying calcification in the sea urchin larva
Wednesday 30 August 2023

8:15 – 12:30 **Topic 4 Imaging methods for biomineralization researches**
(Chairs A. Guignandon and P. Zaslansky)

**8:15** **Topic 4 Keynote lecture**
Paul Zaslansky (Charité - Universitätsmedizin Berlin, Germany)
A darker side to X-ray brightness - assessing calcium mediated structural degradation during bone studies

8:45 (T4 O1) E. Raguin, R. Weinkamer, P.Fratzl
Dynamic interpretation of the bone mineralization process in the chick embryo based on Cryo FIB-SEM images

9:00 (T4 O2) V. Chamard, T.A. Grünewald, H. Dicko, J. Vidal-Dupiol, B. Petton, J. Legrand, A. Campos, E.Tambutté, A. Venn, S. Tambutté, J. Le Luyer, M. Sztucki, M. Burghammer, J. Duboisset
Towards in vivo imaging of the physico-chemistry processes involved in the biomineralization of mollusc shells

9:15 (T4 O3) C. Wendt, A.L. Rossi, J. Cypriano, C. Dilnei de Castro Oliveira, C. Arrouvel, J. Werckmann, M. Farina
Unique features of Caudofoveata (Mollusca) spicules revealed by combined light and electron microscopy techniques

9:30 (T4 O4) S. Amini, T. Zhu, E. Griesshaber, W.W. Schmahl, P. Werner, P. Fratzl
Uncovering the complex world of elastic deformations in biological ceramics

9:45 (T4 O5) R. Guo, A. Somogyi, D. Bazin, E. Letavernier, E. Tang, K. Medjoubi
Study of the role of trace elements on kidney stones pathogenesis with multi-scale and multimodal scanning X-ray tomography

10:00 (T4 O6) M. Hegedűs, V.K. Kis, Z. Kovács
Quantitative evaluation of enamel prism arrangement based on image processing technique

**10:15 – 11:00 Coffee break, posters and group picture**

11:00 – 12:30 **Topic 7 Biomineralization in aquaculture**
(Chairs S. Tambutté and M. Suzuki)

11:00 **Topic 7 Keynote lecture**
Masahiko Awaji (The Graduate School of Agricultural and Life Sciences, the University of Tokyo)
Contribution of Biomineralization Research to the Advancement of Pearl Aquaculture

11:30 (T7 O1) C. Martinand-Mari, E. Potier, E. Gasset, G. Dutto, S. Lallement, C. Bourdy, M. Debiais-Thibaud, E. Farcy
Estradiol and bisphenol A effects on early skeletogenesis in the European sea bass D. labrax

11:45 (T7 O2) P. Ganot, G. Loentgen, F. Marin, L. Plasseraud, D. Allemand, S. Tambutté
An alternative method for extracting the organic matrix from the skeleton of the red coral Corallium rubrum

12:00 (T7 O3) M. Bott, F. Drescher, S. Machill, A. Jantschke
Biogenic organic crystals are solid solutions: HPLC-Quantification of purine-bases

12:15 (T7 O4) Y. Namikawa, M. Suzuki
Carbonic anhydrase activity identified in the powdered nacreous layer of Pinctada fucata

12:30 – 14:30 **Lunch and poster session**
**Meeting of the scientific committee**
14:30 – 17:45 Topic 6 Biomineralization and biomimetics
(Chairs D. Eglin and Y. Ma)

14:30 Topic 6 Keynote lecture
David Kisailus (Materials Science and Engineering, University of California at Irvine)
Biological Adaptations and Blueprints for Extreme Environments

15:00 (T6 O1) R. Zarivach
Structure-function studies of intrinsically disordered proteins-iron oxide interaction

15:15 (T6 O2) N. Luo, B. Lu, X. Chen, F. Chen
The calcium phosphate-based biomineral clusters for rapid remineralization of tooth enamel

15:30 (T6 O3) M. Vitori, V. Srot, L. Korat; B. Bussman, F. Predel, Van Aken Peter A, J. Štrus
Structural and mechanical anisotropy in crustacean claws mineralized with amorphous calcium phosphate

15:45 (T6 O4) M. Burgos-Ruiz, K. Elert, C. Rodriguez-Navarro, E. Ruiz-Agudo
Bio-Inspired Fluorescent Consolidants for the Conservation of Gypsum Plasterwork

16:15 - 17:00 Coffee break and posters

17:00 (T6 O5) A. Kubiak, M. Pajewska-Szmyt, M. Kotula, B. Leśniewski, H. Ehrlich
Iron-based biominerals of marine sponge origin as inspiration for biomimetics

17:15 (T6 O6) Y. Ma
Controlled crystallization of guanine nanocrystals

17:30 (T6 O7) P. Biswas
Role of Polyamines in the condensation of silicic acid for efficient silicification and its study with pH
Thursday 31 August 2023

**8:30 – 12:30** Topic 3 Vertebrate mineralized tissues in health and disease
(M.H. Lafage-Proust and E. Beniash)

**8:30** **Keynote lecture**
Marc D. McKee (McGill University, Faculty of Dental Medicine and Oral Health Sciences, and Faculty of Medicine and Health Sciences, Montreal, Quebec, Canada)
Enzymatic patterning of bone mineralization at the microscale for crossfibrillar mineral tessellation: The Stenciling Principle

9:00 (T3 O1) H. Yamazaki, C.A. Stifler, C. Gabe, A. Thu Bui, L. Lukashova, K. Verdelis, P.U.P.A. Gilbert, H.C. Margolis, E. Beniash
Biological control of enamel mineralization is dependent on phosphorylation of amelogenin

9:15 (T3 O2) R. Hugon, L. Hivert, N. Boutahar, A. Vanden-Bossche, N. Laroche, M. Thomas, M.T. Linossier, L. Vico, M.H. Lafage-Proust, L. Malaval
Low phosphate diet differentially affects bone mineralization in mice deficient in Bone Sialoprotein, Osteopontin or both

9:30 (T3 O3) M. Duclos, A. Gloux, A. Narcy, J. Gautron
Altered bone health in laying hens is associated with FGF23 overexpression in the medullary bone

9:45 (T3 O4) A. Cohen, L. Gotnayer, S. Gal, D. Aranovich, N. Vidavsky
Multicellular spheroids containing synthetic mineral particles to investigate breast precancer malignancy potential according to the mineral type

10:00 (T3 O5) C. Wendt, A. Linhares, C. Santos, V. Zelaya, R. Lopes, M. Farina, A. Rossi
Ultrastructure of mineral ellipsoids at bone-calcium phosphate interface in bone healing process

**10:15 - 11:15** Coffee break and posters

11:15 (T3 O6) A. Berdal, S. Renaud, D. Hotton, L. Laurencena, M. De la Dure-Molla, M. MacDougall
The dental and periodontal mineralization defects related to FAM20A gene mutations

11:30 (T3 O7) Y. Bertache-Djenadi, K. Nguyen, A. Vanden-Bossche, M. Thomas, S. Mundweiler, M.T. Linossier, S. Peyroche, D. Farlay, H. Marotte, L. Vico, N. Rochereau, M. Rousseau
Effect of MOP consumption on calcium metabolism and gut in ovariectomized rat model

11:45 (T3 O8) C.M. Gabe, A. Thu Bui, B.P. Vasquez, E. Beniash, H.C. Margolis
Lack of Amelogenin Phosphorylation Leads To Acidification Of Secretory Enamel

12:00 (T3 O9) C. Dittfeld, P. Metzner, M. Feilmeier, A. Jannasch, K. Matschke, S. Manthey, S. Rammelt, S.M. Tugtekin, G. Steiner
FT-IR spectroscopic imaging of human aortic valve biomineralization and protein secondary structure combining hard and freeze cutting technique

12:15 (T3 O10) I.L. Jaabar, B. Foley, A. Mezzetti, F. Pillier, F. Berenbaum, J. Landoulsi, X. Houard
Dynamics of extracellular matrix remodeling associated with mineralization during the hypertrophic differentiation of articular chondrocytes

**12:30 – 14:00** Lunch and poster session

**14:00-18:00** Discovery cruise to Saint Victor sur Loire

**19:30-00:00** Gala dinner at Dame d4 café
Friday 1 september 2023

8:45 – 10:15 **Topic 2 (Session 2) Biomineral structures and crystallization mechanisms (S. Weiner and H. Coelfen)**

8:45 (T2 O14) A. John Samuel, B. Bellec, I. Zlotnikov
The origin and function of geometric frustration in spicule morphogenesis

9:00 (T2 O15) G. Takahashi, T. Okumura, T. Nagaya, M. Suzuki, Y. Takahashi, T. Kogure
Crystallographic Characteristics of Vaterite in Fish Otolith

9:15 (T2 O16) Zhuanfei Liu, Yunya Niu, Zeyao Fu, Mason Dean, Zhengyi Fu, Yongming Hu and Zhaoyong Zou
3D interrelationship between hierarchical canal network and the gradient mineralization of shark tooth osteodentin

9:30 (T2 O17) J. Yang, T. Willhammar, H. Zeng, J.D. Gale, N. Hedin, D. Gebauer, B.Q. Lu
Novel crystalline phases derived from amorphous calcium hydrogen phosphate

9:45 (T2 O18) A.B. Rodríguez Navarro, S. Madero, M. Greiner, P.A. Rodríguez-Jimenez, C. Jimenez-Lopez, W.W. Schmahl
Changes in bone chemistry, mineralogy and structure during heating

10:00 (T2 O19) F. Grosso Giordano, N. De Belie, N. Boon, C. Rodríguez-Navarro
Infrared and Raman spectroscopy for the fingerprinting of microbially-induced calcium carbonate precipitation

10:15 – 11:00 **Coffee break and posters**

11:00 – 11:45 **Topic 9 Evolution of molecular tool kits**
(Chair F. Marin)

11:00 **Topic 9 Keynote lecture**
F. Marin (UMR CNRS-EPHE 6282 Biogeosciences, Université de Bourgogne, Dijon)
Few thoughts on the evolution of calcifying matrix proteins in metazoans

11:30 (T9 O1) L. Nicolas, C. Martinand-Mari, M Debiais-Thibaud
The vertebrate skeleton through the eye of a cartilaginous fish: Evolutionary perspectives raised by the study of the small spotted catshark Scyliorhinus canicular

11:45-12:30 **Closing remarks**
Presentation of BIOMIN XVIII

12:30 **Farewell and lunch box**
Opening Session

Keynote lecture

Future advances and perspectives in biomineralization research.

Steve Weiner, Ron Shahar and Lia Addadi

Weizmann Institute of Science, Israël

Advances are almost impossible to predict, but we can present our perspectives on future trends. Technological developments have almost always resulted in major breakthroughs in this field. The huge technological progress in the last decade that enables correlating live optical 3D observations with high resolution 3D cryo-electron imaging, opens many opportunities. In this way cells, extracellular matrices and mineral can be visualized in, or almost in their in vivo state. Furthermore, new insights into the 3D structure of mineralized biological materials can improve understanding of their mechanical function, especially when reconsidering how isotropic or anisotropic the structure is. The structural variations in pathological mineralized tissues can be better related to the genetic mutation(s) causing the pathology and how the function of pathological mineralized tissue affects function.

Marine organisms producing abundant calcium carbonate shells are a crucial link in the reduction of atmospheric carbon dioxide that is in part driving climate change. We need therefore to better understand the basic mechanisms involved in mineral formation of Coccolithophoridae, foraminifera, pteropods and dinoflagellate cysts. This we think will lead to a firmer foundation for predicting how these organisms will respond to climate change. The recent discovery that most single-celled marine organisms tested are producing organic crystals such as guanine, raises the novel question whether these organic crystals are directly or indirectly also involved in calcium carbonate formation in some of these organisms? And in general what are the functions of these organic crystals, especially in light manipulation?

Many ions fulfill fundamental processes in cell metabolism. Much more can be learned about their roles by taking advantage of the ability to cryo-fix cells and tissues, thus preserving the ion distributions in their in vivo contexts. Furthermore by studying the cells and tissues that are concentrating, storing and translocating very large amounts of ions during mineralization, the biomineralization field can contribute significantly to this important topic in cell biology.

But one prediction that we can make with certainty is that the future breakthroughs in the field of biomineralization, will not be predicted!

**Keywords:** Mineralized collagen, marine carbonates, calcium
Keynote lecture

Calcareous Mineral Formation — from complex solutes to graded
structures

Stephan E. Wolf

Friedrich-Alexander-University Erlangen-Nuremberg (FAU), Department of Materials Science and
Engineering (WW), Institute of Glass and Ceramics (WW3) Martensstrasse 5 · 91058 Erlangen ·
Germany

Regarding quantity, calcareous minerals formed by biological processes are the most abundant biominerals in the Tree of Life. However, the molecular and physicochemical machinery that drives calcareous mineral formation in organisms is not yet fully decoded. Even “simple” calcite prisms formed by the nacroprismatic bivalve *P. nobilis* require an intricate and diversified set of proteins. Remarkably, their prism morphology can be predicted by standard physical models — which allows for the speculation that significant parts of this “shellome” control mineral assembly processes on lower length scales or at earlier stages. In this contribution, we give an overview of nanoscale assembly processes that drive mineral deposition in calcareous shells and exemplify their ameliorating impact on biomineral properties via structural and functional gradation. On an even lower length scale, we further discuss recent advances in our molecular understanding of mineral solutes interacting with organic additives — be the latter peptidomimetics of biomineralization proteins or even additives of small-molecular weight. These molecular interactions operate beyond classical conceptions as they explicitly rely on the involvement of solute oligomers, often denoted as so-called prenucleation clusters. Our findings provide an explanatory basis for the unexplainedly strong impact of specific organic compounds, calling already for a re-evaluation of the putative role of small molecules in biomineralization processes. Moreover, our findings show that these solute oligomers act as gatekeepers for the chemical composition of amorphous calcium carbonate, the common transient mineral precursor in numerous biomineralization processes. Ultimately, the results point to solute oligomers as the binding partners of “calcareous” biomineralization proteins in solution, breaking new grounds for detailed assessment of protein-mineral interactions.

**Keywords:** mineral formation mechanisms, organo-mineral interactions
## Oral presentations

### T1 O1

#### Bridging biomineralisation fundamentals to paleoclimate reconstructions

A. I. Arns<fnref n="1" />, D. Evans², R. Schiebel<fnref n="1" />, A. Jantschke³, S. E. Wolf⁴, and G. H. Haug<fnref n="1" />,⁵

The biomineralisation of organisms that serve as archives for environmental reconstruction, such as planktic foraminifers, is of particular interest to paleoclimate research. Confident reconstructions over millions of years rely on understanding the mechanistic connection of environmental parameters to trace element and isotopic proxy signals in the shell through biomineralisation, both from a biological and an inorganic perspective.

To explore fundamental abiotic mechanisms, we investigated nucleation and crystallisation processes along with element and isotope fractionation in an in-vitro inorganic experimental model for marine biomineralisation. In this model system, we assume calcification from modified seawater at elevated pH and [CO₃²⁻], with and without the involvement of metastable phases such as amorphous CaCO₃, thereby mimicking processes implied and observed in-vivo.<fnref n="1" />⁻⁴ We observe that in a complex seawater matrix, the dependencies of prenucleation, nucleation and postnucleation regimes in potentiometric titration experiments on the ionic composition of the solutions, as well as the carbonate phases present, differ compared to previous descriptions in compositionally simpler matrices.⁵ Hence, the application of models developed for compositionally simple solutions to carbonate crystallisation processes in seawater is unlikely to yield an accurate description, for example for the presence of transient amorphous phases.⁵

[1] D. Evans, W. Müller, J. Erez, *Geochim. Cosmochim. Acta* **2018**, 236, 198–217.

[2] J. Erez, *Rev. Mineral. Geochemistry* **2003**, 54, 115–149.

[3] A. I. Arns, D. Evans, R. Schiebel, L. Fink, M. Mezger, E. Alig, J. Linckens, K. P. Jochum, M. U. Schmidt, A. Jantschke, G. H. Haug, *Geochemistry, Geophys. Geosystems* **2022**, 23, 1–18.

[4] T. Toyofuku, et al., *Nat. Commun.* **2017**, 8, 6–11.

[5] D. Gebauer, A. Völkel, H. Cölfen, *Science*. **2008**, 322, 1819–1822.

**Keywords**: Carbonate crystallisation, Seawater, Amorphous CaCO₃

<footnote n="1">¹ Max Planck Institute for Chemistry, 55128 Mainz, Germany. ² School of Ocean and Earth Science, University of Southampton, Southampton, UK, ³ Institute of Geosciences, Johannes Gutenberg University Mainz, 55128 Mainz, Germany. ⁴ Department of Materials Science and Engineering, Friedrich Alexander University, 91058 Erlangen, Germany. ⁵ Department of Earth Sciences, ETH Zurich 8092 Zurich, Switzerland.</footnote>
# The biomineralization of intracellular amorphous carbonates by bacteria

Karim Benzerara¹, Juliette Gaëtan¹, Geoffroy Gaschignard ¹,Camille Mangin², Monis-Athar Khan ³, Neha Mehta¹

¹IMPMC, CNRS-Sorbonne Université and MNHN, Paris, France, ²BIAM, CEA Cadarache, ³LBBC, CEA Saclay, France,

Bacteria forming intracellular amorphous carbonates (iAC) have been increasingly discovered, including diverse Proteobacteria, among which some magnetotactic bacteria, as well as Cyanobacteria [1-2]. The iAC grains can be massive and fill a large proportion of the cell volume. They are found in very diverse environments from marine to freshwater, anoxic sediments, soils or thermal water. Bacteria can form iAC even in aqueous solutions significantly undersaturated with all CaCO₃ phases. Sometimes, these bacteria can be locally abundant such as in the case of *Microcystis*, a cosmopolitan bloom-forming cyanobacterium [3]. This suggests a potentially important environmental impact, although efforts to better quantify this are in progress. These biocarbonates can have major and/or trace element composition at odd with what is expected for abiotic carbonates precipitated in the same environment. In particular, one iAC-forming cyanobacterium efficiently sequesters the radioactive ²²⁶Ra isotope intracellularly, even in Ra-diluted solutions and in the presence of competing cations such as Ca, Ba and Sr [4]. This may benefit the future development of efficient ²²⁶Ra bioremediation strategies.

The mechanisms involved in the formation of bacterial intracellular carbonates are under study. They likely differ among the diverse bacteria. Interestingly, a gene with no homologue of known function was discovered, which serves as a good marker of the capability of cyanobacteria to form iAC [5]. Phylogenetic reconstructions suggest that this gene was present in ancestral cyanobacteria with losses in various lineages along the evolution of Cyanobacteria, indicating that this biomineralization pathway is ancient.

[1] Benzerara et al. (2020). *Geobiol.* 19, 199-213.

[2] Monteil et al. (2021). *ISME J* 15, 1-18.

[3] Gaëtan et al. (2023). *Env. Microbiol.*, 25, 751–765.

[4] Mehta et al. (2022). *ACS ES&T Water*, 2, 616–623.

[5] Benzerara et al. (2022). *Genome Biology and Evol.*, 14:evac026.

**Keywords:** cyanobacteria, ACC, intracellular, gene
# Insights into Biosilicification: how does sophisticated biological glass grow

Hermann Ehrlich

*Center of Advanced Technology, Adam Mickiewicz University, Poznan, Poland, herehr@amu.edu.pl*

Our work provides an answer to one of the fundamental questions of science, namely, why biological glass grows past at ambient temperatures and reaches dimensions in the meter range on example of glass sponges. These first metazoans represent an evolutionarily ancient and simultaneously preserved archive of unique nanostructural biosilica. The diversity of shape (more than 80 morphotypes), size (up to 3 meter-long), highly sophisticated network connectivity and ornamentation in skeletal formations of sponges are well recognized. That there is proteinaceous axial, sometimes branched, filament in the extracellular spicules of sponges is known, and we have now shown that this first skeletal pattern generator is in fact actin. Since actin has been perceived as a strictly intracellular protein, no one has ever looked for it and has not been predicted it in such extracellular structures as glassy spicules.

We used a battery of bioanalytical techniques to show that axial filaments in 22, 31 and 2 representatives of poriferan classes Hexactinellida, Demospongiae and Homoscleromorpha, respectively, are made of F-actin. The conserved biomaterials morphology of sponges suggests this is an ancient role that actin has played for at least 545 million years. We suggest that F-actin was localized in an ancestral, intracellular siliceous construct, and as spiculogenesis moved to extracellular spaces the actin continued to play its pattern forming role, leading to a release on a size constraint on fibrous actin complexes. The branching capability of the actin filament bundle was an exaptation that led to the diverse, sometimes very complex composite-based skeletal geometries at the macro level in sponges. Thus, the “epitaxy” of biosilica structures is due to the presence and growth of actin filaments. Being arrested in glass, actin shows new function in sophisticated architecture of biosilica in sponges.

**Keywords:** biosilicification, biomaterials, actin, marine sponges

<figure><img src="image_6.png" /></figure>
Assembly of the intraskeletal coral organic matrix during calcium carbonate formation

Silvia Milita,¹ Tal Zaquin,² Simona Fermani,³ Devis Montroni,³ Iddo Pinkas,⁴ Luisa Barba,⁵ Giuseppe Falini³ and Tali Mass²

¹CNR, via Gobetti 101, Bologna, 40129, Italy. ²Department of Marine Biology, University of Haifa, 3498838, Haifa, Israel. ³Department of Chemistry, University of Bologna, via Selmi 2, Bologna, 40126, Italy. ⁵Department of Chemical Research Support, Weizmann Institute of Science, 76100, Rehovot, Israel. ⁶CNR, I-34100 Trieste, Italy.

Scleractinia coral skeleton formation occurs by a heterogeneous process of nucleation and growth of aragonite in which intra-skeletal soluble organic matrix molecules, usually referred to as SOM, play a key role.¹ Several studies have demonstrated that they influence the polymorphic precipitation of calcium carbonate.² However, the structural aspects that occur during the growth of aragonite have received less attention. In this research, we study the deposition of calcium carbonate on a model substrate, silicon, in the presence of SOM extracted from the skeleton of two coral species representative, which we previously characterized.³ The study is performed by grazing incidence X-ray diffraction with the support of Raman spectroscopy and electron and optical microscopies. The results show that SOM macromolecules once adsorbed on the substrate self-assembled in a layered structure and induced the oriented growth of calcite inhibiting the formation of vaterite. Differently, when SOM macromolecules were dispersed in solution they induced the deposition of amorphous calcium carbonate, ACC, still preserving a layered structure. The entity of these effects was species dependent. In conclusion, we discovered for the first time that the SOM from corals can have a 2D lamellar structure. This structure is preserved when the SOM interacts with ACC but is lost when the interaction occurs with calcite.

References

(1) Zaquin, T.; Pinkas, I.; Di Bisceglie, A. P.; Mucaria, A.; Milita, S.; Fermani, S.; Goffredo, S.; Mass, T.; Falini, G. *Cryst. Growth Des.* **2022**, *22* (8), 5045–5053.

(2) Falini, G.; Fermani, S.; Goffredo, S. In *Seminars in cell & developmental biology*; Elsevier, 2015; Vol. 46, pp 17–26.

(3) Zaquin, T.; Di Bisceglie, A. P.; Pinkas, I.; Falini, G.; Mass, T. *Sci. Rep.* **2022**, *12* (1), 16575.

**Keywords:** Coral skeleton, calcium carbonate, grazing incidence X-ray diffraction, soluble organic matrix, Sty-lophora pistillata, Oculina patagonica
# Amorphous and crystalline precursors to coral skeleton formation in acidifying oceans

Connor A. Schmidt¹, Andrii Hopanchuk¹, Eric Tambutté², Sylvie Tambutté², Pupa U.P.A. Gilbert¹,³,⁴*

¹ Department of Physics, University of Wisconsin, Madison, WI 53706, USA. ² Department of Marine Biology, Centre Scientifique de Monaco, 98000 Monaco, Principality of Monaco. ³ Departments of Chemistry, Materials Science and Engineering, and Geoscience, University of Wisconsin, Madison, WI 53706, USA. ⁴ Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

* Correspondence to: pupa@physics.wisc.edu.

We compared the transient precursor phases on the forming surface of fresh corals grown at different pH values: pH 7.2, 7.4, 7.8, and 8.0 ¹. Using PhotoEmission Electron Microscopy ²,³ at the calcium L-edge we identified the transient metastable precursor phases ⁴⁻⁶ as a function of distance from the surface. We expected to find the most and least metastable precursors at the outermost and innermost layers of the surface, but that is not what we found. One of the intermediate phases penetrates deepest into the skeleton.

1. A. Venn, E. Tambutté, N. Caminiti-Segonds, N. Techer, D. Allemand and S. Tambutté: Effects of light and darkness on pH regulation in three coral species exposed to seawater acidification. *Sci Rep* **9**, 1 (2019).

2. G. De Stasio, M. Capozi, G.F. Lorusso, P.A. Baudat, T.C. Droubay, P. Perfetti, G. Margaritondo and B.P. Tonner: MEPHISTO: Performance tests of a novel synchrotron imaging photoelectron spectromicroscope. *Rev Sci Instrum* **69**, 2062 (1998).

3. G. De Stasio, L. Perfetti, B. Gilbert, O. Fauchoux, M. Capozi, P. Perfetti, G. Margaritondo and B.P. Tonner: MEPHISTO spectromicroscope reaches 20 nm lateral resolution. *Rev Sci Instrum* **70**, 1740 (1999).

4. T. Mass, A.J. Giuffre, C.-Y. Sun, C.A. Stifler, M.J. Frazier, M. Neder, N. Tamura, C.V. Stan, M.A. Marcus and P.U.P.A. Gilbert: Amorphous calcium carbonate particles form coral skeletons. *Procs Natl Acad Sci* **114**, E7670 (2017).

5. C.-Y. Sun, C.A. Stifler, R.V. Chopdekar, C.A. Schmidt, G. Parida, V. Schoeppler, B.I. Fordyce, J.H. Brau, T. Mass, S. Tambutté and P.U.P.A. Gilbert: From particle attachment to space-filling coral skeletons *Procs Natl Acad Sci* **117**, 30159 (2020).

6. C.A. Schmidt, C.A. Stifler, E.L. Luffey, B.I. Fordyce, A. Ahmed, G. Barreiro Pujol, C.P. Breit, S.S. Davison, C.N. Klaus, I.J. Koehler, I.M. LeCloux, C. Matute Diaz, C.M. Nguyen, V. Quach, J.S. Sengkhammee, E.J. Walch, M.M. Xiong, E. Tambutté, S. Tambutté, T. Mass and P.U.P.A. Gilbert: Faster crystallization during coral skeleton formation correlates with resilience to ocean acidification. *J Am Chem Soc* **144**, 1332 (2022).

**Keywords:** coral, amorphous, precursor, acidification
# The molecular basis for pore pattern morphogenesis in diatom silica

Nils Kroger, Christoph Heinze, Iaroslav Babenko, Friedrich Benjamin

*Technische Universität Dresden, Germany*

Diatoms are unicellular, eukaryotic algae characterized by silica-abased cell walls that display complex, species-specific nano- and micropatterns. A hallmark of diatom silica are the hierarchically arranged patterns of pores that endow this material with favorable properties regarding interaction with light, materials transport, and mechanical stability. The porous cell walls are thought to be critical to the evolutionary success of diatoms, and in the materials science community are increasingly utilized to develop new silica-based nanotechnologies. Despite the importance of diatom silica, very little is known about the molecular machinery that enables morphogenesis of this extraordinary material. Recently, we have performed in the model diatom *Thalassiosira pseudonana* the first proteomics analysis of the organelle for silica biogenesis, the silica deposition vesicle (SDV). This led to the identification of 39 proteins that we regard as components of the machinery for silica biogenesis. We have focused on functional characterization of three of these SDV proteins, dAnk1-3, that are widely distributed among the diatoms and contain common protein-protein interaction domains (ankyrin repeats). We demonstrate through gene knockout and rescue experiments together with semi-autonomous, quantitative analysis of electron microscopy images that dAnks control the formation and the patterned arrangement of the pores in the silica. We propose a model that can explain how dAnks control pore formation and provides novel, experimentally testable hypotheses for future studies on silica morphogenesis.

**Keywords:** Silica, Morphogenesis, Proteomics
# Diversity and function of biomineralized materials in ciliates

Marie-Louise Lemlohª

ªUniversity of Stuttgart, Materials Testing Institute, SRF AMICA, Germany

Eukaryotic unicellular organisms such as ciliates (Protist) represent an excellent model system to discover the principles of biomineralization with respect to intracellular mechanisms involved in ion accumulation, vesicular transport, and biomineral formation. Ciliates occur naturally in freshwater, brackish, and marine habitats as well as in extreme environments. Examples of mineral precipitates with known functions include calcium carbonate or barium sulfate biominerals. For other, intracellular mineral-rich inclusions, the function is mostly unknown. To identify mechanisms with specific adaptations involved in mineral formation, we combine dynamic in vivo studies of mineral-forming ciliates together with high-resolution methods such as analytical electron microscopy. A recent example of mineralization that functions in detoxification is the intracellular bioaccumulation of the rare earth element gadolinium in ciliate cells, resulting in the formation and excretion of biogenic particles.

**Keywords:** protist, ciliate, intracellular mineralization, detoxification
Organic architecture of chamber biomineralization in main foraminiferal
calcifiers

TYSZKA Jarosław*¹, GOLEŃ Jan¹, BICKMEYER Ulf², BIJMA Jelle², GODOS Karolina¹, NAGAI Yukiko³, TOYOFUKU Takashi³

¹ING PAN – Institute of Geological Sciences, Polish Academy of Sciences, Research Centre in Cracow, Senacka 1, 31-002 Kraków, Poland, ² Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung, Bremerhaven, Germany, ³ Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Natsushima-cho 2-15, Yokosuka, 237-0061, Japan

*Corresponding author (Email): ndtyszka@cyfronet.pl

Majority of marine foraminifera construct very diverse shells (tests). These unicellular eukaryotes are characterized by anastomosing granular pseudopodial networks (granuloreticulopodia), mostly used for sensing, feeding, attachment and locomotion. However, such networks are not the only pseudopodial structures used by foraminifera to survive in highly competitive and harsh conditions. There are several other pseudopodial structures, such as globopodium and lamellipodia, involved in specific physiological functions. Some of these cytoplasmic structures control formation, shaping and biomineralization of organic or mineral-organic skeletons supported by extracellular organic linings. Simple, monothalamous foraminifera build a single chamber. Much more complex skeletons are constructed by polythalamous foraminifera that form shells by highly algorithmic, iterative additions of chambers. Two main classes of the phylum Foraminifera, i.e. Globothalamea and Tubothalamea, identified on the basis of small subunit rDNA sequences developed distinct morphogenetic strategies (Pawlowski et al., 2013 Mar. Micropal.). The most intriguing feature of foraminiferal shells is their astonishing variety of biomineralizing material. Foraminifera employ synthesis of organic compounds, agglutination of foreign sediment grains, as well as secretion of calcium carbonate mesocrystals. It is worthwhile to stress that foraminifera, constructing calcareous shells, represent one of the main carbonate producers in the oceans with almost 25% of the global oceanic CaCO³ production. Foraminifera provide archives of the past climates and the patterns of evolution. This contribution presents organic architecture and dynamics of subcellular structures employed by the prevalent group of benthic and all planktic foraminifera (Rotaliida, Globothalamea) to secrete their calcareous hyaline tests. Our research is supported by the Polish National Science Center (Grant 2020/37/B/ST10/01953).

**Keywords:** calcification, calcium pathway, mophogenesis, foraminifera, fluorescence staining
Poster presentations

Poster 1

# On identification of Proline-alanine Rich Phospho-Proteins (PARPs) in the sea urchin tooth and their localization relative to the very high magnesium calcite microstructural elements.

Keith Alvares, Guanying Li, Wenle Xu and Derk Joester,

Northwestern University

The sea urchin tooth is a fascinating example of the exceptional level of biological control over microstructure of mineralized tissues and displays a highly hierarchical and functionally graded architecture. Optimized for grazing on hard substrates, its structure comprises both fibrous and platy elements that convey wear resistance, toughness, and self-sharpening to the continuously growing tooth. A unique aspect is that distinct microstructural elements in the tooth differ dramatically in the amount of magnesium incorporated into the calcite (CaCO₃) lattice (1). In fact, concentrations of Mg in the columns (up to % of Ca lattice positions) far exceed the solubility limit of Mg in calcite. Such highly Mg-substituted calcite currently cannot be synthesized in the laboratory. Our working hypothesis is that biomacromolecules occluded in the tooth are involved in the formation of the high magnesium calcite.

Using transcriptomics and proteomics, we have identified a set of proline-alanine rich phospho-proteins (PARPs) that are unique to the *L. variegatus* tooth and do not occur in other sea urchin biominerals, where Mg levels are lower (2). Based on sequence homologies, the 4 PARPs identified could be classified into 2 subgroups. We raised polyclonal antibodies to a 20 amino acid peptide sequence that were unique to two of the PARPS, one in each subgroup. (3) These antibodies were used to localize the PARPs across (a) immature, poorly mineralized and (b) mature, highly mineralized regions of the sea urchin tooth. We will report on our ongoing effort to correlate immuno-histochemical localization of PARPS with microstructural characterization and elemental mapping of the mineralized tissues using SEM-EDS. It appears that the PARPs localize to regions of very high magnesium calcite,

1. Stock, S. R., Seto, J., Deymier, A., Rack, A., and Veis, A. (2018) Growth of second stage mineral in Lytechinus variegatus. *Connect. Tissue Res.* **59**, 345-355

2. Alvares, K., DeHart, C. J., Thomas, P. M., Kelleher, N. L., and Veis, A. (2018) The unique biomineralization transcriptome and proteome of Lytechinus variegatus teeth. *Connect. Tissue Res.* **59**, 20-29

This work was in part supported by NSF DMR-2104759

**Keywords:** Sea urchin tooth
Poster 2

Periostracum formation in *Sepia officinalis* (Sepiidae, Cephalopoda)

Ernesto, Ruiz Villaespesa<fnref n="1" /> Carmen, Salas <fnref n="2" /> Antonio G., Checa<fnref n="1" />

The morphology, composition and mechanical properties of the *Sepia* cuttlebone have been intensively studied. Since the first detailed descriptions by Appellöf (1893), much work has been conducted on the adult shell structure and microstructure. Evolution of the shell from the embryo to the adult was thoroughly detailed by Bandel and Boletzky (1979) and more recent contributions were added by Le Pabic et al. (2016, 2019) and Dauphin et al. (2020). Nevertheless, the shell sac epithelium and his involvement in shell formation have been little studied. At present, there is no clear evidence of how the cells of shell sac epithelium participate in biomineralization. Our goal is to characterize the shell-forming tissues and structures in *Sepia officinalis*, elucidate their role in biomineralization and search possible homologies with other molluscs. Embryos of *S. officinalis* collected from the coast of Málaga were anesthetized and fixed in glutaraldehyde. Histochemical and TEM analyses of the shell sac were conducted in paraffin- and resin-embedded specimens, respectively. Four types of epithelia have been differentiated: simple squamous (dorsal), transitional (lateral bending zones), columnar (lateral fold tips) and simple cuboidal (ventral). The dorsal shield originates at the lateral fold tips where columnar cells secrete numerous vesicles, possibly containing chitin, as indicated by calcofluor dyes. These folds seem deeper than previously depicted and resemble a periostracal groove, from which a periostracum emerges. This periostracum coarsens due to the addition of laminae secreted by the upper and lower epithelia. Thus, the dorsal shield is homologous to the periostracum, which constitutes a support for further mineralization.

**Keywords:** Sepia officinalis, embryo, periostracum, shell sac epithelium, vesicles, chitin

<footnote n="1">¹Departamento de Estratigrafía y Paleontología, Universidad de Granada, 18071 Granada, Spain.</footnote>

<footnote n="2">²Departamento de Biologia Animal, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain</footnote>
# Electron-dense compartments role in calcium transport during coccolithophore biomineralization

Fabio Nudelman, Alexander Triccas

University of Edinburgh, UK

Coccoliths are mineralized scales made of an organized assembly of calcite single crystals grown by ocean dwelling phytoplankton. These structures are a model example of how biological systems control crystallization to produce complex and functional materials. In addition, coccolith production sequesters carbon in ocean sediments, in turn influencing the carbon distribution in the ocean. Given their importance, it is surprising that the mechanisms of coccolith mineralization are still not fully understood, and it is unclear how this process will respond to rising ocean acidity caused by climate change.

One area of uncertainty is how large amounts of calcium are trafficked across the cell during mineralization without raising the free concentration in the cytosol to toxic levels. To avoid this, calcium is stored in electron-dense compartments, although there is no evidence these are involved in coccolith production. It is also unclear how calcium moves in and out of these compartments. A more dynamic imaging technique is therefore needed to quantify how compartments, and the calcium concentrations within, change over the course of calcification.

We imaged electron-dense compartments within the coccolithophore *Chrysotila carterae* using cryo-ptychographic X-ray computed tomography and laser-scanning confocal microscopy. We show compartments changing in both morphology and electron density across calcification, directly linking them to coccolith mineralization. Small compartments are observed at early stages of calcification. During active calcification, compartments become larger and increase in electron density, indicating calcium is being accumulated during coccolith production. Upon completion of calcification, compartments decrease both in size and electron density. These findings improve our understanding on the cellular pathways involved in coccolith production, which will be important in predicting the fate of coccolith mineralization in future acidified oceans.

**Keywords:** coccolithophores, coccoliths, calcium, ptychography
Keynote lecture

Biomimetic Mineralization to investigate Biomineral structure formation
and mineralization mechanisms

Helmut Cölfen, Rui Xiong

*Physical Chemistry, University of Konstanz, Universitätsstr. 10, 78457 Konstanz, Germany*

The structures of Biominerals are often difficult to investigate. It is even more difficult to reveal the structure formation and mineralization mechanisms. In this presentation, 2 biomimetic examples will be discussed, which target to reveal structure formation and mineralization in Biominerals.

The first example is inspired by crab shells with photonic properties. These can be mimicked by crystalline nanocellulose, which self assembles into a chiral structure. This can then be used for infiltration with CaCO₃ for the formation of a hybrid material. Depending on the mineralization time, a strong and flexible material can be formed as well as a hard and stiff material, which leads to outstanding mechanical properties, simultaneously showing photonic effects.

The second example is cellulose nanofibers, which are functionalized with carboxyl groups and subsequently mineralized by CaCO₃. The cellulose nanofiber network mineralized by extremely small amorphous CaCO₃ nanoparticles exhibits outstanding mechanical properties based on multiple toughening mechanisms, which will be discussed.

These toughening mechanisms are also used in Biominerals

<figure><img src="image_7.png" /></figure>
## Oral presentations

### T2 O1

#### The role of lattice distortions in coccoliths morphogenesis

Igor Zlotnikov¹, Dmitry Karpov²

¹ CUBE - Center for Molecular Bioengineering, TU Dresden, ²ESRF, Grenoble, France

Coccoliths—micrometer-sized calcitic scales formed by unicellular algae called coccolithophores—are a prime example of biological regulation over crystal morphogenesis. They have the unique capacity to induce anisotropic growth of calcite single-crystals and thus, to control their final morphology. Whereas calcite is expected to grow as a perfect rhombohedral crystal having identical symmetry-related {104} facets, coccolithophores are able to break the thermodynamically induced symmetry and morph calcite single-crystals into the most intricate shapes in 3D. Our previous work suggests that lattice defects and internal residual strains play an important role in the process of biogenic crystal growth. Therefore, in this work, state-of-the-art X-Ray-based coherent diffraction analysis and imaging methods were used to characterize local lattice properties in single calcitic units in two coccoliths species in 3D. For comparison, two model systems were studied: holococcoliths composed of symmetric calcitic rhombohedral crystals produced by *C. braarudii*; and elaborately shaped heterococcoliths produced by *C. leptoporus*. Combination of Bragg Coherent Diffraction Imaging (Bragg-CDI), which allowed us to visualize local lattice distortions with a spatial resolution of below 10 nm, with high-resolution electron microscopy, provided new mechanistic insights into how coccolithophores break the expected symmetry of single-crystalline calcite and, thus, manipulate its growth.
Intracellular biomineralization of rhombohedral calcite by haploid life phases of coccolithophores

Oz Ben Joseph¹, Lior Aram¹, Diede de Haan¹, Katya Rechav², Assaf Gal¹

¹Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel, ²Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, 7610001, Israel

Organisms can execute tight control over crystal morphology using a cellular toolkit that is not well understood. A prominent example of biological control over crystal morphology is the calcite crystals of coccolith scales produced by marine algae. Interestingly, crystal morphology fundamentally differs in coccoliths produced by the same species' diploid and haploid life-cycle phases, called heterococcoliths and holococcoliths. While calcite crystals of heterococcoliths are highly complex and species-specific, holococcolith crystals are simple rhombohedra, the most common morphology of calcite. In this study, we focus on the growth environment of the simple holococcolith crystals utilizing advanced sample preparation techniques for TEM and FIB-SEM volume imaging. Our results indicate that rhombohedral crystals nucleate and grow intracellularly within voluminous vesicles while experiencing an unconfined, isotropic environment; this environment facilitates the simple morphology of holococcolith crystals. Comparing these findings with the known heterococcolith formation environment suggests that confinement of the crystallization process is a critical factor in shaping biomineral morphology.

**Keywords:** calcite, crystal growth, nucleation, coccolith
Twin-based strengthening mechanism in a crossed-lamellar structure

Katarzyna Berent¹*, Kinga Nalepka², Paweł Czaja³, Łukasz Maj³, Tomasz Machniewicz², Magdalena Bieda-Niemiec³, Krzysztof Sztwiertnia³, Antonio G. Checa⁴,⁵

¹AGH University of Krakow, Academic Centre for Materials and Nanotechnology, 30 Mickiewicza Av., 30059 Krakow, Poland, ²AGH University of Krakow, Department of Strength and Fatigue of Materials and Structures, 30 Mickiewicza Av., 30059 Krakow, Poland, ³Institute of Metallurgy and Materials Science, Polish Academy of Sciences, 25 Reymonta St., 30059 Krakow, Poland,

⁴Universidad de Granada, Departamento de Estratigrafía y Paleontología, 18071 Granada, Spain,

⁵CSIC-Universidad de Granada, Instituto Andaluz de Ciencias de la Tierra, 18100 Armilla, Spain

*kberent@agh.edu.pl

Biological shells typically possess hierarchical structures and carry out multiple functional roles thanks to their remarkable strength and toughness. An example of such a complex hierarchical structure is the crossed-lamellar structure, which is made of the 1st-order lamellae comprising the laths (the 2nd-order lamellae) composed of parallel rods (the 3rd-order lamellae), with relatively low organic content (0.1-1 wt.%). Such biological structures offer a wealth of inspiration for addressing the classic challenge in material design where strength and toughness, two crucial structural properties, are often perceived as mutually exclusive.

Our research focused on understanding the microstructure of the smallest building unit, which forms the crossed lamellar structure. Thin foils were prepared from the *Sinustrombus sinatus* shell by a focused ion beam (FIB) technique in a scanning electron microscope (SEM). High-resolution transmission electron microscopy (HRTEM) was performed to gain more information about the nanoscale structure of the individual rod. The results revealed that the examined lamellas are not single aragonite grains but are divided by densely spaced twin plates. The energetically favorable twin disorientation is formed with an accuracy of 1°. The geometrical phase analysis (GPA) was used to measure strain from HRTEM images. By implementing the original GPA, the recorded crystal lattice was correlated with a reference lattice that includes an ideal twin boundary, allowing for identifying deformation fields within the 3rd-order structural units. It was found that the twin plates experienced strong compression in the direction perpendicular to the longest dimension, resulting in a strain of -0.1%. The high frequency of twin placement in the internal structure of the tertiary lamellae induces prestressing of the basic structural units. This revealed mechanism significantly improves the fracture toughness and the strength of the protective exoskeleton.

**Acknowledgments:** This research was funded by projects: UMO-2018/29/B/ST8/02200 (Polish National Science Center), CGL2017-85118-P and PID2020-116660 GB-100 (Spanish Ministerio de Ciencia e Innovación).

**Keywords:** strengthening mechanism, crossed-lamellar structure, geometrical phase analysis (GPA), twins
# Calcification of the egg capsule in the apple snail: the organic matrix and the calcium-rich granules

Jingliang Huangª, Huan Liuᵇ

ª, Department of Biology, Hong Kong Baptist University, Hong Kong, China, ᵇ, School of Chemical Engineering and Technology, Sun Yat-sen University, Guangdong Province, China.

The apple snail Pomacea canaliculata is an amphibian mollusk that lays aerial eggs above the water level. The hatching larvae is protected from sunlight, desiccation and other damages by a calcified capsule which is composed of vaterite. Vaterite is a metastable calcium carbonate seldom found in inorganic sediment, and the only found stable forms in nature are biominerals in molluscs and corals. Moreover, the egg capsule represents another evolved biomineral after the spiral shell in gastropod. Therefore, the calcified egg capsule provides an interesting biomineralization model to study the crystal polymorph and evolution of calcium carbonate biomineral. We first explored the calcification process of the eggs. It was found that numerous calcium-rich granules were present inside the newly deposited eggs and transported to the outer surface for capsule mineralization, indicating a unique mineralization pathway compared with the exoskeleton shell formation. We then analyzed the organic matrix extracted from the egg capsules and found that the organic matrix could stabilize vaterite and modify the crystal morphology. FTIR revealed that the matrix was composed of proteins and polysaccharides, similar to most molluscan shell matrices. Proteomics analysis showed that the shell proteins contain chitin-binding and calcium binding proteins, and several sulfatases. The sulfatase is unique and may be involved in vaterite deposition via generating sulfate ions from sulfonic polysaccharide. Our work suggests that the calcification of the P. canaliculata egg capsule may be accomplished via a unique pathway which calls for further studies.

**Keywords:** Pomacea canaliculata; egg capsule; organic matrix; shell protein; calcium-rich granule
# Biomimetic transformations of amorphous calcium phosphate to dental and ossifying crystalline hydroxyapatite

Werner E. G. Müller and Xiaohong Wang

ERC Advanced Investigator Grant Research Group, University Medical Center of the Johannes Gutenberg University, Duesbergweg 6, 55128 Mainz, Germany

E-mails: wmueller@uni-mainz.de (W.E.G.M.), wang013@uni-mainz.de (X.H.W.)

Mineral deposition in living organisms is not a passive deposition of the minerals but a highly controlled process starting from the amorphous phases and processed onto organic template(s), into crystalline mature product(s), like enamel or bone. We found that amorphous calcium phosphate (CaP) deposition during bone and teeth mineralization starts with the aggregation of Posner's clusters $Ca_9(PO_4)_6$ into amorphous Ca-phosphate (ACP), which then transforms into crystalline CaP and finally maturates to hydroxyapatite (HA). Using dentin/enamel of human teeth as a model system, we show that the physiological inorganic polymer polyphosphate (polyP), a phosphate donor in mineralization, prevents the transition from amorphous to crystalline CaP at concentrations > 15 wt%. Stabilization of the amorphous phase of CaP by polyP is reversed by hydrolysis of the polymer by alkaline phosphatase (ALP), an enzyme that releases phosphate for mineralization. The ALP is present in calcified enamel and dentin, as shown by immunostaining and enzyme activity measurements. The phase transfer into crystalline CaP can be prevented by ALP inhibitors. The modulating effects of polyP and ALP on the kinetics of the phase transition from amorphous to crystalline CaP are demonstrated. Molecular modeling studies show that the polyP chains are able to penetrate into the channels between the Posner molecules, preventing cluster association to ACP and impairing HA crystal formation.

<figure><img src="image_8.png" /></figure>
It is concluded that during HA development this dynamic interrelation processes, amorphous ↔ crystalline CaP depositions, also run *in vivo* during hard tissue dental organ formation. The ALP exists there and the phosphate deposits, either in phosphorylated proteins or (presumably) also in polyP, are present stage-dependently in enamel-dentine regions.

**Acknowledgements**

This work was supported by an ERC Advanced Investigator Grant (268476) and three ERC-PoC Grants (324564, 662486 and 767234) (W.E.G. M. is the holder of those Grants).

**References**

Müller WEG; Neufurth M; Ushijima H; Muñoz-Espí; Müller LK; Wang S; Schröder HC; Wang XH (2022) Molecular and biochemical approach for understanding the transition of amorphous to crystalline calcium phosphate deposits in human teeth. *Dental Materials* 38. 2014-2029.

Müller WEG, Tolba E, Schröder HC, Muñoz-Espí R, Diehl-Seifert B and Wang XH (2016) Amorphous polyphosphate-hydroxyapatite: a morphogenetically active substrate for bone-related SaOS-2 cells *in vitro*. *Acta Biomat* 31: 358-367.

Müller WEG, Ackermann M, Al-Nawas B, Righesso LAR, Muñoz-Espí R, Tolba E, Neufurth M, Schröder HC, Wang XH (2020) Amplified morphogenetic and bone forming activity of amorphous versus crystalline calcium phosphate/polyphosphate. *Acta Biomat* 118:233-247

Müller WEG, Schröder HC, Wang XH (2019) Inorganic polyphosphates as storage for and generator of metabolic energy in the extracellular matrix. *Chem Rev* 119: 12337-12374.
# Organization of a plywood biomaterial: the calcitic cross-foliated microstructure of limpets

Katarzina Berent¹, Antonio G. Checa²

¹Akademickie Centrum Materiałów I Nanotechnologii, Akademia Górniczo-Hutnicza, Kraków, Poland, ²Departamento de Estratigrafía y Paleontología, Universidad de Granada, 18071 Granada, Spain and Instituto Andaluz de Ciencias de la Tierra, CSIC–Universidad de Granada, 18100 Armilla, Granada, Spain

To construct their shells, molluscs are able to produce a vast array of calcified materials, including granular, prismatic, lamellar, fibrous, foliated and plywood-like. The latter include an aragonitic (the crossed-lamellar) and a calcitic (the crossed foliated) variety, whose modes of formation are particularly enigmatic. We studied the crossed foliated calcitic layers, formed solely by members of the limpet family Patellidae, using scanning electron microscopy and electron backscatter diffraction. In the external layer, the distribution of 1st order lamellae is irregular; toward the interior, the material becomes progressively organized into commarginal 1st order lamellae, with 2nd and 3rd order lamellae dipping in opposite directions in alternating lamellae (the so-called concentric crossed foliated layer). At the same time, the crystallographic texture becomes stronger because each set of 1st order lamellae develops a single orientation for the c-axis, while both sets maintain common orientations for one {104} face (parallel to the growth surface) and one a-axis (perpendicular to the planes of the 1st order lamellae). Each 1st order lamella shows a progressive migration of its crystallographic axes with growth, toward the maxima corresponding to the set to which it belongs. Thus, all 1st order lamellae of the same set show similar orientations in the mature concentric crossed foliated layer. To explain the progressive organization of the material, we hypothesize that a secretional zebra pattern, similar to the one created by the 1st order lamellae on the shell growth surface, is developed on the shell-secreting mantle surface. Cells belonging to alternating stripes behave differently to determine the growth orientation of the laths composing the 1st order lamellae. In this way, we provide an explanation as to how plywood-like materials can be fabricated, which is based mainly on the activity of the mantle cells.

**Keywords:** molluscs, limpets, microstructure, crossed foliated, biogenic calcite, plywood material
Structural and mechanical adaptation of *Lingula anatina* shells

Fabio Nudelman*¹, Andre L. Rossi², Fraser Laidlaw³, Gabriela Graziani⁴, Giuseppe Falini⁵

¹School of Chemistry, University of Edinburgh, Joseph Black Building, The King's Buildings, Edinburgh, UK, ²Brazilian Centre for Physics Research, Rio de Janeiro, Brazil, ³School of Physics and Astronomy, James Clerk Maxwell Building, The King's Buildings, Edinburgh, UK, ⁴Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Polytechnic University of Milan, Milan, Italy, ⁵Department of Chemistry, University of Bologna, Italy

Biominerals are a class of inorganic-organic composite materials that exhibit function-optimized
properties. These properties arise from a hierarchical organization of primary building blocks. While
some biomineral producing organisms can alter these properties in response to environmental stresses,
this generally involves a time-intensive process of resorption and reprecipitation. Here, we report that
the load-bearing shells of the phosphatic brachiopod *Lingula anatina* are an exception to this process.
We observed that these shells are able to dynamically modulate their mechanical properties in response
to a change in environment, i.e. they switch from hard and stiff when dry to malleable when hydrated
within minutes. A passive and stimuli-responsive adaptation sought-after in structural materials. To
understand the mechanisms that underpin these changes in mechanical properties, we first characterised
the structure of the shell at the nano-scale. We used focused-ion beam scanning electron microscopy
(FIB-SEM) to cut 70 nm-thin lamella from the shell for analysis using transmission electron microscopy,
scanning-transmission electron microscopy, electron diffraction and electron energy loss spectroscopy.
We show that the shell, at the nanoscale, is composed of 2-5 nm thick crystals of fluorapatite, interwoven
with chitin and organized into 200 nm rod-like structures. As hydration is known to increase the mobility
of chitin and to weaken its association with the mineral, it is conceivable that the observed organization
of these two major components constitute a mechanism, at the nanoscale, that underpins the changes in
mechanical properties that the shell undergoes as a response to hydration.

**Keywords:** Biomineralization, brachiopod shells, fluorapatite, transmission electron microscopy
# Stabilization of Amorphous Calcium Phosphate by Barium: A Novel Approach Inspired by a Marine Worm

Nina Oehlsen¹, Jörn von Döhren², Steve Weiner³, Jamal Ibrahim³, Linus Stegbauer¹

¹University of Stuttgart, Institute of Interfacial Process Engineering and Plasma Technology, ²Institute for Evolutionary Biology and Ecology - Rheinische Friedrich-Wilhelms-Universität Bonn, ³Weizmann Institute of Science

Amorphous calcium phosphate (ACP) is a transient precursor phase in bone and teeth, transforming into crystalline carbonated hydroxyapatite. Some rare organisms like the Nemertean worm *Amphiporus lactifloreus* [1] produce ACP that is stable throughout the organism's lifetime in its stylet. The stylet is a 100-micron long nail-shaped “tooth” at the end of the proboscis and is used to capture and pierce prey. ACP, although transient under aqueous conditions, could be a valuable mineral phase for tissue-engineering of bone due to its similar elemental composition to carbonated apatite in bone. We aim to study the worm's ACP stability mechanisms to develop methods to stabilize synthetic ACP. We study the 3D structure using nano-CT and FIB SEM, as well as the elemental composition and distribution of stylets extracted from *A. lactifloreus*. The stylet consists of an inner core region, surrounded by an outer lamellar structured layer. In both regions, Ca and P are the main elements. Surprisingly, the concentrations of Ba and Sr are high with Ca/Ba 5:1 by atoms and Ca/Sr 10:1. The outermost layer of the stylet exhibits elemental contrast of heavier elements in nano-CT and is enriched in S and Ba according to EDS. Raman detected SO₄²⁻. Nanoindentation results showed an elastic modulus of 10–28 GPa and a Vickers hardness of 30-160.

We carry out in vitro syntheses with different elemental compositions and monitor the ACP stability by FTIR. The addition of Ba at atomic ratio Ca/Ba = 9:10 is capable of stabilizing ACP and with Ca/Ba = 3:1 and 19:1 can delay crystallization. By substituting Ca in the ACP synthesis with different ratios of Ba and Sr, the crystalline transformation of ACP can be slowed down for weeks. This result shows that Ba can be used to modulate ACP stability. In future this might lead to ACP that can be tailored to various industrial and medical applications in the field of bone repair and replacements.
# Monochromated STEM-EELS analysis of the structure and composition of biogenic calcite reveals the biomineral growth pattern

Marta de Frutos¹, Alejandro B. Rodríguez-Navarro², Xiaoyan Li¹, and Antonio G. Checa³

¹Laboratoire de Physique des Solides, CNRS UMR 8502, Université Paris-Saclay, France,
²Departamento de Mineralogía y Petrología, Universidad de Granada, Spain, ³Departamento de
Estratigrafía y Paleontología, Universidad de Granada and Instituto Andaluz de Ciencias de la
Tierra, Spain

The vast majority of calcium carbonate biocrystals differ from inorganic crystals in that they display a patent nanoughness consisting of lumps of crystalline material (calcite/aragonite) surrounded by amorphous pellicles. Understanding this organization and its formation mechanisms implies to unveil the chemical composition and structure at the nanoscale. Compared to other spectroscopic approaches, STEM-EELS (scanning transmission electron microscopy coupled with Electron Energy Loss Spectroscopy) offers the advantage of an outstanding spatial resolution (at the nanometer scale) in both chemical analysis and imaging. Moreover the use of a latest-generation STEM microscope equipped with an electron monochromator and a direct-detection camera offers the possibility to detect very weak signals with a spectral resolution down to 7 meV, close to those obtained from X-ray based approaches This high spectral resolution gives access to the crystallinity of calcium carbonate phases through the measure of the crystal field splitting (CFS) on Calcium L23-edge.

In the present study (1), high resolution STEM-EELS was used to map compositionally and structurally the calcite secreted by the giant acorn barnacle *Austromegabalanus psittacus*. Our data reveal that the material is composed of irregular lumps (up to two hundred nm in diameter) of crystalline phase (calcite) surrounded by relatively continuous cortexes (up to 20 nm thick) of an amorphous phase composed of calcium carbonate (ACC) and/or nanocalcite plus biomolecules, with a surplus of calcium relative to carbonate. Based on EELS results, we develop a model by which the separation of the crystalline and amorphous phases takes place upon crystallization of the calcite from a precursor ACC. The organic biomolecules are expelled from the crystal lattice and concentrate in the form of pellicles, where they stabilize minor amounts of ACC/nanocalcite. In this way, we change the previously established conception of biomineral structure and growth.

(1) Nanoscale Analysis of the Structure and Composition of Biogenic Calcite Reveals the Biomineral Growth Pattern. Marta de Frutos, Alejandro B. Rodríguez-Navarro, Xiaoyan Li, and Antonio G. Checa ACS Nano 2023 17 (3), 2829-2839; DOI: 10.1021/acsnano.2c11169
<figure><img src="image_9.png" /><figcaption>**Figure 1:** (a,b) Distributions of carbonate (in red) and organic compounds (in green) for two different areas. (c,d) Distributions of crystalline vs amorphous material. The amorphous areas (CFS values <1.1 eV) are represented in green and crystalline ones (CFS values >1.1 eV) in purple.</figcaption></figure>
Anisotropic lattice parameters change in aragonite induced by Na⁺ substitution

Taiga Okumura, Michio Suzuki, Toshihiro Kogure

*The university of Tokyo, Jpan*

Some biogenic aragonites possess larger axial ratios (*a/b* and *c/b* in orthorhombic *Pmcn*) than abiotic counterparts. Our previous research suggested that the anisotropic lattice parameters change is caused by substitution of Na⁺ for Ca²⁺ because the axial ratios correlate with Na content. To verify this hypothesis, we synthesized aragonites *in vitro* in the presence of Na⁺ in the present study. The synthetic crystals were obtained by mixing 50 mM MgCl₂, 10 mM CaCl₂, and 10 mM Na₂CO₃ with different concentrations of NaCl. After stirring the solutions at room temperature for 24 h, precipitates recovered by centrifugation were washed three times with Milli-Q water and once with ethanol, and dried at room temperature. The precipitates were investigated using X-ray diffraction and scanning electron microscopy, confirming that they were all composed of aragonites. When the NaCl concentration was lower, the synthetic aragonites exhibited euhedral columnar shapes and their axial ratios were comparable with those of abiotic ones. However, in the case of higher NaCl concentrations, the aragonites became granular and showed higher axial ratios. The increase in axial ratios was correlated with the Na content in the crystals. The axial ratios decreased by heating at 250 °C probably due to diffusion and segregation of Na⁺ from the crystal lattice of aragonite. Thus, these synthetic experiments indicated that the hypothesis is plausible. Considering relatively lower Cl content in the crystals, the charge compensation for Na substitution can be attributed to the introduction of bicarbonate ions and/or vacancies.

**Keywords** : aragonite, sodium, anisotropy, lattice parameter, axial ratio, synthetic experiment
# Halochromic ACC crystallization in the presence of naphthoquinones unveils biomineral pigmentation in sea urchins

Vaskar Sardhalia,ª Claudio Do Reis Ferreira,ª Shahrouz Amini,ᵇ Peter Fratzl,ᵇ Marta De Frutosᶜ, Delphine Talbotᵈ, Anne Vallée,ᶜ Andy Fitch,ᶜ Stefano Checchia,ᶜ Nadine Nassif,ª Thierry Azaïs,ª and Marie Albéricª*

ªLaboratoire de Chimie de La Matière Condensée de Paris, CNRS, Sorbonne Université, 75005, Paris, France,
ᵇBiomaterials department, Max Planck Institute for colloids and interfaces, 14476, Potsdam, Germany,
ᶜLaboratoire de Physique des Solides, Université Paris saclay, 91405, Orsay, France,
ᵈPhysicochimie des Electrolytes et Nanosystèmes interfaciaux Laboratoire, Sorbonne Université, 75005, Paris, France,
ᵉInstitut Lavoiser de Versailles, CNRS, Université de Versailles Saint-Quentin-en-Yvelines, 78035, France,
ᵉESRF, ID22, Structure of Materials Group ESRF, The European Synchrotron, 38000, Grenoble, France,
ᵉESRF, ID15A, Materials Chemistry, and Materials Engineering, The European Synchrotron, 38000, Grenoble, France

Adult sea urchin biominerals owe their different colors to the presence of 40 different polyhydroxylated naphthoquinone molecules (PHNQs: spinochrome A, B, C, etc.) across species.¹ PHNQs are incorporated within calcite likely during crystal growth,² occurring through amorphous calcium carbonate (ACC) precursors.⁴ Interestingly, spinochrome A displays red color once extracted from purple CaCO₃ *P. lividus* spines in acidic conditions,⁵ suggesting that pH plays a role in color variations as it does in CaCO₃ formation.⁶,⁷ To understand the color variations in biominerals formed through ACC precursor as well as the role of PHNQs in ACC crystallization, we performed ACC precipitation in the presence of synthetic naphthazarin and study its crystallization. Then, the effect of biogenic spinochrome A, B, and E on calcite lattice distortions was investigated. pH was monitored during ACC precipitation and crystallization, *e.i.* after the addition of CaCl₂ to a Na₂CO₃ solution⁸ in the presence of PHNQs. DSC/TGA, XPS, PDF analysis, UV-Vis, STEM-EELS, and ss-NMR spectroscopies were performed to characterize the amorphous and crystalline hybrid pigments. Here, we show that halochromic ACC crystallization may occur in biomineral pigmentation in sea urchins. Indeed, naphthazarin, which is red at acidic pH, turns blue before ACC precipitation, leading to lavender blue calcite due to successive OH deprotonation/protonation during ACC crystallization. However, naphthazarin does not significantly affect ACC structure and stability but does get incorporated into calcite most probably in the form of aggregates. Finally, spinochrome B and E lead to yellowish calcite and spinochrome A to purple calcite whereas the formers induce larger calcite lattice distortions than the latter. The preferential occlusion of spinochrome A in purple *P. lividus* spines may thus be explained by the combination of unaffected atomic structure and obtained intense color.

1. Hou, Y. et al. Naphthoquinones of the spinochrome class: occurrence, isolation, biosynthesis, and biomedical applications. *RSC Adv.* **8**, 32637–32650 (2018).

2. Towe, K. M. Overviews of Biomineralization. *Paleobiology* **16**, 521–526 (1990).

4. Politi, Y., Arad, T., Klein, E., Weiner, S. & Addadi, L. Sea Urchin Spine Calcite Forms via a Transient Amorphous Calcium Carbonate Phase. *Science* **306**, 1161–1164 (2004).

5. Goodwin, T. W. & Srisukh, S. A study of the pigments of the sea-urchins, *Echinus esculentus* L. and *Paracentrotus lividus* Lamarck. *Biochem. J.* **47**, 69–76 (1950).

6. Tobler, D. J., Rodriguez Blanco, J. D., Sørensen, H. O., Stipp, S. L. S. & Dideriksen, K. Effect of pH on Amorphous Calcium Carbonate Structure and Transformation. *Cryst. Growth Des.* **16**, 4500–4508 (2016).

7. Koga, N., Nakagoe, Y. & Tanaka, H. Crystallization of amorphous calcium carbonate. *Thermochim. Acta* **318**, 239–244 (1998).

8. Zou, Z. et al. Opposite Particle Size Effect on Amorphous Calcium Carbonate Crystallization in Water and during Heating in Air. *Chem. Mater.* **27**, 4237–4246 (2015).
Microstructures and biomineralization patterns of gymnolaemate
bryozoans (Bryozoa, Gymnolaemata)

Christian Grenier<fnref n="1" />, Erika Griesshaber<fnref n="3" />, Wolfgang Schmahl<fnref n="3" />, Björn Bernin⁴, Antonio G. Checa*<fnref n="1" />,²

Gymnolaemata is currently the most diverse and widely distributed class among bryozoans, and is mainly comprised of the order Cheilostomata. All cheilostomes are found worldwide and exclusively inhabit marine waters. They produce CaCO₃ skeletons that can be made of either calcite, aragonite, or both. Despite extensive previous research regarding their microstructures and mineralogy, no suitable crystallographic techniques had been applied and their biomineralization processes still remain unclear. We present a detailed study of the microstructures, mineralogy and crystallography of eight extant species of cheilostomes using SEM-EBSD, AFM and micro-CT. We focus on their main microstructures: granular and platy calcite, and fibrous aragonite. The calcitic microstructures are formed by aggregates of crystals that easily change from platy to granular or viceversa. Fibrous aragonite consists of spherulites made of long, thin needles. In both the calcitic and aragonitic microstructures, the crystallographic axes display an axial texture, with the c-axis as the fiber axis, which is stronger in aragonite. The calcite grains sometimes cluster in domains with internal misorientations. Crystal competition is widespread. We reconstruct the biomineralization process in the different species by taking into account the distribution and morphology of the growth fronts of crystals and the theoretical location of the soft tissue. In bimineralic species, calcite formation always predates aragonite formation. In internal walls, growth is bidirectional from the center. In external walls, however, growth proceeds from the cuticle, which acts as a nucleation template, extending outward toward the secretory epithelium. We conclude that biomineralization is seemingly remote in all cases and proceeds in a wide space formed between the cuticle and the soft tissue. The invoked process of crystal growth far from the soft tissue correlates with the inorganic-like appearance of the crystallites.

**Keywords:** Bryozoans, Gymnolaemata, microstructures, biogenic calcite, biogenic aragonite, crystallography, EBSD

<footnote n="1">¹Departamento de Estratigrafía y Paleontología, Universidad de Granada, 18002 Granada, Spain,</footnote>

<footnote>.²Instituto Andaluz de Ciencias de la Tierra, CSIC-Universidad de Granada, 18100 Armilla, Spain,</footnote>

<footnote n="3">³Department of Earth and Environmental Sciences, Ludwig-Maximilians Universität, Munich, Germany, ⁴Institute for Geology, University of Hamburg, Bundesstraße 55, 20146 Hamburg, Germany.</footnote>
# Calcite and guanine formation by the freshwater green alga *Phacotus lenticularis*

Noy Shaked⁺, Sophia Barinova*, Iddo Pinkas&, Sefi Addadi^, Steve Weiner⁺, Lia Addadi⁺

⁺Department of Chemical and Structural Biology, &Department of Chemical Research Support and ^Department of Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot, Israel *Institute of Evolution, University of Haifa, Haifa, Israel

Many unicellular algae produce CaCO₃ minerals in marine and freshwater ecosystems [1]. One freshwater species, *Phacotus lenticularis*, is abundant in lakes and ponds. *P. lenticularis* produces a complex shell composed of aligned crystal plates of calcite. These shells may constitute a significant fraction of basin sediments especially during the bloom period [2].

We investigate the shell formation mechanism in *P. lenticularis* cells collected from a local natural habitat using *in-vivo* fluorescence assays combined with cryo-SEM, which enables the observation of complexes under fully hydrated conditions. During reproduction, 1-3 cell divisions occur to form up to 8 daughter cells that subsequently form a shell within a confined reproduction volume. We observed Ca ion trafficking into the daughter cells, and the formation of small, vesicle- contained intra-cellular crystal. Shell assembly occurs on the cell outermost layer, where biomineralization remains under control by virtue of the protected environment within the reproduction complex. Furthermore, we have identified intriguing indications of signals from the environment that may trigger calcification both in the cell cultures and in natural environments at the onset of the bloom period.

We also observed that cell cultures grown under phosphate stress conditions, produce birefringent crystals that were identified as □□guanine using micro-Raman spectroscopy. Cryo-SEM examination showed that the crystals are enclosed within vehicles, which are dispersed in the cell, as observed using cryo-soft X-ray tomography and cryo-FIB-SEM. Interestingly, guanine crystal deposition was observed in many unicellular species under nitrogen starvation. The occurrence of guanine crystal formation under different triggers may indicate a common reaction to stress response, resulting in nutrient storage.

## References

1. Riding, R., *Calcareous algae and stromatolites*. 1991: Springer Science & Business Media.

2. Müller, G. and M. Oti, *The occurrence of calcified planktonic green algae in freshwater carbonates*. Sedimentology, 1981. **28**(6): p. 897-902.
# The origin and function of geometric frustration in spicule morphogenesis

Abisheik John Samuel*¹, Bellec Bellec², Igor Zlotnikov¹

¹B CUBE – Center for Molecular Bioengineering, Technische Universität Dresde, ²European Synchrotron Radiation Facility, Grenoble, France

Sponges produce complex skeletal structures made out of individual elements called spicules. A subclass of Sponges named Demospongiae consist of spicules made of amorphous silica having various morphologies, ranging from needle-like megascleres to symmetric tripod-like dichotriaenes with multiple branches. All spicules have a central canal consisting of a hybrid crystalline mineral/organic axial filament composed of a protein, silicatein. The filament templates silica deposition and, therefore, plays a crucial role in spicule formation. However, the mechanisms underlying the morphogenesis of the filament, including its branching, have remained elusive.

In this study, we employed advanced X-ray-based techniques to investigate the internal structure of the axial filament in unprecedented resolution. Specifically, we used scanning X-ray Diffraction, Bragg Coherent Diffraction Imaging and nano-Holotomography to visualize the evolution of the crystal habit and to determine the local lattice distortions during filament formation. We demonstrate that during protein crystallization an internal strain is developed as a result of geometric frustration generated by fitting the silicatein units into the crystalline assembly. In turn, at a critical point, the accumulated elastic strain dissipates in the form of branching on specific crystallographic planes, leading to the formation of highly symmetric shapes of the spicules. These findings suggest that spicule morphogenesis is a spontaneous process that is carefully orchestrated by the organism by controlling the conditions in which the filament is self-assembled. This work establishes a novel biomineralization mechanism unique to sponge skeletogenesis.

**Keywords:** Demosponges - Spicule - Morphogenesis - Self assembly – Branching
Crystallographic Characteristics of Vaterite in Fish Otolith

Gen Takahashi¹, Taiga Okumura¹, Takayoshi Nagaya¹, Michio Suzuki², Yoshio Takahashi¹, Toshihiro Kogure¹

¹Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo,
²Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The
University of Tokyo

Teleost fishes have three types of otoliths called lapillus, sagitta, and asteriscus, which adopt different polymorphs of calcium carbonate (CaCO3); lapillus and sagitta are aragonite and asteriscus is vaterite. The reason why metastable vaterite can be formed and stably exist in asteriscus is not yet understood.

In this research, crystallographic characteristics of vaterite in asteriscus have been investigated to elucidate its formation mechanism. Goldfish (Carassius auratus) was selected as a typical teleost fish species, and inorganically synthesized vaterite was also analyzed for comparison. The crystal structure of vaterite was analyzed as a hexagonal system (P63/mmc, a = 4.13, c = 8.49 Å) of the averaged unit cell proposed by Kamhi (1963).

A goldfish asteriscus is almost disk-shaped. Electron back-scattered diffraction (EBSD) analysis of the cross-sections indicated that the ai axes extend radially from the center. This radial structure is composed of domains of about ~10 μm in width. Transmission electron microscopy (TEM) observation of cross-sectional thin films of the domain prepared by focused ion beam (FIB) revealed that the domain is composed of single crystals of 0.5-3 μm separated by small-angle grain boundaries. These crystallographic features such as elongation along the ai axes and mosaic texture were also found in synthesized vaterite, suggesting that vaterite constituting the asteriscus grew according to the intrinsic crystallographic characters of vaterite.

To investigate the nucleation site of the vaterite crystal, asteriscus (~20 μm in size) in goldfish fry was collected and observed by TEM. The center of the asteriscus contains organic matter and a single crystal of vaterite, implying that this organic matter is relevant to the selection of vaterite.
T2 O16

3D interrelationship between hierarchical canal network and the gradient
mineralization of shark tooth osteodentin

Zhuanfei Liu a#, Yunya Niu b#, Zeyao Fu a, Mason Dean c, Zhengyi Fu a, Yongming Hu d,* and
Zhaoyong Zou a,b,*

a State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan
University of Technology, Wuhan 430070, China. b State Key Laboratory of Silicate Materials for
Architectures, Wuhan University of Technology, Wuhan 430070, China. c Department of Infectious
Diseases & Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City
University of Hong Kong. d School of Microelectronics, Hubei University, Wuhan 430062, Hubei,
China.

\# These authors contribute equally

* Corresponding author E-mail address: zzou@whut.edu.cn

Osteodentin is a dominant mineralized collagenous tissue in the teeth of many fishes, with structural and
histological characteristics resembling those of bone. Osteodentin, like bone, comprises osteons as basic
structural building blocks, however, it lacks the osteocytes and the lacuno-canalicular network (LCN),
which are known to play critical roles in controlling the mineralization of the collagenous matrix in
bone. Although numerous vascular canals exist in osteodentin, their role in tooth maturation and the
matrix mineralization process remain poorly understood. Here, high resolution micro-computed
tomography (micro-CT) and focused ion beam-scanning electron microscopy (FIB-SEM) were used to
obtain 3D structural information of osteodentin in shark teeth at multiple scales. We observed a complex
3D network of primary canals with a diameter ranging from ~10 µm to ~120 µm, where the canals are
surrounded by osteon-like concentric layers of lamella, with ‘interosteonal’ tissue intervening between
neighboring osteons. In addition, numerous hierarchically branched secondary canals extended out
radially from the primary canals into the interosteonal tissue, with a decreasing diameter from ~10 µm
to hundreds of nanometers. Interestingly, the mineralization degree increases from the periphery of
primary canals to the interosteonal tissue, suggesting that mineralization begins in the interosteonal
tissue. Correspondingly, the hardness and elastic modulus of the interosteonal tissue is higher than those
of the osteonal tissue. These results demonstrate that the 3D hierarchical canal network is positioned to
play a critical role in controlling the gradient mineralization of osteodentin, also providing valuable
insight into the formation of mineralized collagenous tissue without osteocytes and LCN.
Novel crystalline phases derived from amorphous calcium hydrogen phosphate

Jinqin Yang,† Tom Willhammar,† Hua Zeng,‡ Julian D. Gale,§ Niklas Hedin,† Denis Gebauer,|| Bing-Qiang Lu,‡

† Department of Materials and Environmental Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden, ‡ Tenth People's Hospital, Tongji University, Shanghai No 301, Yanchangzhong Road 200072, China, § Curtin Institute for Computation/The Institute for Geoscience Research (TIGeR), School of Molecular and Life Sciences, Curtin University, GPO Box U1987, Perth, Western Australia 6845, Australia, ||Leibniz Universität Hannover, Institute of Inorganic Chemistry, Callinstr. 9, 30167 Hannover, Germany

Calcium (ortho)phosphate (CaP) is an essential mineral component in organisms and extensively employed as a biomedical material. Different CaP phases have distinct physical/chemical properties, biological effects and physiological applications, which encourages us to explore the new CaPs with novel microstructures and properties. However, since the main crystalline CaP phases were discovered before the 19th century and structurally determined in 20th century, the progress in this field finds little progress.

In this study, by controlling the crystallization of amorphous calcium phosphate (CaHPO₄·xH₂O, ACHP) in the water/organic solvents, we discover five crystalline CaPs (denoted as HLCPs, one of them has been published [1]). With chemical formula CaHPO₄·X, where the X represents a molecule of H₂O or organics, the HLCPs have a layered structure, and the X molecules locates between the CaHPO₄ layers. Different HLCPs are able to transform into each other under specific conditions. The HLCPs show potential to carry the biomedical molecules in the structure as a drug delivery system. Moreover, the HLCPs display a piezoelectricity behavior, which is rarely seen in other CaP counterparts.

This work not only extends the family of CaP, but also provides new candidates for building functional biomaterials.

[1] Lu, B.-Q., et al. "Introducing the crystalline phase of dicalcium phosphate monohydrate." Nature Communications, 2020, 11, 1546
# Changes in bone chemistry, mineralogy and structure during heating

Alejandro B. Rodríguez Navarro<fnref n="1" />*, Sergio Madero1, Martina Greiner<fnref n="2" />, Pablo A. Rodriguez-Jimenez<fnref n="1" />,
Concepción Jimenez-Lopez³, Wolfgang W. Schmahl<fnref n="2" />.

## Background

The study of bone changes during heating is highly relevant for forensic and archeological material analyses. However, bone is a complex nanocomposite material composed of an inorganic phase (nanocrystalline carbonated apatite) mineralizing an organic matrix (largely type I collagen fibrils) and water. Moreover, bone composition and structure is highly heterogeneous and varies in different parts of the skeleton depending on bone function.

## Methodology

We have studied in detail how bone transform during thermal treatments using different analytical techniques such as thermogravimetry (TGA-DSC), electron microscopy, X-ray diffraction and infrared spectroscopy.

## Results and discussion

We show that bone mineral and organic matrix characteristics in specific types of bone tissues strongly influence bone transformation during thermal treatments. For instance, in cortical bone the apatite nanocrystals are integrated within the highly organized collagen fibrils and are coated with phosphorylated proteins (osteopontin). The organic matrix protects the apatite crystals and recrystallization only occurs when organic matter is lost at around 600 C. Also during recrystallization, foreign ions (Mg2+, Na+) are expelled from the apatite lattice to the crystal surface and the degree of preferential orientation of apatite crystals (in cortical bone) increases as larger well oriented apatite crystals grow epitaxially to the expense of smaller randomly oriented crystals. In medullary bone the organic matrix is rich non-collagen proteins and proteoglycans and recrystallization set at much lower temperatures than in cortical bone (around 400 C). Also, the calcination process creates additional microporosity in bone increasing its surface area and reactivity. The information obtained in this study can help to better understand the dynamic of bone transformation during alteration in natural processes (digenesis) and how bone mineral characteristics can be modified for specific applications (bone grafts, implants).

<footnote n="1">¹Departamento de Mineralogía y Petrología, Universidad de Granada, 18002 Granada, Spain,</footnote>

<footnote n="2">²Department für Geo- und Umweltwissenschaften, Ludwig-Maximilians-Universität, 80333 Munich,</footnote>

<footnote>Germany, ³Departamento de Microbiología, Universidad de Granada, 18002 Granada, Spain.</footnote>

<footnote>*Corresponding author: anava@ugr.es</footnote>
# Infrared and Raman spectroscopy for the fingerprinting of microbially-induced calcium carbonate precipitation

Franco Grosso Giordano, Nele De Belie, Nico Boon, Carlos Rodriguez-Navarro

¹ Center for Microbial Ecology and Technology (CMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium, ² Laboratorium Magnel-Vandepitte, Dept. of Structural Engineering and Building Materials, Ghent University, Zwijnaarde 60, B-9052, Gent, Belgium, ³ Department of Mineralogy and Petrology, University of Granada, 18002, Granada, Spain

The use of Fourier Transform Infrared (FTIR) and Raman spectroscopy as identification tools for calcium carbonate (CaCO3⁻) is extensive, spanning inorganic chemistry to mineralogy and marine zoology. A lesser explored field, is the spectroscopic identification of CaCO3⁻ during the self-healing of cracks in construction mortars. Cracks can self-heal over time as CaCO3 precipitates in them. Numerous methods exist to speed this reaction, including the use of microbially-induced carbonate precipitation. Still, it is not possible to readily identify whether the CaCO3 produced is biogenic in origin. Furthermore, the use of FTIR and Raman spectroscopy to fingerprint bacterially precipitated CaCO3 has been limited with little application to explaining its biogenic properties. Nonetheless, in other biomineralization contexts, these spectroscopic techniques are widely used, such as in studies of mollusk shells.

Here we present the potential of FTIR and Raman spectroscopy for differentiating between biotic CaCO3 from two bacteria, Sporosarcina pasteurii and Acinetobacter tjernbergiae, and chemically synthesized CaCO3. Raman spectra identified calcite bands with organic presence due to fluorescence. Furthermore, FTIR spectroscopy shows bacterial CaCO3 as less crystalline and stark interspecies crystal lattice differences. Furthermore, amine and polysaccharide bands were detected between 1700-1550 cm⁻¹ and 1070 cm⁻¹, respectively. Biotic crystals were treated to remove all organic matter to show that the bands are from inter and intra-crystalline occlusions. SEM and TEM was used to identify these occlusions, which to the author's knowledge have not been reported for bacterial carbonates before.

The work applies established analytical solutions in an innovative manner for the fingerprinting of biotic CaCO3 crystals, setting the basis for the use of FTIR and Raman spectroscopy as an identification technique of biotic crystals in mixed environments such as construction mortars. Infrared and Raman spectroscopy for the fingerprinting of microbially-induced calcium carbonate precipitation
Poster presentations

Poster 4

# Diagenetic alterations undergone by Rhynchonelliform brachiopod shells throughout fossilization – Multiscale and analytical approach

Danièle GASPARD

CR2P - Centre de Recherche en Paléontologie – Paris, CNRS - MNHN - Sorbonne Université,
Département Origines et Evolution, Muséum National d'Histoire Naturelle - CP 38, 8 Rue Buffon, F-
75005 Paris.

The atomic force microscopy (AFM) allowed understanding the hierarchical organization of the layers of extant brachiopod shells (Gaspard & Nouet, 2016). But after death, modifications take place leading to light or heavy alterations in these archives throughout the process of fossilization according to free lying or quick burial.

Our observations were carried on equivalent extant as extinct two or three-layered shells. They are: *Compsothyris racovitzae* from Antarctica (recent) and *Cyclothyris difformis* (Early Cenomanian), Rhynchonellida; *Tichosina sp.* from the Caribbean Sea, *Moutonithyris dutempleana* (d'Orb.) (Early Cenomanian), *Sellithyris sella* (Sow.) (Aptian), *Sellithyris cenomanensis* Gasp. (Mid. Cenomanian), *Phaseolina phaseolina* (Lamk) (Upper Cenomanian), respectively a recent and fossils Terebratulida. In the shell thickness the inorganic material is intimately organized with organic materials allowing consider the shell as a “biomaterial”. Several means of observation using the scanning electron microscope (SEM), mapping elements and the (AFM) allow highlight the modifications in these low-Mg calcite shells at different scales. Cross sections in the shells are the supports for these observations.

Early diagenesis can be observed soon after death. It is possible to found fossils with the organic material entirely or partly preserved (S. sella, S. cenomanensis). Recrystallizations appear sometimes lightly (S. sella, S. cenomanensis) or heavily (C. difformis, M. dutempleana, Ph. phaseolina) even both in the same shell. When the organic material disappears, a bacterial activity is often present. In case of secondary calcite or silicic alterations, the nanoparticles, well individualized in the elements forming the inner layers (fibrous and prismatic/columnar) in recent shells, tend gradually to be agglomerated in fossils leading finally to nodules. Physical/chemical palaeoenvironmental context act for these alterations that appear partly or in the whole shell.
# Mechanical origin of the crossed-foliated ultrastructure in mollusks

Richard Johannes Best¹, Dylan Carberry², Tal Cohen², Igor Zlotnikov¹

¹B CUBE – Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany. ²Departments of Civil & Environmental, and Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, USA

Solving the puzzle of what drives of the morphogenesis of various molluscan shell ultrastructures has been the intrigue of biologists, mineralogists and material scientists alike for decades if not centuries. Whereas, historically, the majority of research was focused on the nacreous and the prismatic assemblies, the most common crossed ultrastructures, such as crossed-lamellar and the crossed-foliated architectures, were mostly overlooked. This is because of the spatial complexity of these assemblies that include multi-level hierarchical organization of the mineral building blocks and an intricate crystallographic relationship between them.

In this work, we describe the formation of the crossed-foliated ultrastructure in the shell of the limpet *Patella vulgata*. Detailed electron microscopy based imaging and diffraction analysis in 3D is used to provide a comprehensive structural and crystallographic information on the formation and spatial organization of the mineral building blocks during the growth of this ultrastructure. These data were then used to develop a model for the hierarchical self-assembly of this ultrastructure driven by mechanical forces generated as the result of a complex interplay between the formation of a single mineral unit and the growth of the shell as a whole. The model is supported by first-principles mechanical simulations.
Poster 6

Random crystal orientations in coral skeleton centers of calcification
become more ordered with acidification

Connor A. Schmidt¹*, Eric Tambutté², Sylvie Tambutté², Pupa U.P.A. Gilbert¹,³,⁴

¹ Department of Physics, University of Wisconsin, Madison, WI 53706, USA. ² Department of Marine Biology, Centre Scientifique de Monaco, 98000 Monaco, Principality of Monaco. ³ Departments of Chemistry, Materials Science and Engineering, and Geoscience, University of Wisconsin, Madison, WI 53706, USA. ⁴ Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

* Correspondence to: caschmidt6@wisc.edu

We measured crystallographic c-axis orientations of aragonite crystals in the centers of calcification of *Stylophora pistillata* coral skeletons grown at two different pH values, 7.2 and 8.0¹, using polarization-dependent imaging contrast (PIC) mapping² with PhotoEmission Electron Microscopy (PEEM)³,⁴ at the oxygen K-edge⁵, ⁶. We observed that the crystallites in the centers of calcification of coral skeletons grown at pH 8.0 contained a more even and random distribution of orientations when compared to those grown at pH 7.2. These results are consistent with previously published electron back-scatter diffraction (EBSD) data from the same species of coral⁷, but push the envelope further with 60 nm spatial resolution and quantitative analysis of c-axis orientation distributions across 4 samples.

1. Venn, A.; Tambutté, E.; Caminiti-Segonds, N.; Techer, N.; Allemand, D.; Tambutté, S., Effects of light and darkness on pH regulation in three coral species exposed to seawater acidification. *Sci Rep* **2019**, *9* (1), 1-12.

2. Gilbert, P.; Young, A.; Coppersmith, S. N., Measurement of c-axis angular orientation in calcite (CaCO3) nanocrystals using X-ray absorption spectroscopy. *Proceedings of the National Academy of Sciences* **2011**, *108* (28), 11350-11355.

3. De Stasio, G.; Capozi, M.; Lorusso, G.; Baudat, P.; Droubay, T.; Perfetti, P.; Margaritondo, G.; Tonner, B., MEPHISTO: Performance tests of a novel synchrotron imaging photoelectron spectromicroscope. *Review of scientific instruments* **1998**, *69* (5), 2062-2066.

4. De Stasio, G.; Perfetti, L.; Gilbert, B.; Fauchoux, O.; Capozi, M.; Perfetti, P.; Margaritondo, G.; Tonner, B., MEPHISTO spectromicroscope reaches 20 nm lateral resolution. *Review of scientific instruments* **1999**, *70* (3), 1740-1742.

5. DeVol, R. T.; Metzler, R. A.; Kabalah-Amitai, L.; Pokroy, B.; Politi, Y.; Gal, A.; Addadi, L.; Weiner, S.; Fernandez-Martinez, A.; Demichelis, R., Oxygen spectroscopy and polarization-dependent imaging contrast (PIC)-mapping of calcium carbonate minerals and biominerals. *The Journal of Physical Chemistry B* **2014**, *118* (28), 8449-8457.

6. Sun, C.-Y.; Marcus, M. A.; Frazier, M. J.; Giuffre, A. J.; Mass, T.; Gilbert, P. U., Spherulitic growth of coral skeletons and synthetic aragonite: nature's three-dimensional printing. *ACS nano* **2017**, *11* (7), 6612-6622.

7. Coronado, I.; Fine, M.; Bosellini, F. R.; Stolarski, J., Impact of ocean acidification on crystallographic vital effect of the coral skeleton. *Nature communications* **2019**, *10* (1), 2896.
Poster 7

Ultrastructures and mechanical properties of a flexible plate of calcium carbonate in door snails

Yuri Kurihara,¹ Taro Yoshimura,¹,² Yuya Oaki,¹ Hiroaki Imai,¹

¹ Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan, ² Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 1130-033, Japan

Commonly known as “door snail”, Clausiliidae possess a synapomorphy called a clausilium, a plate attached to the columella by a stalk. Even though clausilium is composed mainly of aragonite-type calcium carbonate, it is characterized by flexibility. However, the microstructures providing the flexible nature have not been clarified. In this study, we examined in detail the microstructure of the shell and the clausilium of *Stereophaedusa japonica* and *Euphaedusa tau* to elucidate their flexible structure.

From detailed observation of the cross-section of the flexible plate and stalk, clausilium is composed of three layers consisting of aragonite blocks with various shapes, such as cylindrical columns shorter than 3 µm, long needles with lengths of several tens of micrometers, and spherulitic grains with diameters of several hundred nanometers. In the outermost layer, the columns are arranged perpendicularly to the plate surface. In the second layer, the long needles that are parallel to the surface are partially stacked like a cross-lamellar structure. The central layer of the plate and stalk is composed of randomly arranged spherulitic grains with a relatively large amount of organic matter. The nano-indentation test showed that the hardness and modulus depend on the microstructure of the layers. The hardness of the central layer is lower than that of the other layer. The combination of various layers would provide unique mechanical properties of clausilium.
# Biomineralization of avian eggshell through the study of abnormal eggshell formation

Arias, J.L., Fernández, M.S

*Faculty of Veterinary and Animal Science, University of Chile, Santiago, Chile*

Remarkable progress in biomineralization knowledge has been reached. However, there are many questions still waiting to be addressed. Among other biomineralized systems, the avian eggshell shows distinctive features, i.e. the absence of cells intermixed with the mineralized structure and the rapidity of mineralization. In fact, 5 g of calcium carbonate are deposited in an 18 h period by a precise arrangement of spatio-temporal sequential deposition of macromolecules and inorganic ions. Three main approaches have been utilized to understand the role of eggshell components for controlling inorganic crystal nucleation, growth, structure and texture of the eggshell: (i) mechanical, crystallographic and microstructure characterization, and chemical composition, (ii) characterization of the assembly process during biological development, including oviduct microstructure, gene expression and sequential secretion, (iii) characterization of the *in vitro* effect of isolated or modified eggshell macromolecules on calcium carbonate nucleation and growth and eggshell reconstitution. Several specific proteins and proteoglycans have been isolated, characterized and postulated to be involved in the control of eggshell biomineralization. However, detailed mechanisms of the interaction of organic specific macromolecules and the inorganic moiety is far from being elucidated. Main studies on eggshell formation have been done with normal egg. However, there are special circumstances in which hens lay abnormal egg. Either changes in the organic composition of such eggshell or the experimental induction of eggshell formation by the administration of determined molecules, are scarcely reported. Here we report the reversible effects of specific molecules mimicking eggshell abnormal formation.

**Keywords:** Biomineralization, chicken, eggshell, misshaped eggs
Poster 9

# Cryo-stopped flow methodology for studying pre-nucleation species via solid-state Nuclear Magnetic Resonance

Ieva Goldberga¹, Tristan Georges¹ and Thierry Azaïs¹*

¹Laboratoire de Chimie de la Matière Condensée de Paris, Sorbonne Université, CNRS, 75005 Paris, France

The nucleation and growth of crystalline solids occurring in precipitation events from aqueous solutions is a central focus impacting many scientific areas, from organic to inorganic chemistry or material science.¹ Recently, the classical nucleation and growth theory (CNT) has been challenged by the observation of stable and soluble pre-nucleation species (PNS) prior to the formation of many solid materials, implying that the non-classical crystallization pathway (NCP) might play a role in this process. PNS are (meta)stable and soluble species – highly dynamic nanometric ionic entities in equilibrium with free ions that spontaneously form in solution before the nucleation of the first solid phase. In a biomineralization context, PNS have been described and reported for calcium phosphates,² carbonates³ and oxalates phases.⁴ Such species are challenging to characterize due to their soluble nature, nanometric size, dynamic behaviour and short lifetime. Thus, new methods are required to monitor these species' evolution in real-time at nanoscale resolution.

Solid-state Nuclear Magnetic Resonance (NMR) has been used to study the structure and composition of biominerals. However, using NMR to access phases and species out of equilibrium, such as PNS, can be challenging. Thus, in this work, we introduce the cryo-stop flow methodology to follow the evolution of calcium phosphate PNS at different time points to gain more insights into their structure and composition. This cryo-stop flow methodology enables the addition and mixing of independent Ca²⁺ and phosphate solutions at controlled concentrations, volumes and rates. The subsequent freezing at defined time points enables to stop the reaction and analyze transient PNS by low-temperature solid-state NMR. Here, we describe the set-up and experimental conditions optimization to study calcium phosphate PNS at the early stages of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) formation, the main component of bones and teeth.

**References:**

1. J. J. De Yoreo, P. U. P. A. Gilbert, N. A. J. M. Sommerdijk, R. L. Penn, S. Whitelam, D. Joester, H. Zhang, J. D. Rimer, A. Navrotsky, J. F. Banfield, A. F. Wallace, F. M. Michel, F. C. Meldrum, H. Cölfen and P. M. Dove, *Science*, DOI:10.1126/science.aaa6760.

2. A. Dey, P. H. H. Bomans, F. A. Müller, J. Will, P. M. Frederik, G. de With and N. A. J. M. Sommerdijk, *Nat. Mater.*, 2010, **9**, 1010–1014.

3. L. Wang, S. Li, E. Ruiz-Agudo, C. V. Putnis and A. Putnis, *CrystEngComm*, 2012, **14**, 6252–6256.

4. E. Ruiz-Agudo, A. Burgos-Cara, C. Ruiz-Agudo, A. Ibañez-Velasco, H. Cölfen and C. Rodriguez-Navarro, *Nat. Commun.*, 2017, **8**, 768.
# Impact of foreign elements on the phase transformation of amorphous calcium carbonate - a bioinspired in vitro study

Christian Liedgens, Anne Jandtscke

*Johannes Gutenberg-Universität Mainz*

Many calcifying organisms use amorphous calcium carbonate (ACC) as a precursor phase which transforms into a stable carbonate phase such as calcite and aragonite [1]. One family of dinoflagellates, the *Thoracosphaeraceae*, forms highly structured calcite shells as part of their life cycle. In these microalgae, intracellular MgCaP-rich bodies take part in the Ca-transport and concentrating mechanism and can be considered a calcite precursor [2]. According to a working hypothesis, these bodies encompass a Mg- and P-containing ACC that transforms into a low-Mg calcite inside the extracellular matrix. So far, the influence of Mg and P on ACC structure and transformation has only been investigated separately [3]. The goal of this project is to investigate the influence of foreign elements on synthetic ACC and its transformation into other polymorphs. Inspired by calcification in dinoflagellates, we synthesised a Mg-P-ACC using different phosphorus sources (e.g., inorganic Pᵢ, polyphosphates) and studied its transformation. This approach will be applied to other elements, such as zinc, iron, and strontium, to gain an understanding of metal incorporation in calcium carbonates. In some organisms, e.g., aragonitic shells of marine bivalves, these serve as a proxy for environmental reconstruction [4]. After optimisation of ACC synthesis, the ACC was transformed into a stable calcium carbonate by heat treatment. Both, the ACC precursor, and the transformed product are comprehensively characterised by vibrational spectroscopy (IR, Raman, and ssNMR), TGA, ICP-OES, and SEM.

Overall, this project aims to constrain the influence of foreign cations on ACC structure and their effect on subsequent phase transformations. These *in vitro* studies provide a synthetic model system that will facilitate the understanding of more complex biological calcification processes.

## Bibliography

[1] Addadi, L., Raz, S., & Weiner, S. (2003). Taking advantage of disorder: amorphous calcium carbonate and its roles in biomineralization. *Advanced Materials*, *15*(12), 959-970.

[2] Jantschke, A., Pinkas, I., Schertel, A., Addadi, L., & Weiner, S. (2020). Biomineralization pathways in calcifying dinoflagellates: Uptake, storage in MgCaP-rich bodies and formation of the shell. *Acta Biomaterialia*, *102*, 427-439.

[3] Albéric, M., Bertinetti, L., Zou, Z., Fratzl, P., Habraken, W., & Politi, Y. (2018). The crystallization of amorphous calcium carbonate is kinetically governed by ion impurities and water. *Advanced Science*, *5*(5), 1701000.

[4] Schöne, B. R., Marali, S., Jantschke, A., Mertz-Kraus, R., Butler, P. G., & Fröhlich, L. (2023). Can element chemical impurities in aragonitic shells of marine bivalves serve as proxies for environmental variability? *Chemical Geology*, *616*, 121215.
# Deciphering the role of organic matter in the biomineralization process of marine sponge spicules: a solid-state NMR investigation

Sylvie Masse *¹, Guillaume P. Laurent ¹, Thibaud Coradin¹, Andrzej Pisera ²

¹ Laboratoire de Chimie de la Matière Condensée de Paris, Sorbonne Université, CNRS, Paris, France, ² Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland

## Introduction

Sponges, one of the most primitive animals, are exclusively aquatic sessile organisms. Siliceous sponges are mainly represented by two classes, Demospongiae (Sollas, 1885) and Hexactinellida (Schmidt, 1870). Their internal skeleton is composed of spicules that serve to stiffen the soft body, helping the animal with protection and anchoring. During the biomineralization process, silica plays a dominant role in skeleton formation, but nothing would be possible without organics intercession.

## Objectives

In this study, we used solid-state NMR spectroscopy to better understand the role of organics during the biosilicification process. Our goal is to more precisely depict the organo-silica interface to discriminate the different species among the great variety of specimen.

## Materials and Methods

Solid-state NMR is one of the most versatile tools of characterization in materials science. However, this technique suffers from low sensitivity and long acquisition times.

Natural specimen of spicules contain a big amount of silicon but a small amount of carbon (e.g., 2-7 wt% C). As no isotopic enrichment is possible and ¹³C isotope is only 1.1 at% nat. ab., it is difficult to collect exploitable data from carbon.

## Results

²⁹Si and ¹³C NMR spectra were compared to assess the importance of the taxon. Then, two-dimensional (2D) experiments showed heteronuclear correlations that depict the organo-mineral interface but suffer from a poor signal-to-noise ratio.

## Conclusion

We now plan to improve the study by using DNP-NMR to enhance the signal and perform a ¹H-²⁹Si-¹³C triple resonance experiment.
# Understanding bone diagenesis: Effect of fluorine substitution under hydrothermal controlled conditions

Héctor Del Valle<fnref n="1" />,<fnref n="2" />, Alejandro B. Rodríguez-Navarro<fnref n="3" />, Isabel Cáceres <fnref n="1" />,<fnref n="2" />

1 Institut Català de Paleoecologia Humana i Evolució Social (IPHES-CERCA), Zona Educacional 4, Campus Sescelades URV (Edifici W3), 43007 Tarragona, Spain, 2 Universitat Rovira i Virgili, Departament d'Història i Història de l'Art, Avinguda de Catalunya 35, 43002 Tarragona, Spain, 3 Departamento de Mineralogía y Petrología, Universidad de Granada, 18002 Granada, Spain.

corresponding author: Héctor Del Valle (hdelvalle@iphes.cat)

Bone diagenesis is a complex phenomenon that involves the modification of the physicochemical, histological, and mechanical properties of bone remains in each burial environment. One of the most common diagenetic processes in archaeological and paleontological sites is the incorporation of fluorine in bone mineral which has been widely recorded on many sites [1-5]. However, information is lacking about how bone mineral changes with ion substitution processes during recrystallization in different environmental conditions. In this study, we induce bone fluoridation under controlled hydrothermal conditions. Fragments of an adult pig femur (Sus scrofa domestica) were treated with sodium fluoride (NaF) solutions (control-0.0, 0.01, 0.1, and 1 M) and at different temperatures (25, 50, 100, and 200°C) for 24 hours. Changes in bone chemistry and structure were characterized using complementary analytical techniques such as infrared spectroscopy (FTIR), X-ray diffraction (XRD) and electron microscopy. The results showed that fluorine is incorporated into the apatite structure and its substitution increases with temperature and NaF concentration as deduced from the contraction of unit cell size (from 532 to 524 Å<fnref n="3" />). Hydrothermal conditions also produced an increase in apatite crystallite size with temperature with is greater in fluorine bearing solutions. Other notable changes were the progressive increase of the 575 cm⁻<fnref n="1" /> band intensity in the v₄(PO₄<fnref n="3" />⁻) region and the inversion of the 605 cm⁻<fnref n="1" /> band relative to 563 cm⁻<fnref n="1" /> bands which the incorporation of fluorine in apatite structure. These results are important for understanding the changes occurring in bone during burial and/or diagenesis in stratigraphic sequences of archaeological and paleontological sites.

**Keywords:** Bone diagenesis, Apatite, Fluoridation, recrystallization, hydrothermal

<footnote n="1">1. Shemesh A. Crystallinity and diagenesis of sedimentary apatites. Geochim Cosmochim Acta. 1990;54:2433-2438.</footnote>

<footnote n="2">2. Geiger SB, Weiner S. Fluoridated carbonatoapatite in the intermediate layer between glass ionomer and dentin. Dent Mater. 1993;9(1):33-36. doi:10.1016/0109-5641(93)90102-V</footnote>

<footnote n="3">3. Wright LE, Schwarcz HP. Infrared and Isotopic Evidence for Diagenesis of Bone Apatite at Dos Pilas, Guatemala: Palaeodietary Implications. J Archaeol Sci. 1996;23:933-944. doi:10.1006/jasc.1996.0087</footnote>

<footnote n="4">4. Keenan SW, Engel AS. Reconstructing diagenetic conditions of bone at the Gray Fossil Site, Tennessee, USA. Palaeogeogr Palaeoclimatol Palaeoecol. 2017;471:48-57. doi:10.1016/j.palaeo.2017.01.037</footnote>

<footnote n="5">5. Stathopoulou ET, Psycharis V, Chryssikos GD, Gionis V, Theodorou G. Bone diagenesis: New data from infrared spectroscopy and X-ray diffraction. Palaeogeogr Palaeoclimatol Palaeoecol. 2008;266(3-4):168-174. doi:10.1016/j.palaeo.2008.03.022</footnote>

<footnote n="6">6. Margariti E, Stathopoulou ET, Sanakis Y, Kotopoulou E, Pavlakis P, Godelitsas A. Journal of African Earth Sciences A geochemical approach to fossilization processes in Miocene vertebrate bones from Sahabi, NE Libya. J African Earth Sci. 2019;149(December 2017):1-18. doi:10.1016/j.jafrearsci.2018.07.019</footnote>
# Magnetotactic Bacteria: more than nanomagnets

Damien Faivre

BIAM, CEA Cadarache, France

Magnetotactic bacteria are known to intracellularly biomineralize the iron oxide magnetite and / or the iron sulfide greigite. Alternatively, metal sulfides are a common group of extracellular bacterial biominerals. Only few cases of intracellular biomineralization have been reported in this group, mostly limited to greigite ($Fe_3S_4$) in magnetotactic bacteria.

Here, we report the intracellular but periplasmic biomineralization of copper sulfide by the magnetotactic bacterium *Desulfamplus magnetovallimortis* (strain BW-1) that is known to mineralize greigite and magnetite ($Fe_3O_4$) in the cytoplasm. BW-1 produces hundreds of spherical nanoparticles, composed of 1-2 nm substructures of a poorly crystalline hexagonal copper sulfide that remains in a thermodynamically unstable state. Differential proteomics suggests that periplasmic proteins, such as a DegP-like protein and a heavy metal-binding protein, could be involved in this process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW-1 reveals previously unknown possibilities for intracellular biomineralization.
APPLICATION OF CALCIUM PHOSPHATES: IN VIVO TEST IN
SHEEP

G.M.L. Dalmônico¹, N.H.A. Camargo² A.L. Dallabrida², Cambra-Moo³, M.A. Rodríguez⁴, A.L. Rossi⁵, A.M.Rossi⁵, M.Farina⁶

¹National Center for Energy and Materials Research - Brazilian Nanotechnology National Laboratory
– CNPEM/LNNano, Brazil, ²Santa Catarina State University (UDESC), Brazil, ³Universidad Autónoma de Madrid, ⁴Instituto de Cerámica y Vidrio - CSIC, Madrid, Spain, ⁵Brazilian Center for Physics Research, (CBPF) Rio de Janeiro, Brazil, ⁶Federal University of Rio de Janeiro.

Treatments for bone loss are research topics and involve different areas of scientific knowledge, engineering, physics, chemistry, biology and biomedicine. The biomaterials that stand out as replacement in bone structure treatments are hydroxyapatite, β and α calcium phosphate, biphasic hydroxyapatite/calcium phosphate β and α and hydroxyapatite matrix nanocomposite biomaterials. These biomaterials stand out as bone substitutes because they present a crystallography similar to that of human skeleton bone apatite, being bioactive and biocompatible. Nanostructured biphasic bioceramics are researched and show potential to be bone substitutes in surgical repairing procedures and reconstruction of bone tissue. This project was developed based on research of granular biomaterials of HA, β-TCP and biphasic composition HA/β-TCP=20/80%. All biomaterials were characterized by different techniques: scanning electron microscopy, atomic force microscopy, confocal microscopy, optical microscopy, polarized light microscopy. The interest of this research was to evaluate the performance of biomaterials in in vivo tests for the time period of 180 days, in relation to osseointegration and the formation of neoformed bone tissue and determine which biomaterials presented potential as bone replacement for biomedical applications. The study was conducted on sixteen healthy half-breed Texel sheeps with 12 months old. Two 4-mm non-critical defects were produced on the medial tibial diaphyseal of each tibia with the aid of a dental drill. For the adequate implantation of the biomaterials, initially the granulated material was deposited in a sterile vat and hydrated with the animal's own arterial blood. The animals were euthanized 180 days after implantation and implanted bone fragments were collected. The results found are encouraging and demonstrate that the granulated microporous biomaterials of calcium phosphate proves to be ability to repair and bone reconstruction for the test in vivo evaluated, revealing the osseointegration and bone formation. The results obtained from the biopsies containing the biomaterials revealed similar osseointegration behaviours, with excellent biomaterial/bone tissue interface and greater bone formation, presenting smaller amounts of remaining biomaterial. It is due to the contribution of porosity and phase composition parameters to the biodegradation process.

**Keywords:** β-calcium phosphate, hydroxyapatite, in vivo testing, bone neoformation.

**Acknowledgments:** The financial support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-BRASIL); the Ministry of Economy and Competitiveness from the Spanish Government (MAT2013-48426-C2-1R) and the Brazilian Center for Physics Research for a financial congress.

[1] Granjeiro, J.M; Soares, G.D.A. 1a ed. VM Cultural Editora Ltda., 2011. [2] Dorozhkin, S.V. Acta Biomat. 8 (2012) 963-977. [3] N.Levandowski Jr., N.H.A.Camargo, D.F. Silva, G.M.L.Dalmônico, P.F.Franczak. Ad Mat Res 936 (2014) 695-700. [4] G.M.L. Dalmônico, D.F. Silva, P.F. Franczak, N.H.A. Camargo, M.A. Rodríguez. Bol Soc Esp Ceram Vidrio 54 (2015) 37-43. [5] Almeida D, Sartoretto SC, Calasans-Maia JdA, Ghiraldini B, Bezerra FJB, Granjeiro JM, et al. (2023) In vivo osseointegration evaluation of implants coated with nanostructured hydroxyapatite in low density bone. PLoS ONE 18(2): e0282067. https://doi.org/10.1371/journal.pone.0282067. [6] Ajami E, Fu C, Wen HB, Bassett J, Park SJ, Pollard M. Early
Bone Healing on Hydroxyapatite-Coated and Chemically-Modified Hydrophilic Implant Surfaces in an Ovine Model. Int J Mol Sci. 2021 Aug 28;22(17):9361. doi: 10.3390/ijms22179361. PMID: 34502270; PMCID: PMC8430562.[7] Makishi, S.; Watanabe, T.; Saito, K.; Ohshima, H. Effect of Hydroxyapatite/β-Tricalcium Phosphate on Osseointegration after Implantation into Mouse Maxilla. Int. J. Mol. Sci. 2023, 24, 3124. https://doi.org/10.3390/ijms24043124
Keynote lecture

Enzymatic patterning of bone mineralization at the microscale for
crossfibrillar mineral tessellation: The Stenciling Principle

Marc D. McKee

*McGill University, Faculty of Dental Medicine and Oral Health Sciences, and Faculty of Medicine and Health Sciences, Montreal, Quebec, Canada*

The Stenciling Principle for extracellular matrix mineralization describes an enzyme-substrate, double-
negative regulatory process (inhibition of inhibitors) that promotes mineralization in bone and other
mineralized tissues, whereas the default condition of inhibition alone prevents mineralization elsewhere
in soft connective tissues. It relates to both small-molecule (e.g. pyrophosphate) and protein (e.g.
osteopontin) inhibitors of mineralization, and promoters (enzymes, e.g. TNAP, PHEX) that degrade the
inhibitors to permit and regulate mineralization. In this process, an organizational microscale motif for
bone mineral arises that we call crossfibrillar mineral tessellation where mineral formations – called
tesselles – geometrically approximate prolate ellipsoids and traverse multiple collagen fibrils (laterally).
Tesselle growth is directed by the structural anisotropy of collagen, being spatially restrained in the
shorter transverse tesselle dimensions. Temporo-spatially, the tesselles abut in 3D (close ellipsoid
packing) to fill the volume of lamellar bone extracellular matrix. Poorly mineralized interfacial gaps
between adjacent tesselles remain discernable even in mature lamellar bone. Tessellation of a same,
small basic unit to form larger structural assemblies results in numerous 3D interfaces, allows dissipation
of critical stresses, and enables fail-safe cyclic deformations in healthy bone. Incomplete mineral
tessellation in osteomalacia may explain why soft osteomalacic bones buckle and deform under loading.
## Oral presentations

### T3 01

# Biological control of enamel mineralization is dependent on phosphorylation of amelogenin.

Hajime Yamazaki¹,²#, Cayla A. Stifler³, Claire Gabe¹,², Ai Thu Bui¹,², Lyudmila Lukashova², Kostas Verdelis²,⁴, P.U.P.A. Gilbert³,⁵, Henry C. Margolis¹,²,⁶, Elia Beniash¹,²,⁷

1 – Department of Oral and Craniofacial Sciences, School of Dental Medicine, University of Pittsburgh, PA, USA, 2- Center for Craniofacial Regeneration, University of Pittsburgh, School of Dental Medicine, Pittsburgh, PA, USA, 3 - Department of Physics, UW-Madison, Madison, WI, USA, 4- Department of Endodontics, University of Pittsburgh School of Dental Medicine, Pittsburgh, PA, USA, 5 - Department of Chemistry, UW-Madison, Madison, WI, USA, 6- Department of Periodontics and Preventive Dentistry, School of Dental Medicine, University of Pittsburgh, PA, USA, 7- Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, PA, USA, # - No longer with the University of Pittsburgh

Amelogenin (AMELX), the major enamel matrix protein, has a sole post-translational modification – phosphorylation at Ser16. *In vitro* mineralization studies revealed that phosphorylated AMELX stabilizes amorphous calcium phosphate (ACP) and inhibits formation of apatite. To assess the role of this sole phosphorylation in amelogenesis we generated a knock-in (KI) mouse model with a Ser16 to Ala substitution. Using multiple approaches, we have characterized structural, chemical and mechanical properties of forming and mature incisal enamel in KI and WT mice. KI mice exhibit a severe enamel phenotype, manifested by a thin enamel layer; the lack of enamel rods, the hallmark of mammalian enamel; and ectopic calcifications. The c-axes of enamel crystals in rod-less inner KI enamel are co-oriented at the Dentin-Enamel Junction (DEJ) and become progressively misaligned toward the surface. The outer KI enamel comprises spherulites, that are structurally consistent with abiotic formation. Our studies of enamel formation also revealed that the rate of transformation of ACP to an apatitic crystal phase is accelerated in KI enamel. Furthermore, we found that mineral density at the onset of enamel formation is twice higher in KI vs WT. These observations are consistent with the results of our *in vitro* mineralization experiments, showing that phosphorylated AMELX controls the rate of mineralization and ACP-apatite transformation. Moreover, while at the onset of enamel secretion ameloblasts look normal, they never develop Tomes' processes, consistent with the absence of enamel rods, and exhibit progressive cell pathology, manifested by loss of polarization and formation of secretory aggregates throughout the ameloblast cell layer. These and other results to be presented demonstrate that the sole phosphorylation of AMELX is crucial for biological control of enamel mineralization, and in maintaining ameloblast phenotype and function. Supported by NIH DE DE029211 (HCM & EB).

**Keywords:** enamel, amelogenin, phosphorylation, biological control
T3 O2

Low phosphate diet differentially affects bone mineralization in mice deficient in Bone Sialoprotein, Osteopontin or both.

HUGON Romain¹, HIVERT Lauriane¹, BOUTAHAR Nadia², VANDEN-BOSSCHE Arnaud¹, LAROCHE Norbert¹, THOMAS Mireille¹, LINOSSIER Marie-Thérèse¹, VICO Laurence¹, LAFAGE-PROUST Marie-Hélène¹, MALAVAL Luc¹.

¹INSERM U1059, Université Jean Monnet, Saint-Etienne, France. ²CHU, INM, Université J Monnet, Saint-Etienne, France.

SIBLING proteins DMP1 & MEPE play a role in phosphate (PO₄) metabolism. We postulated that cognate Bone Sialoprotein (BSP) and Osteopontin (OPN) may also be involved in body PO4 handling. We generated mice deficient (−/−) in either BSP§, OPN or both (DKO)§§, and found that BSP−/− mice are hypophosphaturic while the OPN−/− are not. BSP−/− mice display normal PO₄ serum levels, normal/low serum FGF23 together with features of impaired PO₄ gut absorption. Thus, we hypothesized that a low PO₄ diet would induce a more severe mineralization defect in BSP−/− bones.

5 month-old WT, OPN−/−, BSP−/− and DKO male mice were fed diets containing either normal (ND, 0.55%) or low (LP, <0.05%) inorganic phosphorus content. At d7 they were put in metabolic cages for 1d for urine/feces collection. After killing at d14, femur and blood were sampled. Bone mass was assessed by µCT and bone remodeling by histomorphometry (HM).

Under LP, we found at d7 a dramatic drop in urinary PO₄ levels compared to ND, with lower values in BSP−/− mice while serum levels remained normal in all groups. Bone mass was 38 and 20% lower than ND in OPN−/− and BSP−/− mice, respectively. HM results [median(IQR)] are presented in the table (*p<0.05 vs respective ND, $p<0.05$ vs BSP and OPN under LP).

<table><thead><tr><th rowspan="2">Diet</th><th colspan="2">WT</th><th colspan="2">BSP-/-</th><th colspan="2">OPN -/-</th><th colspan="2">DKO</th></tr><tr><th>ND</th><th>LP</th><th>ND</th><th>LP</th><th>ND</th><th>LP</th><th>ND</th><th>LP</th></tr></thead><tbody><tr><td>OS/BS, %</td><td>12.2 (7.8)</td><td>34.1 (4.2)*</td><td>7.4 (6.6)</td><td>30.9 (23.4)*</td><td>3.9 (2.5)</td><td>22.5 (13.5)*</td><td>3.1 (2.4)</td><td>30.5 (23.5)*</td></tr><tr><td>O.Th, µm</td><td>1.1 (1.1)</td><td>1.7 (1.0)</td><td>2.5 (0.8)</td><td>3.8 (2.0)</td><td>2.7 (0.7)</td><td>6.8 (1.3)*</td><td>1.7 (0.7)</td><td>8.6 (1.8)*§</td></tr><tr><td>MAR, µm/d</td><td>0.99 (0.55)</td><td>0.47 (0.25)</td><td>1.14 (0.21)</td><td>0.31 (0.44)*</td><td></td><td></td><td></td><td></td></tr><tr><td>sLS/BS, %</td><td>5.7 (2.6)</td><td>17.4 (13.8)*</td><td>6.8 (9.5)</td><td>17.8 (89.8)*</td><td></td><td></td><td></td><td></td></tr><tr><td>dLS/BS</td><td>3.5 (2.2)</td><td>6.4 (2.4)</td><td>5.1 (4.3)</td><td>1.9 (2.4)</td><td></td><td></td><td></td><td></td></tr><tr><td>MS/BS, %</td><td>7.4 (3.2)</td><td>15.5 (9.3)*</td><td>9.9 (8.1)</td><td>10.1 (6.3)</td><td></td><td></td><td></td><td></td></tr><tr><td>MS/OS, %</td><td>0.60(0.18)</td><td>0.42 (0.29)</td><td>0.73(0.53)</td><td>0.26 (0.27)*</td><td></td><td></td><td></td><td></td></tr></tbody></table>

**Conclusion:** LP induces a more severe osteomalacia in mice deficient in BSP, OPN or both compared to WT with the worst phenotype in DKO. Overall, our results indicate that both OPN and BSP are involved in PO₄ metabolism, with no/limited vicariance between the 2 proteins

<footnote>§ Granito RN *et al.*, *J. Cell. Physiol.*, 2015, 230: 1342,</footnote>

<footnote>§§ Bouleftour *et al.*, *Bone*, 2019, 120: 411.</footnote>
# Altered bone health in laying hens is associated with FGF23 overexpression in the medullary bone

Michel Duclos, Audrey Gloux, Agnès Narcy, Joël Gautron

INRAE, Université de Tours, BOA, 37380 Nouzilly, France.

michel.duclos@inrae.fr

For the production of each egg, the hen must allocate about 2 g of Ca for eggshell formation, which originates from its diet and from bone mobilisation. As the hen lays between 300 and 500 eggs during its carrier, the challenge to Ca homeostasis is high, and pathological signs such as osteoporosis, deviations or fractures of the wishbone, or even fractures of long bone, show an increasing prevalence at the end of the production cycle. In their less serious forms, they compromise animal welfare, but they can also lead to culling and losses, or to the early termination of a flock. This sets the need to better understand the physiological mechanisms of calcium homeostasis in hens, particularly those involving bone tissue. At sexual maturity the hen develops a specific type of medullary bone, which is subject to daily cycles of bone remodelling and thus serves as a temporary store of Ca. The team's recent work provides new data on the effectors of calcium transport through the intestinal epithelium, on the mechanisms of bone turnover and their regulation. It is consistent with previous ones in highlighting the central role of vitamin D and its metabolites. Moreover, it also highlights a factor that had not yet been studied in details in the laying hen, Fibroblast Growth Factor-23 (FGF23). It is expressed by the medullary bone when eggshell synthesis is active in the uterus and bone mobilisation is maximal. The expression of its putative receptors (FGFRs) and coreceptor (KL) in the kidney suggest that it could exert its action by promoting the urinary excretion of P resulting from osteolysis. In addition, by inhibiting the synthesis of the active form of vitamin D3 or by activating its degradation, it would exert a negative feedback on Ca and P retention. FGF23 is overexpressed during in the late laying hen compared to the peak laying hen. This opens up the possibility of research into whether FGF23 is the cause or only a marker of deregulation of phosphocalcic metabolism
# Multicellular spheroids containing synthetic mineral particles to investigate breast precancer malignancy potential according to the mineral type

Amit Cohen¹, Lotem Gotnayer¹, Sahar Gal¹, Dina Aranovich¹, Netta Vidavsky¹,²

¹Department of Chemical Engineering, Ben-Gurion University of the Negev, Beer Sheva, ²Ilse Katz Institute for Nanoscale Science & Technology, Ben-Gurion University of the Negev, Beer Sheva

## Abstract

Mineral particles that form in soft tissues in association with disease conditions are heterogeneous in their composition and physiochemical properties. Hence, it is challenging to study the effect of mineral type on disease progression in a high-throughput and realistic manner. For example, most early breast precancer lesions, termed ductal carcinoma *in situ* (DCIS), contain microcalcifications (MCs), calcium-containing pathological minerals. The most common type of MCs is calcium phosphate crystals, mainly carbonated apatite; it is associated with either benign or malignant lesions. *In-vitro* studies indicate that the crystal properties of apatite MCs can affect breast cancer progression. A less common type of MCs is calcium oxalate dihydrate (COD), which is almost always found in benign lesions. We developed a 3D tumor model of multicellular spheroids of human precancer cells containing synthetic MC analogs that link the crystal properties of MCs with the progression of breast precancer to invasive cancer. Using this 3D model, we show that apatite crystals induce Her2 overexpression in DCIS cells. This tumor-triggering effect is increased when the carbonate fraction in the MCs decreases. COD crystals, in contrast, decrease Her2 expression in the spheroids, even compared with a control group with no added MC analogs. Furthermore, COD decreases cell proliferation and migration in DCIS monolayers compared to untreated cells and cells incubated with apatite crystals. This finding suggests that COD is not randomly located only in benign lesions—it may actively contribute to suppressing precancer progression in its surroundings. Our model provides an easy-to-manipulate platform to better understand the interactions between mineral particles and their biological microenvironment. A better understanding of the effect of the crystal properties of MCs on precancer progression will potentially provide new directions for better precancer prognosis and treatment.
# Ultrastructure of mineral ellipsoids at bone-calcium phosphate interface in bone healing process

Camila Wendt¹, ⁴, André Linhares¹, Caio Santos³, Victor Zelaya², Ricardo Lopes³, Marcos Farina⁴,
Alexandre Rossi¹

¹Centro Brasileiro de Pesquisas Físicas, Rio de Janeiro, Brasil, ²Laboratório Nacional de Luz Síncrotron, Campinas, Brasil, ³Coppe, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil, ⁴Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil

* camilawendt@cbpf.br

Bone structure at the micro and nanoscale has been revisited in recent years (1-4). An architecture based on sub-micrometer ellipsoid features mineralized within collagen bundles has been proposed as a new hierarchical level of bone (5-6). In this work, we combine synchrotron x-ray (SRuCT) tomography with focused ion beam-scanning electron microscopy (FIB-SEM) and transmission electron microscopy (TEM/HRTEM) to characterize the ellipsoids ultrastructure in the woven bone formed during a healing process in rat tibia defect 21-day implanted with a biodegradable carbonated hydroxyapatite/alginate microspheres. After implantation, microsphere fragmentation improves osteoconduction, and the newly trabecular bone-like network grows, filling the space between the microsphere fragments and attaching to the fragment surface. Mineral tissue is nucleated inside micrometer and sub-micrometer pores. FIB-SEM slice and view tomography showed that the trabeculae volume is filled with osteocyte-lacunae and mineralized ellipsoidal structures oriented in different directions. The presence of these motifs over the entire FIB-SEM series suggests that these structures represent a regular pattern of mineral deposition nucleated in association with collagen bundles. TEM/HRTEM images showed that each ellipsoid comprises a packet of non-ordered mineralized collagen microfibril clusters with a mean length of 1.6 µm and width of 0.7 µm. These structures cover the biomaterial interface, but the ellipsoidal morphology is not identified inside sub-micrometer pores. In this confined condition, the mineralized tissue presents a different ultrastructure than woven trabecular ellipsoids but with a similar electron diffraction pattern. Inside sub-micrometer pores, MT organizes in nearly linear arrangements of mineralized collagen microfibril. The MT nucleation occurs in pores smaller than one micrometer located at more than 5 µm from cells, in a process not apparently controlled directly by cells.

1. Reznikov N. et al., Acta Biomaterialia, 10, 2014.

2. Rustom L. M. et al., Acta Biomaterialia 83, 2019.

3. Bohner M. et al., Materials Today 20, 2017.

4. Binkley D. M. et al., Journal of Structural Biology 212, 2020.

5. Buss D. et al., Journal of Structural Biology, 212, 2020.

6. Micheletti C. et al Acta Biomaterialia 142, 2022.

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação
o Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and INCT REGENERA.
# The dental and periodontal mineralization defects related to FAM20A gene mutations

Ariane Berdal¹, Stéphanie Renaud¹, Dominique Hotton¹, Luc Laurencena¹, Muriel De la Dure-Molla², Mary MacDougall³

¹UFR Odontologie Université Paris Cité, Centre de Référence Maladies Rares CRM R O-Rares, Assistance Publique Hôpitaux de Paris, équipe "physiopathologie orale moléculaire" Centre de recherche des Cordeliers Inserm, Université Paris Cité, Sorbonne Université. France, ²UFR Odontologie Université Paris Cité, Centre de Référence Maladies Rares CRM R O-Rares, Assistance Publique Hôpitaux de Paris, équipe "bases moléculaires et physiopathologiques de l'ostéochondrodysplasie", IHU Imagine, Inserm, Université Paris Cité. Paris, ³UBC Faculty of Dentistry Centre for Oral Dental Disorders (CODED)

ariane.berdal@u-paris.fr

Introduction: FAM20A is a paralog of FAM20C kinase in charge of secreted protein phosphorylation, notably in enamel. FAM20A is a pseudo-kinase which acts indirectly through binding and activating FAM20C. FAM20A loss-of-function variants are associated with Enamel Renal Syndrome - ERS (OMIM# 204690) presenting with amelogenesis imperfecta, tooth eruption impairment and gingival fibromatosis.

Our CRM R Center ensures the follow-up of a ERS patient cohort. Our previous studies on teeth and gingiva suggested that an ectopic mineralization process would intervene in ERS pathophysiology. Here, we explored an animal model, the *Fam20a* knock out (KO) mouse. Heads from wild-type and KO mice (3-5, 7-D PN, 7 week- and 14-month-old) were analyzed (SEM, histology, TEM and amelogenin immunogold labelling). The observed mouse dental phenotype was confronted to our clinical CRM R dataset on ERS patients affected by different *FAM20A* mutations.

KO mouse amelogenesis was biphasic and disordered. The innermost enamel was deposited by ameloblasts, albeit with an abnormal rod-interrod pattern. Starting 5-D postnatally, ameloblasts lose their polarity and intercellular junctions, becoming uncoupled from the forming enamel matrix. They secreted an amelogenin-containing material in ectopic positions : laterally, towards and into the adjoining follicular sac. Ameloblast cell death and embedding in minerals also intervened. The resulting « enamel » aggregated electron-dense amelogenin-positive nodules and cell ghosts. Concurrently, a collagen-rich extracellular matrix was deposited, mineralized and fused to « enamel ».

**Conclusion:** The coronal external layer of ERS teeth would be produced by an ectopic mineralization process involving both the enamel organ and follicular sac. The resulting anchoring of enamel to the periodontium might play a role in tooth eruption arrest, as supported by the characteristic radiological profile of ERS patients with different disease grades.

**Keywords:** ectopic mineralization, enamel, rare diseases
Effect of MOP consumption on calcium metabolism and gut in
ovariectomized rat model

Yacine Bertache-Djenadi¹, Kim Nguyen¹, Arnaud Vanden-Bossche¹, Mireille Thomas¹, Stéphanie Mundweiler¹, Marie-Thérèse Linossier¹, Sylvie Peyroche¹, Delphine Farlay², Hubert Marotte¹, Laurence Vico¹, Nicolas Rochereau³, Marthe Rousseau¹,⁴

1. Université Jean Monnet Saint Étienne, Mines Saint Étienne, INSERM, SAINBIOSE U1059, F-42023, Saint Étienne, France, 2. LYOS, INSERM U1033, UCBL1, UdL, Lyon, France, 3. CIRI, GIMAP, UdL, UCBL1, INSERM U1111, CNRS (UMR 530), CIC1408, Saint Etienne, France, 4. UDL, INSA Lyon, CNRS, MATEIS (UMR 5510), Villeurbanne, France

Osteoporosis is characterized by a bone remodeling imbalance leading to bone mass and mechanical
properties loss, increasing fracture risks. Mother of pearl demonstrated protective effect against bone
loss when ingested by osteoporosis models as ovariectomized (OVX) mice or rats. This may be related
to a direct effect on resorption and bone synthesis as seen on bone cells in vitro. Nevertheless, an indirect
effect through the gut-bone axis or calcium metabolism can also be involved. We therefore investigate
gut-related effect of MOP consumption in ovariectomized rats. 46 animals were sham operated or OVX
at 16-week-old and last ones either fed with normal, 0.25% MOP or calcium (Ca) complemented diet
for 3 months. FGF23, PTH, calcitriol, calcium and phosphate serum levels were quantified and didn't
show any effect other than ovariectomy, except for serum phosphate increase in MOP group compared
to OVX (p<0,025). Intestinal samples went through mRNA extraction and QPCR assessment: CaV 1.3
duodenal expression was significantly higher in MOP fed group than in OVX (p<0,025), SHAM and Ca
groups (p<0,05), TRPV6 significantly lower than SHAM and OVX groups (p<0,05). Cecal cadherin 17
expression decreased significantly in MOP group compared to Ca and SHAM (p<0,05), claudin 12 in
Ca (p<0,05) and MOP (p<0,025) groups compared to OVX and claudin 2 expression increase in Ca
(p<0,025) and OVX (p<0,001) compared to SHAM. Differences between SHAM and OVX groups can
be related to estrogen loss while theses between OVX and Ca groups explained by the 0,12% dietary
calcium increase. Duodenal changes between MOP and Ca groups could result in MOP-induced higher
postprandial calcium absorption and lower reabsorption during fasting while cecal paracellular transport
associated proteins expression decrease could diminish epithelial calcium permeability, thus calcium
absorption as much as its loss. However, theses expression results should be complete by functional
assessments.

Keywords: Calcium absorption; Bone loss; MOP
# Lack of Amelogenin Phosphorylation Leads To Acidification Of Secretory Enamel

Claire M. Gabe¹,², Ai Thu Bui¹,², Brent P. Vasquez¹,², Elia Beniash¹,², Henry C. Margolis¹,²,³

1 - Department of Oral and Craniofacial Sciences, Center for Craniofacial Regeneration, University of Pittsburgh School of Dental Medicine (UPSDM), Pittsburgh, PA, USA; 2 - Center for Craniofacial Regeneration, UPSDM, Pittsburgh, PA, USA; 3 - Department of Preventive Dentistry and Periodontics, UPSDM, Pittsburgh, PA, USA.

Amelogenin (AMELX), the major enamel matrix protein (EMP), has a single phosphorylation on Serine 16. Phosphorylation enhances AMELX's capacity to stabilize amorphous calcium phosphate (ACP) and inhibit apatite crystal formation in vitro [1,2]. To study its effects in vivo we developed a knock-in (KI) mouse model with a Ser16 to Ala substitution in AMELX, which prevents AMELX phosphorylation. KI mice exhibit a severe phenotype with thin hypoplastic enamel, lack of the decussating rod pattern, and a higher rate of ACP to apatite transformation [3].

**Objective:** To test the hypothesis that accelerated enamel mineralization in AMELX-Ser16Ala KI mice induces local acidification that affects the EMPs and the forming enamel mineral phase.

**Methods:** Mandibular incisors from 8-week-old wild-type (WT) and KI mice were isolated and their ameloblast layer was removed. Incisors were freeze-dried and immersed in bromocresol purple (BCP) pH indicator to visualize pH differences in forming enamel of KI and WT. To assess the structural differences in EMPs and enamel mineral phase, we conducted FTIR microspectroscopy in reflectance mode.

**Results:** BCP staining showed the secretory-stage enamel was more acidic in KI vs. WT. A distinct pattern of alternating low and high pH bands typical of maturation-stage was absent in KI enamel. Consistent with the higher initial mineral density in KI enamel (unpublished), the mineral to protein ratio was greater in KI secretory stage enamel. The ratio of acidic phosphate to phosphate was higher in KI enamel, consistent with mineralization in an acidic environment. Differences in Amide I band were also observed in secretory enamel, implying differences in the EMPs conformation in WT and KI. Together, our observations demonstrate that the lack of AMELX phosphorylation leads to acidification of secretory enamel and affects its mineral phase and EMP conformation. Supported by NIH Grant DE029211 (HCM & EB).

1. Kwak SY et al., J Biol Chem., 284:18972-9, 2009. PMID: 19443653; PMCID: PMC270724.
2. Wiedemann-Bidlack et al., J Struct Biol:173:250-60, 2011. PMID: 21074619; PMCID: PMC3293703.
3. Shin NY et al., J Biol Chem., 295:1943-1959, 2020. PMID: 31919099; PMCID: PMC7029122.
# FT-IR spectroscopic imaging of human aortic valve biomineralization and protein secondary structure combining hard and freeze cutting technique

Dittfeld C.¹*, Metzner P.¹*, Feilmeier M.¹, Jannasch A.¹, Matschke K.¹, Manthey S.², Rammelt S.², Tugtekin SM.¹*, Steiner G.³*

1 Department of Cardiac Surgery, Carl Gustav Carus Faculty of Medicine, Technische Universität Dresden, Heart Centre Dresden, Germany, 2 University Center of Orthopaedics, Trauma and Plastic Surgery, Carl Gustav Carus Faculty of Medicine, Technische Universität Dresden, Germany, 3 Department of Anesthesiology and Intensive Care Medicine and Clinical Sensoring and Monitoring, Carl Gustav Carus Faculty of Medicine, Technische Universität Dresden, Germany

* contributed equally, presenting author: P. Metzner

Calcified aortic valve disease (CAVD) is the most common valve disease. Its pathology is beside fibrosis characterized by aortic valve (AV) biomineralization. Dystrophic hydroxyapatite intercalation but also neoosteogenesis are research foci. FT-IR (fourier transformed infrared) spectroscopic imaging provides information on mineral composition and secondary protein structure. Thin sectioning of calcified AV tissue w/o prior decalcification for FT-IR spectroscopy is a challenge and offers insights in AV pathogenesis. Aim of the study is the spectroscopic molecular characterization of AV tissue pathomorphology.

After 5 µm sectioning, FT-IR spectra of explanted, formalin fixed and Technovit 9100 embedded AV tissues were acquired in transmission mode (FT-IR-spectrometer, Vertex 70; IR microscope, Hyperion 3000; Bruker-Optik GmbH). Fuzzy k-means cluster and principal component analysis (PCA) are used to evaluate the spectroscopic data (MATLAB, Mathwork. Inc.). Cusps tissue counterparts were fixed, decalcified and cryosectioned for FT-IR spectroscopy in parallel. Movat's Pentachrom and Masson's trichrome stain were used to visualize tissue pathomorphology.

Technovit penetration varies in strongly calcified AV tissues. Hydroxyapatite characteristics are evaluable in areas not affected by the plastic entry. Areas of the thin section not showing noticeable molecular interaction with the embedding medium were selected. Changes of protein secondary structure as well as variations in the spectral regions of phosphate and carbonate bands were analyzed and interrelated. Decalcified, cryosectioned tissue counterparts can further provide chemical information of protein secondary structure in FT-IR spectroscopy.

Molecular characteristics of AV biomineralization and protein secondary structure can be investigated by hard and freeze cutting techniques in a parallel workflow. Nevertheless, the establishment of alternative embedding strategies is envisioned.
# Dynamics of extracellular matrix remodeling associated with mineralization during the hypertrophic differentiation of articular chondrocytes

Ilhem Lilia Jaabar<sup>a,b</sup>, Brittany Foley<sup>a,c</sup>, Alberto Mezzetti<sup>a</sup>, Françoise Pillier<sup>d</sup>, Francis Berenbaum<sup>b,e</sup>,
Jessem Landoulsi<sup>a</sup>, Xavier Houard<sup>b</sup>

<sup>a</sup> Sorbonne Université, CNRS, Laboratoire de Réactivité de Surface, LRS, F-75005 Paris, France,
<sup>b</sup>Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine, CRSA, F-75012 Paris, France,
<sup>c</sup>Laboratoire de Biomécanique & Bioingénierie, CNRS, Université de Technologie de Compiègne,
BP20529, F-60205 Compiègne Cedex, France, <sup>d</sup>Sorbonne Université, CNRS, Laboratoire Interfaces et
Systèmes Electrochimiques, LISE, F-75012 Paris, France, <sup>e</sup>Rheumatology Department, AP-HP Saint-
Antoine Hospital, 184, rue du Faubourg Saint-Antoine, 75012, Paris, France

## Abstract

The mineralization of cartilage is a pivotal step in the endochondral ossification process, in which cartilage is replaced by bone. Such a process occurs during the growth of long bones and is reactivated in osteoarthritis. It depends on the hypertrophic differentiation of chondrocytes and is associated with a profound remodeling of the cartilage matrix.

Here, we studied the dynamics of matrix mineralization and modifications during the hypertrophic differentiation of articular chondrocytes thanks to an *in vitro* model of progressive differentiation of immature murine articular chondrocytes (iMACs) into prehypertrophic (Prehyp) and hypertrophic (Hyp) chondrocytes. Kinetics of Prehyp to Hyp differentiation were carried out to follow the mineralization of the matrix and its remodeling by immunofluorescence, biochemical and molecular approaches, as well as by physicochemical approaches, including atomic force microscopy (AFM), scanning electron microscopy associated with energy dispersive X-ray spectroscopy (SEM-EDX), attenuated total reflection infrared (ATR-IR) analyses and X-ray diffraction (XRD).

Results show the formation of a mineral phase 7 days after the induction of hypertrophy, which spreads within the matrices to form poorly crystalline carbonated hydroxyapatite. Hyp differentiation also induces a matrix turnover characterized by a rapid drop in type II collagen and aggrecan and the concomitant appearance of type X collagen. This is accompanied by an increase in the concentration and the enzymatic activity of MMP-13, the main collagenase of cartilage. Chemical mapping also shows that phosphorus is concentrated in cellular debris, which may originate partly from apoptosis. These findings provide new insights into the dynamics of matrix remodeling resulting from Hyp differentiation. They also provide relevant guidelines for the understanding of remodeling events that may occur at the pathological osteochondral junction (e.g. during osteoarthritis).

**Keywords:** chondrocyte, hypertrophic differentiation, mineralization, cartilage, collagen, aggrecan
Poster presentations

Poster 15

Chicken eggshell quality in the late phase of the laying cycle

Nicolas Guyot, Joël Gautron*, Magali Chessé, Justine Jimenez, Sandrine Mignon-Grasteau, Yves Nys

INRAE, Université de Tours, BOA, 37380 Nouzilly, France.

*presenting author: joel.gautron@inrae.fr

The laying hen uses large amounts of calcium each day to form the eggshell which is composed of 95% of calcium carbonate on calcitic forms and 3.5% organic matter that directly influence the biomineralization process. Shell mineralisation occurs at night when the animal has no access to feed. The calcium is then taken from the bone which is regenerated during the day. For reasons of sustainability, commercial laying hens are being reared later and later, but as they become older, calcium metabolism deteriorates, leading to shell strength defects and spontaneous bone fractures.

In this work, we investigated the effect of such extension of the laying period on the eggshell quality. Commercial laying hens were reared in cage-free system to 32-34 (peak of lay), 71-73 (current laying cycle) and 94-99 (extended laying cycle) weeks of age. Eggshell quality parameters (breaking strength, dry weight, thickness and toughness) were determined on eggs collected at each period of age. As expected, eggshell breaking strength and thickness significantly decreased during the three tested periods of the laying cycle. More surprisingly, eggshell weight is significantly lower at the oldest age (94-99 weeks). Eggshell toughness is decreased in both 71-73 and 94-99-week-old hens, when compared to 32-34-week-old hens.

Eggshell mechanical properties are due to the mass of materials and to the fabric of the shell. The latter comprises the morphology, size and orientation of crystals that define the shell texture and ultrastructure. As the hen ages, shell mass remains constant, while egg weight increases, resulting in a reduction in shell thickness. The decrease of eggshell mechanical quality parameters observed between 32-34 and 71-73 weeks is likely due to this decrease of shell thickness combined with modifications of the shell fabric and eggshell texture as observed by the decrease in shell toughness. In older hens (94-99 weeks of age), shell mass is reduced. This effect on shell mass was not reported earlier likely due to the absence of measurements at this advanced age. This decrease in mass, combined with changes in the ultrastructure of the calcite crystals making up the shell, leads to a further reduction in the shell biomechanical properties.

Structural and molecular studies must be performed to further understand the mechanisms leading to eggshell degradation at 94-99 weeks and to identify potential levers to lower negative impacts on eggshell and bone quality in extended laying periods.
*In vitro* simulation of the oral consumption of MOP powder and screening of the mineralization capacities of digestion products

Yacine Bertache-Djenadi¹, Kim Nguyen¹, Arnaud Vanden-Bossche¹, Mireille, Thomas¹, Stéphanie Mundweiler¹, Marie-Thérèse Linossier¹, Sylvie Peyroche, ¹, Delphine Farlay², Hubert Marotte¹, Laurence Vico¹, Nicolas Rochereau³, Marthe Rousseau¹,⁴

1. Université Jean Monnet Saint Étienne, Mines Saint Étienne, INSERM, SAINBIOSE U1059, F-42023, Saint Étienne, France, 2. LYOS, INSERM U1033, UCBL1, UdL, Lyon, France, 3. CIRI, GIMAP, UdL, UCBL1, INSERM U1111, CNRS (UMR 530), CIC1408, Saint Etienne, France, 4. UDL, INSA Lyon, CNRS, MATEIS (UMR 5510), Villeurbanne, France

Mother-of-pearl (MOP), the internal layer of mollusks shell, was firstly investigated as a solid and biocompatible bone graft material that led to discover its effect on bone cells. It finally shown in vitro effects on both sides of bone remodeling by repressing bone resorption and stimulating bone formation. That led scientists to investigate it in bone disease characterized by unbalanced remodeling with increased resorption and decreased bone synthesis such as osteoporosis. Indeed, ingested MOP powder or extracts in osteoporotic mice, rat or postmenopausal women showed a limitation of bone loss. However, action mechanism after oral consumption remains unknown. Here, we investigate whether the bone effects of orally ingested MOP could be attributed to previously demonstrated in vitro abilities. MOP dissolution in gastric condition was performed and released organic compounds were extracted and digested according to INFOGEST 2.0's Nature Protocol standardized static digestion protocol. This protocol involves three steps designed to mimic human natural digestion: an oral phase, a gastric phase (pH 3, 37°C, 2h) and an intestinal phase (pH 7, 37°C, 2h). After MOP digestion, products were submitted to absorption assessment on 21 days old intestinal epithelium made of differentiated Caco-2 cells. Undigested, digested and absorbed MOP fractions were then assessed for their pro-mineralization abilities on MC3T3 and primary murine osteoblast. In conclusion some MOP compounds released during gastric dissolution showed a positive effect on mineralization in vitro both before and after their digestion. Absorption assessment didn't allow us to conclude that orally ingested MOP effect on bone loss could be related to a direct effect on bones cells. A fraction of MOP remains undigestible and could be used as a substrate that may influence intestinal microbiota.

**Keywords:** MOP; digestion; bioactive compounds; bone loss
A novel nacreous bioinspired peptide stimulates endochondral
differentiation

Sarah Nahle¹, Ganggang Zhang², Capucine Jourdain De Muizon³, Mireille Thomas¹, Ali Al-Mourabit³, Marthe Rousseau¹,⁴

¹Université Jean Monnet Saint-Étienne, INSERM, Mines Saint Etienne, SAINBIOSE U1059, F-42023, Saint-Étienne, France, ²Department of Orthopedics, The First Affiliated Hospital of Zhengzhou University, China. ³CNRS, Institut de Chimie des Substances Naturelles, Université Paris-Saclay, F-91190 Gif-sur-Yvette, France, ⁴UMR5510 MATEIS, CNRS/Lyon University/INSA-Lyon, Lyon, France

Nacre, also known as mother of pearl, is produced by some molluscs as an inner shell layer. Although nacre is known for its ability to stimulate endochondral differentiation and bone formation, its whole osteogenic composition is still mysterious. Several nacreous proteins have been identified and proven to have an osteogenic power. To our knowledge, there have been no studies exploring the effect of nacre peptides on endochondral differentiation. In this study we aimed to identify nacre peptides that are able to stimulate this biological process. The organic matrix of *Pinctada margaritifera* shell powder has been purified by the cationic approach that we developed previously, and its composition has been analysed. Using tandem mass spectrometry (LC-MS/MS), we found an interesting peptide that has been further produced and several analogs synthesized. The amino acids that may play a role in endochondral differentiation were then highlighted by a new screening tool of a stable cell line expressing an osteogenic reporter gene (ATDC5 pMetLuc2 ColX), that we created. The peptides were tested on a micromass model of ATDC5 cells to investigate their capacity to stimulate endochondral differentiation and mineralization of the extracellular matrix. In conclusion, the present study led to a discovery of a new nacre and synthetic peptides active in endochondral differentiation. Such a discovery can be potentially useful in developing drugs from nacre to improve bone repair.

**Keywords:** nacre, bone, screening, endochondral differentiation
Keynote lecture

Invited Review: A darker side to X-ray brightness - assessing calcium mediated structural degradation during bone studies

Paul Zaslansky

Charité - Universitätsmedizin Berlin, Germany

X-rays are widely used for imaging of bones and other biomineralized tissues. Due to their high penetration power, they traverse rather thick structures yielding important diffraction or tomographic data. The high density of calcium in mineralized collagen and other biogenic matrices almost begs analysis by X-ray radiation methods, where higher resolution and structural sensitivity call for ever increasing high-power and high-energy radiation sources such as synchrotrons. Yet when mineralized samples are exposed, a significant amount of the incoming flux is absorbed by the mineral. Though essential for scattering and image formation, absorption leads to energy deposition in the material.

Absorbed photons induce photon-electron excitations. Synchrotron-based X-ray diffraction and micro-computed tomography methods thus lead to in situ degradation of the collagen fibrils due to primary radiation damage. Following just 40 s of moderate exposure, we observe protein disintegration through dimming of second harmonic generated emission from within the collagen fibers. Diffraction measurements with increasing exposures reveal residual strain relaxation in dry, pre-stressed, apatite nanocrystals, due to expanding c-lattice parameters as collagen is disrupted. We find that ionization of calcium and phosphorous within the nanocrystals yields fluorescence and high-energy scatter electrons that emerge from the mineral to spread multiple micrometers. Ionization then causes structural damage in the organic fibers. Scattered electrons spread beyond regions directly within the field of view of the incident radiation beam. Our observations indicate that damage to bone collagen increases rapidly from the onset of irradiation, suggesting that there is no minimal 'safe' dose that bone can sustain. The talk will present our current understanding of the impact of X-ray methods on the generation and spread of primary radiation damage in bony materials.

**Reference:**

Sauer, K., Zizak, I., Forien, JB. *et al*. Primary radiation damage in bone evolves via collagen destruction by photoelectrons and secondary emission self-absorption. *Nat Commun* **13**, 7829 (2022).
[https://doi.org/10.1038/s41467-022-34247-z](https://doi.org/10.1038/s41467-022-34247-z)

**Keywords:** X-ray, Radiation damage, Collagen backbone destruction
**Dynamic interpretation of the bone mineralization process in the chick embryo based on Cryo FIB-SEM images.**

Emeline Raguin, Richard Weinkamer, Peter Fratzl

*Max Planck Institute of Colloids and Interfaces, Germany*

Cryo focused ion beam with scanning electron microscopy (cryo FIB-SEM) is a powerful tool to image volumes of hydrated frozen specimens at high resolution. Of particular interest, this method enables the visualization of mineral precursors, allowing to shed insights into the bone mineralization process. Here, we focus on the three-dimensional visualization and quantification of the mineral precursors in the femur of the fast-growing chick embryo. Based on volume imaged at different hierarchical with cryo FIB-SEM covering the smallest length scale, we also aimed at providing an interpretation of the mineralization logistics in a dynamic manner, where the speed of transport of the mineral precursors to the site of mineralization is estimated.

In this approach, we used micro-computed tomography (micro-CT) to calculate the osteocytic lacunar density at embryonic day 13 and thus, estimate the volume of bone to be mineralized per cell in one day. 3D volumes were acquired using cryo FIB-SEM to observe the mineral precursors, the cells as well as the mineralized matrix in their close to native states. The results show that numerous vesicles containing mineral precursors are present intracellularly, both in osteocytes and in osteoblasts/pre-osteocytes. The average volume of a vesicle was 0.65 µm3 with a volume of mineral precursor per vesicle of approximately 0.058 µm3. From the micro-CT data, we calculated that a cell is responsible for the mineralization of a volume of about 5100 µm3. Based on the quantification of these 3D snapshots, we estimated that a cell needs to release one vesicle every second to mineralize the required bone volume in one day. Consequently, these vesicles travel at a mean velocity of 0.27 µm/s within the cell.
This dynamic interpretation of the logistics of bone mineralization demonstrates that mineral precursors have to be actively transported within cells before there are shed into the extracellular space, where they move passively via diffusion.

**Keywords:** cryoFIB-SEM, bone, mineral precursors, vesicles
# Towards *in vivo* imaging of the physico-chemistry processes involved in the biomineralization of mollusc shells

Virginie Chamard¹, Tilman A. Grünewald¹, Hamadou Dicko¹, Jeremie Vidal-Dupiol², Bruno Petton³, Jacqueline Legrand³, Andrea Campos⁴, Eric Tambutté⁵, Alex Venn⁵, Sylvie Tambutté⁵, Jérémie Le Luyer⁶, Michael Sztucki⁷, Manfred Burghammer⁷ and Julien Duboisset¹

¹Aix-Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, Marseille, France, ²IHPE, Univ Montpellier, CNRS, IFREMER, Univ Perpignan Via Domitia, Montpellier, France, ³Université de Brest, Ifremer, CNRS, IRD, LEMAR, Plouzané, France, ⁴Aix Marseille Univ, CNRS, Centrale Marseille, FSCM (FR1739), CP2M, 13397 Marseille, France. ⁵Marine Biology Department, Centre Scientifique de Monaco, 98000 Monaco, Principality of Monaco, ⁶Ifremer, UMR 241 Environnement Insulaire Océanien (EIO), Labex Corail, Centre du Pacifique, BP 49, Vairao 98719, French Polynesia, ⁷European Synchrotron Radiation Facility, F-38043 Grenoble Cedex, France

Calcium carbonate biomineralization is widespread across the animal phyla, in particular in the marine environment. It integrates complex physical and chemical processes bio-controlled by the living organisms through ion regulation and organic molecule production. Hard tissues, produced during favourable-condition crystallisation, present a variety of structural, optical and mechanical properties, making the understanding of the exact biomineralization pathways highly desirable. For many species, it has been shown, mostly from post-mortem analysis and biomimetic models, that biomineralization starts with forming an amorphous precursor, which subsequently transforms into a crystal [1]. However, the nature of the transient physico-chemical states and the dynamics of the transformation are still discussed. Direct evidence could be brought by *in vivo* approaches, under the assumption that spatial, temporal and quantitative information is accessible.

Here we present recent developments towards *in vivo* study of calcareous biomineralization:

- To monitor the different chemical states (e.g., amorphous or crystallized carbonates), a highly sensitive coherent Raman microscopy approach is developed. It allows mapping molecular bond concentrations with spatial resolution in the order of 300 nm and demonstrates suitability for the *in vivo* imaging of a shell growing edge (*Pinctada margaritifera*) [2].

- To address the crystallization process, we make use of a novel 4th generation synchrotron source, which allows nanoscale spatially resolved and crystalline-sensitive study of a biomineralizing animal (*Crassostrea gigas* shell). Our results point towards an evolution of the freshly produced crystallites, including growth, coalescence and strain release. Remarkably, the crystallisation occurs without the presence of the mantle, showing that the elements needed for the production of calcite biomineral are already in place in the amorphous precursor [3]. It again highlights the relevance of biomineralization for the design of bio-inspired materials.

[1] J. Duboisset et al., Acta Biomaterialia 2022.

[2] H. Dicko et al., Journal of Structural Biology **214** (2022) 107909.

[3] T. Grünewald et al., in preparation.

**Keywords:** in vivo, microscopy, physico-chemistry, biomineralization, x-rays, Raman, mollusk
# Unique features of Caudofoveata (Mollusca) spicules revealed by combined light and electron microscopy techniques

Camila Wendt<fnref n="1" />, André L. Rossi², Jefferson Cypriano<fnref n="3" />, Cleo Dilnei de Castro Oliveira⁴, Corinne Arrouvel⁵, Jacques Werckmann² and Marcos Farina<fnref n="1" />*

The Caudofoveata is a class of marine worm-like mollusks that live, in their great majority, embedded in the muddy bottom of marine environments. The Caudofoveata tegmentum is composed of a chitinous cuticle [1] and calcareous micrometer sized spicules [2] which are formed extracellularly within a deep invagination of a single cell, where calcium carbonate is secreted on a cup-like organic template. Morphology and thickness of the Caudofoveata spicules have been considered species-specific [3].

In this work, we investigated the calcium carbonate spicules of Caudofoveata (Mollusca), by several methods of microcopy in different length scales, in a correlative way. By analyzing Electron Diffraction patterns of lamellae obtained by Focused Ion Beam (FIB) from spicules, we concluded that the whole structure is composed of a single crystal of aragonite. However, when analyzing SEM images of NaOH or acid treated spicules, it becomes clear that the bulk of these biominerals is formed by a set of alternating plate-like components, with different textures, most probably crystalline, oriented in parallel. When imaging the dorsal surface of spicules by Atomic Force Microscopy (AFM), it was observed a wavy appearance, composed of myriads of tiny, pointed crystallites oriented along the longer axis of the spicules which coincides with the *c*-axis of the aragonite. Besides, AFM showed that the crystallites presented in some areas a twinned appearance. The correlation among the substructures observed after etching (revealed by SEM), the apparent organization of the tiny crystallites as twinned ones (revealed by AFM), and the single crystalline appearance of the whole spicule (revealed by electron diffraction of FIB prepared lamellae), is still an open question and gives strength for further analyses using Caudofoveata spicules as a model for biomineralization studies, including a molluscan phylogeny in the light of still unexplored characters and their organization patterns.

## References:

[1] Beedham, G.E., Trueman, E.R. *J. Zool.* 1968, **154**, 443-451.

[2] Corrêa, P.V.F. Miranda, M.S.; Passos, F.D. *Deep. Res. Part I Oceanogr. Res. Pap.* 2018, **132**, 16-28.

[3] Scheltema, A.H., Schander, C., 2000. *Bioll. Bull. Woods Hole* **198**, 121-151.

**Keywords:** Aragonite spicules; Nanostructure; Caudofoveata; Biomineralization; FIB-SEM

<footnote n="1">¹ Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brasil. ² Centro Brasileiro de Pesquisas Físicas, Xavier Sigaud 150, Rio de Janeiro, Brasil.</footnote>

<footnote n="3">³ Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brasil. ⁴ Instituto de Biologia, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brasil. ⁵ Departamento de Física, Química e Matemática, Centro de Ciências e Tecnologia para a Sustentabilidade, Universidade Federal de São Carlos, Brasil.</footnote>

<footnote>* Correspondence: marcos.farina.souza@gmail.com</footnote>
Uncovering the complex world of elastic deformations in biological
ceramics

Shahrouz Amini¹, Tingting Zhu¹, Erika Griesshaber2, W. W. Schmahl2, Peter Werner¹, Peter Fratzl¹

¹Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, 14476, Potsdam, Germany, ²Ludwig Maximilians University of Munich, Munich 80333, Germany

Biological ceramics are complex materials. Understanding their mechanical behavior requires methods
allowing quantitative imaging of deformations under contact loads - in operando - rather than providing
a snapshot of induced damages. Here, we introduce an imaging method by which mechanical
deformations under contact loads can be imaged in 3D and with sub-micron resolution. By taking
examples of biological ceramics and their geological counterparts, we then unravel how the
crystallographic organization of grains and crystals and organic inclusions influence the formation and
evolution of elastic-inelastic strains. Our technique can pave an important step toward a quantitative
understanding of the formation and development of elastic deformations and their interplay with the
anisotropy and heterogeneity of materials.

**Keywords:** Micromechanics, Elastic strain, Calcitic shells
Study of the role of trace elements on kidney stones pathogenesis with
multi-scale and multimodal scanning X-ray tomography

Ruiqiao Guo<sup>1,2*</sup>, Andrea Somogyi<sup>1</sup>, Dominique Bazin<sup>3</sup>, Emmanuel Letavernier<sup>4,5</sup>, Ellie Tang<sup>4</sup>,
Kadda Medjoubi<sup>1</sup>

<sup>1</sup>Synchrotron SOLEIL, Gif-sur-Yvette, France, <sup>2</sup>Université Paris-Saclay, Gif-sur-Yvette, France,
<sup>3</sup>Institut de Chimie Physique, Université Paris-Saclay, CNRS, Orsay, France, <sup>4</sup>UMR S 1155, Sorbonne
Université, Paris, France, <sup>5</sup>Physiology Unit, Hôpital Tenon, AP-HP, Paris, France

*ruiqiao.guo@universite-paris-saclay.fr*

Urolithiasis is a product of a pathological biomineralization process affecting ~10% of the population. A significant proportion of these stones develops on Randall's plaque (RP), which is a mineral deposit at the tip of renal papillae. However little is known about the processes involved in RP development and especially about those which trigger or influence early-stage pathological calcification. Some results published in the literature have revealed that certain trace metals, e.g., zinc, are significantly enriched in the carbapatite of RP[1]. However, up to our knowledge, the role of these trace metals in the early stage of biomineralization of Randall Plaques has not been addressed yet, partially because this necessitates non-destructive, high analytical sensitivity studies at nanometer resolution.

In this work, thanks to the new developments in multi-length scale and multimodal scanning imaging and tomography at the Nanoscopium beamline (SOLEIL Synchrotron) [2], we studied the complex 3D elemental, crystalline, and morphological structure of Randal papilla and that of micron-sized calcification spheres in RP, an example for incipient calcification. The obtained results will bring nephrologists a step further to understand and prevent pathological calcification processes

**Keywords:** Kidney stones, Randall's plaque, Trace elements, X-ray imaging, Pathological calcifications

**References**

[1] X. Carpentier et al., "High Zn content of Randall's plaque: A μ-X-ray fluorescence investigation," *J. Trace Elem. Med. Biol.*, vol. 25, no. 3, pp. 160-165, Jul. 2011, doi: 10.1016/j.jtemb.2011.05.004.

[2] R. Guo et al., "Towards routine 3D characterization of intact mesoscale samples by multi-scale and multimodal scanning X-ray tomography," *Sci. Rep.*, vol. 12, no. 1, p. 16924, Oct. 2022, doi: 10.1038/s41598-022-21368-0.
# Quantitative evaluation of enamel prism arrangement based on image processing technique

Máté Hegedűsª, Viktória K. Kisᵇ.c, Zsolt Kovácsª

ªDepartment of Materials Physics, Eötvös Loránd University, H-1119 Budapest, Pázmány Péter sétány 1/a, Hungary, ᵇCentre for Energy Research, H-1121 Budapest, Konkoly-Thege Miklós u. 29-33, Hungary, ᶜDepartment of Mineralogy, Eötvös Loránd University, H-1119 Budapest, Pázmány Péter sétány 1/c, Hungary

Tooth enamel is the hardest and most mineralized tissue of vertebrates. The complex microstructure of enamel contains needle-shaped hydroxyapatite (HAP) nanocrystals arranged on several structural level to form the most characteristic unit of mammalian dental enamel, the elongated enamel prism. Although, the c-axis of HAP correlates with the prism direction, prism orientation can not be assigned to any specific crystal orientation This makes quantitative analysis of the prism structure difficult based on diffraction or polarisation based methods.

In present work, we introduce an image processing method based on the analysis of scanning electron microscope (SEM) images of different primary dental enamel sections. The method separates prisms using the systematically changing electron contrast at the prisms' surfaces and approximates different prism sections with ellipses. Output of the method is a set of quantitative parameter, which is able to describe and visualize the local prism orientation for various prism arrangements.

Specific prism orientation maps obtained on distal-mesial cross-sections show the size and growth pathways of enamel producing ameloblast. Additionally, prism orientation in Hunter-Schreger bands, which are mostly identified as periodic dark and light bands under light microscope, was analysed quantitatively and was proved to exhibit nearly identical prism orientations by the prisms arranged into groups. Based on the prism orientation map we determined the orientation dependency of mechanical properties of human dental enamel.

<figure><img src="image_10.png" /></figure>

Hegedűs, Máté; Kis, Viktoria; Szabó, Ábel; Kovács, Ivett; Rózsa, Noémi; Kovács, Zsolt, Gradient Structural Anisotropy of Dental Enamel is Optimized for Enhanced Mechanical Behaviour (submitted)

Hegedűs, Máté; Kis, Viktoria; Kovács, Zsolt, Quantitative evaluation of enamel prism arrangement based on image processing technique (in prep.)
Poster presentations

Poster18

Structural analysis of sub- and supragingival mineralized oral biofilms
using high-resolution imaging techniques

Abdul R. Hamsho<sup>1,2</sup>, Laura Zorzetto<sup>2</sup>, Emeline Raguin<sup>2</sup>, Ernesto Scoppola<sup>2</sup>, Sebastian Paris<sup>1</sup>, Cécile M. Bidan<sup>2</sup>

<sup>1</sup> Centrum für Zahn-, Mund- und Kieferheilkunde Charité - Universitätsmedizin Berlin, Aßmannshauser Str. 4-6 - 14197 Berlin, Germany, <sup>2</sup> Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Am Mühlenberg 1 - 14476 Potsdam, Germany

Biofilms are living materials that appear when bacteria synthesize and assemble extracellular matrix components after colonizing a surface. They constitute a key feature of bacteria survival in biologically, chemically and/or physically challenging environments. In the mouth, the oral microbial community builds biofilms called dental plaque. Oral biofilms are associated with some of the most common dental diseases, such as caries and periodontitis [1]. Dental plaque mineralizes and forms dental calculus, which is generally classified into two categories according to the location. Supragingival calculus is located above the gingival margin. On the other hand, subgingival calculus is located below the gingival margin in the gingival sulcus or periodontal pocket [2].

In this work, we aim to understand the differences in microstructure, mineralization and composition of the two types of dental calculus. We characterized native human teeth ethically harvested from consenting donors. We used X-ray microtomography to investigate the overall morphology of the calculus at the mesoscale. To further characterize the mineralized biofilm, we used Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM). Eventually, using small- and wide-angle X-ray scattering (SAXS and WAXS), we compared the crystalline composition of the two types of calculus. Results obtained by segmenting the microtomography of entire teeth, showed that supragingival calculus is generally porous, whereas subgingival calculus is tightly packed and presents a layered structure. Analysis of the crystalline composition in the different calculi, revealed a variety of different mineral phases present in the two different calculi. The present results may help to better understand the formation of dental calculus and to design new preventive strategies for oral diseases.

References:

[1] Dental calculus: the calcified biofilm and its role in disease development. PERIODONTOLOGY 2000. Akcali A, Lang N P. Vol. 76, Singapore: John Wiley & Sons Ltd, 2018, pp. 109-115.

[2] Subgingival calculus: where are we now? A comparative review. E.A. Roberts-Harry, V. Clerehugh / Journal of Dentistry 28 (2000) 93–102. Elsevier Science Ltd. All rights reserved. PII: S0300-5712(99)00056-1

**Keywords:** mineralized oral biofilm; Subgingival calculus; Supragingival calculus
# Functional membrane interactions regulate silica formation in diatoms

Lior Aram1, Diede de Haan1, Neta Varsano2, Assaf Gal1

1Department of Plant and Environmental Sciences, Weizmann Institute of Science, Israel,
2Department of Chemical Research Support, Weizmann Institute of Science, Israel

The silica cell wall formation in diatoms, a widespread group of unicellular microalgae, is a spectacular example of biological control over mineral formation. Diatom silicification occurs intracellularly in a membrane-bound organelle responsible for silica precipitation and morphogenesis. Despite many years of research, the inorganic-organic interactions that drive silica formation inside this organelle are unclear. This is due mainly to the limitations of traditional TEM techniques in elucidating the structural motifs of this organelle. Here we collected cryo-electron tomography datasets of cryo-FIB milled lamellae from diatom cells at various stages during cell wall formation. We visualize the mineral formation process in-situ with nanometer-scale resolution and reconstruct a timeline of mineral formation inside the cell. Our observations show that the silicification occurs in a highly confined lumen, bordered by the organelle membranes, which are in very close proximity to the plasma membrane. The plasma and organelle membranes interact via membrane contact sites, possibly facilitating continuous transport of lipids from the plasma membrane to the growing organelle, and building blocks for mineralization. These membrane-membrane interactions are manifested in the curvature of the distal organelle membrane, which molds the silica. Our findings reveal a new mechanism that regulates silica growth and shaping through membrane crosstalk.

**Keywords:** Diatom, Silica, cryoEM, Tomography
# Use of fluorescent marker of mineralization dynamics : Application to lactating mice.

Arnaud Vanden-Bossche¹, Emmanuel Play², Ghislaine Roux⁴, Alain Guignandon², Luc Malaval¹, Hubert Marotte²,³, Laurence Vico¹.

¹INSERM, Université Jean Monnet Saint-Étienne, Mines Saint-Étienne, SAINBIOSE U1059, F-42023, Saint-Étienne, France ; ²Université Jean Monnet Saint-Étienne, Mines Saint-Étienne, INSERM, SAINBIOSE U1059, F-42023, Saint-Étienne, France ; ³Université Jean Monnet Saint-Étienne, CHU Saint-Étienne ; ⁴Université Jean Monnet, PLEXAN, F-42023 Saint-Étienne.

Osteoblasts are responsible for bone formation by producing the collagen matrix which gradually mineralizes. In this process, some osteoblasts become enclosed in the matrix and become osteocytes, with their lacunae interconnected by dendrites, forming an important lacuno-canalicular network (LCN).

It has been shown in different models such as PTH injection or lactation (Bonewald et al., 2017) that osteocytes are able to modify the bone matrix in their vicinity through peri-lacunar/canalicular remodeling (PLR). The process has been documented in breastfeeding rodents (Bonewald, 2012, 2020) using bone markers previously validated to label the mineralization fronts (calcein, tetracycline and alizarin) which hardly diffuse throughout the osteocyte network.

Peri-lacunar fixation of these markers, proof of osteocyte remodeling is limited to the first line of young osteocytes and is not able to diffuse in depth to older osteocytes. This method cannot show the existence of this process throughout the osteocyte network at distance from the mineralization front. It would thus be useful to have a label integrated into the young bone matrix.

Our objective was to evaluate the existence of PLR in the LNC of lactating mice, thanks to the use of a temporal multi-labeling with alizarin and a fluorescent molecule circulating throughout the osteocyte network without specific binding to calcium or minerals and spreading into the osteoid tissue and the bone matrix all the more easily as it is little mineralized. During clearance by the kidneys and the digestive tract, only the young bone matrix where mineralization is active remains stained.

During the 10 days following 20 days of breastfeeding, we documented a resumption of bone formation marked by the fluorochrome, at the periosteum and in the entire endosteal surface of the femur cortex, showing a modification of the dynamics of the bone drift compared to non-lactating control mice.

However, we did not observe active mineralization around the lacunae of osteocytes formed before lactation suggesting that lactation/weaning is not associated with in depth PLR.

In conclusion, the fluorochrome used could be useful to study the dynamics of matrix mineralization because classical calcein/alizarin molecules are not able to diffuse through the LCN and the matrix of mineralizing bone tissue.

**Keywords:** Breastfeeding, osteocyte network, dynamic mineralization
# Light Channeling and Damage Localization in *Pinna nobilis* Calcitic Prismatic Layer

Tingting Zhu, Peter Werner, Yannicke Dauphin, Peter Fratzl, Shahrouz Amini

¹Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, 14476 Potsdam, Germany, ²UMR 7205 ISYEB, Museum National d'histoire Naturelle, CNRS UPMC EPHE, 57 rue Cuvier, 75005 Paris, France

*Pinna nobilis* is a marine bivalve mollusk and its shell is composed of outer single-crystal-like calcitic prisms and inner aragonitic nacre. The latter is known for its contribution to the outstanding mechanical property of the shell; hence, its absence at the upper part of the shell, composed solely from the prismatic layer, raises the question of whether the upper part of the shell can serve any functional purposes while sacrificing its mechanical performance. Here, we reveal that the prismatic layer of *Pinna nobilis* exhibits remarkable optical properties, wherein each prism acts as an individual optical fiber that guides light to the inner shell cavity through total internal reflection. This “fiber optic” of prisms enhance spatial resolution and contrast, allowing more precise tracking of moving objects in the surrounding of the shell. We then use our devised “in operando 3D strain mapping” method and show how the architectural organization of the prisms can promote damage localization. Our discovery provides insight into the evolutionary perspective of light sensing in biological materials and offers a conceptual framework for the development of bio-inspired multifunctional ceramics and architecture light-tracking materials.

**Keywords:** *Pinna nobilis*, light tracking, damage localization, in operando 3D strain mapping
Keynote lecture

Invited review: Dynamics of the chicken chorioallantoic membrane and the eggshell during chicken embryonic development: a fine regulation between eggshell decalcification and maintenance of egg defences

Sophie REHAULT-GODBERT

INRAE, University of Tours, BOA, Nouzilly, France

The avian eggshell is a highly ordered mineralized structure that isolates and protects the embryo from environmental fluctuations during its development. This calcitic eggshell, which also contains 3% protein including antimicrobial proteins and peptides, constitutes the first level of egg defence, acting as a physical and molecular barrier against environmental changes, pathogenic microorganisms or dehydration. However, during the second half of the chicken development, the avian embryo uses the minerals of the eggshell, mainly calcium, for the mineralization of its skeleton. The thinning and weakening of the eggshell resulting from the demineralization of its inner surface facilitates chick emergence at the end of incubation but is likely to increase the susceptibility of the embryo to microbial contaminations. The hypothesis is that the antimicrobial proteins occluded in the mineral phase of the eggshell may be released together with the calcium during eggshell solubilisation and may form a local protective proteinaceous film onto the inner surface of the eggshell. Indeed, some authors have pointed out the potential dual role of certain eggshell matrix proteins in both mineralization (during eggshell formation in the hen uterus) and antimicrobial protection (during embryo development).

Decalcification of the eggshell is mediated by the chorioallantoic membrane (CAM), a highly vascularized extra-embryonic membrane that develops on the inner surface of the eggshell from the fifth day of incubation, onwards. It covers the entire inner surface of the eggshell by the eleventh day of incubation. Besides its major role in calcium metabolism and mineral transport, the CAM is likely to play a major role in innate immunity. Indeed, its strategic position (in contact with the inner surface of the eggshell) and its well-developed vascularization allow the local recruitment of immune cells in case of bacterial penetration through a defective eggshell. The CAM also expresses antimicrobial molecules as well as other components of innate immunity (cytokines).

Using the chicken embryo as a model of mineralized structure, this review will shed light on the mechanisms that allowed calcitic eggs to adapt to terrestrial environments, and the fine regulation between the mineralized structures of the egg and the extra-embryonic cellular structures to maintain protection of the embryo throughout incubation.