ICCBMT Abstracts

ICCBMT 14 Hotel De Bilderberg Oosterbeek - The Netherlands International Conferences on the Chemistry and Biology of Mineralized Tissues ICCBMT 14 OOSTERBEEK - THE NETHERLANDS For more information www.iccbmt.org OCTOBER 22-27, 2023 ICCBMT 14 - ABSTRACTS

OCTOBER 2023 ICCBMT 14 M1 - Role of amelogenin phosphorylation in enamel mineral formation during secretory amelogenesis Claire M Gabe1,2, Ai Thu Bui1,2, Lyudmila Lukashova3, Kostas Verdelis2,3, Elia Beniash1,2, Henry C Margolis*1,2,4. 1Dept of Oral and Craniofacial Sciences, University of Pittsburgh School of Dental Medicine (UPSDM), Pittsburgh, PA, USA, 2Center for Craniofacial Regeneration, UPSDM, Pittsburgh, PA, USA, 3Dept of Endodontics, UPSDM, Pittsburgh, PA, USA, 4Dept of Periodontics and Preventive Dentistry, UPSDM, Pittsburgh, PA, USA. INTRODUCTION: We have previously shown that the single phosphorylation site at serine 16 (S16) of amelogenin (AMELX), the predominant extracellular matrix protein in forming enamel, stabilizes amorphous calcium phosphate (ACP) and inhibits apatitic crystal formation in vitro. We have also shown that AMELX phosphorylation plays an essential role in amelogenesis, using a knock-in (KI) mouse model with a substitution of S16 with alanine (AMELXS16A) that prevents AMELX phosphorylation. KI mice, compared to WT, exhibit defective enamel formation, including, a greater rate of ACP to apatite transformation, thin enamel, ectopic calcifications and loss of the enamel rod structure, the key structural component of mammalian enamel. KI ameloblasts also lack Tomes’ processes and develop progressive pathology beginning in early- to mid-secretory stages. PURPOSE: To test the hypothesis that abnormal KI enamel results from the reduced capacity of non-phosphorylated AMELX to slow the initial rate of enamel mineralization, which may lead to local acidification. METHODS: Changes in mandibular incisor enamel mineral density were assessed in 2-8 week old (w/o) KI and WT male and female mice from the onset of enamel formation through maturation by μCT, using a Scanco μCT50 at 2-μm voxel resolution. Changes in enamel pH and composition of freeze-dried incisors were further assessed using pH dyes and FTIR microspectroscopy, respectively. RESULTS: At the onset of enamel formation (first 100 μm) in 8 w/o mice, KI enamel mineral density was significantly greater (by a factor of two) than that of WT mice (n=4-6 / sex & genotype; p <0.00001). No significant differences were found between male and female mice of each genotype. Increases in KI enamel mineral density began to level off at ~mid-maturation, reaching a density of ~1200 mg hydroxyapatite/cm3 at 4.5 mm that was significantly lower than WT enamel, which grew at a significantly greater rate than KI enamel to ~1800 mg hydroxyapatite/cm3. Similarly, at the onset of enamel formation, KI enamel density in 2 and 4 w/o male mice (n=3-5), which exhibit longer secretory stages than 8 w/o mice, was greater than in the WT. The onset of enamel formation from the beginning of dentin in KI male mice was also found to be significantly (p<0.01) delayed by up to 370 μm in 8, 4 and 2 w/o mice, compared to WT mice. Notably, enhanced enamel mineral densification in 8 w/o KI mice was found to coincide with changes in enamel composition. Analyses of freeze-dried incisors (n=3 / genotype) revealed that secretory KI enamel was more acidic than WT enamel. FTIR findings also showed that KI enamel has a higher mineral / protein ratio and a greater acid phosphate (HPO42-) content than the WT. CONCLUSION: Amelogenin phosphorylation slows the rate of secretory enamel mineralization that averts enamel acidification, which could lead to a disruption in ameloblast function and loss of enamel structure. Supported by NIH grant DE029211 (HCM & EB). M2 - Interrogating the multitargeting domain in ameloblastin; implications for its multifunctionality in amelogenesis Natalie C Kegulian#, Gayathri Visakan#, Janet Moradian-Oldak*. Center for Craniofacial Molecular Biology, Dept of Biomedical Sciences, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA. # The authors contributed equally. INTRODUCTION: The extracellular enamel matrix protein Ameloblastin (Ambn) is multifunctional with critical roles in regulation of mineral formation, cell differentiation, cell polarization, and cell–matrix adhesion. We identified a highly conserved amphipathic helix (AH) cell binding domain located adjacent to the C-terminus of Ambn self-assembly and co-assembly Y/F-x-x-Y/L/F-x-Y/F domain (J Dent Res, Aug;99(9):1072-1081, Front Physiol 11, 622086). A recent comprehensive Ambn sequence analysis of 53 species demonstrated that the AH motif is closely related to the emergence of enamel prismatic structure (Mol Biol Evol. 2022 Nov 3;39,11). PURPOSE: To analyze structural changes in Ambn interacting domains during interactions with itself (self-assembly), with amelogenin (Amel), and with cell membrane, to characterize the competitiveness of such interactions, and to examine the function of these interactions in cell polarization and morphology. METHODS: We applied biophysical and spectroscopy techniques, and used recombinant ameloblastin proteins and derived p

OCTOBER 2023 ICCBMT 14 M3 - Effect of polyproline repeat length on enamel crystal formation: of frogs and bulls Mirali Pandya, Gokul Gopinathan, Xianghong Luan, Thomas GH Diekwisch*. Dept of Oral and Craniofacial Sciences, University of Rochester, Rochester, NY. INTRODUCTION: Mammalian tooth enamel is an exquisite bioceramic distinguished by a thick layer of prismatic apatite crystals that does not occur in other vertebrate clades nor in invertebrates. Developing mammalian enamel is also characterized by an abundance of the proline-rich enamel-related protein amelogenin, suggesting that aspects of amelogenin sequence or composition might contribute to enamel apatite crystal and prism growth in mammals. PURPOSE: To determine the effect of polyproline repeat length on mammalian enamel structure. METHODS: In a first set of studies we compared key sequence motifs between amphibian, reptilian and mammalian amelogenins. We then determined the effect of polyproline repeat peptides on self-assembly and apatite versus carbonate crystal growth. In a third and fourth set of studies we generated a frog amelogenin and a bovine amelogenin overexpressing mouse model and compared enamel structure using scanning and transmission electron microscopy. RESULTS: Here we present the results of four studies that examine the role of the elongated stretch of polyproline repeat elements of most mammalian amelogenins in the formation of elongated apatite crystals and organized enamel prisms. (i) Our analysis demonstrated that proline/glutamine ratios and the number of polyproline motifs were significantly higher in mammalian than in reptilian/amphibian amelogenins. (ii) Comparing amelogenin N- and C-terminal peptides, the PXX domain peptides preferentially bound to apatite over carbonate and promoted the formation of elongated and thin apatite crystals. Moreover, long 33mer polyproline peptides promoted self-assembly into smaller subunits than 24mer and 12mer peptides while enhancing apatite crystal elongation. (iii) Enamel of frog amelogenin overexpressors was characterized by a lack of prismatic enamel and increased matrix subunit dimensions in the enamel matrix. (iv) In contrast, enamel of bovine amelogenin overexpressing mice featured smaller enamel matrix subunit dimensions and thinner enamel crystals. CONCLUSIONS: Together, these studies illustrate the far-reaching effects of the elongated polyproline motif of mammalian amelogenin on protein self-assembly and apatite crystal growth. Generous support for these studies by NIDCR grant DE018900 to TGHD is gratefully acknowledged. M4 - High diffusivity pathways and selective ion transport in dental enamel Xingchen Zhao*1, Derk Joester1. 1Dept of Material Science and Engineering, Northwestern University, Evanston, Illinois, USA. INTRODUCTION: Enamel is an acellular, mineralized tissue with limited regenerative potential. Enamel defects are irreversible and ubiquitous, imposing great burdens on society. Ions such as Na+, CO32-, F-, and Mg2+, are important determinants of enamel solubility; some are widely used in the prevention or treatment of defects. Knowledge of the diffusivities of these ions is integral to understanding their uptake and retention in the enamel. Much prior work in this area assumes a homogenous structure of enamel1,2. However, the recent discovery of significant differences in crystallite size, orientation, and composition between rod and interrod enamel and of an amorphous intergranular phase (AIGP) that cements together enamel crystallites and in which F- and Na+diffuse much more rapidly than in bulk hydroxylapatite indicate that multiple diffusion pathways must be considered3-5. PURPOSE: To improve our understanding of transport processes in dental enamel by determining diffusivities that take into account new insights into enamel structure at the nano- and mesoscale. METHODS: Ground and polished sections of human premolars (approximately parallel to the external surface) were treated with aqueous solutions of selected ions (0-1000 ppm, pH 7, 25 ˚C, 24h). Concentration profiles normal to the treated interface were determined using Time-of-Flight Secondary Ion Mass Spectroscopy (TOF-SIMS) in depth profiling mode (for a few μm). Fits were constrained using geometrical parameters and concentrations determined by several other techniques, such as atom probe tomography and micro-X-ray diffraction. RESULTS: Effective and absolute diffusivities for common cations (Na+, K+, Mg2+, Sr2+, Ba2+) and anions (F-, Cl-) were estimated by the fitting of experimental concentration profiles. Effective bulk diffusivities fell into the range between 10-16 to 10-19 cm2/s and decreased in the following order: Sr2+ = F- > Mg2+ = Na+ > Cl- > Ba2+ > K+. Preliminary fitting suggests that effective diffusivities in rod and interrod enamel differ by a factor of up to 10, while that in crystallites and AIGP differs by up to 100 times. CONCLUSION: Depth profiling using SIMS is an effective w

OCTOBER 2023 ICCBMT 14 M5 - Three-dimensional study of early mineralization events in fibrolamellar bone Elena Macías-Sánchez*1, Emeline Raguin2, Nadezda V Tarakina3, Peter Fratzl2. 1Dept of Stratigraphy and Paleontology, University of Granada, Granada, Spain, 2Dept of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany, 3Dept Colloid Chemistry, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. INTRODUCTION: Recently, it has been proposed that mineral deposition in collagen-based materials is driven by a spherulitic-type crystalline growth [1]. Disordered mineral aggregates initially form in the interfibrillar spaces, and subsequently mineral infiltrates adjacent collagen fibrils, which provide the structural framework for the formation of layered spherulites. These spherulites or mineral ellipsoids imbricate forming a new hierarchical level of organization in bone termed tessellation [2]. Although the mechanism has been described in several systems [1, 2, 3], detailed data on the colocalization of the organic and the mineral phases remain scarce. PURPOSE: To demonstrate that the mechanism of crystal growth in early mineralization events is homologous for all type I collagen materials, and to discern differences (if any) with previously described systems, we studied the initial stages of mineralization in lamb fibrolamellar bone. METHODS: The present study combines 3D electron tomography (FIB-SEM serial surface view), scanning/transmission electron microscopy (S/TEM), selected area electron diffraction (SAED) and energy dispersive spectroscopy (EDS) chemical mapping to elucidate crystal distribution and orientation throughout the collagen matrix. RESULTS: The study reveals the internal structure of the forming fibrolamellar bone at nanometer resolution. A pre-deposited, unmineralized matrix of dispersed and non-preferentially oriented collagen fibrils serves as a scaffold for osteocyte alignment. During embryonic development, these osteocytes initiate the mineralization process and become buried in the mineral matrix, which expands both vertically and laterally to form the nascent fibrolamellar units. The collagen fibrils contained in the hypermineralized layer clearly exhibit the 67 nm axial periodicity and are organized in a crisscross orientation. The collagen-mineral interface is characterized by the proliferation of mineral spherulites that grow to confluence and whose profiles are still recognizable in the consolidated mineral layer. The non-mineralized areas between the fibrolamellar units were populated by a large number of cells and vesicular bodies, which may play an active role in the formation of the structure. CONCLUSIONS: Our study confirms that mineral spherulites formation controls mineral deposition in embryonic fibrolamellar bone. High resolution chemical maps provide unambiguous evidence for intrafibrillar mineralization, and how inter- and intrafibrillar mineralization amount to a single process [4]. REFERENCES: 1. Macías-Sánchez et al. Adv. Funct. Mater. 32, 2200504 (2022). 2. Buss et al. J. Struct. Biol. 212, 107603 (2020). 3. Shah et al. Bone Reports 13, 100283 (2020). 4. EMS is supported by a Juan de la Cierva Incorporación fellowship (IJC2020-043639-I) funded by MCIN/AEI/10.13039/501100011033 and European Union NextGenerationEU/PRTR. Authors gratefully acknowledge financial support from the Max Planck Society. M6 - The role of citrate in extrafibrillar mineralization of bone Chenglong Li*1,2,3, Robin HM van der Meijden1,2, Luco Rutten1,2, Yang Li4, Rui Li4, Danielle Laurencin5, Christian Bonhomme6, Melinda Duer4, Nico Sommerdijk1,2. 1Dept of Medical BioSciences (MBS), Radboud University Medical Center, Nijmegen, Netherlands, 2Electron Microscopy Centre, Radboudumc Technology Center Microscopy, Radboud University Medical Center, Nijmegen, Netherlands, 3School of Earth Sciences and Engineering, Nanjing University, Nanjing, China, 4Dept of Chemistry, University of Cambridge, Cambridge, UK, 5CGM, University Montpellier, CNRS, ENSCM, Montpellier, France, 6Laboratoire de Chimie de la Matière Condensée de Paris, UMR Sorbonne Université CNRS, Sorbonne Université, Paris Cedex, France. INTRODUCTION: Bone mineral (apatite) is hierarchically assembled in a collagen matrix, from the nanoscale to the multi-micron level: featuring needle-shaped crystals, platelets, stacks of roughly parallel platelets and eventually mineral aggregates in continuous, cross- fibrillar mineralization. Where collagen is thought to determine the shape and orientation of the intrafibrillar crystals, a key, unanswered question is what determines the mineral shape and orientation in the extrafibrillar space. However, the complex hierarchical structure and the collagen involved in the bone hindered the detail of the extrafibrillar mineralization. Previous studies already showed that citrate is present at the mineral surface in bone, and that (octacalcium phosphate) OCP-citrate/l

OCTOBER 2023 ICCBMT 14 M7 - In-situ model to investigate the effect of collagen overglycosylation on the fibril structure and mineralization Luco Rutten*1,2, Liline Fermin3, Jinge Xu1, Judith Schaart1, Ruud Bank4, Elena Macías Sánchez1,5, Nico Sommerdijk1,2. 1Dept of Medical BioSciences, Research Institute for Medical Innovations, Radboud University Medical Center, Nijmegen, The Netherlands, 2Electron Microscopy Center, Radboudumc Technology Center Microscopy, Radboud University Medical Center, Nijmegen, The Netherlands, 3Dept of Instructive Biomaterial Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, The Netherlands, 4Dept of Pathology and Medical Biology, University of Groningen, Groningen, The Netherlands, 5Dept of Stratigraphy and Palaeontolgy, University of Granada, Granada, Spain. INTRODUCTION: The extracellular matrix of bone consists mainly of collagen, apatite and non-collagenous proteins. Single collagen alpha- helices are synthesized by osteoblasts and folded into the characteristic triple helical structure. During folding, the molecules undergo enzymatic post-translational modifications (PTMs) such as the hydroxylation of proline and lysine residues, and the glycosylation of hydroxylysine. The ability of collagen to mineralize appears to depend on these PTMs, particularly glycosylation. For example, the hypermineralization that characterizes the brittle bones in Osteogenesis Imperfecta (OI) is associated with a higher levels of glycosylation of their collagen fibrils. Previous research has shown that the structure of the collagen is important in directing the mineralization, and therefore changes in the collagen structure could significantly alter the mineralization. We established an in-vitro model to produce overglycosylated single fibrils of which the degree of overmodifications can be tuned and determined. These fibrils will be used to investigate the mineralization mechanism using advanced electron microscopy. PURPOSE: We aim to elucidate the role of overglycosylation in collagen mineralization. Understanding the mechanism of impaired mineralization due to overglycosylation will result in fundamental information about bone formation in pathological stages. METHODS: Overglycosylation was induced by supplementing Cyclosporin A (CsA) to a mouse osteoblast cell culture. CsA inhibits the cis-trans isomerase of proline which subsequently reduces the folding rate of the collagen triple helix and thereby increases the degree of glycosylation. Fluorescent microscopy confirmed the production and secretion of collagen. High purity intact fibrils were thereafter isolated from the cell culture and high-performance liquid chromatography (HPLC) was used to determine the degree of modification, as well as the degree of crosslinking. High resolution cryo-TEM imaging was used to evaluate the structure of the collagen fibril and its mineralization in near-native hydrated state. RESULTS: HPLC showed that both the degree of modification and the degree of crosslinking were significantly increased by CsA treatment. Cryo-TEM revealed both a decrease in fibril diameter and changes in the (sub-)banding pattern of the overglycosylated fibrils. In-vitro mineralization of the isolated fibrils will show how these modifications influence the mineralization mechanism. CONCLUSIONS: We have established an in-vitro model to investigate the effect of glycosylation on collagen fibril mineralization. Using this model, we show that overglycosylation alters the fibril structure. Cryo-TEM will reveal the changes in mineralization, as the collagen structure is essential in directing the mineral growth. M8 - The critical roles of a novel GTPase regulator, Din, in the homeostasis of MSCs in bones, sutures and teeth Changchun Dong, Bikash Lamichhane, Xiaofang Wang*. Dept of Biomedical Sciences, Texas A&M University School of Dentistry, Dallas, TX, USA. INTRODUCTION: The postnatal growth, turnover and injury repair in bones and teeth rely on proper homeostasis of MSCs. Rho GTPases are binary molecular switch regulating downstream signaling pathways and cytoskeleton reorganization to regulate cell proliferation, differentiation, polarization and motility. So far, little is known about GTPase roles in dental and skeletal MSCs, and the upstream inputs for GTPase signaling have not been well understood. With a newly generated gene-knockout mouse model, we found that a predicted gene, Din (4930453N24Rik), plays key roles in governing MSC homeostasis by regulating Rho GTPases in bones, sutures and teeth. METHODS: Din-KO and Din-flox mice were created through Gene-Trap. Din expression pattern was determined by LacZ staining, RNAScope and scRNA-Seq. K14-Cre, Wnt1-Cre and 2.3k Col1-Cre mice were used for conditional knockout of Din from epithelium, ectomesenchyme and osteoblasts/odontoblasts. Bone and teeth were characterized by u-CT, X-ray, histology and double labeling. Transcripto

OCTOBER 2023 ICCBMT 14 M9 - Effects of circadian clock disruption in dental mineralized tissues formation Raed Said1*, Helya Mortazavi1, Takashi Yamashiro2, Petros Papagerakis1,2, Silvana Papagerakis2,3. 1Dept of Anatomy, Physiology and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Canada, 2Dept of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Osaka University, Japan, 3Dept of Surgery, College of Medicine, University of Saskatchewan, Saskatoon, Canada. BACKGROUND: The circadian clock controls the diurnal rhythms of almost all physiological mammalian processes through the differential expression of specialized transcription factors called clock genes. The main clock genes include the core circadian activator Brain and muscle Arnt-like protein-1 (BMAL1) and the circadian suppressors period genes (PER1, 2 & 3) and cryptochrome genes (CRY1 & 2). Dental mineralized tissues (i.e., enamel and dentin) are formed incrementally following twenty-four-hour cycles which strongly suggests that the process of dental tissues secretion and mineralization (i.e., odontogenesis) is temporally regulated by the circadian clock. OBJECTIVES: Our aim was to examine the role of the main circadian clock genes in regulating odontogenesis using three circadian knockout (KO) models resulting in three different circadian phenotypes; Per2 KO (shortened circadian period and arhythmic), Cry1 KO (shortened circadian period) and Cry2 KO (longer circadian period) mice. METHODS: The dental phenotype in the three KO and their corresponding wild-type (WT) littermates were examined through gross morphology, SEM imaging, X-ray imaging and μCT analysis in young (3 weeks old) and older mice (7 weeks old). Changes in gene and protein expression levels of several key genes were evaluated by qRT-PCR and immunohistochemistry to elucidate how each circadian clock gene affects the process of dental tissue formation. RESULTS: Our results showed that circadian clock disruption results in abnormal tooth development. Both Per2 and Cry2 KO mice presented with shortened incisors and reduced teeth volume compared to WT. Per2 deletion resulted in an abnormal enamel phenotype with a significant delay in enamel secretion and mineralization associated with abnormal ameloblasts morphology and increased expression levels of the main enamel matrix protein amelogenin (AMEL) and the enamel protease kallikrein-4 peptidase (KLK4). Cry2 KO mice also showed an abnormal albeit less pronounced enamel phenotype with no significant difference in AMEL expression and overexpression of enamel matrix metalloprotease 20 (MMP20). Regarding dentin, Cry2 KO showed the most significant reduction in dentin volume and mineral density compared to WT and other circadian KO models. Enamel or dentin phenotypes were found normal in Cry1 KO mice. CONCLUSIONS: Our data strongly suggest that the circadian clock exerts significant effects in regulating dental mineralized tissue development and function. Biorhythm disorders could be involved in the development of dental malformations. Dental tissues chronobiology may [provide new ways of dental disease prevention and treatment using physiological interventions and/or small molecules targeting the circadian clock. M10 - Unravelling the relationship between isolated bone matrix vesicles and forming mineral at the nanometer scale Marcos A E Cruz*1,2, Luco Rutten2,3, Martijn Martens2,3, Pietro Ciancaglini1, Anat Akiva2,3, Ana P Ramos1, Nico Sommerdijk2,3. 1Dept of Chemistry, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil. 2Dept of Medical BioSciences, Radboud University Medical Center, The Netherlands. 3Electron Microscopy Centre, Radboudumc Technology Center Microscopy, Radboud University Medical Center, The Netherlands. INTRODUCTION: MVs play a role in initial HAp deposition during bone mineralization, but the underlying mechanisms are not well understood. In vitro studies propose two pathways for HAp precipitation: one involves enhanced phosphatase activity within MVs to generate Pi from organophosphate compounds, and the other depends on membrane components and luminal Ca2+ and Pi ions to trigger mineralization in a phosphatase-independent manner. However, the relevance of these pathways and the relationship between MVs and forming mineral in such in vitro experiments remain unclear. PURPOSE: Our goal is to investigate the content and action of MVs during in vitro mineralization by cryoTEM to understand the role of vesicles in mineral formation. METHODS: Crude MVs were extracted from chicken bones via collagenase digestion and differential ultracentrifugation, and then purified through isosmotic density gradient fractionation to separate low and high-density contents. Crude and low-dense MVs were then characterized through enzymatic activity, DLS, turbidimetry, and cryoTEM imaging to determine their content and compare their ability to

OCTOBER 2023 ICCBMT 14 M11 - Accelerated biomineralization and osteocyte maturation on-a-chip: A 3D bioprinting and microfluidics approach Rahul Madathiparambil Visalakshan1,4, Mauricio Sousa1,4, Anthony Taheri1,4, Avathamsa Athirasala*1,4, Luiz Bertassoni1,2,3,4. 1Knight Cancer Precision Biofabrication Hub, Cancer Early Detection Advanced Research (CEDAR), Knight Cancer Institute, OHSU, Oregon, USA, 2Center for Regenerative Medicine, OHSU School of Medicine, Oregon, USA, 3Dept of Biomedical Engineering, OHSU School of Medicine, Oregon, USA, 4Division of Biomaterials and Biomechanics, Dept of Restorative Dentistry, OHSU School of Dentistry, Oregon, USA. INTRODUCTION: Bone defects pose a significant challenge in the medical field, and current treatments such as autologous bone grafting are not always suitable for severe injuries due to potential complications. Developing biomaterials that are osteoinductive with live cells is essential for treating bone defects. Current bone scaffolds fail to mimic the characteristics of native bone, including the calcified microenvironment and cell-laden 3D matrix. We have developed a strategy to mimic these key characteristics of native bone by performing nanoscale mineralization of cell-laden hydrogels. PURPOSE: The purpose of this study is to develop a bone-like structure that is biomineralized to the nanoscale level and embedded with human mesenchymal stem cells (hMSCs) that undergo osteogenic differentiation without conventional supplements. We also aim to create a microfluidic cell culture system to expedite the mineralization process and enhance regenerative capacity. METHODS: We utilized a Digital Light Processing (DLP) printer to 3D bioprint microgels of gelatin metacryloyl GelMA encapsulated with hMSCs. These microgels were mineralized under flow within 3 hours with calcium and phosphate rich media in a custom-made chip. Characterization with alizarin red and von Kossa staining confirmed biomineralization, while live/dead staining showed hMSCs viability after mineralization underflow. OCN, RUNX2 & PDPN expression indicated successful osteogenic stem cell differentiation in the presence of fluid shear stress (FSS). RESULTS: Our microfluidic system recapitulates biomineralization and early osteogenesis on injectable microgels, and we were able to fabricate a mineralized bone-like structure with osteogenesis in just 3 hours compared to the conventional 21-day process. OCN and PDPN expression after 1 day of culture indicated that the MSCs were committed to the osteocyte-like lineage and actively forming bone tissue. Our novel approach offers an effective strategy for developing treatments for bone defects by recapitulating biomineralization and osteogenesis on an injectable microgel using 3D bioprinting and microfluidics. CONCLUSIONS: In conclusion, our study demonstrates a promising approach to treat bone defects by developing biomaterials that closely mimic the natural bone microenvironment. Our approach offers several advantages, including the ability to expedite the mineralization process and enhance regenerative capacity. This strategy has the potential to revolutionize the treatment of bone defects and accelerate the development of new therapies. M12 - Transcriptome analysis indicates a stimulatory role of DMP1 in periodontal ligament stem cells and promotes osteoblast differentiation Cassandra Villani*1, Yinghua Chen1, Anne George1. 1Dept of Oral Biology UIC College of Dentistry, Chicago IL. INTRODUCTION: Periodontitis is a progressive disease that can result in bone erosion and subsequent tooth loss. The proximity of periodontal ligament stem cells (PDLSCs) to areas of common bone erosion and their osteoblast differentiation potential makes them a promising target for regenerative periodontal treatments. Dentin matrix protein 1 (DMP1) is an extracellular matrix protein and plays a dual functional role, both as an extracellular mediator of hydroxyapatite deposition and an intracellular regulator of osteoblast-specific gene transcription. Glucose regulatory protein 78 (GRP78), a master regulator of endoplasmic reticulum stress, interacts with DMP1 and facilitates endocytosis. PURPOSE: To evaluate the transcriptomic profiles of PDLSCs and PDLSCs overexpressing GRP78 treated with DMP1 to identify enriched pathways associated with osteoblast differentiation. METHODS: Control PDLSCs and PDLSCs OE-GRP78 were treated with DMP1 for 0 and 48 hours under growth conditions or under differentiation conditions. Total RNA was isolated and subjected to mRNA sequencing. Bioinformatic principal component and differential expression analyses were conducted on all sample groups using the edgeR Bioconductor package in R. Normalized edgeR gene counts were subjected to one-way Anova testing to determine differentially expressed genes with adjusted p-values < 0.05. Upregulated genes were subject to pathway analysis using the Database for Annotation, Visualization, and Integrated Disc

OCTOBER 2023 ICCBMT 14 M13 - Runx2 is required for hypertrophic chondrocyte mediated cartilage degradation and bone resorption Caris Smith*, Harunur Rashid, Vashti Convers, Katelynn Clark, Haiyan Chen, Amjad Javed. Dept of Oral and Maxillofacial Surgery, University of Alabama at Birmingham School of Dentistry, Birmingham, AL, USA. INTRODUCTION: Global or resting chondrocyte specific deletion of Runx2 gene results in failure of chondrocyte differentiation, endochondral ossification, and perinatal lethality. Importantly, Runx2 expression increases progressively from resting to hypertrophic chondrocytes (HCs). Terminal HCs regulate endochondral ossification by secreting matrix resorbing enzymes and angiogenic factors, however, the role of Runx2 in HCs during the majority of chondrogenesis takes place postnatally and remains unknown. METHODS: Runx2 gene was deleted in HCs using the type-X collagen-Cre. Progression of endochondral ossification was analyzed at birth and in 10-weeks- old littermates using molecular, biochemical, and histological approaches. RESULTS: Homozygous (Runx2HC/HC) mice survive to adulthood but show limb dwarfism and enlarged growth plates. A significant decrease in apoptosis of Runx2-defecient HCs resulted in doubling of the HC zone in the growth plate. Consistent with these observations, expression of HC markers was significantly increased in Runx2HC/HC mice. Biochemical staining revealed a significant increase in the adult articular cartilage, as well as growth plate cartilage that extended into the mid-diaphysis. Interestingly, the expression of collagen type 2 and the major proteoglycans was unchanged. In contrast, expression of collagenases and aggrecanases were markedly decreased, indicating impaired resorption of the cartilage matrix in Runx2HC/HC mice. Both histomorphometry and μCT analyses revealed a significant increase in trabecular BV/TV, trabecular number, thickness, and a concomitant decrease in trabecular space in Runx2HC/HC mice. To identify if the increased bone mass is linked to osteoblast activity, expression of early and late markers was evaluated and found to be comparable amongst the newborn and adult littermates. Consistent with these findings, dynamic bone synthesis showed no differences in mineral apposition or bone formation rates. Surprisingly, 3-point-bending test revealed bone fragility despite an increased trabecular bone mass in Runx2HC/HC littermates. Similar bone synthesis prompted examination of bone resorption in adult littermates. TRAP staining showed a significant decrease in number and surface of osteoclasts in Runx2HC/HC mice. Runx2 deficient HCs showed a significant decrease in Rankl expression and Rankl/Opg ratio. Finally, the bone marrow culture from Runx2HC/HC mice showed few, smaller osteoclasts with low expression of markers of mature osteoclast. CONCLUSIONS: Runx2 controls endochondral ossification by regulating the expression of cartilage resorbing enzymes and osteoclast differentiation factors in hypertrophic chondrocytes. T1 - The potential role of internal stresses for the mechanical properties of bone Peter Fratzl1*, Victoria Schemenz2, Wolfgang Wagermaier1, Paul Zaslansky2. 1Dept of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany, 2Dept for Operative, Preventive and Pediatric Dentistry, Charité - Universitätsmedizin Berlin. INTRODUCTION: Huge internal stresses with tens of megapascal have been observed in bone [1] and dentin [2]. Mineral particles are under compression by collagen molecules that shrink during mineralization and the resulting stresses correspond to almost half the tensile strength of bone. Recent x-ray measurements reported that damage to collagen in dry bone leads to a reduction of the compressive stress on the mineral [3], indicating the important role of an intact organic bone matrix. Moreover, in-vitro mineralization experiments of collagen fibers show that collagen contracts gradually with the intrafibrillar deposition of mineral [4]. RESULTS: The presentation reviews these experimental findings and develops a model of mineralized collagen fibrils that allows estimating how internal stresses influence the properties of a mineralized fibril. In particular, the main beneficial effect is that mineral particles rarely experience tension, as they start from an initial state of compression. This built-in stress within the particles counteracts external tensile loads, thereby protecting mineral from premature brittle failure. REFERENCES: [1] R. P. Hoo & al., Acta Biomaterialia 7, 2943–2951 (2011). [2] J.-B. Forien & al., Nano Letters 15, 3729–3734 (2015). [3] K. Sauer & al., Nature Communications 13, 7829 (2022). [4] H. Ping & al., Science 376, 188–192 (2022).

OCTOBER 2023 ICCBMT 14 T2 - Biomineralogical signatures of pathological mineralization Lara A. Estroff*1, Jennie A.M.R. Kunitake1, Stephan Sutter1, Brenda Javier-Boodhan1, Derek Hu1, Jonathan Butcher2, Claudia Fischbach2, Admir Masic3. 1Dept Materials Science and Engineering, Cornell University, Ithaca, NY, 2Dept Biomedical Engineering, Cornell University, Ithaca, NY, 3Dept of Civil Engineering, MIT, Cambridge, MA. INTRODUCTION: Pathological calcification is a wide-spread phenomenon in the human body, in which calcium minerals form in soft tissues, often associated with a disease state. Two examples are: microcalcifications (MCs), which are primarily biological apatite and occur in cancerous and benign breast pathologies, and calcific aortic valve disease (CAVD), in which calcium phosphate mineral forms in the aortic valve leaflets. PURPOSE: The objective of this study is to investigate the association of calcification mineral properties, e.g., the biomineralogical signatures, and pathological parameters, both at the local (i.e., the tissue surrounding the calcification) and patient level (i.e., the overall patient diagnosis). METHODS: In this study, frozen human breast tumor tissue and fixed human aortic valve leaflets were embedded and sectioned. To interrogate the effects of physiochemical heterogeneity on calcification composition, we take an omics- inspired approach: we acquire spatially registered Raman and IR microscopy and energy dispersive spectroscopy (EDS) mapping measurements on the tissue sections and collectively define a biomineralogical signature for each calcification, together with serial histopathology employed to classify the surrounding pathology and tissue environment. For example, for the MCs, Raman mineral metrics included carbonate-to-phosphate and the ν1 phosphate peak full width at half maximum. Calcification organic matrix metrics included calcification lipid-to-protein and phenylalanine-to-phosphate. EDS analysis metrics included Ca/P, Na/Ca, Mg/Ca, Al/Ca, Fe/Ca and Zn/Ca. RESULTS: For the MCs in breast tissue, we observe that 1) calcifications cluster into physiologically relevant groups reflecting tissue type and local malignancy; 2) carbonate content exhibits substantial intratumor heterogeneity; 3) trace metals including zinc, iron, and aluminum, are enhanced in malignant-localized calcifications; 4) the lipid-to-protein ratio within calcifications is lower in patients with poorer prognosis, suggesting that expanding diagnostic metrics to include “mineral-entrapped” organic material may hold prognostic promise. For the aortic valve tissue, carbonated hydroxyapatite was found to be the predominant phase, with traces of whitlockite and octacalcium phosphate. Calcifications were extremely heterogeneous across and among patients in terms of size, matrix composition, etc. For example, some calcifications were associated strongly with lipids, while others calcifications with collagen. The association of specific matrix species with calcifications could reflect different events in the pathological progression of the disease (e.g., inflammation and fibrosis), and warrant further investigation. CONCLUSIONS: This multimodal methodology lays the groundwork for establishing biomineralogical signatures of pathological mineral deposits associated with a range of different diseases. T3 - 3D correlative live-to-cryo microscopy shows collagen development in zebrafish scale Robin HM van der Meijden1,2, Marit de Beer1,2, Rona Roverts1,2, Deniz Daviran1, Luco Rutten1,2, Judith M Schaart1,2, Merijn van Erp1,2, Matthijn Vos3, Elena Macías-Sánchez1,2,4, Jurriaan R Metz5, Nico Sommerdijk1,2, Anat Akiva*1,2. 1Electron Microscopy Center, Radboudumc, Nijmegen, 2Dept of Medical BioSciences, Radboudumc, Nijmegen, 3NanoImaging Core Facility, Centre de Ressources et Recherches Technologiques (C2RT), Institut Pasteur, France, 4Dept of Stratigraphy and Paleontology, University of Granada, Granada, 5Dept of Animal Ecology and Physiology, Radboud Institute for Biological and Environmental Sciences, Faculty of Science, Radboud University, Nijmegen. INTRODUCTION: The functioning of tissues is strongly determined by the extracellular matrix (ECM). Fibrillar collagen is a major component of almost all ECMs and of which the properties are dependent on fibril thickness, degree of cross-linking and the presence of mineral crystals. METHODS: To study how collagen structure is adapted to local needs we have developed a unique 3D multi-modal multi-scale imaging approach, which includes live fluorescence and Raman microscopy, spatially correlated with cryo-FIB/SEM and cryo-TEM, to obtain a map of structural, chemical and biological details at any chosen location in the tissue. As a model system, we use the zebrafish scale, which is known to have a highly organized ECM that contains both mineralized and non-mineralized collagen layers. RESULTS: The development of the partially mineralized collagen matrix of the zebrafish

OCTOBER 2023 ICCBMT 14 T4 - Evolutionary materials optimization? Neutron tomography reveals differences in water permeability between osteocytic and anosteocytic bone Andreia Silveira*1, Nikolay Kardjilov2, Henning Markötter3, Elena Longo4, Imke Greving4, Ron Shahar5, Paul Zaslansky1. 1Dept for Restorative and Preventive Dentistry, Charité-Universitaetsmedizin, Berlin, Germany, 2Institute of Applied Materials, Helmholtz Centre for Materials and Energy, Berlin, Germany, 3Bundesanstalt für Materialforschung und -Prüfung (BAM), Berlin, Germany, 4Institute of Materials Physics, Helmholtz- Zentrum Hereon, Geesthacht, Germany, 5Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot, Israel. INTRODUCTION: Bone tissue is mainly known for the nanofibers of collagen protein and nanoparticles of carbonated hydroxyapatite. The nanocomposite also contains traces of noncollagenous proteins (γ-carboxyglutamate- containing proteins, proteoglycans (PGs), and glycoproteins) and almost 20% of water. The structural and mechanical properties of bone are determined by the hierarchical arrangement of these components at different length scales. Water occupies various domains in bone, most notably inside the vascular system (> 20 micrometers in diameter), and the lacuna-canalicular network (LNC) (< 10 micrometers diameter) but also within collagen and at the surfaces of hydroxyapatite mineral. Water is a key player in transduction of changes in bone loading conditions via a cell-mediated sensing process that is vital for bone remodeling, leading to continuous resorption and formation of bone tissue throughout life. PURPOSE: To understand the role of water and minor organic components in bone. We hypothesize that the bony nanocomposite design, housing an LCN, makes it possible to successfully sense mechanical loads in a manner that is poorly understood. To this end, we compare osteocytic bone (zebrafish) with anosteocytic bone (medaka) to examine the interplay between the material constituents contrasting and comparing bone-housing versus bone-cell-deficient materials. METHODS: We compare the vertebrae of similarly-sized fish - medaka (Oryzias latipes) and zebrafish (Danio rerio) using complementary advanced methods using electrons, X-rays and neutrons. We exploit the high sensitivity of neutron imaging to the presence of hydrogen to compare water flow through bones with or lacking an inbuilt LCN. Water exchange was quantified by 3D analysis of hydrated bone samples and the same samples were imaged after infusion with deuterated water (D2O). The contrast difference was used to assess water diffusion, which we correlated with the micro-nano structure, using phase-contrast enhanced nanotomography, Raman spectroscopy and complementary histological staining. RESULTS: Unexpectedly, far more water was expelled from medaka bone (∼60%) as compared with zebrafish bone (33%∼39%). In the osteocytic bone, the diffusion of water seems to be restricted to flow within the LCN. In contrast, anosteocytic bone is amenable to D2O diffusion, despite the absence of LCN porosity. Raman spectroscopy and histology reveal that zebrafish bone has a stronger PG fingerprint as compared with medaka bone. CONCLUSIONS: Though PGs have been associated with water retention in mammalian bones, they have never been associated with water permeability. Our findings raise the possibility that osteocytic bone has a PG-based mechanism to restrict water diffusion, possibly essential for keeping water in the LCN as part of a poroelastic mechanotransduction system. In contrast, anosteocytic bone structure allows the free diffusion of water in the matrix, which may be an evolutionary alternative to LCN system. T5 - Structural description of CaCO3 prenucleation clusters through 13C MAS-DNP NMR Thierry Azaïs,*1 Tristan Georges,1 Vinavadini Ramnarain,2 Christel Gervais,1 Clément Sanchez1, Ovidiu Ersen2. 1Laboratoire de Chimie de la Matière Condensée de Paris, Sorbonne Universite & CNRS Paris, France, 2 Institut de Physique et Chimie des Matériaux Strasbourg & ICFRC, Strasbourg, France. INTRODUCTION: Biomineralization is a fascinating and complex biological process leading to the formation of mineral phases in vivo. Calcium carbonate (CaCO3) is one of the most significant biominerals due to its implication in the CO2 cycle and is found in very diverse species and mineralized tissues including the skeletons of sea urchins, cuticles of crustaceans, mollusks shells or corals. Contrary to the classical nucleation theory, it was recently shown that CaCO3 crystallization is occurring through a non-classical nucleation pathway for which various intermediate phases are involved. In particular, it was shown that before the nucleation of the first solid, prenucleation clusters (PNC) are in equilibrium with free ions in aqueous solution. These PNC are yet poorly understood due to their nanometric size, dynamic behavior and transient nature leading to short lifetim

OCTOBER 2023 ICCBMT 14 T6 - Studying collagen mineralization dynamics using in-situ Raman spectroscopy together with in-situ small and wide angle X-ray scattering Emma Tong1, Julia E Parker2, Laurie Gower3, Roland Kröger*1. 1School of Physics, Engineering and Technology, University of York, UK, 2Diamond Light Source, UK, 3Dept of Materials Engineering, University of Florida. INTRODUCTION: Collagen mineralization is central for bone formation resulting in a fractal-like organization of the mineral phase within the collagen matrix with the control of the crystal growth being essential for bone to obtain its unique mechanical properties [1]. However, many aspects of the of the mineralization dynamics remain unclear. PURPOSE: To shed light on these dynamics we applied the polymer- induced liquid precursor (PILP) process [2] using osteopontin as an active protein polymer to mimic this process in vitro using rat-tail derived collagen. METHODS: Mineralization was carried out in situ in a custom-made heatable flow-cell allowing for Raman microspectroscopy as well as small-angle and wide-angle X-ray scattering (SAXS, WAXS. The mineralized collagen fibers were subsequently analysed using nano-X-ray fluorescence (nano-XRF) to map the distribution of Ca and P and transmission electron microscopy (TEM) for microstructural investigations. RESULTS: In situ Raman spectroscopy allowed to monitor the mineralization dynamics indicating the presence of an intermediate calcium phosphate phase before hydroxyapatite dominates the spectra. Using this flow cell, we performed in situ SAXS obtaining vital information on the impact of mineralization on the collagen D-banding (a periodic pattern caused by repeat sequences of overlap and gap regions in the staggered collagen molecule organization) revealing initial expansion upon infiltration with subsequent contraction during nucleation followed by expansion once the crystal growth phase formed. With in situ WAXS we studied the time dependent evolution of crystallinity monitoring the {002} lattice planes in the hydroxyapatite mineral phase with a gradual reduction of lattice plane spacing over time related to the increased degree of crystallinity. The in vitro mineralization experiments show significant similarities to the mineralization patterns observed in bone with Raman microscopy and SAXS/WAXS data being in strong agreement. The mineralization pattern observed by nano-XRF reveals a tessellation motif similar to human and murine bone with the crystal morphologies observed by TEM indicating a similar crystal organization pattern compared to that found in bone [3]. CONCLUSIONS: This confirms a strong similarity of the physico-chemical processes controlling collagen mineralization in our in vitro system compared to that found for bone. We reveal the occurrence of at least one intermediate mineral phase within the first two hours of mineralization and a change of the D-banding indicating the infiltration, nucleation and growth phases of the calcium phosphate formation within the collagen matrix. [1] N. Reznikov, R. Kröger et al., Fractal-like hierarchical organization of bone begins at the nanoscale, Science 360, science.aao2189 (2018). [2] M.J. Olszta et al., Connective Tissue Research 44 (1), 326 (2003). [3] D. Buss, N. Reznikov, M. McKee, J. Struct. Biol. 212 107603 (2020). T7 - X-ray primary radiation damage spreads in bone via collagen destruction due to photoelectron ionization and secondary emission self-absorption Katrein Sauer*1, Ivo Zizak2, Jean-Baptiste Forien3, Alexander Rack4, Ernesto Scoppola5, Paul Zaslansky1. 1Charité - Universitätsmedizin Berlin, Dept for Operative, Preventive and Pediatric Dentistry, Aßmannshauser Straße 4-6, Berlin, Germany, 2Helmholtz-Zentrum Berlin, Dept for Structure and Dynamics of Energy Materials (SE-ASD), Albert-Einstein-Straße 15, Berlin, Germany, 3Lawrence Livermore National Laboratory, Materials Science Division, Livermore, CA, USA, 4ESRF-The European Synchrotron, Structure of Materials Group-ID19, Grenoble Cedex 9, France, 5Max Planck Institute of Colloids and Interfaces, Dept of Biomaterials, Am Mühlenberg 1, Potsdam, Brandenburg, Germany. INTRODUCTION: X-rays are widely used for imaging and sterilization of bones. Due to their high penetration power, X-rays are effective for radiation therapy to eliminate unwanted pathogens, and are used in tissue banks and for transplants such as corneal xenografts or bone allografts. However, the high-energy photons induce ionization, destroying organic building blocks, such as proteins. Bone material is made of a mineralized collagen matrix, and X-ray radiation studies are frequent with ever increasing use of high-power and high-energy synchrotron sources. When exposed to irradiation, some of the radiation is absorbed by the bone ingredients, mainly the mineral nanocrystals, which inevitably results in scattering and energy deposition. There have been reports and recommendation doses designed to circu

OCTOBER 2023 ICCBMT 14 T8 - Precision remineralising technologies to advance dental enamel health Palwinder Kaur1*, Helen Chappell2, Mohammed Al-Mosawi1, Maisoon Al-Jawad1, Ursulla McDonnell3, Christabel Fowler3. 1School of Dentistry, University of Leeds, UK, 2School of Food Science and Nutrition, University of Leeds, UK, 3Dept of Oral Health Research and Development, Haleon, Weybridge, UK. INTRODUCTION: Erosion of dental enamel is becoming a significant problem, affecting a significant percentage of children and adults in Europe. Several studies have been conducted to understand the underlying mechanism behind enamel erosion, and efforts have been made to develop therapeutic techniques capable of remineralising the eroded enamel. However, experimental data from these studies are limited in providing detailed elemental information about the demineralised and remineralised enamel. For this reason, theoretical methods, including first-principles modelling, have become essential to mineral studies to comprehend and predict their properties. Combining computational modelling with Synchrotron X-Ray Diffraction (S-XRD) will allow us to understand and predict the crystallographic orientation and the chemistry of dental enamel as a function of acid attack and subsequent remineralisation processes. METHODS: We employed the first principles-based code, CASTEP, to carry out structural optimisations of enamel bulk models. Elemental substitutions with strontium (Sr2+), magnesium (Mg2+) and tin (Sn2+) ions were performed with bulk models, and their formation energies (Ef) were calculated. For the laboratory experiments, healthy human tooth specimens were divided into three groups: healthy enamel (control); artificially demineralised erosive surface lesion; remineralised lesion using a remineralising solution containing: i) Ca2+ and PO43- ii) plus Sr2+ ions. Tooth samples were analysed using a scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS). Later, tooth samples were sliced and carefully polished to ~50 μm for S-XRD measurements. Regions of interest near the enamel surface were mapped using XRD with an incident energy of 15 keV and a beam spot size of ~4 μm. The 002 reflection azimuthal curves from each diffraction pattern were fitted using the Gaussian function with an in-house built software. The crystallites’ alignment was determined using the peak width analysis. RESULTS AND DISCUSSION: Serial replacements of Ca2+ ions in enamel models were performed, and their Ef values indicated that substitutions of Mg2+ and Sn2+ are thermodynamically unstable, while the Sr2+ substitution is favourable. Following the positive developments in computational modelling, Sr2+ was used as a remineralising ingredient. SEM images showed that, compared to the control, the crystallites formed were much broader following the treatment with a remineralisation (Sr2+ ions) solution. EDS analysis of these newly formed crystallites exhibited an Sr2+ peak, indicating the incorporation of Sr2+ in the remineralised enamel surface. CONCLUSION: In this study, we employed first-principles methods and advanced laboratory techniques to study the effect of substituting different ions in the hydroxyapatite lattice. The best candidate for enamel repair is Sr due to the negative formation energy and formation of acid-resistant crystallites. W1 - 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, Edinburgh, UK, 2Brazilian Centre for Physics Research, Rio de Janeiro, Brazil, 3School of Physics and Astronomy, University of Edinburgh, Edinburgh, UK, 4Instituto Ortopedico Rizzoli, Bologna, Italy, 5Dept of Chemistry, University of Bologna, Bologna, Italy. INTRODUCTION: For hundreds of millions of years, nature has evolved a large assortment of organic-inorganic hybrid materials that are optimally adapted for a wide range of functions, including navigation, protection, mechanical support and protection. These materials not only exhibit exceptional material properties but also display multifunctionality, including features such as adapting, sensing and self- healing. Among the most remarkable biominerals found in nature are the shells of the phosphatic brachiopods Discinisca tenuis and Lingula anatina. These shells are constituted of an inorganic phase, composed of carbonate-substituted fluorapatite in the form of francolite that comprises ~68 % of the shell weight and an organic fraction that is composed of proteins, glycosaminoglycans and chitin and comprises the remaining 32% of the shell weight. We previously reported in D. tenuis that the shells switch from harder than bone when dry, to flexible when hydrated. Although the L. anatina shells exhibit similar behaviour upon hydration, they have different structures to D. tenuis and as such, the mechanisms that underpin such changes in m

OCTOBER 2023 ICCBMT 14 W2 - Metabolic profiling of modern and fossilized mineralized tissues: The crystallite/mineral niche Timothy G Bromage*1,2, Christiane Denys3, Christopher Lawrence De Jesus4, Hediye Erdjument-Bromage4, Ottmar Kullmer2, Friedemann Schrenk2, Daniel J Buss5, Marc D McKee5, Eran Ittah6, Natalie Reznikov6, Gail M Ashley7, Oliver Sandrock8, Sher B Poudel1, Shoshana Yakar1, Bin Hu1, Sasan Rabieh1, Thomas A Neubert4. 1Dept of Molecular Pathobiology, New York University College of Dentistry, New York, USA, 2Dept of Paleoanthropology, Senckenberg Research Institute and Natural History Museum, Frankfurt, Germany, 3Inst of Systematics and Evolution of Biodiversity, National Museum of Natural History, Paris, France, 4Dept of Cell Biology, New York University Grossman School of Medicine, New York, USA, 5Dept of Anatomy and Cell Biology, and Dental Medicine and Oral Health Sciences, McGill University, Montreal, Canada, 6Dept of Bioengineering, McGill University, Montreal, Canada, 7Dept of Earth and Planetary Sciences, Rutgers, the State University of New Jersey, New Brunswick, New Jersey, USA, 8Earth and Life History, Hessisches Landesmuseum Darmstadt, Darmstadt, Germany. INTRODUCTION: Metabolic profiling is typically performed on whole blood/plasma and its transudates urine and saliva. However, it can also be performed on hard tissues such as bone, where mineral crystallite niches harbor metabolites once in the circulation. Remarkably, metabolites in these niches represent a serum transudate entombed within the mineralized extracellular matrix of bone during its formation and that putatively survive in the nanoscopic pool of structural “bound” water occurring in these ultrastructural locations. PURPOSE: To innovate metabolism-characterizing biomolecular strategies for metabolic profiling of modern and paleontological bone and tooth tissues through ultrafine-scaled characterization of mammalian metabolisms that i) reflect internal physiologic responses to environmental conditions, and ii) reveal dietary fuelling of metabolic rate. METHODS: Modern and fossil specimens were subject to acid extraction protocols preserving biomolecule integrity. Liquid samples were centrifuged and the methanolic supernatant transferred to Liquid Chromatography-Tandem Mass Spectrometry for analysis. We used a QExactive HF-X mass spectrometer coupled directly to a Vanquish UHPLC system (ThermoScientific) operated in positive and negative ion modes in data-dependent acquisition mode. Raw mass spectrometry data were subject to database searches and all metabolites comprehensively annotated. Ingenuity Pathway Analysis (IPA; QIAGEN) was employed for analyses of mammalian endogenous and non-mammalian exogenous metabolites (e.g., from food). Soil from fossil sites was analyzed for metabolite contaminants. RESULTS: Modern and 1-3 million year old African fossil bones and teeth were analyzed from several mammal species. Roughly 2000-7000 metabolites were detected from <500 mg of material from many samples; with quality control steps and corrections for contaminants, this was reduced to hundreds for IPA. Modern and fossil endogenous metabolites reflect biological functions, and the paleometabolome showed hits for fur coloration, growth, and health. In one fossil ground squirrel, metabolites related to estrogen biosynthesis and signaling, confirming it to be a female. Exogenous metabolites of laboratory- reared mice highlight coupling of metabolic profiles to their food consumption. From fossils, the exogenous paleometabolome indicated soil bacteria, a parasitic infection (Trypanosoma brucei), and insect, fungi, and monocotyledon and dicotyledon consumption. Taken together, reconstruction of the ancient environment indicates temperatures of 16-35 deg. C, 500-700 mm annual rainfall, well-drained loamy pH 6-8 soils of various textures, and open sunny conditions. CONCLUSIONS: Benefits of metabolic profiling from mineralized tissues will inform organismal paleoecology, particularly of those species without modern analogues. In addition, it will enhance reconstructions of locomotor and feeding niches afforded by informed environmental reconstruction. W3 - Mineral and fiber/organic assemblies in the gekkotan eggshell in 3D as characterized by submicron X-ray tomography and FIB-SEM serial sectioning Joseph Deering*1, Valentin Nelea1, Marc D McKee1,2. 1Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC, Canada, 2Dept of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada. INTRODUCTION: Mineralized eggshells oviposited by birds and reptiles provide a rigid and durable exterior, resist pathogen ingress, and facilitate water and gaseous exchange for the growing embryo. Whereas avian eggshells are highly mineralized with well-organized columnar calcite structure emanating from their fibrous organic membrane, the mineralized eggshells of many squamate reptiles lie in stark contrast to those of avi

OCTOBER 2023 ICCBMT 14 W4 - The shell calcitic prismatic layer of Pinna nobilis and its protein repertoire Frédéric Marin*1, Benazir Khurshid1,2, Daniel J Jackson3, Matt Harrington4, Sébastien Motreuil1, Cédric Broussard5, Asher Schmidt6, Stephan Wolf7, Jonathan Perrin2, Daniel Vielzeuf8. 1UMR CNRS-EPHE 6282 Biogeosciences, University of Burgundy, Dijon, France, 2Synchrotron SOLEIL, Gif-sur-Yvette, France, 3Dept Geobiology, Georg-August University, Gottingen, Germany, 4Dept Chemistry, McGill University, Montreal, Canada, 5Plateforme proteomique 3P5, University Paris Cité, Paris, France, 6Schulich Faculty of Chemistry, Technion, Haifa, Israel, 7Dept Materials Science & Engineering, Friedrich-Alexander-University, Erlangen, Germany, 8Laboratoire CINaM-CNRS, Aix-Marseille University, Marseille, France. INTRODUCTION: Mollusk shells are characterized by the superimposition of two to four calcified layers of well-defined microstructure. The “simple calcitic prisms”, as defined by the classical treatise on shell microstructures from Taylor, Kennedy and Hall (1969), represent one of them. They have been particularly well studied in the Mediterranean fan mussel, Pinna nobilis: recent theoretical models on the formation of simple prismatic layers give a prominent place to physical principles without involving ‘biology’. Although these views tend to describe satisfyingly the geometry of simple prisms layers, they ignore the presence of associated organics around and within the prisms. PURPOSE: The scope of our study is to identify as thoroughly as possible the protein constituents of the prismatic layer of Pinna nobilis. METHODS: Few living juvenile specimens of Pinna nobilis were collected and kept and fed in aquariums for few days before being sacrificed. Their mantle tissues were collected and used for the production of a transcriptome of high quality. In parallel, the shell matrix associated to the prismatic layer was extracted and proteomics was performed on different extracts (soluble and insoluble). In addition, one specific protein, accripin11, was further studied. RESULTS: We obtained the full sequences of hundreds of proteins that are associated to the prismatic layer of Pinna nobilis. While some proteins can be assigned a putative function owing to their sequences, many of them - in particular proteins with domains of low complexity (i.e., domains that exhibit biased amino acid composition) - do not exhibit clear homology with proteins of better-known systems. CONCLUSION: In a counterintuitive manner, the deposition of the 'simple' calcitic prisms of the Mediterranean fan mussel Pinna nobilis requires an extremely complex assemblage of proteins, many of which exhibiting compositionally biased sequences. Our data stress the inadequacy of existing models, emphasize novel molecular functions in biomineralization, urge to conciliate biology and physics and raise questions on the functions of proteins with low complexity domains. W5 - Spiralled structure of narwhal tusk studied by multimodal hierarchical imaging A Rodriguez-Palomo*1,2, J Palle2, E Garde3, PA Vibe2, TEK Christensen2,4, NK Wittig2, MRV Jørgensen2,5, I Kantor5,6, M Burghammer7, J Liu7, K Jakata7, P Cook7, JT Avaro8, C Appel9, LC Nielsen1, MP Heide-Jørgensen3, M Liebi1,9,10, H Birkedal2. 1Chalmers University of Technology, Gothenburg, Sweden, 2Aarhus University, Aarhus, Denmark, 3Greenland Institute of Natural Resources, Nuuk, Greenland, 4Sino-Danish Center, University of Chinese Academy of Sciences, 5MAX IV Laboratory, Lund University, Lund, Sweden, 6Technical University of Denmark, Lyngby, Denmark, 7European Synchrotron Radiation Facility (ESRF), Grenoble, France, 8Swiss Federal Laboratories for Materials Science and Technology (EMPA), St. Gallen, Switzerland, 9Paul Scherrer Institute, Villigen, Switzerland, 10Federal Polytechnique University of Lausanne (EPFL), Lausanne, Switzerland. INTRODUCTION: Narwhals are fascinating animals whose left-handed spiral tusk has caught the attention of humankind for centuries and has been associated with the unicorn myth. The twisted tusk, which grows principally in males, has highly anisotropic mechanical properties and an extraordinarily high impact resistance [1]. We hypothesize that the orientation of the biological nanostructure reflects the spiral macro-structure, which defines the mechanical properties. METHODS: We combined 2D and 3D imaging techniques using X- rays and polarised visible light to study its hierarchical structure at multiple length scales. Small-angle X-ray scattering and diffraction imaging [3], X-ray fluorescence, and birefringence microscopy were used to investigate the anisotropy of the tusk building blocks (i.e., mineralised collagen fibrils) from the nano to the macro scale. RESULTS AND DISCUSSION: Narwhal tusks have a central pulp chamber surrounded by primary dentine and a layer of cementum. Unlike human teeth, no enamel is present [2]. The building blocks of the narwhal cementum a

OCTOBER 2023 ICCBMT 14 W6 - New insights into the nature of osteodentin Ron Shahar*1, Josh Milgram1, Senthil Thangadurai1, Paul Zaslansky2, Emeline Raguin3. 1Laboratory of Bone Biomechanics, Koret School of Veterinary Medicine, Faculty of Agriculture, The Hebrew University of Jerusalem, 2Dept for Restorative and Preventive Dentistry, Charité- Universitaetsmedizin, Berlin, Germany, 3Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. INTRODUCTION: The teeth of actinopterygian fish, like those of mammals, consist of a thin outer hyper-mineralized layer (enamel or enameloid) that surrounds a core of dentin. While all mammalian species have a single type of dentin (called orthodentin), various dentin types have been reported in the teeth of fish. The most common type of fish dentin is orthodentin. However, the second most common type of fish dentin, called osteodentin and found in several teleost species and in many Selachians, is structurally radically different from orthodentin. Osteodentin, comprising denteons and inter-denteonal matrix, is grossly similar to mammalian osteonal bone (hence its name), although it lacks cells and a lacuno-canalicular system. The current consensus is that although osteodentin is morphologically different from orthodentin, it is a true dentinal material, the product of odontoblast cells. PURPOSE: To characterize osteodentin at nm resolution in 3D in order to gain insights into the identity of this material – bone, dentin or an intermediate tissue. METHODS: We performed a high-resolution study of osteodentin found in the teeth of the Atlantic wolffish, Anarhichas lupus, using a variety of microscopy techniques, including various microscopy methods, high resolution microCT scans and FIB/SEM. RESULTS: The results provide a detailed description of the three-dimensional structure of osteodentin at several length-scales. We found that the 3D arrangement of the mineralized collagen fibrils in osteodentin is remarkably similar to their arrangements in osteonal mammalian bone. We also detected a dense network of nanopore/nanochannels, as has been recently reported for the bone material of several mammals. CONCLUSIONS: We conclude that it is much more likely that osteodentin is bony tissue rather than true dentin, or an intermediate tissue, although definitive proof would require identification of the cells secreting the osteo-dentinal matrix – whether osteoblasts or odontoblasts. These findings confirm the close evolutionary origin of dentin and bone. Acknowledgements: This work was funded by the Israel Science fund (ISF), grant # 700/17. W7 - Specific genes for calcification of the red coral corallium rubrum: Identification and evolution deduced from tissue expression and phylogeny Philippe Ganot*1,2, Markus Fritz4, Tobias Rausch4, Didier Zoccola2, Didier Aurelle5, Anne Haguenauer5, Vladimir Benes4, Denis Allemand1,3, Sylvie Tambutté1,2. 1Unité de Recherche sur la Biologie des Coraux Précieux CSM - CHANEL, 2Laboratoire de Physiologie et Biochimie, Centre Scientifique de Monaco, 3Centre Scientifique de Monaco, Monaco, 4European Molecular Biology laboratory, Germany, 5Institut Méditerranéen de la Biodiversité et d’Ecologie marine et continentale, CNRS, Marseille, France. INTRODUCTION: During evolution, the genetic path leading a tissue to acquire new function includes a) Horizontal Gene Transfer (HGT, integration of an exogenous gene acquired from another organism), b) co-option (sharing the product of a gene originally expressed in another tissue), c) sub-functionalization (duplication and expression of a gene from a gene expressed elsewhere), and/or d) neo- functionalization (production of a novel gene function, e.g. by associating previously unrelated protein domains in the same gene or by accumulating mutations). PURPOSE: The precious red coral Corallium rubrum (Octocorallia) acquired calcification independently from the reef-building corals (Hexacorallia) during post-Ediacaran evolution, but how did the red coral acquired the faculty of calcifying an axial skeleton? In this study, we restricted our analysis to our previously identified 102 proteins components of the skeletal Organic Matrix (OM). METHODS: After tissues dissection (calcifying and non-calcifying) and RNA extraction, we assembled and annotated the reference C. rubrum transcriptome. Using RNAseq with stringent cut-off criteria, we deduced tissue gene expression. Genes expressed in the calcifying epithelium were sorted according to being ubiquitous (expressed everywhere), preferential (also expressed in other tissues, albeit to a much lesser extend) and specific (not expressed in other tissues). Next, using sequence homology and phylogeny, we reconstructed individual gene families. Combined with gene expression profiles, we were then able to deduce evolutionary scenario of our genes of interest. RESULTS: Among the total 28,310 annotated C. rubrum transcripts, 510 were preferentially expressed in the calcifyi

OCTOBER 2023 ICCBMT 14 W8 - Amorphous phase distribution in the side of the stomatopod dactyl club Thorbjørn Erik Køppen Christensen*1,2,3,4, Innokenty Kantor2,5, Mads Ry Vogel Jørgensen1,2, Olof Gutowski6, Ann-Christin Dippel6, Peter Alling Strange Vibe2, Nina Kølln Wittig2, Maja Østergaard2, Alexander Bernthz Jensen2, Henrik Birkedal2. 1Dept of Chemistry and interdisciplinary Nanoscience center (iNANO), Aarhus University, Aarhus Denmark, 2DanMAX, MAX IV Laboratory, Lund University, Lund, Sweden, 3Section for Visual Computing, Dept of Applied Mathematics and Computer Science, Technical University of Denmark, Lyngby, Denmark, 4Sino-Danish Center, University of Chinese Academy of Sciences, 5Dept of Physics, Technical University of Denmark, Lyngby, Denmark, 6Deutsches Elektronen- Synchrotron DESY, Hamburg, Germany. INTRODUCTION: The stomatopod is a fascinating animal. Accelerating its weaponized appendage clubs with more than 100 000 m/s^2 allows it to break the shells of its prey [1]. While the shell and club are fundamentally made of the same components, the intricate structure of the dactyl club allows it to withstand multiple uses overcoming the tremendous forces of each hit. There are four major structural zones of the dactyl club, the impact region, impact surface, the striated region, and the periodic region [1,2]. The impact surface and region consists of a mix of hydroxyapatite (HAp) and chitin, while the periodic and striated region consists of mixture of chitin, amorphous calcium phosphate (ACP) and amorphous calcium carbonate (ACC). We hypothesize that the distribution of ACC and ACP in the periodic region is not uniform across the region, but rather that it is highly controlled between different areas of the periodic region. METHODS: Using a combination of lab-based μ-computed tomography (CT), X-ray diffraction (XRD) mapping, and XRD-CT [3], multiple clubs were investigated. The high brilliance of synchrotrons enables the study of the distribution of ACC and ACP through pair distribution function (PDF) analysis of XRD data while XRD-CT makes it possible to map the 3D distribution of crystalline phases. three clubs were scanned using XRD and PDF mapping at DanMAX at MAX IV, and one was scanned using XRD-CT at P07 at DESY. Lab-based μ-CT was used to scan multiple clubs, and identify differences in density across their periodic regions. RESULTS: Mapping of the periodic regions of the dactyl club showed that the periodic region sometimes forms large calcite crystallites, however the morphology and distributions of the crystallites vary between specimen, as does whether they appear at all. This indicates that the structure of the dactyl club has some flexibility, as it only needs to last through one molting cycle. Further investigation of the amorphous phases, show that the amorphous material in the periodic region in the side of the club, also called the lateral periodic region, differs from the periodic region in the front of the club, also called the medial periodic region. This difference in the amorphous phase could explain why large calcite crystallites are only seen in the lateral periodic region. The club having HAp, calcite, ACP, and ACC in such close proximity to each other shows a great level of control over the different phases in the club. CONCLUSIONS: These results show that the distribution of amorphous phase is controlled in the stomatopod dactyl club. This allows locally higher and lower levels of ACC and ACP depending on the needs. This control is present even though there is a high level of flexibility in the structure of the dactyl club. [1] S.N. Patek et al, 10.1038/428819a [2] J.C. Weaver et al, 10.1126/science.1218764 [3] S. Frølich et al, 10.1107/S1600576715022633 W9 - A unique type of syndromic amelogenesis imperfecta sheds light on the mechanism that leads to enamel rod decussation Olivier Duverger*1, Sean K Wang1, Quynh C Nguyen1, Priyam H Jani1, Fabian Mandoza1, Yan Wang2, Daniel Martin3, Pamela A Frischmeyer- Guerrerio4, Janice S Lee1. 1Craniofacial Anomalies and Regeneration Section, 2Mass Spectrometry Facility, 3Genomics and Computational Biology Core, NIDCR, NIH, Bethesda, MD, USA. INTRODUCTION: Amelogenesis Imperfecta is generally characterized by reduction of enamel thickness or mineralization, or a combination of both. We recently reported that patients with Loeys-Dietz syndrome type II, caused by mutations in TGFBR2 (gene encoding TGF-beta receptor 2), exhibit severe enamel anomalies with unclear etiology. PURPOSE: The objective of this study was to further characterize the nature of the enamel defects caused by mutations in TGFBR2 and to use a mouse model to investigate the cellular and molecular mechanism that leads to these defects. METHODS: Panoramic X-rays from patients before and after second molar eruption were evaluated. Deciduous teeth from patients and teeth from a mouse model of the disease were analyzed by micro-computed tomography, scanning electron mic

OCTOBER 2023 ICCBMT 14 W10 - Heterozygous variants in MGP lead to endoplasmic reticulum stress causing spondyloepiphyseal dysplasia Ophélie Gourgas1 , Gabrielle Lemire2,3,4, Alison L Eaton2,5, Sultanah Alshahrani6, Angela L Duker7, Jingjing Li1, Ricki S Carroll7, Stuart Mackenzie7, Sarah M Nikkel8, Michael B Bober7, Kym M Boycott2,3, Monzur Murshed1,6,9. 1Dept of Medicine, McGill University, Montreal, QC, Canada, 2Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, ON, Canada, 3Dept of Genetics, Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada, 4Broad Center for Mendelian Genomics, Program in Medical and Population Genetics, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA, 5University of Alberta, Edmonton, AB, Canada, 6Faculty of Dentistry, McGill University, Montreal, QC, Canada, 7Nemours Children’s Health, Wilmington, DE, USA, 8University of British Columbia, Vancouver, BC, Canada, 9Shriners Hospital for Children, McGill University, Montreal, QC, Canada. INTRODUCTION: Matrix Gla protein (MGP) is a vitamin K-dependent post-translationally modified protein, highly expressed in vascular and cartilaginous tissues. It is a potent inhibitor of extracellular matrix mineralization. Biallelic loss of function variants in the MGP gene cause Keutel syndrome, an autosomal recessive disorder characterized by widespread calcification of various cartilaginous tissues and skeletal and vascular anomalies. We recently reported four individuals from two unrelated families with two heterozygous variants in MGP, both altering the Cysteine 19 residue to phenylalanine or tyrosine (C19F or C19Y, respectively). These individuals presented with a spondyloepiphyseal skeletal dysplasia characterized by short stature with a short trunk, diffuse platyspondyly, midface retrusion, progressive epiphyseal anomalies and brachytelephalangism. In this study, we examined the underlying cellular and molecular mechanisms by which the novel C19F mutation causes abnormal calcification of the growth plates and a new form of skeletal dysplasia. PURPOSE: To investigate the cellular and molecular effects of one of the novel heterozygous deleterious variants (c.MGP 56G>T; p.MGP C19F) of MGP using both cell culture and genetically modified mouse models. METHODS: We transfected ATDC5 chondrogenic cells and HEK293 cells with vectors expressing C19F MGP and examined intracellular localization, secretion and processing of the mutant protein using immunofluorescence, Western blotting and mass spectrometry. Using a CRISPR/Cas9-based approach, we introduced the MGP 56G>T mutation in mice. The skeletal anomalies including premature calcification of the growth plates were investigated using micro-CT, histomorphometry, marker gene analyses and cell death assays. RESULTS: Heterozygous ‘knock-in’ mice expressing C19F MGP recapitulated most of the skeletal anomalies observed in the affected individuals carrying the same mutation. We demonstrated that the main underlying mechanism leading to the observed skeletal dysplasia is endoplasmic reticulum stress-induced apoptosis of the growth plate chondrocytes. In addition, premature calcification of the growth plates and increased differentiation of chondrocytes may act as secondary causes leading to skeletal deformities and low bone mass accrual. CONCLUSION: Our findings support that heterozygous variants in MGP altering Cys19 residue cause autosomal dominant spondyloepiphyseal dysplasia, a condition distinct from Keutel syndrome both clinically and molecularly. W11 - Aortic valve mineralization: A detailed look using correlative Raman-EM imaging Robin HM van der Meijden*1,2, Carolien Kuster1,3, Amber van Broekhoven3, Wieteke Broeders4, Niels Riksen4, Jan-Hein Cornel3,5,6, Saloua El- Massaoudi3, Nico Sommerdijk1,2, Anat Akiva1,2. 1Dept of MedicalBioSciences, Radboudumc, Nijmegen, the Netherlands, 2Electron Microscopy Centre, Radboudumc, Nijmgen, The Netherlands, 3Dept of Cardiology, Radboudumc, Nijmegen, The Netherlands, 4Dept of Internal Medicine, Radboudumc, Nijmgen, The Netherlands, 5Dutch Network for Cardiovascular Research (WCN), Utrecht, The Netherlands, 6Dept of Cardiology, Northwest Clinics, Alkmaar, The Netherlands. INTRODUCTION: Aortic valve stenosis (AS) is the most common valve disease in Western countries and is mostly considered a disease of the elderly. To date, invasive aortic valve replacement is the only curative treatment for AS. Therefore, there is a clinical need to identify new therapeutic targets in the pathophysiology of AS to find an effective medical intervention to prevent or slow down the disease. Stenotic aortic valves present features such as lipid accumulation, inflammation, fibrosis, and progressive calcification. However, molecular details of the extracellular matrix (ECM) and the progressive calcification of the diseased tissue remain poorly understood. METHOD: In this study we correlate Raman micros

OCTOBER 2023 ICCBMT 14 W12 - Comparison of the effects of PTH (1-34), PTHrP (1-36) and abaloparatide (ABL) on the murine osteoblast transcriptome Michael J Mosca1,2, Zhiming He1, Florante R Ricarte1, Carole Le Henaff1, Nicola C Partridge*1. 1Dept of Molecular Pathobiology, New York University College of Dentistry, 2Vilcek Institute of Graduate Biomedical Sciences, New York University Grossman School of Medicine, New York, NY, USA. INTRODUCTION: Osteoporosis is a prevalent disease with substantial morbidity/mortality among the aging population. Due to gaps in knowledge, current therapeutics are limited in their ability to prevent degeneration of bone while also stimulating formation of new bone. Teriparatide (hPTH (1-34)) and its analogs PTHrP (1-36) and abaloparatide (ABL), have been utilized for treatment of osteoporosis but have significant limitations in efficacy over long-term use. PURPOSE: Research from our laboratory has shown PTH (1-34), PTHrP (1-36), and ABL exert time and dose-dependent differential responses in the osteoblast, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. METHODS: Differentiating mouse calvarial osteoblasts were treated with 1 nM of hPTH (1-34), PTHrP (1-36) or ABL for 4 h, RNA collected and either bulk RNAseq or qRT-PCR performed. Differential gene expression was performed, followed by Gene Ontology analyses for gene expression significantly above ±1.0 log2FC and a false discovery rate <0.05 after treatments. RESULTS: RNA-Sequencing revealed that hPTH (1-34) regulated 367 genes, 194 were unique; PTHrP (1-36) regulated 117 genes, 15 were unique; ABL regulated 179 genes, 20 were unique. There were 74 genes shared only among PTH(1-34) and ABL; 16 genes were shared only among PTH (1-34) and PTHrP; and 83 genes were shared among all three peptides. Gene ontology analyses show biological processes are similar between the three peptides but have some pathway-specific differences. Further analysis of the data illuminated that the three peptides increased expression of Vitamin D receptor (Vdr), Phosphodiesterase 10a (Pde10a), Cbp/p300-interacting transactivator 1 (Cited1), Wnt family member 11 (Wnt11), and Secreted frizzled related protein 4 (Sfrp4) mRNAs similarly to Rankl expression, i.e., in a differential expression pattern. These findings were confirmed via qRT-PCR of additional cultured samples of mouse calvarial osteoblasts, treated with 1 nM of hPTH (1-34), PTHrP (1-36), or ABL for 4 h prior to harvest. Additionally, we analyzed mRNA abundance of several genes of interest, including Wnt4, Wnt7, Dkk1, Kcnk10, Hdac4, Epha3, Tcf7, Fzd5, Pp2r2a, and Dvl3 based on genes and pathways chosen from ontology analyses to be significantly regulated by each peptide. CONCLUSIONS: Our findings highlight the complexity of the genetic and functional events triggered by PTH (1-34) and its analogs. Many studies have examined PTH signaling in the osteoblast/osteocyte; ours is the first to examine global effects of these three peptides on the osteoblast transcriptome. Further delineation of which signaling events are attributable to PTH (1-34), PTHrP (1-36), and ABL exclusively and which are shared among all three will further our understanding of the effects these peptides have on the osteoblast and lead to refinement of PTH-derived treatments for osteoporosis. W13 - Zinc in microscopic calcifications isolated from thyroid fine needle aspiration may serve as a biomarker of thyroid nodule malignancy Lotem Gotnayer1, Dina Aranovich1, Merav Fraenkel2,3, Uri Yoel2,3, Netta Vidavsky1,4*. 1Dept of Chemical Engineering, 2Faculty of Health Sciences, 4Ilse Katz Institute for Nanoscale Science & Technology, Ben-Gurion University of the Negev, Beer Sheva, Israel, 3Endocrinology, Soroka University Medical Center, Beer Sheva, Israel. INTRODUCTION: Thyroid nodules (TNs) are common neck ultrasonography (US) findings, yet only 5-10% of these nodules harbor thyroid cancer (TC). When US characteristics are consistent with an intermediate or high suspicion for TN malignancy, fine needle aspiration for cytology (FNAC) is indicated. The main limitation of FNAC is that cytological results can be indeterminate in up to 30% of cases, necessitating reevaluation through repeated FNAC, expensive molecular testing, or diagnostic thyroid lobe resection. As such, there is a need for further refinement of current diagnostic algorithms for TNs without subjecting patients to additional invasive procedures. PURPOSE: As calcifications detected during thyroid US are considered a high-risk feature for malignancy, we hypothesized that microcalcifications (MCs) could be obtained from the material remaining in the syringe at the end of the FNA procedure. Hence, we aimed to isolate these FNA MCs and analyze their crystal properties according to the TN malignancy. METHODS: Clinical samples were collected during routine FNAC procedures conducted at the endocrine unit of SUMC for patients with a

OCTOBER 2023 ICCBMT 14 W14 - Compromises in osteocyte lacunar canalicular network with diabetes and correlations with matrix properties Morgan Bolger, Tara Tekkey, David Kohn*. University of Michigan, Ann Arbor, MI. INTRODUCTION: Type 1 diabetics (T1D) have a 7X greater risk of hip fracture. The slightly lower BMD is insufficient to account for this increased risk, suggesting other bone properties are critical determinants. PURPOSE: We hypothesized that material properties are perturbed in diabetes and related to changes in the osteocyte lacunar canalicular network (OLCN). This study aimed to define the sequence of OLCN changes with diabetes and how these changes relate to bone composition and mechanical properties in the perilacunar zone and at the whole bone level. METHODS: 12 week C57BL/6J mice were administered STZ for 5d and sacrificed at baseline, 3 or 7 weeks after diabetic onset, with age matched controls. Right femora were stained with rhodamine, imaged and analyzed confocally for OLCN morphology using a Matlab script that converted images into skeletonized maps of topology. Z-stack images of lacunae were were computationally cleared and characterized using open-source code. Left femora were scanned via μCT and subjected to 4-point bending. Additional right femora were subjected to nanoindentation, distinguishing the perilacunar (PL) from intracortical (IC) regions. Raman measurements were taken in the PL and IC regions. 2-way ANOVAs assessed effects of disease and time and linear regressions related mechanical, compositional and OLCN metrics. RESULTS: Osteocyte canaliculi in bones of diabetic mice exhibited lower branch tortuosity relative to baseline. Branch order is a measure of how many bifurcations exist exiting the lacunae. Diabetic mice exhibited significantly lower # of 3rd, 4th and 5th order branches relative to baseline and controls. The total # of nodes (canalicular intersections) was significantly lower in diabetic mice relative to baseline. At the whole bone level, the # of nodes explained 45% of the variance in Ct.BV/TV and 31% of the variance in ultimate load. At the local level, OLCN measures were more predictive of PL mechanical properties and composition compared to the IC regions. The # of branches explained 22% and 30% of the variance in elastic work at the IC and PL regions, respectively. The # of branches explained 12%, and 16% of the proline/hydroxyproline ratio at the IC and PL regions, respectively. CONCLUSIONS: The OLCN is reduced in diabetes: fewer higher order branches around lacunae, decreased branch tortuosity and # of nodes. These compromises to the OLCN were absent changes in lacunae density or morphology and occurred prior to a reduction in BFR. The data suggest a temporal sequence of PL remodeling that initiates with OLCN alterations. The # of nodes in the OLCN positively correlated with BMD and ultimate load highlighting the importance of the OLCN in driving whole bone properties. At the local level, the # of branches surrounding individual lacunae negatively correlated with elastic work and correlated to proline/hydroxyproline, highlighting the interplay between the OLCN, local composition and mechanics in diabetes. W15 - The paradox of fragile but dense bones in Type 2 diabetes Eve Donnelly*1,2, Sashank Lekkala1, Heather B Hunt1, Kendall F Moseley3. 1Dept of Materials Science and Engineering, Cornell University, Ithaca, NY, USA, 2Research Institute, Hospital for Special Surgery, New York, NY, USA, 3Division of Endocrinology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. INTRODUCTION: Bone fracture risk is one of the myriad clinical complications of type 2 diabetes mellitus (T2DM). Counterintuitively, the increased fracture risk in T2DM occurs despite greater bone mineral density (BMD) and persists after accounting for potential confounders like body mass index, neuropathies, and falls. Although the mechanisms that underlie bone fragility in T2DM are not yet well established, a multiplicity of factors, including hyperglycemia, altered bone remodeling, and accumulation of advanced glycation endproducts (AGEs) in the bone matrix are implicated. PURPOSE: In this work we elucidate the factors that influence fragility in T2DM by characterizing the biochemical, material, microarchitectural, and mechanical properties of bone from clinical populations of men and postmenopausal women with and without T2DM (T2DM: n=25 women [age: 65 ± 7 y], n=31 men [65 ± 8 y]; non-DM: n=35 women [64 ± 5 y] n=34 men [age = 62 ± 9 y]). METHODS: AGEs were assessed using high-performance liquid chromatography to measure pentosidine and using multiphoton microscopy to spatially resolve the density of fluorescent AGEs (fAGEs). Mechanical properties were analyzed with compression testing (cancellous bone) and nanoindentation, and composition was analyzed with FTIR and Raman imaging. RESULTS: Across study populations, bone tissue from patients with T2DM had greater concentratio

OCTOBER 2023 ICCBMT 14 W16 - Alterations in bone matrix mineralization caused by the coexistence of osteogenesis imperfecta and hypophosphatasia Nadja Fratzl-Zelman*1,2, Agnès Linglart3, Kim Bin4, Frank Rauch5,6, Stéphane Blouin1,2, Régis Coutant7, Aurélie Donzeau7. 1Ludwig Boltzmann Institute of Osteology at Hanusch Hospital of OEGK and AUVA Trauma Centre Meidling, 1st Med. Dept Hanusch Hospital, Vienna, Austria, 2Vienna Bone and Growth Center, Vienna, Austria, 3AP-HP, Paris Saclay University, INSERM, OSCAR filière, EndoERN and BOND centers, Endocrinology and Diabetes for Children, France, 4Pediatric Orthopedic Surgery Angers University Hospital, Angers, France, 5Shriners Hospital for Children, Canada, Montreal, Canada, 6Dept of Human Genetics, McGill University, Montreal, Canada, 7Dept of Pediatric Endocrinology and Diabetology, Angers University Hospital, Angers, France. INTRODUCTION: Osteogenesis imperfecta (OI) and hypophosphatasia (HPP) are rare heritable skeletal disorders. OI is usually caused by mutations in or COL1A2 resulting in quantitative or structural abnormalities in type I collagen. HPP is caused by loss-of-function mutations in the gene encoding tissue-non-specific isoenzyme of alkaline phosphatase (ALPL). Both conditions lead to bone fragility with opposite effects on matrix mineralization: hyper- and hypomineralization in OI and HPP, respectively. Here we present bone tissue characteristics in a family with coexistence of OI and HPP. APPROACH & METHODS: The father and his 3 children had growth retardation, low bone mass and recurrent fractures. The most severely affected child presented with 9 fractures prior to 3 years, his twin siblings with 1 tibial fracture and cascades of vertebral fractures or with 4 fractures before the age of 6 years. None of them had blue sclera or dentinogenesis imperfecta. Genetic analysis revealed pathogenic mutations in COL1A2 (c.838G>A) and in ALPL (c.13333T>C) for all. After multidisciplinary meeting, diagnostic transiliac bone biopsy was indicated for the children. Bone tissue phenotype was assessed by histomorphometry and quantitative backscattered electron imaging (qBEI). RESULTS: Histomorphometry revealed in all three biopsy samples reduced cortical thickness and low trabecular bone volume, typical for OI. However, unlike OI, osteoid thickness, volume and mineralization lag time were highly increased in the very small and isolated trabeculae, indicating severe osteomalacia typical for HPP. Cortices consisted of well- mineralized lamellae aligned with the periosteal surfaces and few osteons with focal osteoid. Numerous osteoblasts and osteoclasts appeared on different cortical surfaces. Despite excessive osteoid formation, qBEI demonstrated that the mean trabecular calcium concentration was either normal, slightly decreased or even increased, compared to reference values for healthy children. However, the mineralized volume reported by combining osteoid and mineralized bone volume was similar in all three patients and lower than in healthy children or children with OI. Cortical bone matrix mineralization was similar in the three patients and higher than in healthy children. Osteocyte lacunae density was increased as in OI bone. CONCLUSIONS: Our bone analyses reveal classical features of OI such as low trabecular volume and elevated osteocyte lacunae density but also severe osteomalacia, a classical feature of HPP. The differences in bone matrix mineralization and osteoid deposition between cortical and trabecular bone indicate a shift from an OI phenotype during primary bone formation in the cortices to a very pronounced HPP phenotype in trabecular bone suggesting that the lack of ALP activity affects more severely mineralization of remodeled bone. Altogether this led us to treat the children first with recombinant alkaline phosphatase then with bisphosphonates. W17 - Physico-chemical characterization of minerals in cardiovascular tissues of senior body donors sheds light on cardiovascular calcification progression Raphaela Allgayer1, Adi Orlov2, Geoffroy Noël3, Marta Cerruti*1. 1Dept of Mining and Materials Engineering, McGill University, Montreal, Canada, 2Dept of Bioengineering, McGill University, Montreal, Canada, 3Dept of Surgery, University of California, San Diego, USA. INTRODUCTION: Cardiovascular calcification (CC) is the pathological deposition of calcium phosphate minerals on cardiovascular tissues. This significantly increases risk of heart failure, especially in seniors and patients affected by diabetes, chronic kidney diseases and atherosclerosis. There is no cure for CC; the only treatment in severe cases is surgery. Differences in underlying pathology and sex affect the localization of CC as well as mineral amount, morphology and phases. This complexity hinders our understanding of the mechanism of CC. Also, most studies rely on animal or simplified in-vitro models. Studies based on humans mostly analyze surgically removed explants, whic

OCTOBER 2023 ICCBMT 14 TH1 - Large scale comparison of mineralized tissues in wildtype and mutant mouse jaws: from semantic segmentation to extraction of metrics Victoria Cooley1, Ethan Suwandi1, Denis Keane1, William Guise2, Viktor Nikitin3, Pavel Shevchenko3, Adya Verma4, Tomas Wald4,5, Jeffrey O Bush5,6, Ophir Klein4,5,7,8, Stuart Stock9,10, Derk Joester*1. 1Dept of Materials Science and Engineering, Northwestern University, Evanston, IL, USA, 2DND-CAT Synchrotron Research Center, Northwestern University, Evanston, IL, USA, 3Advanced Photon Source, Argonne National Laboratory, Lemont, IL, USA, 4Dept of Orofacial Sciences, University of California San Francisco, San Francisco, CA, USA, 5Program in Craniofacial Biology, University of California San Francisco, San Francisco, CA, USA, 6Dept of Cell and Tissue Biology, University of California San Francisco, San Francisco, CA, USA, 7Dept of Pediatrics, University of California, San Francisco, San Francisco, CA, USA, 8Dept of Pediatrics, Cedars-Sinai Medical Center, Los Angeles, CA, USA, 9Dept of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA, 10Northwestern University, Simpson Querrey Institute, Northwestern University, Chicago, IL, USA. INTRODUCTION: Dental developmental defects, whether congenital, acquired, or environmental in origin, are associated with a significant cost to society and have profound psychological impacts.1 Despite significant progress over the last decade, the developmental processes that give rise to dentin and enamel remain incompletely understood. A recent, concerted effort to create an integrated, multi-modal set of tools, protocols, and reference materials/data for amelogenesis research resulted in a large set of mouse models including stage-specific Cre drivers, reporters, and conditional knockout and knock-in mutants.2 As part of this project, we collected more than 600 micro- computed tomography data sets at isotropic voxel sizes been 0.67 μm and 6 μm, using both synchrotron (85%) and lab (15%) sources, for a total of more than 250 reconstructions of jaws of 12 distinct genotypes in male and 16 genotypes in female mice.5. PURPOSE: To develop a workflow that ensures unbiased and reproducible analysis and comparison of 3D reconstructions across experimental groups at this scale. METHODS: Because mineral density gradients in the incisor prevent conventional approaches, we trained, validated, and deployed a 2D convolutional neural network (CNN) with u-net[3] architecture to semantically segment mouse jaws.[4] We further developed code to extract and compare metrics describing entire classes, such as bone or molar dentin, and, separately, the secretory and maturation stages of the continuously growing, curved incisor. RESULTS: Trained CNNs were found to segment wildtype enamel, and mutants with hypoplastic and hypomineralized enamel as well as ectopic mineralization with high fidelity (>95% overlap and boundary accuracy). Automated comparison of class features such as mean values and mineral density distributions was successful. Automated extraction of the radius of curvature of the incisor and the position of landmarks enabled rapid comparison of the onset, duration, and rate of enamel deposition in secretory stage and of mineral densification in secretory stage as a function of the arc length. CONCLUSION: CNN-based segmentation and automated quantification is a versatile tool that we hope will empower enamel researchers, delineate mechanisms of disease, and enable the development of new approaches of intervention. REFERENCES [1] Klein, et al. Int J Oral Sci 2017, 9, e3; [2] EnamelBase: A Consortium of Amelogenesis Models, 2023, in https://www.nidcr.nih.gov/sites/default/files/2022-04/2022-UG3- EnamelBase-Flyer.pdf; [3] Ronneberger et al., MICCAI 2015, Springer International Publishing, Cham, 2015, pp. 234-241; [4] Cooley et al, in SPIE Opt. Eng. + App. Vol. 11840, SPIE, San Diego, CA, 2021. [5] This work was supported in part by NIH UG3DE028872 and NSF DGE- 1842165, and used resources of the Advanced Photon Source, a U.S. Dept of Energy (DOE) Office of Science User Facility operated by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. TH2 - Osteopontin-derived phosphopeptide inhibits in vivo calcium oxalate formation in Drosophila melanogaster Polycronis P Akouris1,2, John A Chmiel1,2, Gerrit A Stuivenberg1,2, Wongsakorn Kiattiburut1,2, Jennifer Bjazevic3, Hassan Razvi3, Bernd Grohe1, Jeremy P Burton1,2,3, Kait F Al1,2, Harvey A Goldberg*4. 1Canadian Centre for Human Microbiome and Probiotics, 2Dept of Microbiology and Immunology, 3Division of Urology, Dept of Surgery, 4Depts of Dentistry and Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON, Canada. INTRODUCTION: Kidney stone disease affects nearly one in ten individuals and has a recurrence rate of >50%. The majority of stones are calcium based with approximately

OCTOBER 2023 ICCBMT 14 TH3 - Role of Claudin-10 in amelogenesis The Nghia Nguyen*1, Sandy Ribes1, Caroline Andrique1, Jerome Bouchet1, Magalie Requin2, André Le Bivic2, Pascal Houillier3,5, Dominik Muller4, Claire Bardet1, Thibaud Coradin6, Fernando Ramirez Rozzi1,7, Tilman Breiderhoff4, Catherine Chaussain1,5. 1URP2496, Laboratory Orofacial Pathologies, Imaging and Biotherapies, Dental School, Université Paris Cité, France, 2IBDM, UMR 7288, Faculté des Sciences de Luminy, Marseille, France, 3Centre de Recherche des Cordeliers, INSERM, CNRS, Université de Paris, France, 4Dept of Pediatric Nephrology, Charité University School of Medicine, Berlin, Germany, 5AP-HP, Reference Centers for rare diseases of the kidney and of the metabolism of calcium and phosphorus, Paris, France, 6Sorbonne Université, CNRS, Laboratoire de Chimie de la Matière Condensée de Paris, France, 7Eco-anthropologie (EA), Muséum national d’Histoire naturelle, CNRS, Université de Paris, Paris, France. INTRODUCTION: Claudin-10, a tight junction (TJ) protein, is expressed in the thick ascending limb of Henle’s loop, in the skin and in salivary glands. We previously reported that six patients from two unrelated families with the HELIX (Hypohidrosis, Electrolyte imbalance -renal loss of NaCl with secondary hyperaldosteronism and hypokalemia-, HypoLacrymia, Ichthyosis, Xerostomia) syndrome due to mutations in CLDN10 gene displayed severe enamel wear. PURPOSE: To determine whether enamel wear associated with the HELIX syndrome is secondary to Xerostomia or electrolyte dysbalance or also caused by abnormal enamel formation due to the absence of claudin-10 in the enamel organ, we investigated 1) the pattern of claudin-10 expression in the forming tooth, and 2) the consequences of its absence on amelogenesis. METHOD: We studied amelogenesis in Cldn10 KO mice, which die at post-natal day 1 (PND1) by combining several methods (histology and immunohistochemistry, stimulated emission depletion (STED), transmission electron microscopy (TEM) with immunogold, RNA sequencing after laser capture microdissection). We also implanted KO and WT molar germ in the kidney capsule of adult WT male mice and characterized the enamel at 2 months upon implantation (scanning electron microscopy (SEM), Energy Dispersive X-Ray Analysis (EDX), Raman micro-spectroscopy). RESULTS: Immunohistochemistry performed on WT molars and incisor showed that claudin-10b was expressed in the enamel organ at all the stages of amelogenesis, mainly in the stratum intermedium and, at a much lesser degree, in ameloblasts. These observations were confirmed by STED and TEM. Histological analyses revealed that, in PND1 Cldn10 KO mice, the ameloblast layer displayed a chaotic organization; TEM further showed that the proximal TJs were significantly enlarged compared to WT(p<0.001). The expression of enamel matrix proteins was similar in WT and Cldn10 KO mice. RNA sequencing analysis showed that the Notch and Wnt pathways were changed in the stratum intermedium in KO samples. Tooth germ culture of Cldn10 KO mice were next carried in the kidney-capsule of adult WT mice to study enamel formation independently of kidney and saliva dysfunction. At 2 months, SEM showed comparable enamel formation in KO and WT germs, with well-distinguishable prism decussation in both groups. In addition, EDX showed similar Ca/P ratio between the two groups (1.43 vs 1.53; p=0.132) while Raman micro-spectroscopy did not evidence differences in enamel mineral composition. CONCLUSION: claudin-10 is expressed in the enamel organ and amelogenesis is impaired in KO mice but not in KO germs implanted in the kidney capsule, suggesting that the enamel defects observed in patients with CLDN10 mutations may mainly result from the renal dysfunction or insufficient saliva. PERSPECTIVE: The ongoing generation of tamoxifen-inducible KRT14-Cre Cldn10 KO mice will allow the characterization of all the stages of amelogenesis in the absence of Claudin-10. TH4 - Tooth root organoids as models to study dental tissue regeneration Tia Calabrese*1, Kristi Rothermund*2, Claire M Gabe*1, Elia Beniash*1-3, Lance Davidson*1-6, Fatima N Syed-Picard*1-3. 1Dept of Oral and Craniofacial Sciences, University of Pittsburgh, 2Dept of Bioengineering, University of Pittsburgh, 3McGowan Institute of Regenerative Medicine, University of Pittsburgh, 4Dept of Developmental Biology, University of Pittsburgh, 5Dept of Computational and Systems Biology, University of Pittsburgh, 6Biomedical Engineering, Carnegie Mellon University. INTRODUCTION: Challenges in regenerating the tooth root lie in controllably rebuilding its the multiple unique constituent tissues in a spatially-organized manner and achieving functional integration with surrounding bone. Organoids are three-dimensional (3D) engineered constructs generated by stem cells that self-assemble into multiple spatially-organized tissues. Our group has previously developed dentin- pulp complex and periodontal

OCTOBER 2023 ICCBMT 14 TH5 - Lactation is associated with changes in mouse bone cellular and sub-cellular network architecture T Tang*1, A Munoz2, J. Huo1, A Gourrier3, A Carriero2, K Grandfield1. 1Dept of Materials Science and Engineering, McMaster University, ON, Canada, 2Biomedical Engineering Dept, The City College of New York, NY, USA, 3University Grenoble Alpes, CNRS, LIPhy, Grenoble, France. INTRODUCTION: Being the major reservoir for mineral, our bone is under constant remodeling to adapt to different biological and mechanical demands. This renewal process is effectively orchestrated by bone cells, in particular osteocytes. (Bonewald JBRM 2011) One unique characteristic of osteocytes is their formation of a highly organized lacunocanalicular network (LCN), a fluid-filled unmineralized structure that perfuses bone matrix and consists of osteocyte lacunae interconnected by nanometer-sized canaliculi. (Buenzli & Sims Bone 2015) Studies have shown that the LCN may play a role in remodeling the perilacunar/canalicular space, a process known as osteocytic osteolysis (OO) that contributes to bone homeostasis. (Teti & Zallone Bone 2009) However, the concept of OO has been long debated and whether osteocytes can directly resorb and replace the local bone matrix remains controversial. PURPOSE: To better understand the OO process and the underlying mechanisms, the current study aims to examine the potential architectural changes in bone LCN in three- dimension (3D) using lactating mice that have been shown to exhibit large reversible mineral depletion. METHODS: Three groups of female C57BL/6 mice were used for the study, including pregnancy (14-wk-old), lactation (16-wk-old), and recovery (17-wk-old). All groups have age-matched controls. Backscattered electron imaging (BEI) was first used to examine the transverse section of the mouse femoral cortical bone. Selected areas in endosteal and mid-cortical bone regions were further investigated with 3D focused ion beam-scanning electron microscopy (FIB-SEM) to reveal the structural features at the sub-cellular level (spatial resolution of ~5 nm, imaging volumes of ~15 x 5 x 5 μm3). RESULTS: Backscattered electron imaging has revealed altered and disorganized LCN on the anterior aspect of endosteal cortical bone in the lactation and recovery groups compared to the pregnancy group. 3D FIB-SEM imaging found a sub-canalicular, nanochannel network structure that is much more extensive and refined than the well-known osteocyte canaliculi (~30 nm vs 300 nm in diameter) in all examined samples. This unmineralized, interconnected nanostructure resembles the recently reported nanochannels in mineralizing turkey leg tendon, (Zou et al. PNAS 2020) mouse bone-cartilage interface, (Tang et al. Adv NanoBiomed Res 2022) and human femoral cortical bone. (Tang et al. JBMR 2022) Interestingly, increased volume percentage of nanochannels were found in endosteal cortical bone compared to mid-cortical bone in all three groups. Further, the lactating mice showed overall elevated nanochannel volume percentage compared to the other two groups. CONCLUSIONS: These preliminary data provide new evidence for a heterogeneous nanoscale mineral tissue distribution associated with lactation and yield new insight into potential calcium exchange and transport in bone associated with osteocytic osteolysis at the sub-canalicular level. TH6 - Native and recombinant bone sialoprotein improves alveolar bone healing in mice Michael B Chavez*1,2, Michelle H Tan1, Natalie L Andras1, Tamara N Kolli1, Emily Y Chu3, Harvey A Goldberg4, Brian L Foster1. 1Division of Biosciences, The Ohio State University College of Dentistry, Columbus, OH, USA, 2University of Iowa College of Dentistry and Dental Clinics, Iowa City, IA, USA, 3Dept of General Dentistry, Operative Division, University of Maryland School of Dentistry, Baltimore, MD, USA, 4Dept of Dentistry and Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Canada. INTRODUCTION: Bone sialoprotein (Gene: Ibsp/protein: BSP) is an extracellular matrix protein associated with mineralized tissues and shown to promote mineralization via evolutionarily conserved amino acid domains. BSP includes a collagen-binding motif, 2-3 polyglutamic acid hydroxyapatite-nucleating domains, and an RGD integrin-binding sequence; post-translational modifications (PTMs) including serine phosphorylation and O/N-linked glycosylations also contribute to mineralization functions. Ibsp knockout (Ibsp-/-) mice exhibit defective alveolar bone mineralization and healing following the challenge of first molar tooth extraction. We aimed to determine whether exogenous BSP could affect socket healing in mice. We hypothesized BSP would rescue defective alveolar bone healing in Ibsp-/- mice. METHODS: First maxillary molars were bilaterally extracted from 42 days postnatal Ibsp-/- and WT mice. Collagen gel with or without BSP was delivered to sockets. BSP included nat

OCTOBER 2023 ICCBMT 14 TH7 - Crispr-engineered conditional knock-in mouse model to study osteogenesis imperfecta type V Pierre Moffatt*1,2, Marie-Helene Gaumond1, Lisa Lamplugh1, Hadil Al Jalad1. 1Shriners Hospitals for Children - Canada, Montreal, QC, Canada, 2Dept of Human Genetics, McGill University, Montreal, QC, Canada. INTRODUCTION: Osteogenesis imperfecta (OI) is a rare heritable disorder most often caused by dominant mutations in COL1A, leading to skeletal fragility, reduced height, and fractures. OI type V (OI-V) is the only other autosomal dominant form that is caused by a recurrent mutation (c.-14C>T) in the IFITM5/BRIL (Bone-Restricted Ifitm-Like) gene. This mutation leads to addition of 5 a.a. (MALEP) onto the N- terminus of BRIL, a small osteoblast-specific transmembrane protein. OI-V resembles other OI types with regards to many skeletal characteristics mentioned above, but has unique clinical features, such as formation hyperplastic calluses and interosseous calcification. It is still obscure how the mutant BRIL cause such variable defects in the affected patients. PURPOSE: Because the germline knock-in mouse for OI-V is neonatal lethal, our goal was to engineer a Cre-inducible mouse model to allow a strict spatio-temporal expression of the mutant Bril allele and post-natal phenotyping. METHODS: Crispr-Cas9 was used to insert a STOP-cassette upstream of the Bril gene, along with the c.-14C>T mutation. Expression of the mutation would be conditional upon Cre-mediated excision of the STOP cassette, resuming in full ‘physiological’ expression. Mice were crossed with Prx1-Cre to induce expression in the appendicular skeleton and collected at embryonic day E18.5, birth, and at 12-weeks for skeletal phenotyping. RESULTS: The Ifitm5fl/+ mice were normal, but the Ifitm5fl/+;Prx1- Cre presented with a variable phenotype at all ages. Excision of the STOP cassette and expression of MALEP-BRIL was confirmed by sequencing and western blotting. At E18.5, staining with alizarin red and alcian blue revealed a wide spectrum of skeletal deformities, ranging from very mild to extremely severe. The stylopods (femur, humerus) were more affected than the zeugopods (tibia, ulna/radius), being misshapen and bent, with midshaft fractures. In some embryos, however, both limbs appeared normal. Histology and immunohistochemistry indicated that the E18.5 bent femurs had a cartilaginous midshaft, with almost no COL1A1 production. Picrosirius red staining indicated a woven-type COL1A1 extracellular matrix. Gene expression monitoring showed elevated Ptgs2, Nr4a3, Inhba, and Msmp, but reduced Col1a1, Bglap, Mmp8, Stfa3, Acp5, and Sost, suggesting induced inflammation and blunted osteoblastogenesis. Many mutant mice were found dead at birth, which displayed severely affected long bones, while others survived to adulthood without apparent problems. In 12-week-old mutant mice, femurs were shorter, and tibias bent. uCT scanning of the femur cortices indicated a change in geometry (elliptic shape) and increased porosity. 3-point-bending analysis revealed statistically reduced strength, ductility, and toughness, but only in females. CONCLUSIONS: The current model seems to recapitulate the human disease more faithfully, showing a variable phenotype. TH8 - Attaching organic fibers to mineral: The case of the avian eggshell Daniel J Buss1, Natalie Reznikov1,2,3, Marc D McKee*1,3. 1Dept of Anatomy and Cell Biology, 2Dept of Bioengineering, 3Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC, Canada. INTRODUCTION: In birds, mineralized eggshell formation in the oviduct shell gland is preceded by fiber assembly into an interconnected and branching meshwork (the eggshell membrane) at the surface of the egg "white" (albumen). This membrane forms the substratum for avian eggshell mineral deposition and growth. Incorporation of some of the outermost membrane fibers into the early shell structure provides for attachment between the shell and the membranes, the structural integrity of which is critical to chick development; chick growth and hatching fail if the membrane detaches from the shell. PURPOSE: To provide a multiscale 3D structural analysis of how organic fibers of the avian eggshell membrane are organized and attached at the shell-membrane interface. METHODS: Using submicron X-ray micro-computed tomography (X-ray microscopy), TEM and FIB-SEM electron microscopy methods for volume imaging and lamella production of conventional- and cryo- (high pressure freezing) prepared samples, coupled with deep learning-based segmentation and reconstruction and quantification using Dragonfly software, we report here on a multiscale 2D and 3D analysis of full-thickness eggshell membrane and its interdigitation with the mineralized eggshell of the domestic chicken (Gallus gallus domesticus). RESULTS: We describe (in unfertilized and unincubated eggs) the nature and extent of eggshell membrane fiber interdigitation

OCTOBER 2023 ICCBMT 14 TH9 - Deletion of Prkar1a subunit in osteoblasts cause severe bone pathology with impairment of osteoblast differentiation and increased osteoclast activity Carole Le Henaff*1, Brandon Finnie1, Zhiming He1, Joshua Johnson1, Maria Pacheco1, Yasaman Nahaei1, Juhee Jeong1, Henry M Kronenberg3, Lawrence S Kirschner2, Nicola C Partridge1. 1Dept of Molecular Pathobiology, New York University College of Dentistry, New York, NY, 2Dept of Cancer Biology and Genetics, Division of Endocrinology, Diabetes, and Metabolism, Dept of Internal Medicine, The Ohio State University, Columbus, OH, 3Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA. INTRODUCTION: Parathyroid hormone (PTH) was the first osteoanabolic hormone for treating osteoporosis. PTH acts through Parathyroid Hormone Receptor 1 (PTHR1) and Protein Kinase A (PKA) activation to regulate osteoblastic gene expression. However, hyperactive PTHR1 and its downstream signaling pathway (Gsα) are involved in several bone diseases including hyperparathyroidism, Jansen’s metaphyseal chondrodysplasia or Fibrous Displasia-McCune-Albright Syndrome. PKA is an enzyme whose activation is dependent on the activity of Gsα and cellular levels of cyclic AMP. Hyperactive PKA due to a mutation in PKA regulatory subunit 1A (Prkar1a) is found in Carney disease and has been linked to benign tumor formation while its impact on bone formation or remodeling remains unknown. PURPOSE: Our study aimed to elucidate the effects of increased PKA activity and better understand the actions of PTH in bones. METHODS: Tamoxifen (1mg/10g) was injected weekly to 1-month-old C57Bl/6J male and female col1CREERT/Prkar1afl/fl mice or Prkar1afl/fl mice as controls for 3 weeks to delete Prkar1a in osteoblasts and increase PKA activity in these cells. RESULTS: In both sexes, col1CREERT/Prkar1afl/fl mice demonstrated bone pathologies in their skulls, femurs and vertebrae and tumors in their tails. MicroCT showed cortical bone breakdown with apparent trabecular bone in the cortical area in femurs, vertebrae, tails and skulls. Surprisingly, the col1CREERT/Prkar1afl/fl skulls showed thicker but more porous calvariae, shown by alizarin red staining and μCT. No change was observed in mandibles or teeth. Col1CREERT/Prkar1afl/fl mice had tumors in their tails evident by an invasion of stromal and osteoclastic cells but with intact growth plate, cartilage and intervertebral discs. Deletion of Prkar1a increased bone turnover with a huge increase in osteoblast activity shown by serum- P1NP levels (6.5-13 fold), only single fluorescent labeling and a substantial increase in osteoclast activity shown by CTX levels (4.4-12 fold) and TRAP staining (showing an increased osteoclast number and size). In both sexes, cortical and trabecular bone RNAs showed a large increase in bone sialoprotein mRNA levels (3 to 6 fold) with a sharp decrease in osteocalcin (0.2-0.4 fold) showing a change in osteoblast differentiation. Furthermore, PTH-responsive-genes were significantly changed: Sost expression was decreased to 0.1-0.2 fold, Rankl was upregulated by 2-3 fold (causing the increased osteoclastogenesis) and Mmp13 was increased by at least 3 fold. RNA-Seq was conducted in trabecular bone. The data confirmed an impairment of osteoblast differentiation with a defect in ossification. It was evident that there was a profound modification of gene expression of chemokines and Wnt members together with enhanced osteoclastogenesis. CONCLUSION: High PKA activity in osteoblasts appears to be involved with high bone turnover and pathological events mimicking diseases from a hyperactive PTHR1/PKA pathway. TH10 - Development of a bone-on-a-chip to study bone formation in health and disease Judith Schaart*1, Dorothee Wasserberg2, Wouter Nijhuis3, Harrie Weinans3, Pascal Jonkheijm2, Andries van der Meer2, Nico Sommerdijk1,4, Anat Akiva1,4. 1Dept of Medical BioSciences, Radboudumc, Nijmegen, The Netherlands, 2TechMed Centre, MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands, 3Dept of Orthopaedic Surgery, University Medical Centre Utrecht, Wilhelmina Children’s Hospital, Utrecht, The Netherlands, 4Electron Microscopy Center, Radboudumc, Nijmegen, The Netherlands. INTRODUCTION: In the past decades the complexity of bone structure, dynamics, and composition have been studied extensively. This revealed that bone is a hierarchically organized organ, in which the aligned and mineralized collagen matrix is continuously renewed by the bone cells. The aligned organization of the bone matrix is crucial to the mechanical properties of bone. However, the pathways underlying matrix formation and mineralization in the developing bone remain elusive. PURPOSE: The understanding of these mechanisms of collagen deposition, alignment and early mineral deposition, can be crucial to understand the origin of bone related diseases, such as osteoporosis or osteogenesis imperfecta, f

OCTOBER 2023 ICCBMT 14 TH11 - Application of the mineral-binding protein fetuin-A for the detection and treatment of calcified lesions Robert Dzhanaev*1, Christian Hasberg1, Andrea Gorgels1, Carlo Schmitz1, Camilla Franziska Winkler1, Hanna Malyaran1,2, Steffen Gräber1, Anouk Gentier3, Armand Jaminon3, Stijn Agten3, Tilman Hackeng3, Asim Cengiz Akbulut3, Leon Schurgers3, Felix Manuel Mottaghy4,5, Claudia Goettsch6, Nina Petrova7, Cathy Shanahan8, Willi Jahnen-Dechent1. 1Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Hospital, Aachen, Germany, 2IZKF - Interdisciplinary Center for Clinical Research, RWTH Aachen University Hospital, Aachen, Germany, 3Dept of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands, 4Dept of Nuclear Medicine, University Hospital Aachen, RWTH Aachen University, Aachen, Germany, 5Dept of Radiology and Nuclear Medicine, Maastricht University Medical Centre, Maastricht, The Netherlands, 6Dept of Internal Medicine I, Cardiology, Medical Faculty, RWTH Aachen University Hospital, Aachen, Germany, 7Division of Diabetes and Nutritional Sciences, King's College London, London, UK, 8British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, London, UK. INTRODUCTION: Calcium plays an essential role in the biology of vertebrates. Calcium content in body fluids is maintained within a narrow physiologic range by feedback control. Phosphate is equally important for metabolism and is likewise controlled, albeit over a wider range. This results in a nearly supersaturated state of calcium phosphate in body liquids driving mineral precipitation in soft tissues, which is actively prevented by calcification inhibitors. The hepatic plasma protein fetuin-A is a circulating mineralization inhibitor regulating calcium phosphate crystal growth and calcified matrix metabolism. Ectopic mineralization is associated with many pathological conditions aggravating their outcome. Current diagnostic methods lack sensitivity towards microcalcifications representing the initial stages of the process. Given the irreversibility of established calcifications, novel theranostic tools capable of detecting and eliminating nascent calcium phosphate deposits are highly desirable. METHODS: We designed fluorescent fusion proteins consisting of fetuin-A coupled to a green or red fluorescent protein derivate (mEmerald or mRuby3) for the detection of mineralization and calcification in living cells. In parallel, we produced fusion proteins consisting of murine fetuin-A and RANKL to locally generate osteoclasts at sites of calcification. Fluorescent proteins were tested for their ability to detect microcalcifications in cell cultures and tissue sections retrieved from calcification-prone mice. The chimeric cytokine was tested for its ability to generate functional osteoclasts from progenitor cells. RESULTS: We employed novel fetuin-A-based fusion to detect and reverse ectopic calcifications. We show that fetuin-A-based imaging agents are non-toxic and suitable for live imaging of microcalcifications beyond the detection limit of conventional staining techniques. The ability of fetuin-A to preferentially bind nascent calcium phosphate crystals allowed the resolution of histopathological detail of early kidney damage that went previously undetected. Endogenous expression of fetuin-A fluorescent fusion proteins allowed extended live imaging of calcifying cells with unprecedented sensitivity and specificity. Stimulation of osteoclasts precursors with the novel fetuin-A-based cytokine but not with RANKL alone promoted the production of functional osteoclasts on plates precoated with hydroxyapatite. CONCLUSION: Ectopic microcalcifications represent a major clinical concern lacking effective diagnostic and treatment options. In this paper, we describe novel highly sensitive fetuin-A-based fusion proteins for imaging and reversing microcalcifications. We show that fusion proteins consisting of a fetuin-A mineral binding moiety and a fluorescent protein are superior to the routine methods for detecting calcifications. Fetuin-A-based cytokine is the first tool for targeted osteoclast activation to date. TH12 - Interfibrillar mineralization of three-dimensional chitin scaffolds derived from mushrooms Edward de la Uz1, Jo Laura1, Vivian Merk1*. 1Dept of Chemistry & Biochemistry and Dept of Ocean & Mechanical Engineering, Florida Atlantic University, Boca Raton, FL, USA. INTRODUCTION: The polysaccharide chitin constitutes the framework of various biomineralized tissues, including the exoskeleton in arthropods or the nacre of mollusk shells. In many biological systems, chitin is closely associated with hard minerals that enhance the mechanical properties of the soft organic matrix. PURPOSE: To emulate the skeletal structures observed in nature, we generated biomimetic hybrid composite materials from inexpensive and

OCTOBER 2023 ICCBMT 14 TH13 - A novel biofilm inhibitor & TTO modulate dental bacteria involvement & enhance tertiary dentin formation to synergistically prevent & delay caries Ibrahim Hoja*1, Liubov Lobanova1, Zoi Daskalaki2, Clarence Geyer3, Hidemitsu Harada4, Silvana Papagerakis6,7, Petros Papagerakis1,5,7. 1College of Dentistry, University of Saskatchewan, Saskatoon, SK, Canada, 2Dept of Paediatric Dentistry, School of Dentistry, School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece, 3Dept of Pathology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada, 4Dept of Anatomy, Division of Developmental Biology & Regenerative Medicine, Iwate Medical University, Iwate, Japan, 5Dept of Anatomy, Physiology and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada, 6Dept of Surgery, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada, 7Dept of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Osaka University, Osaka, Japan. INTRODUCTION: Dental caries is a chronic disease and a major health burden. In recent decades, dentistry has increasingly focused on enhancing preventive care and regenerative treatments. Our recent study discovered that Biofilm Inhibitor Molecule (C2011; patent pending) and Tea Tree Oil (TTO) present strong synergistic antimicrobial properties that have been effective against a wide range of bacteria in vitro. PURPOSE: The purpose of this study is to examine the efficacy of C2011 molecule and TTO in preventing and/or delaying the development and progression of dental caries lesions. We also aim to examine the potential impact of the C2011 and TTO combination on dental pulp stem cells (DPSCs) and tertiary dentin formation as well as in preventing and treating destruction of gingival epithelium upon bacterial infection. METHODS: For this study, we used Sprague-Dawley rats (n=32) divided into four groups, and all the groups received the same combination of bacteria suspension (S. mutans and S. sobrinus) and the same cariogenic diet (rich in sugar). The control group did not receive any treatment (group 1). The second and third groups were treated with TTO and C2011, respectively. Group 4 was treated with a combination mixture of TTO and C2011. On day 1 of the study, cariogenic microorganisms, and treatment products were applied directly to tooth surfaces twice weekly. Following 12 weeks of treatment, by using micro-CT imaging and histology, we are assessing the progression of the carious lesions on each tooth/rat. The biological tertiary dentin formation and DPSCs activity is being evaluated using histological staining and immunohistochemistry. In parallel, C2011 and TTO are also being evaluated using a junctional epithelium (JE) cell line we established, before and after treatment with LPS. Results: Our data reveal that rats received the TTO or C2011 treatment developed less caries overall compared to the control group. Our results also showed that a combination of C2011 and TTO treatment displayed statistically significant reduction of the percentage of molars with dental caries compared to the control group. All caries detected in the control group were deeper and showed closer proximity to dental pulp chamber while caries in rats treated with C2011 and TTO remained mostly superficial. CONCLUSION: Our study shows that combination of C2011 molecule and TTO reduced and prevented caries progression by inhibiting S. mutans and S. sobrinus. Additional studies are ongoing to evaluate the potential anti-bacterial roles of these molecules in preventing JE detachment. The potential effects of these molecules in attracting and modulating the activity of DPSC and gingival stem cells remain also to be elucidated. TH14 - Development of protein-based matrices for enamel regeneration Sara Gamea*1,2, Elham Radvar1, Dimitra Athanasiadou,3,4, Ryan Lee Chan5, Sunie Kundi1, Shwan Horamee1, Shuaib Hadadi1, Ka Lung Andrew Chan6, Nicola Pugno7,8, Paul Sharpe9, Karina Carneiro4,5, Sherif Elsharkawy1,10. 1Centre for Oral, Clinical, and Translational Sciences, Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College London, London, UK, 2Dept of Restorative Dentistry, Faculty of Dentistry, Tanta University, Tanta, Egypt, 3Dept of Physics, Chalmers University of Technology, Gothenburg, Sweden, 4Faculty of Dentistry, University of Toronto, Toronto, ON, Canada, 5Institute of Biomedical Engineering, University of Toronto, Toronto, ON, Canada, 6 Institute of Pharmaceutical Science, King's College London, UK, 7Laboratory for Bioinspired, Bionic, Nano, Meta, Materials & Mechanics, Dept of Civil, Environmental and Mechanical Engineering, University of Trento, Trento, Italy, 8School of Engineering and Materials Science, Queen Mary University of London, London, UK, 9Centre for Craniofacial and Regenerative Biology, Faculty of Dentistry, Oral & Craniofacial Sciences, King's College London, London,

OCTOBER 2023 ICCBMT 14 P1 - Modeling cardiovascular calcification: in vitro collagen calcification at pathological fetuin A levels replicates characteristic morphology and phases Raphaela Allgayer*1, Diego Mantovani2, Marta Cerruti1. 1Biointerface Lab, Dept of Mining and Materials Engineering, McGill University, Montreal, QC, Canada, 2Laboratory for Biomaterials and Bioengineering, CRC-I, Dept of Mining Metallurgical and Materials Engineering, Regenerative Medicine, CHU de Québec Research Center, Laval University, Quebec City, QC, Canada. INTRODUCTION: The formation of calcium phosphate (CaP) minerals on collagen matrices is the common feature of many physiological and pathological processes, such as bone mineralization or cardiovascular calcification. While the mineralization of collagen in bone results in highly organized composites with CaP crystals aligned along collagen fibrils, cardiovascular calcifications (CCs) consist mainly of ~1 μm- sized spheres and larger calcific nodules, along with some fibrillar calcifications. The different morphologies of the minerals in bone and CC suggest that the two processes follow different mechanisms. Indeed, while both in bone mineralization and CC CaPs initially deposit as amorphous material and mature into apatite, the intermediate phases found in the two cases are different: octacalcium phosphate and small amounts (< 1%) of dicalcium phosphate dihydrate (DCPD) have been reported in bone mineralization, while DCPD is the most common intermediate in CC, accounting for up to 40% of the minerals. The causes for CC are complex and vary with the underlying pathology, which hinders our understanding of CC and the development of relevant treatments. In vitro models for pathological collagen calcification would be excellent platforms to study CC and test new methods for CC detection and treatment. However, current in vitro models mostly focus on collagen calcification in bone formation. A key difference between bone mineralization and CC are the lower levels of calcification inhibitors in the calcifying tissue of patients with CC compared to healthy individuals. So far, in vitro collagen mineralization was only studied with physiological calcification inhibitor levels. Pathological calcification inhibitor levels and mineral phase transformations in collagen matrices have not been investigated. PURPOSE: We aim at creating a collagen-based model for CC, by studying how sub-physiological concentrations of the most common calcification inhibitor, fetuin A, affect CaP mineralization in pathological conditions. METHODS: We calcify collagen gels in simulated body fluid with different levels of fetuin A and analyze CaPs deposited on them with advanced materials characterization techniques to study their morphologies and phases. RESULTS: The collagen gels calcify faster with decreasing fetuin A concentration. At sub-physiological fetuin A concentrations, CaPs in the collagen gels morphologically resemble CCs. Mineralized fibers become more abundant and thinner, and the prevalence of spherical deposits decreases with increasing fetuin A concentration. After 7 d and 14 d of incubation, DCPD is more common at sub-physiological than at physiological fetuin A levels. CONCLUSIONS: At sub-physiological fetuin A levels the CaP morphologies and phases resemble those of human samples with CC, while the CaPs in the physiological solution approach the characteristics of bone mineral. Thus, a collagen gel with appropriately tuned fetuin A level can be a good model for CC. P2 - New insights into the effects of a metabolic disorder on the crystallography of dental enamel Mohammed Al-Mosawi*1, Owen Addison2, Christian Hendriksz3, Maisoon Al-Jawad1. 1Division of Oral Biology, School of Dentistry, University of Leeds, Leeds, UK, 2School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK, 3Birmingham Children’s Hospital, Birmingham Children’s Hospital NHS Foundation Trust, Birmingham, UK. INTRODUCTION: Mucopolysaccharidosis IV type A (MPS IVA), also known as Morquio syndrome, is an inherited metabolic disorder characterised by the abnormal accumulation of glycosaminoglycans (GAGs) in various cell types, including the enamel matrix secreting cells, ameloblasts. Reports suggest that GAGs accumulation in ameloblasts can lead to dental abnormalities such as enamel thinning and enamel-dentine delamination at the enamel-dentine junction (EDJ). PURPOSE: Here, we characterise the crystallography of enamel at the EDJ in MPS IVA affected teeth using synchrotron X-ray diffraction (S-XRD) to elucidate the mechanisms of hard tissue malformation observed in MPS IVA and potentially other metabolic diseases. METHODS: A permanent maxillary first molar from a patient affected by MPS IVA and a type-matched control were obtained from Birmingham Children’s Hospital NHS Foundation Trust, following ethical approval. The samples were sectioned into 20 μm thick slices and analyse

OCTOBER 2023 ICCBMT 14 P3 - Dentin mineralization alteration in mice exposed to Di(2-ethylhexyl) phthalate (DEHP), a widespread endocrine disruptor Ai Thu Bui1, Sophia Loiodice1, Lotfi Slimani2, Sophia Houari1,2, Elsa Vennat3, Nicolas Roublier3, Sakina Mhaouty-Kodja4, Sylvie Babajko*1,2. 1Centre de Recherche des Cordeliers, INSERM UMRS 1138, Université Paris Cité, Sorbonne Université, Laboratory of Molecular Oral Pathophysiology, 2Université Paris Cité, UP 2496, Laboratory of Orofacial Pathologies, Imaging and Biotherapies, Montrouge, 3CNRS, Centrale- Supélec, Université Paris-Saclay, Laboratory of Mechanics of Soils, Structures and Materials, Châtenay-Malabry, 4CNRS, Inserm, Sorbonne Université, Neuroscience Paris Seine–Institut de Biologie Paris-Seine, Paris, France. INTRODUCTION: Endocrine disruptors are environmental toxicants that contribute to fertility trouble, many chronic pathologies such as endocrine-dependent cancers and diabetes, but also alter brain development and IQ level. Our recent data showed that rodents chronically exposed to low-dose endocrine disruptors (EDs) widely used by plastic industry, bisphenol A or Di(2-ethylhexyl) phthalate (DEHP), present enamel hypomineralization suggesting ED contribution to developmental defects of enamel (DDE) such as MIH (Molar Incisor Hypomineralization). PURPOSE: Analyze the incisor dentin of mice exposed to low-dose DEHP. METHODS: Adult C57BL/6 mice were exposed daily to 0.5 to 50 μg/kg/d DEHP in food for 12 weeks. Dentin mineralization and hardness were analyzed by μCT and nanoindentation, respectively. Gene expression was measured by RT-qPCR analysis of RNA extracted from microdissected mesenchymal tissue. C1 mouse oral mesenchymal cell line was used for in vitro studies. RESULTS: Clinical macroscopic observations showed various dental lesions such as opacities, scratches and enamel breakdown in 30 % males and 15 % females, among whom 18 % and 9 % respectively had broken incisors. DEHP-exposed mouse dentin showed a 9% decrease in mineral density whatever the region of analysis ranging from 360 mg/cm3 for controls to 337 mg/cm3 for exposed mice. The severity of observed structural alterations was dependent on the DEHP doses. Nanoindentation showed a dose-dependent decrease in dentin hardness and Young’s modulus values from the dentin-enamel- junction to pulp chamber for all samples. Dentin of mice exposed to 50 μg/kg/d DEHP (D-50) presented similar profiles compared to controls with a systematic reduction by 5 to 10 %. BSP and DMP-1 expression levels were significantly reduced in dose-dependent manner. Similar data were obtained in vitro with cells treated with MEHP, the active metabolite of DEHP, suggesting a direct effect of these phthalates on dental cells. CONCLUSIONS: These data demonstrate that chronic exposure to low-dose DEHP contributes to disrupt structural, mechanical and biochemical properties of rodent dentin. These effects on dentin in addition to those previously reported on enamel alter mineralization of dental tissues explaining their fragility and broken teeth. P4-F - Exploring site-specific functions of bone sialoprotein in mineralization using conditionally ablated mouse models Natalie L Andras*1, Michael B Chavez1,2, Michelle H Tan1, Tamara N Kolli1, Brian L Foster1. 1Division of Biosciences, The Ohio State University College of Dentistry, Columbus, Ohio, USA, 2University of Iowa College of Dentistry and Dental Clinics, Iowa City, IA, USA. INTRODUCTION: Bone sialoprotein (Ibsp gene; BSP protein) is a multifunctional, extracellular matrix protein in skeletal and dental mineralized tissues. In mice, global BSP ablation (Ibsp-/-) contributes to delayed mineralization and reduced long bone size. The craniofacial phenotype of Ibsp-/- mice reveals alveolar bone hypomineralization and periodontal breakdown associated with cementum defects. The tissue-specific functions of BSP remain unclear, due in part to, the interrelationship between skeletal and dentoalveolar development as well as the presence of BSP in systemic circulation. PURPOSE: We aimed to define cell- and tissue-specific functions of BSP in craniofacial and postcranial bone, which vary in their origin (ectomesenchymal vs. mesodermal) and mode of ossification (intramembranous vs. endochondral). We hypothesized BSP is more critical for mineralization of the dentoalveolar complex compared to long bones. METHODS: To test tissue-specific functions of BSP, we developed a floxed Ibsp mouse model (Ibspfl/fl) and conditionally deleted Ibsp from 1.) neural crest-derived ectomesenchyme (contributing to alveolar bone) using Wnt1-Cre2 mice and 2.) limb mesenchymal progenitors using Prx1- Cre mice. Skulls, mandibles, and femurs were harvested from Wnt1-Cre2+; Ibspfl/fl and Prx1-Cre+; Ibspfl/fl conditional knockout (BspWnt1- cKO and BspPrx1-cKO, respectively) and wild-type (WT; Ibspfl/fl) control mice (n=6/group/age) at 30- and 90-days-post-natal (dpn). We additionally challenged BspWnt1-c

OCTOBER 2023 ICCBMT 14 P6 - 3D printing of fibrillar collagen scaffolds with native-like organization Paula Vena1,2, Lena Stöcker1, Marcela García Jimenez1, Miguel Castilho1,2. 1Dept of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands, 2Dept of Orthopeadics, University Medical Center Utrecht, Utrecht, The Netherlands. INTRODUCTION: The network organization of collagen fibres (Col) within the extracellular matrix (ECM) is tissue-specific. This organization, including density, dimensions, alignment and porosity, plays a crucial role in determining the mechanical and biological properties of the tissue. Unfortunately, it cannot be fully mimicked in vitro. This is mainly because most studies on Col organization neglect the physiological conditions, in particular, the macromolecular crowded environment and confinement forces that affect in vivo processes. PURPOSE: We aim to recapitulate in vitro the in vivo organization of Col in the ECM of different tissues. To achieve this, we explore the role of molecular crowding on the density and dimensions of Col fibers, while study whether the flow and respective shear forces induced during extrusion- based 3D printing guide the organization and dimensions of Col fibers. METHODS: Col constructs are fabricated by extruding Col-based inks into a gelatin support bath. The Col-based inks, composed of either Col molecules (sCol) or a dispersion of Col native-like fibers (dCol), with or without the addition of other macromolecules (polyethylene glycol (PEG) or Ficoll). Key printing conditions are systematically varied to study collagen organization and dimensions. The effect of the Col-based ink and the printing parameters on the final organization of the Col constructs is studied by optical, fluorescence and scanning electron microscopies. RESULTS: 3D Col constructs were successfully obtained by extrusion of the different inks (sCol, sCol + MMC, dCol and dCol + MMC) in a gelatin support bath. The printed sColl filaments are 2-3 times thicker and less defined compared to dColl. sColl constructs degrade faster over time than dCol constructs. The addition of MMC to the dColl ink leads to increased stability and higher resolution of the printed filaments under the same printing conditions: (183±3) μm for dCol and (138±2) μm for dColl+MMC. Col fibers of the dColl+MMC constructs are aligned, while for sColl and dColl, they present an isotropic organization. In all cases, the collagen fibres show the typical D-banding pattern. CONCLUSIONS: We show that the macro- and microstructure organization of Col fibers within Col constructs can be tuned by optimizing the composition of Col-based inks (type of Col precursor and presence of MMC) and the printing parameters. Extrusion 3D printing in a gelatin support bath allows for the creation of Col structures with complex shapes and defined porosities, while the addition of MMC to the Col ink leads to denser constructs (better resolution) where the Col fibers are aligned parallel to the printing direction. This strategy proves to be successful to obtain 3D Col constructs with tunable properties, which opens new axis to study biological processes such as biomineralization or cell-matrix interactions. P7 - Ultrastructural and chemical analysis of aortic valve calcification in a rabbit model Dimitra Athanasiadou1 ⸹*, Nikolaos Anousakis-Vlachochristou2,3 ⸹, Sherif Elsharkawy4, Marianne Liebi1,5,6, Manolis Mavroidis3, CD Anagnostopoulos3, D Cokkinos3, Konstantinos Toutouzas2,3. 1Dept of Physics, Chalmers University of Technology, Gothenburg, Sweden, 2National and Kapodistrian University of Athens, Medical School, First Dept of Cardiology, Athens, Greece, 3Cardiovascular Translational Research Unit, Center for Clinical, Experimental Surgery & Translational Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece, 4Faculty of Dentistry, Oral & Craniofacial Sciences, King's College London, London, UK, 5Photon Science Division, Paul Scherrer Institute, Villigen, Switzerland, 6Institute of Materials, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland. ⸹equal contribution INTRODUCTION: Calcific aortic valve stenosis (CAVS), the most prevalent valvular heart disease, remains without pharmaceutical therapy. Osteoblastic transformation and heavy calcification of the valve constitute the hallmarks of the disease. Since its natural course may exceed a decade, we need reliable in vivo models to study the early calcification stages. Previously, we have modified and validated a rabbit model with equivalence to human disease. Here, we sought to investigate the structure and chemical composition of the calcified lesion using a rabbit model. METHODS: Aortic valve stenosis was induced in New Zealand rabbits using a diet supplemented with 1% cholesterol and 3500 IUs/kg Vitamin D2 in oil, daily. Calcification was quantified using 18F-NaF microPET/CT. After 2 and 7 weeks, valve tissue f

OCTOBER 2023 ICCBMT 14 P8-F - Semantic segmentation of enamel caries using convolutional neural networks Sarah Boyer*1, Victoria Cooley1, Robert Free1, Stuart R Stock2, William Guise3, Denis Keane1, Pavel Shevchenko4, Derk Joester1#. 1Dept of Materials Science and Engineering, Northwestern University, Evanston, IL, USA, 2Dept of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA, 3DND-CAT Synchrotron Research Center, Northwestern University, Evanston, IL, USA, 4Advanced Photon Source, Argonne National Laboratory, Lemont, IL, USA, * presenting author # to whom correspondence should be addressed: d-joester@northwestern.edu INTRODUCTION: Tooth decay (caries) affects nearly everybody, has a large impact on quality of life, and is associated with great cost to society.1-2 A major bottleneck to innovation in prophylaxis and treatment is the lack of understanding of the etiology. Our goal is to better understand the formation of the surface zone, a highly mineralized, yet dynamic layer that covers subsurface lesions, and prevents remineralization. We recently demonstrated, using synchrotron micro-computed tomography (SMCT), that surface zones develop in the “gold standard” rat caries model.3 Following up, we are considering the impact of fluoride and sucrose on lesion formation and structure.4 Towards this goal, we need to identify surface zones in rat molars using SMCT for further analyses at high spatial resolution. PURPOSE: To develop an automated workflow that ensures unbiased and reproducible segmentation, analysis, and comparison of caries and healthy enamel in rat molars. METHODS: Eight female Wistar weanlings per group were placed in a study with a two-factor design (dietary fluoride: low or high; cariogenic stimulus: none or high) and evaluated at two time points (14d or 28d post weaning).5 Using SMCT, 64 reconstructions of M1 molars at 1.73 μm isotropic voxel size were obtained. As threshold-based segmentation fails due to similar attenuation of dentin and lesions and with recent success using convolutional neural networks (CNNs) to segment mouse jaws6, a 2D CNN with U-Net7 architecture was trained to segment molars. We further developed code to extract and compare metrics describing healthy enamel and caries lesions across experimental groups. RESULTS: Trained CNNs segmented caries lesions and healthy enamel with high specificity (>99%). Automated extraction of number, volume, surface area, overall mineral density, and density profiles was successful. Preliminary analysis reveals that indicators for lesion number and severity follow trends expected given the treatments. Evaluation of mineral density profiles is ongoing. CONCLUSIONS: CNN-based segmentation and automated quantification is a versatile tool that we hope will empower cariologists to help understand the development of surface zones and enable advancements of new intervention approaches. REFERENCES: [1] in Oral Health in America: A Report of the Surgeon General, U.S. DHHSS, Rockville, MD, 2000; [2] National Health Expenditure Data, 2021; [3] Free et al. J Synchrotron Radiat 2017, 24; [4] This work was supported in part by NIH (DE028872, DE025702, DE026952, TL1TR001423), NSF (DGE-1842165), used resources of the Advanced Photon Source, a U.S. Dept of Energy (DOE) Office of Science User Facility operated by Argonne National Laboratory under Contract No. DE-AC02-06CH11357; [5] Animal experiments were approved by Northwestern Univeristy’s IACUC under protocol IS00001464; [6] Cooley et al, in SPIE Opt Vol. 11840 San Diego, CA, 2021; [7] Ronneberger et al. MICCAI 2015, pp 234-241. P9-F - Transcriptome profiling of DPSCs stimulated with DPP identifies key signaling networks responsible for odontoblast-specific lineage differentiation Yinghua Chen*, Cassandra Villani, Amudha Ganapathy, Anne George. Dept Oral Biology, UIC College of Dentistry, Chicago, IL. USA. INTRODUCTION: Dental pulp stem cells (DPSCs) are a promising source of adult stem cells for regenerative medicine applications, therefore understanding the differentiation mechanism of DPSCs is necessary. The predominant noncollagenous protein in the dentin matrix is dentin phosphophoryn (DPP). Silencing DPP leads to several dental anomalies such as Dentinogenesis Imperfecta Type II. Previous studies have demonstrated the role of this protein in collagen assembly and in the nucleation of hydroxyapatite in the extracellular matrix. In the ECM, DPP binds to integrins and activates the non-receptor tyrosine kinase focal adhesion kinase, thereby promoting cell adhesion. Recently, we have reported that DPP can induce odontogenic differentiation of DPSCs through NF-κB pathway. To identify gene regulation targets related to postnatal odontoblast differentiation, transcriptome analysis was carried out to correlate genomic information to molecular signaling pathways. PURPOSE: In this study, RNA-sequencing and bioinformatics analyses were performed to ide

OCTOBER 2023 ICCBMT 14 P10 - Under hypocalcemia, Irisin deletion protects young and aged female mice from bone loss but worsens bone loss in aged compared to young males Anika Shimonty1,2, Fabrizio Pin1,2,3, Matt Prideaux1,2, Gang Peng1,4, Lynda F Bonewald*1,2,3. 1Indiana Center for Musculoskeletal Health, 2Dept of Anatomy, Cell Biology, and Physiology, 3Simon Comprehensive Cancer Center, 4Dept of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA. INTRODUCTION: Irisin, a hormone generated by the proteolytic cleavage of Fibronectin type III Domain Containing protein 5 (FNDC5) has been described to have beneficial effects on brain and fat, but effects on bone are contradictory (Tsourdi et al, Clin. Med, 2022) as some studies have found positive effects but our studies have shown that irisin deletion protects against bone loss due to ovariectomy (Kim et al, Cell, 2018). Recently we have shown that irisin deletion also protects against bone loss with lactation and low calcium diet in females but exacerbates male mice bone loss due to a low calcium diet. PURPOSE: To determine if irisin plays a role in aging bone loss. METHODS: 5 and 20 mo old mice wildtype, WT, and irisin knockout, KO, male and female were compared using microCT, dexa, histology, serum measurements, and RNAseq analysis of osteocytes from the 5 mo old animals. RESULTS: No differences were observed between WT and FNDC5 global KO female mice, however, male KO mice, 5 and 20 mo, had higher BV/TV but less biomechanical strength compared to WT males. With low Ca diet, both 5 and 18 mo female KO were partially protected against bone loss and weakness: (BV/TV;43.2% KO:40.4% WT), ultimate force (17.8N KO:15.7N WT) and stiffness (59.8Nmm KO:43.4 Nmm WT). In contrast, both 5 and 18-mo male KO mice lost more bone and had lower bone volume (BV/TV; 48.6% KO:55.4% WT) on a low Ca diet and lower ultimate force (14.9N KO:18.4N WT). To begin to identify responsible molecular mechanisms, RNAseq was performed on osteocyte enriched bone from 5 mo old animals as two major functions of the osteocyte are to remove their perilacunar matrix to provide calcium and to produce RANKL to activate osteoclasts. Sex differences showed that the female WT osteocyte transcriptome had higher expression of genes responsible for osteocytic osteolysis including Acp5, Ocstamp, Dcstamp, Ctsk, Tnfsf11, Mmp13 compared to WT males. The male WT transcriptome had higher expression of genes related to steroid, lipid, and fatty acid metabolism. There were few differences between WT females and KO females except for lower expression of the osteocytic osteolysis genes in the KO. With calcium deficiency, female KO osteocytes have lower while male KO osteocytes have higher expression of osteocytic osteolysis genes such as Tnsfs11 and Ocstamp compared to WT. CONCLUSIONS: In young females irisin aids or primes the osteocyte to release calcium under calcium demanding conditions most likely to ensure offspring survival, however in the aging female, this effect of irisin on osteocyte function becomes detrimental with either sex hormone and/or calcium deficiency. In contrast, irisin appears beneficial for males at any age, but especially with aging. P11 - Inner structure and composition of cultured black pearls from Pinctada margaritifera Yannicke Dauphin*1, Frédéric Jamme2, Christophe Sandt3, Jean-Pierre Cuif4. 1ISYEB, UMR 7205, MNHN Paris, France, 2DISCO beamline, synchrotron SOLEIL, France, 3SMIS beamline, synchrotron SOLEIL, France, 4CR2P, UMR 7207, MNHN Paris, France. INTRODUCTION: Pearls produced by molluscs are used for jewellery since centuries. Natural pearls are rare and not perfectly spherical. Multiple attempts have been made to obtain pearls from cultured animals (e.g. Pinctada, the pearl oyster). Mikimoto was successful in using the graft procedure of Mise & Nishikawa in 1893. Optical microscope examination of the pearl surfaces concluded that nacre in both natural and cultured pearls were not optically different (Paris trial 1924). At that time, it was admitted that in cultured pearls, the pearl layer was nacre only, but it has been repeatedly shown that its basal part comprises various structures not found in the shell. This indicates that various alterations of the mineralizing metabolism have occurred in the cells producing minerals after grafting. Here, we describe the structural and compositional modifications occurring during the growth of the pearl layer (from nucleus to the outer surface). METHODS: Sections of pearls show that multiple structures overlap in the 2 mm thick pearl layer. High resolution in situ analyses are needed to elucidate their composition and evolution during the growth of the pearl. Optical micrographs were obtained using bright field, dark field and UV fluorescence illumination on polished surfaces. Raman and FTIR spectra and maps allowed us to determine the mineralogy and the presence of organic components. Deep UV (

OCTOBER 2023 ICCBMT 14 P12 - Assessing traumatic injuries in a bone ex vivo model Miruna Chipara1,2, Richard Moakes1, Melissa Finlay1,2, Amy Naylor2, Alexander Stoll3, Liam M Grover1. 1Healthcare Technologies Institute, School of Chemical Engineering, University of Birmingham, Birmingham, UK, 2Institute of Inflammation and Ageing, Medical School, University of Birmingham, Birmingham, UK, 3Defence Science and Technology Laboratory, Ministry of Defence, Salisbury, UK. INTRODUCTION: While gunshot and bomb-related injury care remains sadly relevant to this day, injury profile and intervention practices are still poorly understood and limited. Bone damage following traumatic wounding results in the mechanic destruction and death of tissue at the site of injury. Surgeons debride the tissue, removing any necrotic regions but bone continues to turn gradually necrotic, thus the wound remains open, increasing the risk of infection. Currently there are no methods that enable full characterisation of such fragments in anatomically relevant positions within the fracture. We have worked on the use of a structured or fluid-gel materials as support matrices in which cell bearing gels can be immobilised, allowing for the construction of large and complex tissues. We have recently explored the possibility of using these materials to support and process tissue fragments such that they could be maintained in an anatomically relevant configuration ex-vivo. EXPERIMENTAL METHODS: Ex-vivo model: Fresh rat and mouse fractured bones were added to a polyethylene glycol (PEG) fluid gel support medium. A fibrin gel was injected in between the fracture site. Additionally, to understand better cell migration and mineralisation within the fibrin gels, bone chips in fibrin constructs were analysed separately. RESULTS AND DISCUSSION: Ex-vivo models are of utmost importance in the case of traumatic injuries where in vitro cultures are unable to replicate the wounding scenario and in vivo experiments are difficult to conduct. The support medium plays a vital role in maintaining the viability of the tissue ex-vivo, therefore, to test the capability of PEG gels to deliver nutrients to the encapsulated bones, as well as to eliminate metabolic waste, we analysed the rheological and diffusion properties. The fluid gel sheared at 700 rpm displayed most favourable properties due to a smaller particulate system that allows for a better diffusion. A fluid gel has the property to self-heal, thereby providing support to another gelling material to be printed by dispersing it into the interstices of the supporting fluid gel particles. This enables complex structuring and preserves the bony ends in a relevant anatomical configuration. Fibrin gel was selected as the bridging material due to its structural and biochemical similarities to the microenvironment of the callus formed early in fracture healing. Cells were able to migrate into the fibrin gel within a few days and displayed an early osteocyte marker, podoplanin as early as one week, to three weeks in culture. Moreover, after 25 days, extracellular matrix surrounding an organised cell network resembled mineral deposition in bone and it is currently being analysed further. CONCLUSION: In conclusion, traumatic injury field could highly benefit from the usage of biomaterials, for both a deeper understanding of injury profile through more accessible models and improvement of early intervention practices. P13 - Surface modification of biomimetic self-assembling peptide scaffolds on their potential to promote de novo nucleation of hydroxyapatite Robert PW Davies1,2,*, J Jing Kang1, SJ Brooks1, J Kirkham1. 1Division of Oral Biology, School of Dentistry, St James’s University Hospital, University of Leeds, 2Bragg Centre for Materials Research, University of Leeds, Leeds, UK. INTRODUCTION: Dental caries is the most prevalent disease world-wide, presenting clinically as white or brown lesions. Lesions have the capacity to repair to some degree due epitaxic HA overgrowth on the remaining enamel crystals by virtue of saliva being super saturated with respect to hydroxyapatite (HA). Despite this, the spontaneous re-nucleation of HA to seed new crystals is highly unlikely. Untreated caries can progress requiring drilling and filling. As an alternative self-assembling peptide P11-4 has demonstrated efficacy in treating lesions as a non-evasive approach. The peptide diffuses through the surface of the lesion and forms hierarchical structures. It has been postulated that he surface chemistry of the hierarchical structures are responsible for its mechanism of action by growing de novo HA from within the lesion. PURPOSE: Modulate the surface chemistry of the hierarchical structures of P11-4 and develop more efficient self- assembling motifs for the treatment of caries lesions, by changing the peptides’ primary structure. To assess the efficacy of each peptide using an ex-vivo model. METHODS: The physicochemical and ma

OCTOBER 2023 ICCBMT 14 P14 - Bone diagenesis at early stage followed-up during 12 months by Raman spectroscopy Guillaume Falgayrac1*, Raffaele Vitale2, Yann Delannoy1,3, Hélène Behal4, Guillaume Penel1, Cecile Olejnik1, Ludovic Duponchel2, Thomas Colard5,6. 1Univ. Lille, CHU Lille, Univ. Littoral Côte d’Opale, ULR 4490 - MABLab- Adiposité Médullaire et Os, Lille, France, 2Univ Lille, CNRS, UMR 8516-LASIRe-Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l’Environnement, Lille, France, 3Univ Lille, CHU Lille, ULR 7367-UTML&A-Unité de Taphonomie Médico-Légale & d’Anatomie, Lille, France, 4Univ Lille, CHU Lille, ULR 2694-METRICS: Évaluation des technologies de santé et des pratiques médicales, Lille, France, 5Univ Bordeaux, CNRS, MCC, PACEA, UMR 5199, Pessac, France, 6Univ Lille, CHU Lille, Dept of Oral and Maxillofacial Radiology, Lille, France. INTRODUCTION: For the forensic anthropologists, bone remains encapsulate many features of the individual’s life history. The study of post-mortem alteration of bone is needed to differentiate the diagenetic processes from the physiological or pathological processes. The bone diagenesis is defined as all interactions that occur between the bone and the burial environment [1]. Diagenetic mechanisms are complex especially in the early stages because numerous processes take place which depend on the burial environment (weather, soil, scavengers, …). Numerous studies focused on the evaluation of the diagenetic processes over archaeological periods, but few focused over forensic period [2]. PURPOSE: The aim of this study is to evaluate the molecular alterations induced by diagenesis on human bone during 12 months using Raman microspectroscopy. METHODS: Six ribs from 6 subjects were placed in burial environment which consists in a plastic bin filled with clay soil from north of France. Plastic bins were placed outside and sheltered. Each month, a bone slice (<5 mm) was sampled from each rib. The remaining ribs are placed back in their burial environment. This procedure was repeated each month during 12 months. The bone slices were analysed on Raman microscope LabRAM (HR800) and equipped with a 785 nm laser diode. Forty spectra were acquired per rib and per month. The composition was assessed by 5 physico-chemical parameters calculated from Raman spectra. RESULTS: The mineral/organic ratio and the carbonation type-B are decreased at a rate of -0.02 (p=0.015) and -0.03×10-3 (p<0.001) per month, respectively. The crystallinity is increased at a rate of +0.04×10-3 (p<0.001) per month. The modifications observed in the mineral matrix were in agreement with previous studies. The hydroxyproline/proline ratio is not modified. The collagen cross-links are decreased at the rate of -0.012 (p < 0.001) per month. The collagen cross-links give an assessment of the bonds that contribute to the stabilization of the helicoidal structure of collagen fibrils. This finding is in agreement with the mathematical simulation of the collagen hydrolysis [3]. CONCLUSIONS: In our conditions, the mechanism of diagenesis is driven by the hydrolysis of the collagen for bone remains buried during 12 months. [1] R.E.M. Hedges, Archaeometry, 44 (2002) 319. [2] N. Procopio, C.A. Mein, S. Starace, A. Bonicelli, A. Williams, Biology, 10 (2021). [3] M.J. Collins, M.S. Riley, A.M. Child, G. Turner-Walker, J. Archaeol.Science, 22 (1995). P15 - Biomimetic mineralization using seriated ALP-functionalized multilayer systems Brittany Foley*1,2, Mohamed Selmane3, Alberto Mezzetti1, Antoine Miche1, Frédéric Nadaud4, Karim El Kirat1, Jessem Landoulsi1,2. 1Laboratoire de Biomécanique & Bioingénierie, CNRS, Université de Technologie de Compiègne, France, 2Sorbonne Université, CNRS, Laboratoire de Réactivité de Surface, Paris, France, 3Fédération de Chimie et Matériaux de Paris-Centre, Paris, France, 4Service d’analyse physico-chimique, Université de Technologie de Compiègne, Compiègne, France. INTRODUCTION: The enzyme alkaline phosphatase (ALP) is a key player in the formation of mineralized tissues and is increasingly used in biomimetic mineralization and remineralization systems. In vivo, ALP hydrolyzes PPi to yield phosphate ions for the precipitation of calcium phosphate, however, the use of alkaline phosphatase for in vitro biomineralization requires the selection of a phosphate-containing substrate, whose structure may affect subsequent formation of calcium phosphate mineral. PURPOSE: Our objective is to use ALP to construct functional coatings for biomimetic mineralization of calcium phosphate at the surface, and to apply these coatings to native biomineral surfaces for applications in mineralized tissue repair. METHODS: Enzymatic activity is measured using UV/Vis spectrophotometry. Mineralized surfaces are characterized by attenuated total reflectance Fourier-transform infrared spectroscopy (ATR- FTIR), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and X-ray p

OCTOBER 2023 ICCBMT 14 P16 - Raman spectroscopy assessement of the mineral produced by human osteoblasts differentiated on the extracellular matrix of bone marrow adipocytes Laura Entz1, Guillaume Falgayrac*1, Christophe Chauveau1, Gilles Pasquier1, Stéphanie Lucas1. 1Univ Littoral Côte d’Opale, Univ Lille, CHU Lille, ULR 4490 - MABLab- Adiposité Médullaire et Os, Lille, France. INTRODUCTION: Bone marrow adipocytes (BMAds) have been revealed as important contributor cells in bone homeostasis. These lipid- laden cells primarily arise in close vicinity of trabeculae within bones and dramatically accrue in various types of osteoporosis, such as due to aging, menopause, and type 2 diabetes (T2D). In T2D, skeletal fragility is associated with several changes in bone quality that are incompletely understood. In this pathophysiological context, the phenotype of BMAds is altered and their quantity increases in relationship to poor glycemic control. Yet, whether BMAds can produce a specific extracellular matrix (ECM) which could interfere with the mineralization process of osteoblasts is barely studied. While various techniques exist to assess the bone quality, Raman microspectroscopy has been shown to be the technique of choice for analyzing the quality of the osteoblast-formed mineral. PURPOSE: In complementarity of molecular approaches, we aimed to determine in an in vitro model the quality of the ECMs following BMAd removal and the contribution of these ECMs on mineralization quality in the context of chronic hyperglycemia. METHODS: Human Bone Marrow Mesenchymal Stromal Cells (BM-MSCs) were differentiated for 21 days in adipogenic medium containing either a normoglycemic (LG, 5.5 mM) or a high glucose concentration (HG, 25 mM). The ECMs are laid down by BMAds. The BMAds were removed by hypotonic shock. The ECMs were analyzed using Raman microspectroscopy to assess their integrity and basic composition. BM-MSCs were seeded on the BMAd ECMs and differentiated toward osteoblastogenesis in LG and HG conditions for 16 days. The quality of the mineral formed by osteoblasts was evaluated by Raman microspectroscopy through the measurements of the mineral/organic ratio (amount of mineral compared to organic), the type-B carbonate content and the crystallinity (the perfection/length of the crystal). The quality of the organic part was assessed with 2 collagen crosslink ratios. RESULTS: Raman spectroscopy analyses demonstrated that the BMAd ECMs displayed a similar composition compared to the ECM underlying the nondecellularized BMAds, indicating the good integrity. These analyses also showed several differences in the composition of ECM according to the glucose concentration, which further guides the identification of BMAd ECM components. Raman spectrometry revealed that culturing on BMAd matrices specifically prevents type-B carbonate substitution and favors collagen crosslinkings, in contrast to exposure to HG concentration alone. Moreover, the mineral to organic ratio was disrupted according to the presence of BMAd ECM and the glucose concentration used for BMAd or osteoblast culture. HG concentration and BMAd ECM thus led to different defects in mineralization quality. CONCLUSIONS: This finding shed light on the involvement of BMAds, which should be considered in the compromised bone quality of T2D and osteoporosis patients more generally. P17 - Investigating the role of self-assembling peptides in guided enamel remineralisation on the micro- and nanoscale using synchrotron X-ray techniques Reham Gonnah*1,2, Julia Parker2, Robert Davies1, Maisoon Al-Jawad1. 1Division of Oral Biology, School of Dentistry, University of Leeds, Leeds, UK, 2Diamond Light Source Ltd, Harwell Science & Innovation Campus, Oxfordshire, UK. INTRODUCTION: Enamel is the hardest mineralised tissue in the human body, composed of highly ordered hydroxyapatite (HA). It is this organisation/texture within the HA crystallites that gives enamel its exceptional mechanical properties. Despite its strength, enamel is still prone to disease; dental caries (DC). One new approach to treat DC is the biomimetic self-assembling peptide (SAP) P11-4 which has been shown to promote enamel remineralisation; however, P11-4’s mechanism of action is yet to be elucidated. PURPOSE: To better understand the role of P11-4 in enamel remineralisation in order to optimise the motif for more effective treatment strategies. METHODS: Artificially induced caries lesions (80-120 μm in depth) were produced on sectioned sound teeth following ethical approval (ref: 161221/RG/340) from the Dental Research Ethics Committee (School of Dentistry, University of Leeds). Enamel samples were remineralised with and without P11-4 for 5 days, and were subject to X-ray tomography and X-ray diffraction mapping on the DIAD beamline at Diamond Light Source (DLS) after sample preparation. The mineral density (MD) was extracted from reconstructed tomography image stacks, and the azimuthal spread of

OCTOBER 2023 ICCBMT 14 P18-F - Unraveling the complexity of cave bear molars: The influence of enamel distribution and enamel-dentine junction shape Mebin George Varghese*1, Otto Stenberg1, Susanna Sova1,2, Jukka Jernvall1,2. 1Institute of Biotechnology, 2Dept of Geosciences and Geography, University of Helsinki, Finland. INTRODUCTION: Tooth enamel is the most mineralized tissue in the mammalian body. It does not get repaired or remodeled like bones and is rarely uniformly distributed across the occlusal surface, thereby giving rise to variations in tooth morphology. Bears are a fascinating group with a varied feeding ecology and different tooth shapes. This difference in tooth shape among species, especially within the tooth crown, has been informative about the functional properties of teeth, taxonomy, and life history. Cave bear (Ursus spelaeus), an iconic extinct bear species that inhabited Europe and Western Asia until the last glaciation, evolved a specialized dentition with large molars with broad masticatory surfaces and many low, rounded cusps. As tooth shape correlates with diet and changes in feeding behavior are usually interpreted as adaptations to exploit food resources, progressive complications in the cheek teeth are thought to imply a heavy dietary reliance on tough plant matter. PURPOSE: As the final tooth morphology is the result of two developmental phases: patterning and enamel deposition, it raises the question of how much of the exceptionally high complexity of the cave bear molars can be attributed to the enamel distribution or, alternatively, to the shape of the underlying enamel-dentine junction (EDJ). Also, how different would they be when compared to the molars of other extant bear species? METHODS: We scanned the second upper molar teeth of the bears with 3D X-ray microtomography (μCT). Both the enamel and EDJ shapes were reconstructed using the μCT scans. The shapes were compared using Orientation Patch Count (OPC), Dirichlet Normal Energy (DNE), and enamel thickness. Nutrient-limited model of enamel matrix secretion and geometric extrapolation of the enamel surface from EDJ were used to examine variations in enamel distribution. RESULT: In cave bears, the high complexity is not a simple geometric extrapolation of the enamel from the EDJ. Rather, subtle features of the EDJ are magnified on the enamel surface. CONCLUSION: This suggests that the high complexity of cave bear molars is largely accomplished during the enamel matrix secretion phase of tooth formation, creating a series of cusps and valleys that increase the surface area available for grinding and shearing tough plant material. However, the shape of the underlying EDJ also plays a crucial role in determining tooth morphology. The EDJ is the boundary between the enamel and dentine layers of the tooth and influences how the tooth develops and shapes itself. It can create variations in tooth shape and size that are independent of the enamel distribution, and these can also contribute to the complexity of the tooth. P19-F - Evaluating amelotin-coated hydroxyapatite nanoparticles for the remineralization of artificial carious lesions in vitro Kelsey O'Hagan-Wong1, Joachim Enax3, Frederic Meyer3, Laurent Bozec1, Bernhard Ganss*1,2. 1Faculty of Dentistry, 2Institute of Biomedical Engineering, University of Toronto, Toronto, ON, Canada, 3Research Dept Oral Care Dr. Kurt Wolff GmbH & Co. KG, Bielefeld, Germany. INTRODUCTION: Hydroxyapatite (HAP) toothpastes contain HAP particles in micro or nanocrystalline form, which deposit into and restore demineralized enamel surfaces. As such, they are currently being explored as a fluoride-free anti-caries agent. Amelotin (AMTN) is a protein expressed during the maturation process of amelogenesis that has been found to be an important promoter in the mineralization of dental tissues. Recent work from our lab has demonstrated that AMTN-coated HAP nanoparticles (AMTN-HAP) promote collagen mineralization in vitro and that the pre-treatment of dentin with these nano complexes improves the bond strength of resin restorations. Given its proven ability to promote hydroxyapatite mineralization, AMTN-HAP may be a potential therapeutic to reverse incipient caries lesions. PURPOSE: The main objectives of this present study were: 1- to compare artificially induced enamel white spot lesions (WSL) with naturally occurring WSL and 2- to investigate the effect of AMTN-HAP on the remineralization of artificial WSL in human-extracted molars. METHODS: Healthy extracted human third molars were demineralized in a lactic acid buffer (pH= 4.5) for 7 days at 37 °C to create artificial non-cavitated WSL. The WSL (n=4) were treated with recombinant AMTN-HAP for 2 hours in artificial saliva. Conditions in which the WSL were untreated and treated with AMTN and HAP alone were used as controls. Samples were imaged and assessed using micro-CT and optical coherence tomography (OCT). RESULTS: The non-cavitated WSL lesio

OCTOBER 2023 ICCBMT 14 P20-F - Texture distribution changes in dental enamel with KLK4 mutation: Implications for understanding amelogenesis imperfecta pathogenesis Asmaa A Harfoush*1, Robert P Davies1,2, Neil H Thomson1,2,3, Maisoon Al-Jawad1,2. 1Division of Oral Biology, School of Dentistry, St James’s University Hospital, University of Leeds, 2Bragg Centre for Materials Research, University of Leeds, 3School of Physics and Astronomy, University of Leeds, Leeds, UK. INTRODUCTION: KLK4 is a gene that encodes for an enzyme called Kallikrein-related peptidase 4, which plays a critical role in the proper formation of tooth enamel. Without functional KLK4, enamel matrix proteins cannot be properly cleaved and processed, which leads to enamel defects and the development of the hereditary disease Amelogenesis Imperfecta (AI). PURPOSE: To investigate over multiple length scales the impact of a homozygous frameshift mutation (c.632delT) affecting the KLK4 gene on the crystallography, mineral density and microstructure of enamel and compare it to healthy enamel. METHODS: Five deciduous KLK4 teeth belonging to the same individual were scanned whole with high resolution micro-computed tomography (μ-CT) in order to assess mineral density changes. Teeth were then sectioned to obtain a mid labio-lingual slice and scanned by synchrotron X-ray diffraction for texture analysis at BM-28 beamline, at the the European Synchrotron Radiation Facility (ESRF). The same teeth slices were then prepared and examined using scanning electron microscopy (SEM) in order to correlate crystallographic changes to microstructure disruptions. RESULTS: Our results showed decreased mineral density in the KLK4 samples particularly in the molar teeth where the bulk of the enamel seemed significantly affected. The average enamel density of the molars measured was 2.10 g/cm3 compared to 2.73 g/cm3 in healthy matching controls. Enamel microstructure was also disrupted in the KLK4 samples, presenting thin and poorly defined enamel rods, wide interprismatic spaces and fusion of the crystallites across the enamel thickness. Our 2D texture maps indicate the co-existence of two populations (POP1 and POP2) of crystallites differing in terms of dominance and relative orientation, with angular separation ranging between 20-55◦apart. Crystallite organization in both populations was generally more disrupted in the KLK4-affected enamel, with most disruption occurring toward the inner enamel near the EDJ, especially at the lingual side of the tooth. In healthy enamel, higher texture was present at the surface gradually decreasing toward inner enamel. CONCLUSION: Our combined μ-CT, SXRD and SEM results indicate that the c.632delT variant impacts proper enamel formation and function. From our texture data we conclude that KLK4 is not only a critical protein for proper maturation and mineralization but also for hydroxyapatite crystallite organization particularly near the EDJ. P21 - Environmental pH modulates organic-inorganic interactions to regulate hierarchical mineralization Abshar Hasan*1,2,3, Gabriele Greco4,5, Sherif Elsharkawy6, Nicola M Pugno5,7, Alvaro Mata1,2,3. 1School of Pharmacy, University of Nottingham, Nottingham, UK, 2Biodiscovery Institute, University of Nottingham, Nottingham, UK, 3Dept of Chemical & Environmental Engineering, University of Nottingham, Nottingham, UK, 4Dept of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden, 5Laboratory of Bio-Inspired, Bionic, Nano, Meta, Materials & Mechanics, Department of Civil, Environmental and Mechanical Engineering, University of Trento, Trento, Italy, 6Faculty of Dentistry, Oral & Craniofacial Sciences, King's College London, London, UK, 7School of Engineering and Materials Science, Queen Mary University of London, London, UK. INTRODUCTION: Nature generates hierarchical biomineralized structures such as bones and dental enamel from highly regulated environments comprising dynamic organic matrices and environmental factors such as pH, temperature etc. Despite advances in our understanding of biomineralization processes, there remains a knowledge gap in our understanding of how underpinning organic-inorganic interactions can regulate hierarchical mineralization. Thus, we have previously reported on a mineralizing platform based on elastin-like recombinamers (ELRs) that through modulation of order and disordered regions can trigger the growth of fluorapatite (FAp) nanocrystals in a hierarchical manner (Nat. Commun. 9, 2018). PURPOSE: Inspired by the role of pH fluctuations in the amelogenin matrix during enamel development (Front. Physiol., 13, 2022), here we explored how pH changes can affect the supramolecular organization of the ELR matrix and consequently its physical (e.g., stiffness, swelling) properties, ultimately having direct implications on the hierarchical growth of FAp nanocrystals. METHODS: ELR solution (5% w/v) was crosslinked and dried to

OCTOBER 2023 ICCBMT 14 P22-F - Understanding structure and aggregation of amelogenin under various conditions Bojana Ginovska*1, Hoshin Kim1, Sebastian Mergelsberg1, Garry Buchko1, Jinhui Tao1, Susrut Akkineni2. 1Pacific Northwest National Lab, Richland, WA, USA, 2University of Washington, Seattle, WA, USA. INTRODUCTION: Amelogenin proteins play a crucial role in forming enamel in teeth and controlling the growth of hydroxyapatite (HAP). It has been known that primary to tertiary structures of amelogenin and the way they aggregate directly influences the formation and growth of enamel. However, in spite of this importance, structural details of amelogenin proteins are poorly understood as they are disordered proteins and only a fraction of amelogenin proteins at certain conditions have been experimentally studied. PURPOSE: The goal of this research is to characterize the structure of the amelogenin protein and its aggregates under various conditions. APPROACHES: Using experimental tools such as SAXS and computational tools - from atomistic simulations to deep learning-based structural prediction tools and experiments we explored the monomeric to oligomeric amelogenin structure. RESULTS: SAXS measurements were done across a systematic range of pH values, and concentrations to determine the size of oligomers of ameogenin in solution. Concurrent molecular dynamics (MD) studies for the amelogenin monomer were done to identify structured regions in the protein and determine size and shape of the monomer. Simulated SAXS data was obtained from the computational structures to elucidate specific features in treasured spectra. From the MD different level of structuring was identified under different condition, providing insight on how the secondary structure is affected by the environmental conditions. CONCLUSION: Contrary to previous understanding, where the monomer only exists at low pH values, we find that monomeric structure of the protein are likely to coexists with oligomers in a dynamic equilibrium across the pH range 3 to 8. P23 - Structure-function analysis of Fetuin-A Christian Hasberg1, Camilla Winkler1, Steffen Gräber1, Robert Dzhanhaev1, Carlo Schmitz1, Willi Jahnen-Dechent1. Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Hospital, Aachen, Germany. INTRODUCTION: Mineral crystals are incorporated into the matrices of living creatures through the controlled process of biomineralization, creating a solid framework for the remaining body components. As calcification is physiologically limited to the collagenous matrix of bones and teeth, a variety of local and circulatory calcification inhibitors actively prevent this in soft tissues. Fetuin-A, a plasma protein produced from the liver, controls mineralization. Fetuin-A is a systemic inhibitor of ectopic calcification. By generating colloidal protein-mineral complexes, fetuin-A stabilizes saturated mineral solutions. PURPOSE: The structural basis for fetuin-A's ability to inhibit calcification is the binding of mineral clusters to an extended ß-sheet in the protein's cystatin-like amino-terminal CY1 domain, which is rich in acidic amino acids. We propose that the mineral binding domain of CY1 is initially covered by an intrinsically disordered C-terminal region (CTR). Consequently, for CY1 to interact with calcium phosphate mineral, the CTR domain needs to be opened prior to mineral binding. The effects of phosphorylation, glycosylation, and disulfide bridges on mineral binding were studied. METHODS: Chinese hamster ovary CHO cells were used to produce recombinant fetuin-A disulfide, glycosylation and phosphorylation variants. The proteins were purified using immobilized metal affinity chromatography (IMAC). Their ability to bind minerals was evaluated using a calcium phosphate precipitate inhibition assay. This assay measures calcium precipitates formed from a supersaturated calcium and phosphate solution. In the presence of fetuin-A, calcium and phosphate form mineral clusters, potentially ripening into crystallization nuclei. By mineral deposition, the surface of the nuclei facilitates further growth into a crystal. Using cryo-EM the structure and function of fetuin-A will be experimentally validated, especially to pinpoint structural elements involved in the biomineral metabolism. RESULTS: The crystal nuclei are bound to fetuin-A forming colloidal protein-mineral complex preventing further growth and precipitation of mineral crystal nuclei. Fetuin-A’s strong affinity for mineral nuclei varies depending on post-translational modification. Notably, negative charge imposed by Ser/Thr phosphorylation sites and acidic amino acids enhance mineral binding and thus inhibitory activity. CONCLUSION: The CTR domain of fetuin-A may harbors mineral binding motifs in addition to the acidic amino acids contained in the amino-terminal in be involved in fetuin-A mineral binding, at least indirectly, according to the structure-function analysis. Aci

OCTOBER 2023 ICCBMT 14 P24 - DNA assemblies guide calcium phosphate mineralization Ryan Lee Chan*1, Liza DiCecco2, Kathryn Grandfield2,3, Karina M M Carneiro1,4. 1Institute of Biomedical Engineering, University of Toronto, Toronto, ON, Canada; 2Dept of Materials Science and Engineering, McMaster University, Hamilton, ON, Canada; 3School of Biomedical Engineering, McMaster University, Hamilton; 4Faculty of Dentistry, University of Toronto, Toronto, ON, Canada. INTRODUCTION: DNA assemblies are promising biomaterials as they can fold into complex and highly ordered nanoarchitectures capable of mimicking naturally occurring structures. Further, they can act as scaffolds and be functionalized at specific locations. To date, there remains limited research on DNA assemblies as guides for calcium phosphate (CaP) mineral growth and mineralized tissue regeneration. We aim to investigate and enhance the molecular mechanisms that occur during the formation of hydroxyapatite (HA) on DNA surfaces in vitro. PURPOSE: The objective is to functionalize DNA assemblies (DNA origami rectangles) with extracellular matrix (ECM)-derived mineral-promoting peptides. It is hypothesized that controlled, enhanced and selective CaP mineral growth will occur on DNA nanostructures through strategic patterning of mineral-promoting peptides. METHODS: To do so, mineral-promoting peptides with an additional cysteine amino acid (thiol source) were covalently attached to DNA-maleimide through a thiol-ene click chemistry reaction and purified by column chromatography. Peptide-DNA were site-specifically incorporated onto DNA origami rectangles and were characterized by gel electrophoresis and AFM. Mineral was selectively grown on the DNA origami rectangle surfaces in mineralizing conditions and characterized by AFM and TEM. Liquid in situ TEM, SAED and XRD were employed to characterize the extent and control of the mineral grown. RESULTS: Peptide-DNA conjugation was achieved with a ~30-60% yield and was successfully attached at five site-specific locations on the DNA origami rectangle surface. In mineralizing solution, mineral deposition following the shape of the underlying scaffold of the functionalized DNA assemblies was observed, with a significant height increase. CaP crystals were obtained upon incubation in mineralizing solution. CONCLUSIONS: Functionalized DNA assemblies were designed and successfully synthesized. DNA assemblies were shown to guide and enhance CaP crystal growth; therefore, the hypothesis is accepted. Future work will test alternate compositions and ratios of mineral-promoting peptides and DNA assemblies. SIGNIFICANCE: There is a need for new approaches in bone repair and implant integration within bone that promote osteogenesis and mineralization to improve the quality of treatment outcomes. Insight into HA biomineralization, nucleation and growth using DNA assemblies may contribute to the understanding of bone and teeth formation, pathological calcification mechanisms and designing biomimetic DNA-based materials for mineralized tissue regeneration. P25 - The sacroiliac joint: a sensitive tool to highlight altered bone phenotype in murine models of skeletal disorders Stéphane Hilliquin*1,2, Volha Zhukouskaya1,3, Olivier Fogel2, Chahrazad Cherifi4, Lotfi Slimani1, Karine Briot2, Rik Lories5,6, Catherine Chaussain1,3,4, Corinne Miceli-Richard2, Claire Bardet1. 1Université Paris Cité, Institut des maladies musculo-squelettiques, Laboratory Orofacial Pathologies, Imaging and Biotherapies URP2496 and FHU-DDS-Net, Dental School, and Plateforme d’Imagerie du Vivant, Montrouge, France, 2Dept of Rheumatology, Cochin Hospital, Université Paris Cité, Paris, France, 3Centre de référence des maladies rares du métabolisme du calcium et du phosphate, Plateforme d’expertise maladies rares Paris Saclay, filière OSCAR, EndoRare and BOND ERN, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France, 4AP-HP Reference Center for Rare Disorders of the Calcium and Phosphate Metabolism, Dental Medicine Dept, Bretonneau Hospital, GHN, Paris, France, 5Division de Rhumatologie, University Hospitals Leuven, Leuven, Belgium, 6Dept of Development and Regeneration, Skeletal Biology and Engineering Research Centre, Skeletal Biology and Engineering Research Centre, KU Leuven, Leuven, Belgium. INTRODUCTION: Bone disorders may affect the skeleton in different ways, some bones being very impaired and others less severely. In translational studies using murine models of human skeletal diseases, the bone phenotype is mainly evaluated at the distal femur or proximal tibia. The sacroiliac joint (SIJ), which connects the spine to the pelvis, is involved in the balanced transfer of mechanical energy from the lumbar spine to the lower extremities. Because of its role in biomechanical stress, the SIJ is a region of particular interest in various bone diseases. OBJECTIVE: Here we aimed to characterize the SIJ in several murine models to develop a highly reliable tool for stu

OCTOBER 2023 ICCBMT 14 P26-F - Impact of therapeutic strategies on dentoalveolar phenotype in the murine model of X-linked hypophosphatemia: what about gene therapy? Elis J Lira dos Santos*1, Amandine François1,2, Julien Po1, Volha V Zhukouskaya1,2,3, Agnès Linglart4, Giuseppe Ronzitti2, Fanny Collaud2, Catherine Chaussain1,4, Claire Bardet1. 1Université de Paris, Institut des maladies musculo-squelettiques, Laboratory Orofacial Pathologies, Imaging and Biotherapies URP2496 and FHU-DDS-Net, Dental School, and Plateforme d’Imagerie du Vivant (PIV), Montrouge, France, 2Université Paris-Saclay, Univ Evry, Inserm, Genethon, INTEGRARE Research Unit UMR_S951, Evry, France, 3Paris-Saclay University, INSERM U1185, AP-HP, DMU SEA, Endocrinology and Diabetes for Children, Reference Center for Rare Diseases of the Calcium and Phosphate Metabolism, OSCAR filière, EndoRare, and BOND ERN, Bicêtre Hospital, Le Kremlin-Bicêtre, France, 4AP-HP, Reference Center for Rare Disorders of the Calcium and Phosphate Metabolism, Dental Medicine Dept, Bretonneau Hospital, GHN-Université de Paris, Paris, France. INTRODUCTION: X-linked hypophosphatemia (XLH) is the most common form of genetic rickets (1/20 000 births) and is associated with elevated levels of fibroblast growth factor 23 (FGF23). Mainly diagnosed during childhood by growth retardation and deformities of the lower limbs, this disorder also affects dentoalveolar mineralization resulting in higher susceptibility to spontaneous dental necrosis and periodontitis. The FGF23-neutralizing antibody burosumab was approved for clinical use as an alternative to conventional treatment (daily intake of phosphorus and 1,25-(OH)2-vitamin D) as it was shown to improve the long bone phenotype and dental abscesses. We recently demonstrated proof of concept of gene therapy using Adeno-associated virus (AAV) vectors on long bone phenotype. PURPOSE: Here, we study the gene therapy impact on the dentoalveolar features associated with XLH to assess its efficacy. METHODS: Hyp-Duk mice were treated with AAV-cFGF23 injections from 4 weeks to 4 months after birth and compared to untreated Hyp-Duk and wild-type (WT) controls (n=4-6 per group). Dentoalveolar phenotype was analyzed by micro-computed tomography (micro-CT) and histology. RESULTS: AAV- cFGF23 treatment improved alveolar bone volume fraction and mineral density in Hyp-Duk mice. Additionally, this treatment restored dentin/cementum volume, resulting in a rescue similar to WT levels. Enlargement of pulp chambers was also recovered with a reduced pulp volume in treated Hyp-Duk mice. Histological analyses highlighted the positive outcomes on mineralization of different dental tissues such as dentin, alveolar bone, and dental cementum in Hyp-Duk mice. CONCLUSIONS: Dentoalveolar tissues are responsive to gene therapy treatment for XLH. We highlight substantial improvements confirming the relevance of therapeutic strategies targeting FGF23. P27 - Developing a novel bone explant model to investigate physiological influences on bone health Mikayla Moody*1, Sydney Whittaker1, Tannin Schmidt1, Alix Deymier1. 1Dept of Biomedical Engineering, University of Connecticut Health Center, Farmington, Connecticut, USA. INTRODUCTION: Bone is primarily composed of a high substituted calcium phosphate apatite mineral. Due to its ability to exchange ions with the surrounding fluid, bone mineral composition can change with body fluid pH and ionic composition. Such changes will affect bone mineral crystallinity, solubility, and mechanics, thus affecting whole bone function. Due to the multi-organ interactions of the skeletal system in vivo, simplified explant models are ideal for investigating these body fluid-bone interactions. Current explant models use neonatal bone due to increased perfusion and cell viability. However, neonatal mineral is underdeveloped, making it ill-suited for these studies. Therefore, it is necessary to develop a relevant adult explant model to explore fluid mediated effects on bone composition and function. PURPOSE: Develop a viable adult bone explant model to examine the compositional and mechanical effects of changes in body fluid. METHODS: Femurs were dissected from 8-week-old male CD-1 mice and cultured up to 7 days in low glucose DMEM supplemented with 10% FBS, 0.1% ascorbic acid, 1% non-essential amino acids, 1% pen-strep, 1% amphotericin B and maintained at pH 7.4 with bicarbonate. Femurs were either placed in culture intact, with removal of the proximal epiphysis, or with removal of both epiphyses. Media was changed every 2 days. Metabolic activity was determined via AlamarBlue. Osteocyte viability was examined via qRT-PCR using primers for DMP-1 and SOST. 3-point bend mechanical testing determined tissue mechanical properties. Statistics and regression models were performed using Prism. RESULTS: Cell metabolic activity, as measured via AlamarBlue fluorescence normalized by bone length, increased over the span of seven days for a

OCTOBER 2023 ICCBMT 14 P28 - Promising strategy to enhance biomineralization for bone repair and regeneration: Bioactive tissue engineered scaffold Hemalatha Kanniyappan1, Varun Gnanasekar2, Yani Sun3, Rong Wang4, Aftab Merchant1, Mathew T Mathew1. 1Regeneratve Medicine and Disability Research Lab (RMDR), Dept of Biomedical Science, University of Illinois College of Medicine Rockford, Rockford, IL, 2University of Wisconsin-Madison, Madison, WI, 3Dept of Material Science, University of Illinois at Chicago, Chicago, 4Dept of Chemistry, Illinois Institute of Technology (IIT), Chicago, IL. INTRODUCTION: Conventional bone grafts are difficult to procure and are often rejected by the recipient's body. Synthetic grafts are another option, but their usefulness is limited due to insufficient mechanical strength, biomineralization, bioactivity (vascularization), and biocompatibility to replicate a bone. An amalgam of pectin, polyvinyl alcohol (PVA), and chitosan could provide such an interpenetrating polymer network IPN scaffold. Pectin improves gelation, osteogenic differentiation, and bioactivity; polyvinyl alcohol increases mechanical strength; chitosan enhances bioactivity and hemostasis and is bacteriostatic and fungistatic. PURPOSE: We hypothesize that a pectin- based IPN scaffold is a suitable grafting biomaterial for bone tissue engineering that will promote bone repair and regeneration. METHODS: The scaffolds were fabricated with 1% w/v polymeric blend by lyophilization. The mineralization property of the fabricated scaffolds was determined using the Alizarin Red Staining with MG-63 cells for the time intervals of 7 and 14 days. Also, the osteogenic differentiation potential was determined using the alkaline phosphatase activity for the period of 7 and 14 days. In vivo CAM assay was performed to determine the vascularization potential of the fabricated IPN scaffolds. The mechanical strength of the scaffolds was analyzed using a UTM. All the experiments were performed in triplicates. RESULTS: SEM image (Fig 1a) exposes the porous with highly interconnected nature of the fabricated IPN scaffold. In vitro cytocompatibility (Fig 1b) proves the scaffold is non-toxic and effectively supports cell proliferation. Also, the alizarin red staining (Fig 1 c) shows enhanced biomineralization with calcium deposition. Osteogenic differentiation is analyzed by the ALP assay (Fig 1d), which showed improved ALP activity. In vivo CAM assay (Fig 1e) proves the angiogenic nature of the IPN scaffolds. Finally, the young’s moduli and compressive strength of the scaffold (Fig 1f) were found to be 80, and 2.5 MPa lies in the range of cancellous bone. The statistical analyses were performed using GraphPad Prism Software, and the results were assessed with ordinary two-way ANOVA followed by a Tukey post hoc test. A value of p < 0.05 was considered significant. DISCUSSION & CONCLUSIONS: Our findings demonstrate that the pectin-based IPN scaffold enhances the biomineralization process. The in vitro analysis confirmed that the IPN scaffolds are nontoxic and induce bone cell adhesion, mineralization, migration, and proliferation. The CAM assay showed that the scaffolds promote arteriogenesis and angiogenesis. ACKNOWLEDGEMENTS: NIH, Blazer foundation, Michael Reese Award, Bridge-Grant Award. REFERENCES: [1] K Dulany et al.,2019, [2] A Oryan et al.,2014, [3] Lin et al., 2013. P29-F - Miniaturized device for assessing calcification propensity of implant materials using simulated body fluid calcification medium Aaron Morgan*1, Andrea Gorgels1, Jan Ritter2, Johanna C Clauser2, Felix Stockmeier3,4, Lucas Stüwe3, John Linkhorst3, Matthias Wessling3,4, Ulrich Steinseifer2, Willi Jahnen-Dechent1. 1Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Hospital, Aachen, Germany, 2Dept of Cardiovascular Engineering, Institute of Applied Medical Engineering, Helmholtz Institute Aachen, RWTH Aachen University, Aachen, Germany, 3Chemical Process Engineering AVT.CVT, RWTH Aachen University, Forckenbeckstraße 51, Aachen, Germany, 4DWI - Leibniz- Institute for Interactive Materials, Forckenbeckstraße 50, Aachen, Germany. INTRODUCTION: Biohybrid implants are a promising advancement for the treatment of many diseases, but remain difficult and expensive to develop, test, and validate. Large testing devices use accelerated pump rates, require large material patches and medium volumes, and are often incompatible with sterile cell culture conditions. As many advanced materials are difficult or impossible to produce at large scale, these requirements preclude the investigation of many constructs. PURPOSE: To develop a miniaturized, dual-channel flow chamber that allows for detection, monitoring, and visualization of material calcification in real-time and simulates key aspects of the physiological environment. The mineral chaperone protein fetuin-A, which has a high binding affinity to calcium, is used to identify calcifications within the materi

OCTOBER 2023 ICCBMT 14 P30-F - Structural and molecular characterization of an Scpppq1 knock out mouse Pierre Moffatt1, Katia Julissa Ponce2, Rima Wazen2, Dainelys Guadarrama Bello2, Antonio Nanci*2,3. 1Shriners Hospitals for Children, Montréal, Montréal, QC, Canada, 2Laboratory for the Study of Calcified Tissues and Biomaterials, Dept of Stomatology, 3Dept of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, QC, Canada. BACKGROUND: The Scpppq1 (secretory calcium-binding phosphoprotein proline-glutamine rich 1) gene is located within the Secretory Ca- binding PhosphoProtein (SCPP) cluster, next to the ancestral Sparcl1. It encodes a small, secreted protein that, depending on the species, ranges between 87 and 95 amino acids, including the signal peptide. The mature protein is rich in proline (~20 %), leucine (~17 %), glutamine (~12 %) and phenylalanine (~10 %). Apart from a putative serine phosphorylation motif (SSSE) at position 26–29 conserved among most species, SCPPPQ1 presents no homology to other proteins. The protein is expressed only in late maturation stage ameloblasts and in junctional epithelium (JE) cells where it conspicuously accumulates in the specialized basal lamina (sBL) at the interface between these cells and the tooth. Besides having Ca-binding properties that could mediate cell adhesion to the tooth, there is no functional information on this unique protein. PURPOSE: To better understand the role of SCPPPQ1 we have created a knockout (KO) mouse and we present here a preliminary report of some phenotypic changes observed. METHODS: A “global” Scpppq1 KO mouse was generated using CRISPR-Cas9. Comparative electron microscope structural analyses, immunogold labeling and RT-qPCR were carried out on wild type (WT) and KO incisors and maxillary molar JE. RESULTS: No expression of SCPPPQ1 was observed in the KO, confirming inactivation of the gene. Mice reproduced and grew (size and weight) normally, as compared to the WT littermates. Consistent with the expression of SCPPPQ1 during the late maturation stage, scanning electron imaging showed no difference in the rod shape and decussation pattern. While focal detachment of the enamel organ from the tooth surface and formation of cystic structures were occasionally seen in WT mice, these were more frequent and larger in the KO. In absence of SCPPPQ1, the dentogingival junction appeared to detach from the tooth surface and descend along the tooth root. JE cell changes including focal regions of parakeratinisation, an overall paucity of ODAM labeling, and a notable increase in the size of lysosomal-like elements immunoreactive for AMTN were observed. RT-qPCR pointed lower levels of expression of AMTN and ODAM in the incisor but in the JE there was actually an apparent increase in AMTN. Both in the maturation stage and JE, there were changes in the appearance of the sBL, being thicker at variable degrees and less dense. Labeling for ODAM was present throughout the thickened sBL but abnormally concentrated near the tooth surface. CONCLUSIONS: These observations indicate that that SCPPPQ1 plays a role in structuring the sBL and in maintaining cell adhesion to the tooth surface. While alteration of this relationship in late maturation has no major impact on enamel structure, adhesive integrity of the maturation enamel organ and JE appeared perturbed. P31 - Triple function of amelogenin peptide-chitosan hydrogel for dentin repair J Cai, J Moradian-Oldak*. Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, LA, USA. BACKGROUND: Biomimetic strategies like peptide-guided collagen mineralization promise to enhance the effectiveness of dentin remineralization. We recently reported that rationally designed amelogenin-derived peptides P26 and P32 promoted apatite nucleation, mineralized collagen, and showed potential in enamel regrowth and dentin remineralization. AIM: To facilitate the clinical application of amelogenin-derived peptides and to uncover their effectiveness in repairing dentin, we have now implemented a chitosan (CS) hydrogel for peptide delivery and have investigated the effects of P26-CS and P32-CS hydrogels on dentin remineralization using two in situ experimental models that exhibited different levels of demineralization. METHODS: The efficacy of the peptide-CS hydrogels in dentin repair was evaluated by characterizing the microstructure, mineral density, mineral phase, and nanomechanical properties of the remineralized samples. The new strategy of atomic force microscopy (AFM) PeakForce Quantitative Nanomechanical (QNM) mapping was utilized for direct visualization and nanomechanical analysis of repaired dentin lesions across the lesion depth. RESULTS: Results from the two models indicated the potential triple functions of peptide-CS hydrogels for dentin repair: building a highly organized protective mineralized layer on dentin, occluding dentinal tubules by p

OCTOBER 2023 ICCBMT 14 P32-F - Understanding the craniofacial abnormalities in the C19F variant and two models lacking the conserved functional residues of Matrix Gla protein Sultanah Alshahrani1, Kyoungmi Bak2, Ophélie Gourgas3, Abhinav Parashar3, Juliana Marulanda3, Monzur Murshed1,3,4*. 1Faculty of Dentistry, McGill University, Montreal, QC, Canada, 2Dept of Anatomy and Cell Biology, Faculty of Medicine, 3Dept of Medicine, McGill University, Montreal, QC, Canada, 4Shriners Hospital for Children, McGill University, Montreal, QC, Canada. BACKGROUND: Matrix Gla protein (MGP), a secreted phosphoprotein produced by vascular cells and chondrocytes, is a potent inhibitor of soft tissue calcification. MGP undergoes two post-translational modifications – three conserved serine residues are phosphorylated, while four conserved glutamic residues are -carboxylated. The loss of these residues affects MGP’s anti-mineralization function. Recently, individuals with a heterozygous variant in MGP (c.MGP 56G>T; p.MGP C19F) with skeletal deformities and craniofacial anomalies have been reported. In MGP-deficient (Mgp-/-) mice, a model for human Keutel syndrome, midface hypoplasia is associated with nasal septum calcification as well as premature closure of the spheno-occipital synchondrosis. Currently, it is not known whether the same or different causes are responsible for the midface hypoplasia seen in Mgp+/56G>T mice expressing C19F MGP. In addition, we do not know whether MGP’s two sets of conserved residues contribute to its role as a regulator of midface development. OBJECTIVES: First, to investigate the mechanisms underlying midface hypoplasia in Mgp+/56G>T mice, and second, to examine the role of MGP’s conserved residues in craniofacial development. METHODS: We generated 3 different mouse lines carrying the heterozygote Mgp 56G>T mutation (Mgp+/56G>T mice), homozygous ‘knock-in’ mutation to replace MGP’s conserved serine residues to alanine (MgpS3mut/S3mut mice), and Mgp-null mice producing a variant of MGP in chondrocytes in which the conserved glutamic acid residues have been mutated to alanine (Mgp-/- ;Col2a1-GlamutMgp mice). We examined their craniofacial abnormalities using cell and molecular biology techniques, micro-CT-based cephalometric analyses, and bone histomorphometry. RESULTS: We show that the cranial growth plates are prematurely calcified with massive deaths of prehypertrophic and hypertrophic chondrocytes in Mgp+/56G>T mice. Interestingly, unlike in Mgp-/- mice, the nasal septum Mgp+/56G>T mice are not calcified. Our work suggest that the cranial growth plate anomalies are the major cause of midface hypoplasia Mgp+/56G>T mice. The two other models also show premature calcification of the growth plates impairing craniofacial development. CONCLUSION: C19F MGP affects facial development by promoting chondrocyte deaths and premature closure of the growth plates. Our work establishes that MGP is a major regulator of midface development, and both sets of its conserved residues are required for this function. P33 - The ability of recombinant amelogenin protein compared to poly glycolic acid to regenerate lost dental tissue in immature teeth with pulp necrosis Ali Alharthy#, Madawy Kehely1, Jamaluddinsyed2, Maha MF Mounir*. #Intern at the Faculty of Dentistry, King Abdulaziz University, 1Assistant Professor, Oral Diagnostic Science, Faculty of Dentistry, King Abdulaziz University, 2Assistant Professor, Oral Basic and Clinical Sciences, Faculty of Dentistry, King Abdulaziz University, *Professor, Oral Biology, Faculty of Dentistry, King Abdulaziz University. INTRODUCTION: Recombinant DNA-produced amelogenin protein RAP was compared to Poly Glycolic acid PGA in a study of immature apex closure conducted in 12 young mongrel dogs. METHODS: Root canals of maxillary and mandibular premolars (n = 120) were root canal treated with the complete removal of the pulp tissue and left open for 14 days. Canals were cleansed, irrigated, and split equally for treatment with RAP (n = 60) or PGA (n = 60). RESULTS: After 1 and 3 months, the animals were sacrificed and the treated teeth were recovered for histological assessment and immune detection of protein markers associated with odontogenic cells. After 1 month, amelogenin-treated canals revealed calcified tissue formed at the apical foramen and a pulp chamber containing soft connective tissue; amelogenin-treated canals assessed after 3-month intervals further included apical tissue functionally attached to bone by a periodontal ligament PDL. In contrast, although a calcified apical tissue was formed in the PGA group neither soft connective tissue within the pulp chamber nor the periodontal ligament was observed. CONCLUSION: The findings suggest that recombinant amelogenin protein is the component that can signal for pulp, periodontal regeneration, and dentin bridging via activation of the Wnt signaling pathway. PGA initiates the regeneration of the dentin apical barrier

OCTOBER 2023 ICCBMT 14 P34 - A computational model accounting for physicochemical aspects of bone mineralization Hossein Poorhemati1,3*, Svetlana Komarova1,2,3. 1Dept of Biological and Biomedical Engineering, McGill University, Canada, 2Faculty of Dental Medicine and Oral Health Sciences, McGill University, Canada, 3Shriners Hospital for Children, Montreal, QC, Canada. INTRODUCTION: Bone consists of an organic, primarily collagenous matrix with embedded crystals of calcium phosphate in the form of hydroxyapatite. The quality and quantity of mineralization determine the bone material properties. Abnormal mineralization of bone matrix results in severe clinical problems, including bone deformities and fractures, such as those observed in osteogenesis imperfecta and vitamin D deficiency. Hydroxyapatite formation in bone occurs at restricted nucleation sites provided by specific extracellular matrix proteins and is controlled by multiple mineralization inhibitors. Physicochemical factors, such as the availability of required ions and the aqueous environment's saturation status, are also important in biological mineralization. PURPOSE: Mathematical modeling provides important insights into the complex dynamic of mineralization, and models addressing the biological aspects of bone mineralization have been developed. However, there is still a lack of a mathematical description of the physicochemical processes that occur in the bone vicinity during bone formation or resorption and during pathophysiological conditions. This study aimed to use mathematical modelling to describe the complex physicochemical environment permissive to the precipitation of biological hydroxyapatite. METHODS: We simulated the processes occurring in the interstitial fluid (ISF) in the bone vicinity. The ISF was defined as an aqueous environment containing seven commonly reported chemical components (calcium, phosphate, carbonate, sodium, potassium, magnesium, and chloride) that form 30 chemical species. We simulated reversible equilibrium reactions among these chemical species and calculated supersaturation for hydroxyapatite as well as precipitation rate using kinetic theory. RESULTS: The simulated ISF was of correct ionic strength and predicted the equilibrium component concentrations consistent with the experimental findings. The supersaturation index S of physiological ISF was ~5-10 when S >1 defines the solution that supports precipitation. These data are consistent with prior findings that mineralization inhibitors are required to prevent spontaneous mineral precipitation. The predicted initial hydroxyapatite precipitation rate was faster than observed experimentally, suggesting a strong influence of biological regulators. Among all the chemical components, calcium and phosphate affected the precipitation process the most, followed by carbonate, which also had the strongest influence on system pH. CONCLUSIONS: This model will allow in silico studies of complex clinical scenarios associated with alterations in ISF ion composition, such as osteomalacia, rickets, hypophosphatemia and chronic kidney disease. Moreover, with minor adaptations, it could be used to understand other mineralized tissues, such as dentin and enamel. P35 - Identification of candidate pathways and pharmacological drugs that mediate pathological skeletal remodelling in spinal osteoblasts Ellie Northall*1, Tom Nicholson1, Nicola Foster2, Alicia El Haj2, Liam Grover2, Helen M McGettrick1, Amy J Naylor1, Matthew Newton Ede3, Simon W Jones1. 1Institute of Inflammation and Ageing, MRC Centre for Musculoskeletal Ageing Research, University of Birmingham, 2School of Chemical Engineering, University of Birmingham, 3Royal Orthopaedic Hospital NHS Trust, Birmingham, UK. BACKGROUND: Osteoblast activity is a key determinant in the development and maintenance of healthy skeletal tissue with ageing. Indeed, dysregulation of osteoblast activity is associated with abnormal bone remodelling, leading to the development of musculoskeletal disorders including osteoporosis and scoliosis. We previously reported that spinal osteoblasts at the curve apex in adolescent idiopathic scoliosis (AIS) patients exhibit a differential metabolic and proliferative phenotype, compared to osteoblasts from non-curve spine, indicating that intrinsic differences in osteoblast phenotype might drive abnormal skeletal development in scoliotic patients. AIMS: To identify candidate pathways and candidate pharmacological drugs as mediators of skeletal development in scoliotic patients. METHODS: Spinal tissue was collected perioperatively from (i) the concave and (ii) convex side at the curve apex and (iii) from outside the curve (non- curve) from 6 AIS female patients (age 13-18 years). Osteoblast phenotype was determined by RNA sequencing with candidate pathways and targets identified using bioinformatic pathway analysis (Ingenuity). Target modulation of a candidate pathway was performed using a small molecule

OCTOBER 2023 ICCBMT 14 P36-F - Matrisome proteomic profiling between young and old dentin identifies age- and sex-differences Michelle Blyumin1, Mona Omar1, Sarah B Peters1. 1Division of Biosciences, College of Dentistry, The Ohio State University. OBJECTIVE: Dentin is primarily composed of hydroxyapatite crystals within an organic matrix that contains a variety of sequestered bioactive molecules. These matrisomal contents are released to the pulp tissue via bacterial secretions and/or dental procedures and have been shown to induce angiogenesis, neurite outgrowth, and reparative dentinogenesis. While it is well-established that the aging process of appendicular and axial skeletons differs between the sexes, little is known about whether this also occurs in teeth and how this affects the ability to withstand mechanical load and/or repair dentin. A recent study reported differences in the longevity of dental restorations between the sexes, with female procedures surviving longer than they did in male patients, implying differences in fracture susceptibility and/or bonding or restorations. We hypothesize that the continuous apposition of dentin changes with age and between the sexes and could affect tooth matrix composition and regenerative capacities. The objective of this project is to characterize the dentin proteome in young and old male and female rats to identify age- and sex-related changes. METHODS: First, second, and third molars were extracted from maxillae and mandibles from 3 young (3 months) and 3 old (24 months) male and female rats. Crown and root portions were separated, and pulpal tissue was removed. The mineralized tissue was cryogenically milled, proteins were extracted, and proteomics analysis was performed. Proteome categories were divided into collagens, extracellular matrix (ECM) glycoproteins, proteoglycans, ECM regulators, ECM-affiliated proteins, and secreted factors. RESULTS: Proteomics analysis demonstrated significant differences in identified proteins, protein quantities, and fold-change increases/decreases with time between male and female dentin. Interestingly, the crown dentin contained several proteins that significantly decreased with age in male, whereas only decorin changed in female crowns with age. The root dentin contained several proteins that increased with age and were significantly different in identity and quantity between male and female samples. In particular, male root collagens demonstrated no change whereas COL5A1 and COL12A1 increased 10-fold and 4- fold in female roots, respectively. Female root dentin also contained more secreted factors with age. For instance, female root dentin demonstrated >40-fold and 5.5-fold increases in TGFB2 and TGFB1, respectively whereas no members of the TGFbeta family changed in male root dentin. CONCLUSIONS: This preclinical study is the first to established age- and sex-related changes in the dentin content. Subsequent studies will profile mechanical testing with nanoindentation and morphology with micro-computed tomography. We believe these studies could identify biomarkers of tooth aging and be used to identify the crucial signals regulating mechanical integrity and pulp vitality of teeth with age. P37-F - Insights into mineral transport within the embryonal chick femur using cryo-FIB SEM 3D volume imaging Emeline Raguin*1, Richard Weinkamer1, Peter Fratzl1. 1Dept of Biomaterials, Max Planck Institute of Colloids and Interfaces Am Mühlenberg 1, Potsdam 14476, Germany. INTRODUCTION: Bone growth and mineralization requires the transport of large amounts of calcium ions from the blood stream to the sites of mineralization. However, calcium concentration is highly regulated and must be maintained at very low concentrations both in the serum and the cytosol of the cells to ensure proper intracellular signaling function without disrupting the normal metabolism. Thus, the transportation path raises a logistical challenge that remains elusive. PURPOSE: Our study focusses on the three-dimensional visualization and quantification of the mineral precursors in the femur of the fast-growing chick embryo. The aim is also to provide a dynamic interpretation of the mineralization process based on the 3D volumes by estimating the speed at which mineralization precursors have to be transported to the sites of mineralization. METHODS: Osteocytic lacunar density was established using micro-computed tomography (micro-CT). At the nanoscale level, focused ion beam with scanning electron microscopy (FIB-SEM) and the serial surface view method (SSV) was used in cryogenic condition to observe the mineral precursors, the cells as well as the mineralized matrix in their close to native states. RESULTS: Cryo FIB-SEM has revealed the presence of numerous intracellular vesicles containing mineral precursors in osteocytes as well as in osteoblasts/pre-osteocytes. An interconnected network of nanochannels of approximately 40nm in diameter and in relation wi

OCTOBER 2023 ICCBMT 14 P38-F - Optical photothermal infrared (O-PTIR) spectroscopy and imaging of bone mineralization at submicron scale William Querido*1. 1Dept of Bioengineering, Temple University, Philadelphia, PA, USA. INTRODUCTION: As a beautifully hierarchical structure, the basis of bone quality and strength relies on its submicron scale building blocks— mineralized collagen fibrils and bundles on the order of ~500 nm. Vibrational spectroscopic techniques such as Fourier transform infrared (FTIR) spectroscopy have been widely used to investigate bone mineralization. However, with these approaches the spatial resolution of analysis is limited to several micrometers (~10 μm), which cannot capture properties of individual tissue features at the submicron scale. Recently, a new modality of infrared spectroscopy, optical photothermal infrared (O-PTIR) spectroscopy, has been developed to allow data collection at 500 nm spatial resolution. PURPOSE: Our recent research focuses on the novel application of O-PTIR spectroscopy and imaging to assess bone tissue composition at the submicron scale, aiming to advance this method for the analysis of mineralized tissues. METHODS: We analyzed two different types of bone samples to demonstrate the feasibility and unique application of this new method. Importantly, minimal sample preparation was necessary other than cutting the samples into slices that would fit onto the microscope stage. We used thick (~2-4 mm) slices of unembedded human femoral neck and of mouse tibia embedded in poly(methyl methacrylate) (PMMA). Spectra were acquired using the state-of-the-art mIRage submicron infrared microscope (Photothermal Spectroscopy Corp.) with spot size of 500 nm. RESULTS: Spectra of unembedded cortical and trabecular bone tissues showed typical peaks from bone apatite (PO4, CO3, HPO4) and collagen (amide I). Moreover, it was possible to map the distribution of specific tissue components throughout individual osteons (cortical bone) and trabeculae at submicron scale resolution. Interestingly, quantification of mineral content demonstrated the typical alternating lamellae across the osteon, as well as the trabeculae mineralization pattern aligned to its longitudinal axis. Additionally, O-PTIR spectra from PMMA-embedded mouse tibia cortical bone showed only minor influences of PMMA, presenting clear typical peaks necessary to quantify relevant tissue properties, demonstrating the feasibility to assess composition of embedded samples. Moreover, by obtaining hyperspectral images, we could gather a vast data collection comprising thousands of spectra acquired at 500 nm spatially- defined spots, which can provide a rich and detailed assessment of submicron scale compositional properties and distribution throughout the tissue area. CONCLUSIONS: This study highlights the novel application of O-PTIR spectroscopy and imaging to assess bone mineralization at submicron scale in thick samples without the need for cumbersome thin-sectioning of calcified bone. Further advance of O-PTIR spectroscopy will contribute to understanding bone biomineralization and in investigating the submicron scale origins of bone diseases such as osteoporosis. P39-F - Bone matrix mineral content regulates early-stage metastasis by altering mesenchymal stem cell fate Nicole Sempertegui*1, Stephanie Lux1, Matthew Whitman1, Siyoung Choi1, Claudia Fischbach1. 1Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA. INTRODUCTION: Reduced bone mineral density predicts increased risk for bone metastasis, the primary reason for morbidity and mortality in patients with advanced breast cancer. Nevertheless, it remains poorly understood how bone matrix mineral content regulates bone metastasis and which roles mesenchymal stem cells (MSCs) play in this process. Understanding these connections is critical as bone matrix mineralization has been shown to alter MSC fate in regenerative settings and because MSCs are critical regulators of tumor cell phenotype during early (i.e., non-osteolytic) stages of metastasis and thus, determinants of tumor cell dormancy or outgrowth. PURPOSE: Here, we studied the hypothesis that reduced matrix mineralization promotes tumor growth during early stages of bone metastasis by reducing MSC differentiation into osteoblasts, while promoting differentiation into cancer-associated fibroblasts (CAFs). METHODS: Our group has developed biofunctional bone matrix models in which we can selectively control mineral content using both synthetic methods and controlled dissolution of mineral from decellularized bone scaffolds. Using these models in the presence and absence of tumor-conditioned media (TCM) collected from MDA-MB231 breast cancer cells, we analyzed MSC differentiation into osteoblasts and CAFs through colorimetric, immunofluorescence, and ELISA-based assays. Moreover, we determined the contribution of mineral-dependent changes in MSC mechanosignaling

OCTOBER 2023 ICCBMT 14 P40 - Characterization of biomineralization in shark vertebral cartilage Dawn Raja Somu*1, Malena Fuentes1, Lihua Lou2, Arvind Agarwal2, Imke Greving3, Vivian Merk1. 1Dept of Chemistry & Biochemistry and Dept of Ocean & Mechanical Engineering, Florida Atlantic University, Boca Raton, FL, USA, 2Dept of Mechanical and Materials Engineering, Florida International University, Miami, FL, USA, 3Institute of Materials Physics, Hemholtz-Zentrum Hereon, Geesthacht, Germany. INTRODUCTION: The interaction between inorganic and organic materials in a biological system can result in interesting biomechanical properties. Shark cartilage, a biomineralized tissue, is composed of hydroxyapatite mineral, collagen, and glucosaminoglycans (sugars). PURPOSE: We seek to establish the link between structure, orientation, and amount of organo-mineral content in shark vertebral cartilage and macroscale biomechanical properties, especially in terms of the swimming efficiency of sharks. METHODS: To explore the biomechanical role of vertebral cartilage micro- and nanostructure, we focused on shark vertebrae from selected species of sharks, namely Carcharhinus limbatus (Blacktip, a carcharhiniform shark showing block-like mineralization within vertebrae) and Isurus oxyrinchus (Shortfin Mako, a lamniform shark with lamellar mineralization within vertebrae). We characterize ultrastructural features and nanoscale compositional differences using techniques such as Scanning Electron Microscopy (SEM), synchrotron X-ray nanotomography, Atomic Force Microscopy (AFM), Polarized Light Microscopy (PLM), and Raman spectroscopy. These studies were complemented by nanoindentation measurements of shark vertebrae collected in Ringers’ solution to mimic the natural hydrated state of the cartilage. RESULTS: SEM and Nano-CT data showed the arrangement of collagen fibers and minerals in shark cartilage at the micro- and nanoscale. AFM topographical analyses also provided further insight into the structure. Raman spectroscopic mappings helped detail the microscale organization of vertebral cartilage and the chemical composition within the mineralized collagen network and along the interface between mineralized and unmineralized cartilage. We were also able to examine the arrangement of collagenous fibers in shark cartilage using PLM. Additionally, our nanoindentation data revealed profound variations in Young’s moduli between mineralized and unmineralized regimes, linking local compositional variations to biomechanical function. We also found interspecific differences in mechanical properties of the unmineralized regions of vertebrae. Through the gathered data, steps are taken to address the gaps in knowledge of the ultrastructure of vertebral cartilage and the influence of its nanomechanical properties on the observed macroscale mechanical properties. CONCLUSIONS: A deeper understanding of the macroscopic implications of micro- and nanostructural arrangement and interaction of cartilage components will aid in the design and development of bio-inspired cartilaginous material, which could be used in tissue engineering to create artificial biocompatible composite materials. P41 - Live imaging of mineralization and calcification in cell cultures Luca Reicher*1, Aaron Morgan1, Robert Dzhanaev1, Andrea Gorgels1,2, Claudia Goettsch2, Willi Jahnen-Dechent2. 1Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Hospital, Aachen, Germany, 2Dept of Internal Medicine I, RWTH Aachen University Hospital, Aachen, Germany. INTRODUCTION: Mineralization is a physiological event in bone and tooth cells. Calcification is an age and disease associated pathologic event in many soft tissue cells particularly in the vasculature, causing high morbidity and mortality. The molecular mechanisms underlying the process of osteogenic mineralization vs. vascular calcification are not fully understood. PURPOSE: To investigate the initiation and progression of physiological mineralization vs. pathological calcification, cell cultures of osteoblast-like cells and of vascular cells were cultured in osteogenic media and calcification media, respectively. The mineral chaperone protein fetuin-A, which has a high binding affinity for nascent calcium phosphate mineral, was used as an imaging agent to identify the earliest stages of mineralization and calcification. The use of a live imaging system provided temporal resolution to monitor the progression of mineralization, calcification, and cell health allowing cell sampling for detailed molecular analysis at meaningful time points. METHODS: Professional mineralizing osteoblast cells and non-professional mineralizing calcifying vascular cells were cultured in osteogenic and calcification media. Fluorescence-labelled fetuin-A was added to the culture medium to identify newly formed mineral. Throughout the study, live imaging was used to monitor cell morphology and health as well as mineralization and calcif

OCTOBER 2023 ICCBMT 14 P42 - The effects of CK2 alpha 1 conditional knockout on mineralization of skeletal bone and teeth Charles Sfeir1,2,3, Elia Beniash 2,3, Kai Liu 1,2. 1Dept of Periodontics and Preventive Dentistry, University of Pittsburgh, 2Dept of Oral Craniofacial Sciences, University of Pittsburgh, 3McGowan Institute for Regenerative Medicine, University of Pittsburgh. INTRODUCTION: The protein kinase CK2 is a constitutively active, highly conserved and ubiquitously expressed. Over 300 substrates known for CK2 which is involved with different cellular processes. The mechanism by which a single kinase regulates these different activities remains to be well elucidated. PURPOSE: CK2a1 is a catalytic subunit of CK2 tetramer. The present research was designed to explore the effects of CK2a1 on the mineralization of skeletal bone and teeth. METHODS: 1. CK2a1 conditional knockout (CKO) mice was established with CK2a1 fl/fl and Sp7 cre mice. 2. Micro CT was conducted to confirm the phenotype changes of skeletal bone and teeth. 3. Histology techniques were applied for general information of skeletal bone. 4. FTIR spectrum was performed to depict molecule structure of organic and inorganic components in teeth that reflect tissue mineralization. 5. Micro indentation was applied to observe the characteristics of tissue hardness. RESULTS: The CKO mice were successfully established. Micro CT data shows: in CKO mice, the crown of 2nd molar-BV/TV ↓, BV (Dentin)/TV ↓; the root of 2nd molar- BV/TV ↓, TMD ↓; the alveolar of 2nd molar- BV/TV ↓, Tb.Th ↓; TMD ↓ and BS/BV ↑. Micro CT 3D data shows: less thickness of enamel, root and alveolar in CKO mice; less mineralization and disform in CKO incisor. In CKO mice femur, BV/TV ↓, Conn-Dens ↓, Tb.Th ↓ and BS/BV ↑ in trabecular bone; BV/TV ↓, Apparent Density ↓ & TMD ↓ and Porosity ↑ in cortical bone. 3D data shows less thickness of femur bone tissue. In FTIR spectrum data of 2nd molar dentin, CKO mice have significantly higher ratio values of Amide/Phosphate in both peak area and height. Those results indicate that the molar dentins in CKO mice have less. P43-F - Nanostructure of regenerated bone in critical-size defects imaged by X-ray scattering Adrian Rodriguez-Palomo*1, Elisa A Casanova2, Lisa Stähli2, Olivier Gröninger3, Yvonne Neldner2, Simon Tiziani2, Ana Perez Dominguez4, Manuel Guizar-Sicairos5, Zirui Gao5, Christian Appel5, Leonard C Nielsen1, Marios Georgiadis6, Franz E Weber4, Wendelin Stark3, Hans- Christoph Pape2, Paolo Cinelli2,7, Marianne Liebi1,5. 1Dept of Physics, Chalmers University of Technology, Gothenburg, Sweden, 2Dept of Trauma Surgery, University of Zurich, University Hospital Zurich, Zurich, Switzerland, 3Institute for Chemical and Bioengineering, ETH Zurich, Zurich, Switzerland, 4Oral Biotechnology and Bioengineering, Dept of Cranio-Maxillofacial and Oral Surgery, Centre for Dental Medicine, University of Zurich, Zurich, Switzerland, 5Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland, 6Dept of Radiology, Stanford School of Medicine, Stanford, CA, USA, 7Centre for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Zurich, Switzerland. INTRODUCTION: The healing process in large bone defects is a frequent source of problems for orthopaedic surgery which remain unsolved. Bioengineered bone grafts and scaffolds are an alternative in which achieving a continuous interphase between the host bone and the graft is still a major issue. Bioinspired composite scaffolds that resemble the nanostructure of bone are especially interesting as they offer a viable environment for cells to migrate, differentiate and regenerate tissue. One such example is PLGA fibres in combination with amorphous calcium-phosphates nanoparticles (aCaPs), which lead to enhanced osteoconductivity and biocompatibility. The use of aligned fibres and cell-seeded scaffolds have been proven to be effective in improving the migration and proliferation of cells. In this work biocompatible 3D PLGA/aCaP scaffolds with oriented and aligned fibres, containing only extracellular matrix, or seeded with adipose- derived mesenchymal stromal cells, were tested in a mouse model for critical size bone defects. METHODS: Small-angle X-ray scattering (SAXS) has become a key method for studying the anisotropic nanostructure of bone as an arrangement of mineralized collagen fibrils. By raster scanning a macroscopic sample with a microbeam and measuring a scattering pattern at each point, the distribution of nanostructural heterogeneities of complex structures can be mapped with micrometric spatial resolution. To assess the integration and quality of regenerated bone growth in such scaffolds scanning SAXS and SAXS tensor tomography (SASTT) were used at cSAXS beamline at the Swiss Light Source (Paul Scherrer Institute, Switzerland). RESULTS AND DISCUSSION: Scaffolds with aligned fibres showed a higher degree of osseointegration and bone growth in vivo, with nanostruct

OCTOBER 2023 ICCBMT 14 P44 - Shear-dependent self-assembly of calcium pyrophosphate nanostructures DC Bassett1, Thomas E Robinson1*, Reghan J Hill2, Liam M Grover1, Jake E Barralet3. 1Healthcare Technologies Institute, University of Birmingham, UK, 2Dept of Chemical Engineering, McGill University, Canada, 3Dept of Dentistry, McGill University, Canada. INTRODUCTION: The transition from amorphous precursor to stable crystal structure is paramount to understanding biological mineralisation. This applies to the physiological formation, remodelling and regeneration of bones and teeth, as well as pathological mineralisation. One such condition is pseudogout, also known as calcium pyrophosphate deposition disease, where crystals of this material are painfully deposited in joints. Calcium pyrophosphate also forms a fascinating array of intermediate structures while transitioning from amorphous to crystalline, with nanostructures favourable for a number of commercial applications such as drug delivery. PURPOSE: To explore the transition of calcium pyrophosphate from amorphous precipitate to final crystal. METHODS: Calcium pyrophosphate was precipitated by combining stoichiometric ratios of calcium and pyrophosphate at ambient temperature. The temporal structures were visualised using SEM, the ionic concentrations followed over time, and the crystallinity determined through XRD. RESULTS: Under quiescent conditions, an initial spherical precipitate is formed, which combine together to form a network of nanofibres. These fibres bundle to form discrete microspheres, while maintaining their nanostructure. These fibres continue to elongate, with ever increasing aspect ratio, until crystallisation if left for long time periods. If the system is agitated, the initial precipitate transitions directly to nanotubes in a short time, which align and form bundles. The bundles densify through aqueous sintering, then crystallise. Interestingly, agitation not only leads to crystallisation in a much shorter time (20 mins vs 12 hours), but results in a different final crystal phase. CONCLUSIONS: It is hypothesised that the initial precipitate forms spheres, as the most kinetically favourable state for an ionic cluster, and then transitions to the most thermodynamically stable structure, the anisotropic crystal. These transitions, which include deprotonation of the pyrophosphate and expulsion of sodium, thus lead to increased aspect ratio in each of the intermediate phases. In addition to being interesting in its own right, and providing a viable route to industrial scale nanostructure production, these observations may have implications for the formation of hydroxyapatite in vivo. P45-F - What regulates the enamel matrix distribution? Normal and abnormal enamel distribution in human molars Susanna Sova*1,2, Teemu Häkkinen3, Leo Tjäderhane4, Jukka Jernvall1,2. 1Institute of Biotechnology, University of Helsinki, Helsinki, Finland, Finland, 2Dept of Geoscience and Geography, University of Helsinki, Helsinki, Finland, 3University of California, San Francisco, US, 4Dept of Oral and Maxillofacial Diseases, Helsinki University Hospital, University of Helsinki, Helsinki, Finland. INTRODUCTION: Correctly shaped teeth are crucial for most mammals. The occlusal morphology is initially determined during the tooth patterning, and is then finalized with the secretion of enamel matrix layer. The enamel thickness has been studied for decades, especially among primates, but the mechanistic basis of how enamel distribution is regulation during matrix secretion remains poorly understood. In our previous work (Häkkinen et al. 2019), we studied the species-specific enamel distribution in suid and primate molars. Based on the topographic analyses, we presented a computational model that simulates the secretion of enamel matrix in 2D. This model successfully mimics the enamel distribution of domestic pig, human and orangutan molars, and we proposed that the diffusion of nutrients during enamel matrix secretion could be the key factor driving the species-specific distribution of enamel. PURPOSE: In the current work, we study variation of enamel thickness among human wisdom teeth and molars affected by rickets, thereby contrasting subtle and severe variation in enamel distribution. Our aim is to test if the variation in the occlusal surface morphology can be explained only by the morphology of the enamel-dentine junction (EDJ), or it is affected by other factors, such as nutrient-limited secretion of enamel matrix. METHODS: Enamel distribution analysis compared local thickness and surface properties of enamel to that of the underlying EDJ. These analyses were based on 3-dimensional microCT reconstructions of the molars. The computational simulations of enamel matrix secretion are based on 2-dimensional sections of the. RESULTS: The enamel distribution of normal molars is mostly explained by variation in the underlying EDJ. In contract, enamel pits of the ric

OCTOBER 2023 ICCBMT 14 P46 - Secondary hyperparathyroidism in nephrectomized rats: changes to osteocyte lacunar volume distribution through an X-ray computed tomography study Anastasiia Sadetskaia*1, Nina Kølln Wittig1, Maja Østergaard1, Jesper Skovhus Thomsen2, Søren Egstrand3, Maria L Mace3, Henrik Birkedal1. 1Dept of Chemistry and iNANO, 2Dept of Biomedicine, Aarhus University, Aarhus, 3Dept of Nephrology, Rigshospitalet, Copenhagen, Denmark. INTRODUCTION: Chronic kidney disorder (CKD) is a quite common disease, accounting not only for kidney failure, but also for secondary hyperparathyroidism phenomenon (2HPT) – elevated PTH levels as the result of vitamin D deficiency and, hence, low calcium levels in blood. As the consequence of 2HPT, the bone – the biggest source of calcium in the body – undergoes resorption of its mineral content, leading to high porosity. Usually this is due to osteoclast activity – bone resorbing cells. However, there have been indications that during high PTH levels, the mineralized matrix around osteocytes (lacunar spaces) might also change due to additional resorption (osteocytic osteolysis). PURPOSE: To the best of our knowledge, no work was done regarding the study of changes in osteocyte lacunar spaces during CKD with usage of 3D imaging techniques to eliminate possible cell-orientation effects. We hypothesize that changes in osteocyte lacunae are present in nephrectomised rats and propose that high-resolution synchrotron radiation X-ray computed tomography (SR-CT) study would reveal whether it is the case. METHODS: The mid-diaphysis of nephrectomised and healthy rat femur samples were cut in the diamond saw and milled into cylindric rods (ø~800 μm, length 10-15 mm) for the SR-CT experiment. The number of samples was 28 in total, each taken from a different animal. In order to reach the necessary resolution, SR-CT images were taken on synchrotron TOMCAT facility, PSI, with an X-Ray energy of 18 keV and 0.325 μm voxel size. The SR-CT reconstructed images consisted of 2500x2500 pixels. Custom-written MATLAB code was used for analysis. RESULTS: As expected, the images analysis revealed high porosity in the femur and PTH levels were elevated in nephrectomised rats. The lacunar spaces were separated, and their mean volumes compared, which did not reveal differences. However, samples with highly enlarged lacunae were also observed as well as apparent new bone formation in nephrectomised rats near the porosities. Furthermore, the lacunae seem to be larger near porosities than in bulk bone. These findings suggest that the apparent lack of significant changes in osteocyte sizes may be caused by differences in the relative amounts of newly formed and old bone. CONCLUSIONS: Our results show that, on the first glance, no significant changes in osteocyte lacunar spaces were observed for nephrectomised rats. However, there are indications that continuing bone turnover might camouflage the effects of nephrectomy on osteocytes and its surrounding matrix. P47-F - Mineralized and vascularized bone-like organoid created with high-throughput bioprinting Genevieve E Romanowicz*1, Ethan Dinh1, Kelly Leguineche1, Samuel Lester1, Ella Leeson1, Angela Lin1, Ramesh Subbiah2, Luiz E Bertassoni2, Robert E Guldberg1. 1Knight Campus for Accelerating Scientific Impact, University of Oregon, Eugene, OR, USA, 2Cancer Early Detection Advanced Research Center (CEDAR), Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA. OBJECTIVES: The highly mineralized collagen and cellularity of native bone, until recently, has been difficult to replicate in vitro. Utilizing our previously published mineralization protocol, we sought to develop a bone-like organoid for use as an in vitro model system of bone or as an alternative to bone autografts. In this study, we outline high-throughput fabrication of mineralized, cell-laden, injectable, bone- like microdots using robotic bioprinting. METHODS: Microdots were fabricated by printing small volumes of rat-tail type I collagen containing human mesenchymal stromal cells (hMSCs) and self-contracted over 3 days. Microdots were mineralized with media containing osteopontin, calcium and phosphate. Mineral quality was determined with Fourier transform infrared spectroscopy (FTIR)(n=4/group). Injectability and viability was demonstrated by in vitro injection through an 18G needle with live/dead staining post-injection(n=3/group). Spatial mineral patterning was determined via high-resolution x-ray microcomputed tomography (Zeiss XRadia, 0.7 um resolution) after 21 days in culture and immunofluorescent staining for RUNX2 (early osteoblasts), osteocalcin (mature osteoblasts), and sclerostin (mature osteocytes) at both 7 days culture (n=3/group). Vascularized constructs were similarly created using a mixed population of fibroblast like- cells and vascular fragments from rats adipose tissue. These constructs were then stained for isolectin to visualize the v

OCTOBER 2023 ICCBMT 14 P48 - Microstructure quantification of shark vertebral mineralized cartilage SR Stock*1, KC James2, MS Passerotti3, PD Shevchenko4, JS Park4, JD Almer4, LJ Natanson3, A Gomez5, H Kierdorf6, U Kierdorf6. 1Dept of Cell & Developmental Biology, Feinberg Sch of Medicine, & Simpson Querrey Inst, Northwestern Univ, Chicago, IL, USA, 2Southwest Fisheries Sci Cent, Natl Marine Fisheries Serv, NOAA, La Jolla, CA, USA, 3Northeast Fisheries Sci Cent, Natl. Marine Fisheries Serv., NOAA, RI, USA, Advanced Photon Source, Argonne Natl Lab, Lemont, IL, USA, 5Dept of Pathology, Univ Cadiz, Cadiz, Spain, 6Dept of Biology, Univ Hildesheim, Hildesheim, Germany. INTRODUCTION: The skeletons of elasmobranchs, which include sharks and rays, consist of cartilage (type 2 collagen). This is related to but somewhat different from the type 1 collagen found in bone. Some elasmobranch skeletal cartilage is mineralized with a bioapatite related to hydroxyapatite, and the centra (i.e., vertebral bodies) of sharks possess remarkable resistance to large bending strains in vivo strains, strains that exceed ±4%. Further, these centra do not fracture even after experiencing millions of loading cycles over a shark’s lifetime. PURPOSE: It appears that these centra’s fracture resistance is related to a hierarchy of structures, and this talk focuses on the bioapatite micro-organization of the tissue of centra of shark orders Carcharhiniformes and Lamniformes. Earlier results of lab microCT on entire centra provided a 3D quantitative picture of the macrostructure and of microstructure down to 20 μm. METHODS: This poster focuses on synchrotron microCT of blocks cut from centra, on x-ray diffraction and small angle scattering of the mineral phase in these blocks and on novel 3D energy dispersive diffraction mapping of crystallographic texture in intact centra. RESULTS: These data with volume elements (voxels) <1 μm reveal that the centra tissue consists of closely spaced, mineralized trabeculae, and the talk focuses on 3D quantification of trabecular thickness Tb.Th, trabecular spacing Tb.Sp and volume fraction of mineralized tissue MV/TV. Other microstructure contributions to centrum functionality are described. X-ray diffraction and small angle scattering show the shark mineral and its nanostructural organization are somewhat different from those in bone. Crystallographic texture varies within the centra. CONCLUSION: Much remains to be done. P49-F - Bone matrix and lacuno-canalicular network is altered in a mouse model for Marfan Syndrome Victoria Schemenz*1,2 , Ghazal Hedjazi3, Markus A Hartmann3, Peter Fratzl2, Bettina M Willie4, Elizabeth Zimmermann4, Wolfgang Wagermaier2. 1Dept for Operative, Preventive and Pediatric Dentistry, Charité - Universitätsmedizin Berlin, Germany, 2Dept of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany, 3Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of OEGK and AUVA Trauma Centre Meidling, 1st Medical Dept Hanusch Hospital, Vienna, Austria, 4Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal , Canada. INTRODUCTION: Marfan syndrome (MFS) is a multisystem disorder caused by mutations in the fibrillin-1 (Fbn1) gene affecting all connective tissues. Typical skeletal manifestations are long bone overgrowth, low bone mass, low bone mineral density (osteopenia) and increased fracture risks. Together these data suggest that Fbn1 plays a major role in regulating skeletal homeostasis. At the same time, our recent studies point towards an active involvement of osteocytes on bone mineralization and quality. Therefore, this study is characterizing the correlations of the bone material properties and architecture of the LCN in mice with MFS compared to healthy littermates. METHODS: The Fbn1^{mgr/mgr} (mgr/mgr) mouse model is one of the most severe mouse models of MFS. The homozygous mice die of aortic aneurysm after 2-6 months and exhibit other skeletal manifestations that are similar to human MFS. The tibiae of mgr/mgr mice and their healthy littermates were imaged from week 7 to 12 using in-vivo μCT to study bone growth. Additionally, twelve week old mice were sacrificed to investigate lacuno-canalicular-network (LCN) architecture and bone ultrastructural properties at the submicron level. Therefore a combination of techniques was used: (i) Confocal laser scanning microscopy (CLSM) combined with rhodamine staining was used for three-dimensional visualization of the LCN, (ii) second harmonic generation microscopy (SHG) shows the alignment and orientation of collagen fibrils, (iii) quantitative back-scattered electron imaging (qBEI) was employed to quantify the local mineral content, while (iv) synchrotron small and wide angle x-ray scattering (SAXS and WAXS) allowed determination of bone mineral thickness and length. RESULTS: The mgr/mgr mice show longer tibiae than their healthy littermates already from an age of 7 weeks. In con

OCTOBER 2023 ICCBMT 14 P50-F - Just average: Constructing a 3D digital anatomical atlas of the human distal femur Benjamin Zvi Rudski*1, Natalie Reznikov1,2. 1Dept of Quantitative Life Sciences, 2Dept of Bioengineering, McGill University, Montreal, QC, Canada. INTRODUCTION: Despite overall similarities, homologous bones in different individuals exhibit internal and external variation. These differences prevent a direct comparison of bone architecture amongst individuals. To compare bones, a generalizable and representative reference is required. This reference is an anatomical atlas – a super-average that may not physically exist but is sufficiently representative of the group. In this proof-of-concept work, we have constructed a 3D digital atlas of the human distal femur that accurately reflects common anatomical features. METHODS: We obtained 3D micro-computed tomography (μCT, voxel 50 μm) images of the left and right distal femur of 11 adult males from the Forensic Anthropology Center at the Texas State University. We constructed a 3D digital atlas using the Iterative Centroid Approach (ICA) [1] in the Anima library [2]. This method, inspired by geometry, progressively builds an atlas by adding one new image at a time [1]. We segmented the images and constructed atlases of subsets at various levels of downsampling, with or without digital filling-in. Dragonfly (Object Research Systems Inc.) was used for visualisation. RESULTS: The surface-rendered atlas at the highest extent of downsampling resulted in 3D image quality comparable to that of a clinical CT image. This low-resolution reference demonstrated high anatomical accuracy with well-defined epicondyle topography (tuberculum adductorium, sulcus popliteus), sharp condylar and patellar surface outline, and characteristic nutrient foramina. Higher resolution atlas images produced a high-fidelity rendering of universal trabecular features such as the remnants of the fused growth plate and the enhanced anisotropy of the cruciate ligament attachment sites, along with high-resolution surface features. These preserved features represent the phylogenetic reference. Ontogenetic features such as osteophytes and individual-specific trabecular trajectories gradually vanished with the addition of more individual images to the averaged atlas reference. CONCLUSION: The 3D digital atlas constructed preserves anatomically accurate external and internal features of the human distal femur. This high accuracy will enable future comparison of distinct reference groups. Certain limitations remain to be resolved, such as the high computational cost of atlas construction. In future work, we will devise a simple and intuitive metric to quantify the deviation of individual 3D bone images from the atlas, thus describing the variation within a population. This approach will be beneficial to zoology, comparative anatomy, medical imaging, and biological and forensic anthropology. REFERENCES: [1] Legouhy, A., Rousseau, F., Barillot, C., & Commowick, O. (2022). An iterative centroid approach for diffeomorphic online atlasing. IEEE Transactions on Medical Imaging, 41(9), 2521–2531. https://doi.org/10.1109/TMI.2022.3166593 [2] https://anima.irisa.fr, RRID:SCR_017017, SCR_017072. P51 - The consequences of dehydration-hydration on bone anisotropy and implications on the sublamellar organization of mineralized collagen fibrils Feride Sermin Utku*1, Onur Cem Namlı2. 1Dept of Biomedical Engineering, 2Dept of Mechanical Engineering Dept, Yeditepe University, Faculty of Engineering, Istanbul, Turkey. INTRODUCTION: Bone consists of hydroxyapatite, Type I Collagen, mucopolysaccharides and bone fluid, which associates with bone constituents and improves the mechanical properties of bone. PURPOSE: In this study, the effect of dehydration-hydration based sublamellar dimensional change on bone anisotropy was used as a tool to understand sublamellar organization of mineralized collagen fibrils. METHODS: Knowing that dehydration causes dimensional changes comparable to those observed in the mechanical testing of a bone sample, here, the dehydrated organic component of lamellar bone was modelled to contract towards the mineral, forming a contraction vector as the surface normal of the mineral plate. The amount of dehydration based contraction in rotated collagen fibrils was calculated for two models of sublamellar arrangements, namely A and B, where the mineral plate of the 0 (axial, [0 0 1]) sublamellar collagen fibril was oriented respectively along either (0 1 0) or (0 0 1) planes. Projections of sublamellar contraction vectors were denoted as u, v and w displacements at 10–20–30 angles and summed to give the lamellar total. Using the total displacements, anisotropy ratios of properties in directions parallel (W) versus perpendicular (U or V) to the osteonal axis were calculated. RESULTS: With dehydration, the osteonal lamellae in Model A (behaving as positive Poisson’s ratio material) may display

OCTOBER 2023 ICCBMT 14 P52-F - Evaluating the role of the N-terminus, histidine-rich region, and C-terminus on the Interaction of amelogenin with hydroxyapatite Wendy J Shaw*, Jinhui Tao, Garry Buchko, Emma Hanson, Alice Dohnalkova, Barbara Tarasevich. Pacific Northwest National Laboratory, Richland, WA, USA. INTRODUCTION: Enamel is one of the hardest minerals known. Its exquisite properties are in part due to the proteins present during formation. Amelogenin proteins comprise >90% of these proteins are are demonstrated by null mice and by single point variants which cause malformed enamel to be necessary for proper enamel formation. Mechanistic controls of amelogenin over crystal growth are not well understood, although secondary, tertiary, and quaternary structures are all thought to contribute to the resulting enamel structure. PURPOSE: We are focused on providing quantitative insight into the structure of amelogenin in solution and bound to a hydroxyapatite (HAP). The goal of these studies was to understand how the protein-HAP interface is controlled. We created a series of “mechanistic probes” to identify residues important in controlling binding to HAP, removing the charged groups in the N-terminus, the C-terminus, and the hydrophobic central region. METHODS: We evaluated the secondary, tertiary, and quaternary structures of these probes and their impact on HAP nucleation and growth using solution state NMR, solid state NMR, HAP nucleation and growth studies, and in situ atomic force microscopy (AFM). RESULTS: Reducing the charge in the N-terminus and the hydrophobic region affected the oligomer quaternary structure by changing protein-protein interactions which in turn, affected protein binding, while, as expected based on previous studies by others, the C-terminus is the dominant force in binding the protein to HAP. We also found the modified proteins affected the induction time to form amorphous calcium phosphate and also the time to transform to HAP. CONCLUSION: These studies are allowing us to providing fundamental insights into protein-crystal interactions that are not possible to obtain in vivo. Further studies will focus on evaluating molecular level structural details. This work is funded by NIDCR, NIH, grant # DE-015347. P53 - The patchiness of the osteocyte lacunocanalicular network in trabecular bone of human vertebrae Maximilian Rummler1*, Chloe Jones2, Markus Hartmann2, Stéphane Blouin2, Andrea Berzlanovich3, Richard Weinkamer1. 1Max Planck Institute of Colloids and Interfaces, Potsdam, Germany, 2Ludwig Boltzmann Institute of Osteology at Hanusch Hospital of OEGK and AUVA Trauma Centre Meidling, 1st Med. Dept Hanusch Hospital, Vienna, Austria, 3Unit of Forensic Gerontology, Center of Forensic Medicine, Medical University of Vienna, Vienna, Austria. INTRODUCTION: Bone can adapt its shape to external cues such as changes in mechanical environment. This ensures the mechanical competence and fracture resistance of bone during daily activities. This mechanical adaptation of the tissue is thought to be orchestrated by osteocytes which reside in pores called lacunae within the bone matrix. These osteocytes are connected to one another by sub- micrometer wide canals called canaliculi and thus forming a network, the osteocyte lacunocanalicular network (LCN). In osteons, found within cortical bone, the LCN is organized with most canaliculi running radially towards the Haversian canal [1]. However, much less is known about the organization of the LCN in trabecular bone. PURPOSE: Using our methodology to image and quantify the three- dimensional architecture of the LCN, we characterize the short-range and long-range connectivity of the LCN and correlation of network architecture with trabecular shape and loading. METHODS: Lumbar Vertebrae (L1) from women of different age (56 and 95 years) were dissected and stored in ethanol. The trabecular architecture was imaged using microCT. From the vertebrae, rods of 5x5 mm side length were prepared and stained using rhodamine 6G dissolved in ethanol. Small plates were sectioned along the rod and polished. The stained LCN of trabecular bone was imaged using confocal laser scanning microscopy and the images postprocessed and translated into a mathematical network using an in-house custom software package, TINA [1]. To assess heterogeneity within the LCN, network parameters are calculated in cubic subvolumes of 8 μm edge length. Lacunar parameters, such as lacunae degree, as well as volume, surface and shape were calculated. RESULTS: The low volume fraction of trabecular bone and thus an intense fluorescent signal from the pore space, makes imaging of the LCN in trabecular bone more challenging. However, we succeeded to obtain an image quality, which allows a structural analysis of the LCN architecture. Compared to cortical bone, a sparse network within trabeculae was found with lower canalicular density and less branching. First LCN quantification in trabecular

OCTOBER 2023 ICCBMT 14 P54-F - X-ray microtomography imaging of gene expression in mineralizing tissues Vilma Väänänen*1, Mona Christensen1, Heikki Suhonen2, Jukka Jernvall1,3. 1Institute of Biotechnology, University of Helsinki, Finland, 2Dept of Physics, University of Helsinki, Finland, 3Dept of Geosciences and Geography, University of Helsinki, Finland. INTRODUCTION: X-ray microtomography (μCT) has gradually developed into a widely adopted 3D imaging method of mineralizing tissues due to its high, uniform resolution and noninvasiveness, but it remains limited in molecular imaging applications. Several approaches have demonstrated detection of a specific protein using μCT imaging, but methods for detection of gene activity on the level of mRNA are just emerging. PURPOSE: Enzyme-mediated silver reduction visualizes protein distribution in intact samples using μCT. In this work, we apply similar approach to in situ hybridization with addition of gold enhancement step to visualize mRNA using μCT imaging. METHODS: We applied horseradish peroxidase-assisted reduction of silver and catalytic gold enhancement of the silver deposit to in situ hybridization in order to detect expression patterns of collagen type II alpha 1 (Col2a1) and sonic hedgehog (Shh) in embryonic mouse tissues. Resulting expression patterns were compared to patterns generated by standard chromogenic detection by means of light microscopy and the samples were then imaged with μCT for 3D visualization. RESULTS: Expression patterns of Shh and Col2a1 are comparable with the standard detection and visible using standard benchtop μCT imaging. Additionally we show that the approach is compatible with prior phosphotungstic acid staining, a contrast enhancement approach in μCT imaging of soft tissues. CONCLUSIONS: We show that combination of enzyme mediated reduction of silver and gold enhancement can be used to visualize gene expression patterns using μCT imaging. Being a modification of standard in situ hybridization approach, the method does not require transgenic mouse lines and can be used when conventional probe design of in situ hybridization is possible. Additionally, the approach can be integrated with existing laboratory routines of contrast enhanced μCT imaging. Future applications include 3D characterization of selected genes expressed during late crown morphogenesis of a mouse. P55-F - In vitro models of calcific aortic valve disease to evaluate the effect of mineral phase on aortic valve cell populations Stephan Sutter*1, Jennie AMR Kunitake1, Alexander Cruz2, Jonathan T Butcher2, Lara A Estroff1. 1Dept of Materials Science and Engineering, 2Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA. INTRODUCTION: Calcific aortic valve disease (CAVD), which affects 26% of people over the age of 65, involves the formation of mineralized nodules in the leaflets of the aortic valve. Mineralization causes valve leaflets to stiffen and impairs their ability to regulate blood flow into the aorta, leading to an increased risk of heart failure. PURPOSE: The purpose of this work is to study how the presence and properties (composition, phase, morphology) of mineral nanoparticles in the leaflets influence the formation of calcific nodules, with the particular goal of developing in vitro models that recapitulate key aspects of early stages of CAVD that precede the onset of symptoms (and are therefore not typically seen in surgically excised human aortic valves). We hypothesize that the presence of calcium phosphate nanoparticles, and particularly their aforementioned properties, affects the formation and growth of calcific nodules in the aortic valve. We explore these interactions through tissue engineering models mimicking human aortic valve leaflets. METHODS: 3D collagen constructs are seeded with synthetic calcium phosphate nanoparticles of varying phase – hydroxyapatite (HAp; Ca5(PO4)3OH) and whitlockite (Wh; (Ca,Mg)3(PO4)2). These particles aim to mimic those found in human valve calcifications, and are made by aqueous precipitation. The tissue models are cellularized in vitro models of an aortic valve leaflet, consisting of a collagen hydrogel containing valve interstitial cells (VICs) with a surface layer of valve endothelial cells (VECs) in osteogenic media. The hydrogel is cast into a PDMS well with an embedded circular spring providing uniform static stress to the gel. Seeding with nanoparticles and incubation in osteogenic media leads to rapid (~1 week) formation of calcific nodules within the constructs; these nodules and their environment are characterized by Raman mapping, immunofluorescence, and histology. RESULTS: We have successfully synthesized nanoparticles of HAp and Wh (phase validated by x-ray diffraction and Raman spectroscopy),and incorporated them into spring gel constructs to create a 3D in vitro model of calcific aortic valve lesions. Raman mapping of these spring gels has revealed that nodule formation

OCTOBER 2023 ICCBMT 14 P56-F - Development and initial uses of a rat model of cortical bone matrix maturation during remodeling D Rick Sumner*1,2,3 Ryan D Ross1,2. 1Dept of Anatomy & Cell Biology, 2Dept of Orthopedic Surgery, 3Center for Integrated Microbiome & Chronobiology Research, Rush University Medical Center, Chicago, IL, USA. INTRODUCTION: Multiple factors affect bone mechanical integrity, including the arrangement of the tissue in space and the composition of the extracellular matrix. Bone drugs can affect both, for instance by increasing bone mass and/or altering matrix composition. In most, if not all, in vivo models it is not possible to separate the structural and compositional effects of a given treatment because of the interdependent relationships between bone resorption and formation. PURPOSE: We sought to develop a rat model to investigate the effects of bone drugs on matrix maturation of bone tissue independent of the drug effects on structure. METHODS: We adapted a rat model in which lactating animals are given a low calcium diet. We administered the low-calcium diet during lactation (induction phase) followed by a return to a normal calcium diet for various periods of time (recovery phase). In an experiment to determine the effects of certain osteoporosis drugs, we treated rats who had been subjected to the low calcium regimen during lactation with vehicle (saline), sodium fluoride (NaF), zoledronic acid (ZA), or sclerostin antibody (Scl-Ab) for either 7 or 28 days during recovery. RESULTS: Animals given the lactation/low-Ca diet had elevated cortical porosity at the end of the induction phase in the diaphysis of the femur and tibia. We focus on the distal femoral diaphysis because this site had the greatest amount of induced porosity in the size range of rat secondary osteons. The mineral-to-matrix ratio in the bone formed during recovery was unaffected by the lactation/low-Ca induction method. We also found that dietary Ca restriction during lactation did not affect post-weaning bone formation kinetics or serum Ca and phosphate levels. Intracortical crystallinity was increased in the ZA group at day 10 compared with vehicle. At the endocortical surface, the mineral-to-matrix ratio was increased at days 5 and 10 following NaF treatment and endocortical crystallinity was increased at day 5 following ZA treatment compared with vehicle. CONCLUSIONS: Because the lactation/low-Ca model increases intra-cortical remodeling in adult rats without simultaneously adversely affecting matrix maturation, we used the model to study the effects of select osteoporosis drugs on matrix maturation. The low calcium regimen during lactation did not affect bone formation kinetics or mineral metabolism during the recovery phase, justifying the use of this model to study matrix maturation in the adult skeleton. The tested drugs affected matrix maturation more strongly at the endocortical then the intracortical envelope. Synchronization of bone formation at multiple sites in this model facilitates the study of how drugs affect matrix maturation independent of their effects on the initiation of remodeling. P57 - Influence of measurement parameters on determination of osteocyte lacunar morphology with laboratory X-ray micro-CT Nina Kølln Wittig*1, Maja Østergaard1, Henrik Birkedal1. 1iNANO and Dept of Chemistry, Aarhus University, Aarhus, Denmark. INTRODUCTION: Osteocytes are interconnected cells residing in the lacuno-canalicular network (LCN) within the mineralized bone matrix. They are orchestrators of bone remodeling and may even take direct part in remodeling of the peri-lacunar bone.1,2 The morphology of osteocyte lacunae has been suggested to depend on/change with many factors including age,3 bone type,4 and health status.5 In turn, the lacunar morphology is likely important for osteocyte function and bone integrity.6 Proper determination of lacunar morphology is therefore essential to assess the roles of osteocytes in bone biology and health. To this end, X-ray micro-computed tomography (μ-CT) is becoming commonly implemented.7 The quality of such a measurement governs whether the lacunar morphology can be extracted, but short measurement times can be determining for a study’s ability to draw statistically relevant conclusions. PURPOSE: This study therefore investigates how adjustment of measurement parameters to tune measurement time versus quality affects determination of lacunar properties using laboratory X-ray μ-CT. METHODS: Tomograms of a human bone sample were acquired at various settings using a ZEISS Xradia 620 Versa 3D microscope and at the TOMCAT beamline at the Swiss Light Source. RESULTS: Increasing image resolution and quality systematically shifted the lacunar volume distribution towards smaller volumes as the resolution was improved. However, emergence of canalicular protrusions in the TOMCAT data complicated segmenting out the lacunae only and therefore shifted the lacunar volume distribut

OCTOBER 2023 ICCBMT 14 P58-F - Nanoscale analysis of osteonal bone tissue using 3D electron microscopy Alyssa Williams*1, Tengteng Tang2, Aurélien Gourrier3,4, Nabil Bassim1,2,5, Kathryn Grandfield1,2. 1School of Biomedical Engineering, McMaster University, Hamilton, ON, Canada, 2Dept of Materials Science and Engineering, McMaster University, Hamilton, ON, Canada, 3Univ Grenoble Alpes, LIPHY, Grenoble, France, 4CNRS, LIPHY, Grenoble, France, 5Canadian Centre for Electron Microscopy, McMaster University, Hamilton, ON, Canada. INTRODUCTION: The remarkable mechanical properties of bone derive from not only a well-balanced ratio between its content of mineral and collagen but also appropriate spatial relationships between its components. While the structure of mineralized collagen fibrils has long been studied by transmission electron microscopy (TEM) and electron tomography little is known about the three-dimensional (3D) organization of collagen fibrils with respect to newly discovered mineral ellipsoidal deposits[1,2]. In part, the challenge lies in the requirements for nanometer resolution and a large field of view in the range of tens of micrometres in 3D. In this regard, recent advances in focused ion beam-scanning electron microscopy (FIB-SEM) in serial sectioning or nanotomography mode allow for simultaneous visualization of nanoscale collagen fibrils and their associated microscale mineral ellipsoids. PURPOSE: This study aims to couple FIB-SEM nanotomography techniques with innovative image-processing tools to understand the spatial relationship between collagen fibrils and mineral ellipsoids in human lamellar bone. METHODS: Human tibial bone was retrieved with ethical approval, and glutaraldehyde fixed, dehydrated, and embedded in Embed812. Three osteons with clear lamellar distinctions in the mid-cortex were chosen for further analysis. FIB-SEM nanotomography was acquired on a Zeiss Crossbeam 540 and NVision with an isovoxel size of 5-10 nm on volumes ranging in size from 4- 12μm. Image processing tools including deep learning and u-net segmentation from ORS Dragonfly and fast Fourier transform (FFT) analysis using python were applied to the YZ reconstructed plane to visualize mineral ellipsoids and collagen banding. To enhance the visualization of collagen fibrils, inverse FFT analysis was applied on YZ images using python. RESULTS: The FFT and subsequent inverse FFT analysis enhanced the appearance of the collagen fibril banding pattern. 3D segmentation of mineral ellipsoids and collagen fibrils highlighted fibrils with classic D-banding were present across the mineral ellipsoids, from the highly mineralized center to the less mineralized and almost porous-looking periphery. This 3D view corroborates analysis from 2D TEM that suggested dark circular regions at the periphery of mineral ellipsoids (rosettes) were collagen fibrils in cross-section, not pores [3]. CONCLUSION: This work highlighted an effective image processing method to amplify the visualization of collagen fibrils in 3D, showing the presence of mineralized collagen fibrils across the full diameter of ellipsoidal mineral clusters and notably, also at the periphery of them in regions that appear less mineralized. This approach may be helpful for probing the fraction of collagen or nanoporosity in bone. References: [1] D. J. Buss et al., J Struct Biol 2020, 212, 107603.;[2] D. M. Binkley et al., J Struct Biol 2020, 107615.;[3] K. Grandfield et al., Calcified Tissue Int 2018, 103, 606. P59 - Fetuin-A Phosphorylation regulates mineral binding Camilla Winkler*1, Christian Hasberg1, Carlo Schmitz1, Steffen Gräber1, Willi Jahnen-Dechent1. 1Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Hospital, Aachen, Germany. INTRODUCTION: Fetuin-A is a plasma protein involved in a variety of processes in animals. Fetuin-A may play a role in inflammation and mineralization. In presence of Fetuin-A amounts far above the solubility product of calcium and phosphate can be stabilized and thereby transported through the body. In this case, Fetuin-A forms caliprotein monomers (CPM) together with mineral first. A single Fetuin-A molecule with clusters of calcium and phosphate is involved. Presumably, this is the physiological form that is prevalent in all of us. However, if CPMs can aggregate, which may be the case in patients with for example chronic kidney disease, they mature into primary and later secondary Caliproteinparticles (CPP) which are cyototoxic. PURPOSE: The determination of the essential structural properties of the protein for its function as a mineral carrier. Fetuin-A could act as an inhibitor of unwanted mineralization e.g. in patients with vascular calcification. METHODS: The mineral binding depends on the structure of the protein. By cryo electron microscopy, CPM should be visualized for the first time and thus the areas of mineral binding identified. This is challenging because the CPM is expected to have an approxima

OCTOBER 2023 ICCBMT 14 P60 - Aberrations of the crosslink of collagen type I and bone structure organization in osteogenesis imperfecta Wouter H Nijhuis1, Zhiming Wu*1, Behdad Pouran1, Stefan Smit1, Ruud Bank4, Kelly Warmink1, Mies van Steenbergen2, Harrie Weinans1,3, Ralph J Sakkers1. 1Dept of Orthopedic Surgery, University Medical Center Utrecht, The Netherlands, 2Dept of Pharmaceutics, Utrecht University, The Netherlands, 3Dept of Biomechanical Engineering, Faculty of Mechanical, Maritime and Materials Engineering, Delft University of Technology, The Netherlands, 4MATRIX Research Group, Dept of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, The Netherlands. INTRODUCTION: Patients with osteogenesis imperfecta (OI) suffer from brittle bones, frequent fractures, short stature, and skeletal deformities. Currently, there is no clear insight on the alterations in bone matrix in OI and their relation to the clinical outcome of the patient. PURPOSE: To investigate the hypothesis that the genetic defect in type I collagen of OI patients leads to altered hydroxylysylpyridinoline (HP) and lysylpyridinoline (HP) cross-links. METHODS: Bone specimens were collected over a period of more than twenty years from children who underwent surgery at Wilhelmina Children’s Hospital, UMCUtrecht, the Netherlands. Only specimens originating from long bones of the lower extremities, i.e. femur, tibia and fibula, were included. Control bone samples were from children of the same age who underwent surgery for fractures. The samples were weighed, hydrolyzed, and derivatized using FMOC chemistry for amino acid analysis. The pyridinoline crosslinks HP and LP were measured by ultra-performance liquid chromatography with ACQUITY UPLC BEH C18 column. RESULTS: Assuming the presence of 300 Hyp residues per collagen molecule to calculate the amount of collagen in the sample, the bone samples of patients with OI showed significantly decreased collagen content (0.6475±0.08148), indicating higher mineralization levels, compared to the controls (0.8485±0.06427). We also found elevated levels of Hyl in bone samples from OI patients (14.49±5.263 compared to control 8.371±1.543), as well as significantly elevated levels of the enzymatic collagen cross-link hydroxylysylpyridinoline (HP, 4.462±1.440 to 3.137±0.6990 normalized by the amount of collagen) and to some extent elevated lysylpyridinoline (LP, 1.365 (0.4375-5.215) to 1.214 (0.6923-2.318)). The molecular HP/LP ratios were significantly higher in OI bone (3.698±2.064 to 2.537±0.6784). CONCLUSIONS: Our study revealed increased HP crosslinks and abnormal HP/LP crosslink ratios; the latter is due to the lysine hyper-hydroxylation of the triple helix of type I collagen in bone tissue from OI patients. Indeed, we found high Hyl levels of collagen in OI samples. The overhydroxylation probably results in steric hindrance between collagen molecules, leading to wider spacing between the molecules within the fibrils. Our study also revealed that patients with OI showed significantly decreased collagen content per dry bone, indicating higher mineralization levels in the bone samples. A previous publication mentioned an increase of Hyl and glycosylated Hyl results in collagen fibrils with a smaller diameter, whereas the low hydroxylysine collagen forms thick fibrils on a background of thin ones. The thinner fibrils contain collagen molecules that are more widely spaced from each other, allowing more crystals to be accommodated between the collagen molecules. This leads to an abnormal high bone matrix mineralization and potential brittleness. P61 - Alterations of the carbonate environment with Na or K substitution in biomimetic apatites Stephanie Wong*1,2, Christophe Drouet2, Alix Deymier1. 1Dept of Biomedical Engineering, University of Connecticut Health Center, Farmington, Connecticut, USA, 2CIRIMAT, University of Toulouse, Toulouse, France. INTRODUCTION: Changes in body fluids enveloping bones and teeth cause constant dissolution/recrystallization of the carbonate apatite (CAp) mineral component. This allows substitutions, such as CO3, Na, and K, within the nanocrystals. Bioapatite naturally has 2-6 wt% CO3 depending on the tissue type, in which CO3 substitutes for the OH- (A-type) and PO43- (B-type) sites. These CO3 types can drastically change the solubility, structure, and mechanics of apatite. In addition, Na easily co-substitutes with CO3 in the Ca sites for crystal charge balance and promotes the uptake of both ions, which changes the CO3 chemical environment. Na-incorporated CAp also exhibits reduced bulk modulus as CO3 increases. While K/CO3 co-substitution also occurs, albeit at lower amounts, little is known about K and CO3’s relationship in nanocrystalline apatites and their mechanics. Therefore, it is necessary to understand the mechanism of Na and K co- substitutions on CO3 incorporation to elucidate the changes of bioapatite properties in patho

OCTOBER 2023 ICCBMT 14 P62 - Morphological characterization of the osteocytes lacunocanalicular network (LCN) at osteolytic tumorous lesions in murine tibia Mahdi Ayoubi*1, Matthew Aaron Whitman2, Jun Sun3, Chongshan Liu4, Matthew B Greenblatt3, Claudia Fischbach2, Lara A Estroff1. 1Dept of Materials Science and Engineering, Cornell University, Ithaca, NY, USA, 2Dept of Biomedical Engineering, Cornell University, Ithaca, NY, USA, 3 Dept of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA, 4Dept of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY, USA. INTRODUCTION: Bone is known as one of the earliest and most frequent targets of breast cancer metastasis, which is the leading cause of death in breast cancer patients. Perturbed bone remodeling, and consequently, creation of osteolytic lesions are hallmarks of bone metastasis. Up to now, it is not fully understood how metastasized tumor cells interact with osteoclasts and osteoblasts to disrupt bone remodeling. In healthy bone, osteocytes, the most abundant cells comprising 90-95% of bone cells, are known as the orchestrators of normal bone remodeling thanks to their mechanosensitive characteristics. PURPOSE: We hypothesize that in the presence of bone- resident tumor cells, the function of osteocytes to orchestrate remodeling is adversely affected resulting in disrupted remodeling cycles and additional resorption of bone, which allows the progression of tumor growth in osteolytic lesions. Such changes might be reflected in the morphological properties of the lacunocanalicular network (LCN), which houses osteocytes and their processes. METHODS: To test our hypothesis, we study an in vivo murine model of metastasis, which is prepared by caudal artery injection of Py8119 mouse mammary carcinoma cells into immunocompetent C57BL/6 mouse. After sacrifice, micro computed tomography (μCT) is used to image the tibia, a frequent site of metastasis, and to localize the osteolytic lesion. After rhodamine staining, the osteocytic LCN is imaged using confocal laser scanning microscopy (CLSM). Based on the 2D overview images, higher resolution 3D images of smaller regions of interest (ROIs) near to and far away from the tumorous lesion are obtained. Morphological analysis of the lacunae as well as connectomics analysis are performed after segmentation and skeletonization of the LCN. RESULTS: μCT image of the tumor-containing tibia reveals a large (a few millimeters wide) osteolytic lesion at the metaphyseal bone adjacent to the growth plate. 2D CLSM image of the same region shows that almost half of the metaphysis including trabecular and cortical bones is eroded by the tumorous lesion. Our preliminary higher resolution images of the LCN within the selected ROIs reveal that morphological properties of the osteocytes lacunae, i.e., volume, aspect ratio and orientation, as well as the canalicular density have been altered near the tumor cells. While adjacent to the tumorous lesion the lacunae are larger, ellipsoidal, and randomly oriented, the lacunae far away from the tumor cells are smaller, elongated, and aligned with the long axis of the tibia. Also, the canalicular network is looser near the tumorous lesion. CONCLUSIONS: Altered LCN morphology suggests that tumor cells can affect the osteocytes and their LCN within the bone matrix adjacent to the tumorous lesion. It is likely that tumor-affected osteocytes do not behave like normal osteocytes to orchestrate bone remodeling, and therefore, extra resorption occurs at the tumorous lesion. P63-F - An in vitro model for preferential gap zone collagen mineralization Liyang Zhong*1, Ruixin Gao1, Eli Sone1,2,3. 1Institute of Biomedical Engineering, 2Dept of Material Science & Engineering, 3Faculty of Dentistry, University of Toronto, Toronto, ON, Canada. INTRODUCTION: Mineralization of collagen is a complex and highly controlled process that occurs in bone and other mineralized connective tissues. In native mineralization, hydroxyapatite minerals are formed within the collagen fibrils, and aligned with the fibril axis. Although several in vitro models have been developed to mimic collagen mineralization, most fail to reproduce the preferential distribution of HA minerals in the gap zones of collagen fibrils. This is a crucial property of mineralization as the distribution of minerals may impact the mechanical properties of fibrils. Here, we report on an in vitro model of collagen mineralization based on chemically crosslinked collagen fibrils. This model can highly mimic native collagen fibril mineralization by reproducing intrafibrillar, highly aligned, and preferentially distributed minerals. PURPOSE: The objective is to determine the role of chemical crosslinking in collagen mineralization, and whether preferential gap zone mineralization is related to changes in mechanical properties of crosslinked fibrils. METHODS: Acid soluble collagen was extracted from rat tail tendons, fo

OCTOBER 2023 ICCBMT 14 P64 - Characterisation of Ti implant surfaces: Coated with self-assembling peptide (SAP) P11-4 Dina G Abdel Fattah*1, Reem Elgendy1, Robert P Davies1,2. 1Division of Oral Biology, School of Dentistry, St James’s University Hospital, University of Leeds, 2Bragg Centre for Materials Research, University of Leeds. INTRODUCTION: Osteoporosis is one such metabolic condition that occurs when excessive bone resorption is not compensated by a concomitant increase in bone formation. The increased fracture rates and impaired healing associated especially with post-menopausal osteoporosis has become a significant dental challenge. Lack of osseointegration will lead to implant loosening can be countered by optimizing the osteointegration capacity of implant surfaces. PURPOSE: To develop a multicomponent bioactive coating for Ti implant surfaces using SAP P11-4 as the main component in combination with a hydroxy apatite layer to enhance bone integration and regeneration around the implant surface under osteoporotic conditions. METHODS: Sandblasted, large grit, acid-etched treated Ti discs were prepared and divided into six groups. G1 was used as the control group. G2 Positive control Electrodeposited CaP layer. G3 Dip coated P11-4. G4 and 5 coated with a sublimed gel of P11-4 alone with a concentration 10 and 30 mg/ml respectively. G6 P11-4 (30mg/ml) sublimed on previously electrodeposited CaP layer. The physicochemical properties were investigated via Fourier-transform infrared spectroscopy (FTIR) to analyze the secondary structure of peptides β-sheet. X-ray Diffraction (XRD) used to study the crystalline and semi- crystalline structure of all samples and Scanning electron microscopy with energy-dispersive X-ray spectrometer (SEM/EDX) to investigate surface morphology and Ca/P ratio of Calcium phosphate and hybrid P11-4 /HA coatings. RESULTS: By FTIR the SAP conformation was rich in β-sheet in all coatings. Peaks centered around 1625 cm-1 and 1682 cm-1 are indicative of β-sheet component, with the unordered component centered around the 1649 cm-1. The highest amount of β-sheet was detected in G6 then G5. However, G3 and G4 revealed the least amount. XRD confirmed that presence of β-sheet in all P11-4 coated samples. EDX analysis revealed HA phase with Ca/P ratio 1.76 in hybrid and electrodeposited CaP coatings. G2 showed a needle like structure of HA under SEM, while in G6 showed a striking difference in the crystal morphology with needle-like crystals having a shorter and flatter morphology with undulated and rounded crystals. The lamination is very characteristic for G4 and 5 with more spreading and uniformity in the latter while in G3 SEM images showed non- homogenous covering of the surface. CONCLUSIONS: P11-4 has demonstrated an ability to provide a templated heterogeneous nucleation site for HA and bone, therefore has the potential to be used as a functional coating. We show that the incorporation of P11-4 onto electro deposited CaP maintains the correct peptide secondary structure. Our study indicates an increased presence of β-sheet in the Ti implant covered in CaP vs Ti alone. Our hypothesis is that the hierarchical structures formed by P11-4 are intrinsically attracted to the Ca ions in the HAp, increasing persistence of the fibrils. P65 - Injectable biomimetic mineralized cell-free tissue for biomineralization model and tissue repair Marion Merle*1, Milena Lama1, Camila Bussola Tovani1, Nadine Nassif1. 1Sorbonne Université/CNRS/Collège de France, UMR7574, Paris, France. INTRODUCTION: Type I collagen is the main structural protein found in human biological tissues. Various fibrillar organizations of the protein are found within the extracellular matrices but all exhibit dense 3D anisotropic structures. Since type I collagen molecules possess liquid-crystal properties in vitro [1], it was suggested that such properties could also be shared by procollagen in vivo [2]. In addition, the 3D molecular arrangements are preserved during the formation of collagen fibrils [3]. Finally, mineralization would be partly driven by collagen fibrillar confinement allowing the co-alignment between collagen fibrils and apatite platelets [4]. PURPOSE: This work intends to present an innovative and bioinspired synthesis to elaborate an injectable mineralized tissue-like material. By matching the composition and microstructure of the natural tissue, we aim to build competitive tuneable substitutes for specific tissues as well as to gain a better comprehension of in vivo complex physico-chemical mechanisms of tissues’ mineralization. APPROACHES: The injectable cell-free material is an organic/inorganic composite. The organic part consists of spray-dried non-denatured and dense collagen microparticles, and the mineral one of biomimetic hydroxyapatite nanocrystals. The collagen particles provides, after being mixed with an aqueous solvent, the opportunity to form highly concentrated gels by injection, although

OCTOBER 2023 ICCBMT 14 P67 - Comparative study on the osteogenic potential of subchondral and fibrocartilage cells of the TMJ Alejandro Almarza1, Xudong Dong1, Pinero Carlos1*. 1Center for Craniofacial Regeneration, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA, USA. INTRODUCTION: The Temporomandibular Joint (TMJ) is the unique ginglymo diarthrodial synovial joint of the body, and a vital component of the stomatognathic system. Despite the limited regenerative ability of cartilage, recent studies on goats have demonstrate that the TMJ cartilage, which is histologically defined as fibrocartilage, possess an innate regenerate ability in vivo. These studies have determined that the subchondral fibrocartilage interface of the TMJ condyles contains a heterogeneous cell population that includes stem cells/progenitor cells. PURPOSE: This study aims to explore the osteogenic potential of the cell populations of the subchondral bone and fibrocartilage of the TMJ, and go further on understanding the tissue biology of these compartments. This work seeks to better understand the cells involved in subchondral bone remodeling and fibrocartilage regeneration in the context of TMJ tissue engineering. METHODS: Cells from the surface and cartilage layers (separately) were compared to osteoblast from the mandibular condyle for the osteogenic potential. Cells were cultured in 2D for 21 days in media containing inorganic phosphate and 10% FBS. Then all cultures were stained for alkaline phosphatase and alizarin red for mineral deposition. RESULTS: Results showed that all three cell types were able to deposit copious amounts of mineral. A non-osteogenic media control was used. CONCLUSIONS: We demonstrate that the subchondral bone and fibrocartilage compartments of the TMJ condyles contain a heterogeneous cell population. These populations showed different mineralization activity after cultured under osteogenic condition. We conclude that a putative subchondral stem cells can be harnessed through tissue engineering to guide subchondral bone mineralization and remodeling, as well as fibrocartilage regeneration of the TMJ condyles.