Skeletal dysmorphology and mineralization defects in Fgf20 KO mice.

Introduction: Fibroblast growth factor 20 (Fgf20), a member of the Fgf9 subfamily, was identified as an important regulator of bone differentiation and homeostasis processes. However, the role of Fgf20 in bone physiology has not been approached yet. Here we present a comprehensive bone phenotype analysis of mice with functional ablation of Fgf20. Methods: The study conducts an extensive analysis of Fgf20 knockout mice compared to controls, incorporating microCT scanning, volumetric analysis, Fgf9 subfamily expression and stimulation experiment and histological evaluation. Results: The bone phenotype could be detected especially in the area of​ the lumbar and caudal part of the spine and in fingers. Regarding the spine, Fgf20-/- mice exhibited adhesions of the transverse process of the sixth lumbar vertebra to the pelvis as well as malformations in the distal part of their tails. Preaxial polydactyly and polysyndactyly in varying degrees of severity were also detected. High resolution microCT analysis of distal femurs and the fourth lumbar vertebra showed significant differences in structure and mineralization in both cortical and trabecular bone. These findings were histologically validated and may be associated with the expression of Fgf20 in chondrocytes and their progenitors. Moreover, histological sections demonstrated increased bone tissue formation, disruption of Fgf20-/- femur cartilage, and cellular-level alterations, particularly in osteoclasts. We also observed molar dysmorphology, including root taurodontism, and described variations in mineralization and dentin thickness. Discussion: Our analysis provides evidence that Fgf20, together with other members of the Fgf9 subfamily, plays a crucial regulatory role in skeletal development and bone homeostasis.

Improvement of osteoblast adhesion, viability, and mineralization by restoring the cell cytoskeleton after bisphosphonate discontinuation in vitro.

OBJECTIVE: Bisphosphonates are prescribed to treat excessive bone resorption in patients with osteoporosis. However, its use is associated with potential adverse effects such as medication-related osteonecrosis of the jaw, prompting the introduction of the drug holiday concept in patients prior to dentoalveolar surgery. Furthermore, bisphosphonate discontinuation has been studied in vivo, in humans, and in animal models. However, it is not known whether this approach could affect bone cells in vitro. Therefore, the objective of this study was to investigate the potential effects of bisphosphonate discontinuation on pre-osteoblast and osteoblast activities in vitro. METHODOLOGY: Pre-osteoblasts (MC3T3) and osteoblasts were treated with bisphosphonate (alendronate) at concentrations of 1, 5, and 10 µM. Alendronate was then withdrawn at different time points. The negative control consisted of untreated cells (0 µM), while the positive control consisted of cells incubated with alendronate throughout the experiment. Cell viability, cell adhesion, cell cytoskeleton, mineralization, and gene expressions were investigated. RESULTS: Pre-osteoblasts and osteoblasts showed a decrease in cell viability after treatment with 5-10 µM alendronate for 4 days or longer. Two days of alendronate discontinuation significantly increased cell viability compared with the positive control. However, these levels did not reach those of the negative control. Bone nodule formation was reduced by alendronate. Discontinuation of alendronate regained bone nodule formation. Longer periods of discontinuation were more effective in restoring nodule formation than shorter periods. Addition of alendronate resulted in an increase in the percentage of dead cells, which, in turn, decreased when alendronate was discontinued. Alendronate affected the cell cytoskeleton by disassembling actin stress fibers. Cell adhesion and cell morphological parameters were also affected by alendronate. Discontinuation of alendronate restored cell adhesion and these parameters. Overall, the highest improvement after alendronate discontinuation was seen at 10 µM. However, alendronate treatment and discontinuation did not affect osteoblast gene expression. CONCLUSION: Discontinuation of alendronate helps to reverse the negative effects of the drug on cell viability, cell adhesion, and mineralization by restoring the cell cytoskeleton. Our data suggest the benefits of drug holiday and/or intermittent strategies for alendronate administration at the cellular level.

Birc5 and Nudc are screened as candidate reference genes for RT-qPCR studies in mouse cementoblast mineralization using time-series RNA-seq data.

BACKGROUND: The robustness and credibility of RT-qPCR results are critically dependent on the selection of suitable reference genes. However, the mineralization of the extracellular matrix can alter the intracellular tension and energy metabolism within cells, potentially impacting the expression of traditional reference genes, namely Actb and Gapdh. OBJECTIVE: To methodically identify appropriate reference genes for research focused on mouse cementoblast mineralization. MATERIALS AND METHODS: Time-series transcriptomic data of mouse cementoblast mineralization were used. To ensure expression stability and medium to high expression levels, three specific criteria were applied to select potential reference genes. The expression stability of these genes was ranked based on the DI index (1/coefficient of variation) to identify the top six potential reference genes. RT-qPCR validation was performed on these top six candidates, comparing their performance against six previously used reference genes (Rpl22, Ppib, Gusb, Rplp0, Actb, and Gapdh). Cq values of these 12 genes were analyzed by RefFinder to get a stability ranking. RESULTS: A total of 4418 (12.27%) genes met the selection criteria. Among them, Rab5if, Chmp4b, Birc5, Pea15a, Nudc, Supt4a were identified as candidate reference genes. RefFinder analyses revealed that two candidates (Birc5 and Nudc) exhibited superior performance compared to previously used reference genes. LIMITATIONS: RefFinder's stability ranking does not consider the influence of primer efficiency. CONCLUSIONS AND IMPLICATIONS: We propose Birc5 and Nudc as candidate reference genes for RT-qPCR studies investigating mouse cementoblast mineralization and cementum repair.

Cellular morphological trait dataset for extant coccolithophores from the Atlantic Ocean.

Calcification and biomass production by planktonic marine organisms influences the global carbon cycle and fuels marine ecosystems. The major calcifying plankton group coccolithophores are highly diverse, comprising ca. 250-300 extant species. However, coccolithophore size (a key functional trait) and degree of calcification are poorly quantified, as most of our understanding of this group comes from a small number of species. We generated a novel reference dataset of coccolithophore morphological traits, including cell-specific data for coccosphere and cell size, coccolith size, number of coccoliths per cell, and cellular calcite content. This dataset includes observations from 1074 individual cells and represents 61 species from 25 genera spanning equatorial to temperate coccolithophore populations that were sampled during the Atlantic Meridional Transect (AMT) 14 cruise in 2004. This unique dataset can be used to explore relationships between morphological traits (cell size and cell calcite) and environmental conditions, investigate species-specific and community contributions to pelagic carbonate production, export and plankton biomass, and inform and validate coccolithophore representation in marine ecosystem and biogeochemical models.

Functions of Epimedin C in a zebrafish model of glucocorticoid-induced osteoporosis.

Epimedium is thought to enhance the integrity of tendons and bones, ease joint discomfort and rigidity and enhance kidney function. Although glucocorticoids are commonly used in clinical practice, the mechanism by which the active compound Epimedin C (EC) alleviates glucocorticoid-induced osteoporosis (GIOP) is not well understood. The therapeutic potential of EC in treating GIOP was evaluated using alizarin red S staining, calcein immersion and fluorescence imaging, and bone mineralization, bone mass accumulation and bone density in zebrafish larvae were determined. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the key signalling pathways related to bone development were identified. A protein-protein interaction network (PPIN) was constructed to identify osteoclast characteristic genes and the findings were verified using real-time quantitative PCR (RT-qPCR). The bone tissue damage caused by prednisolone was reduced by EC. It also altered physiological processes, improved bone density, boosted mineralization and increased bone mass and activity. Subsequent empirical investigations showed that EC impacted the major signalling pathways involved in bone development, such as osteoclast differentiation, oestrogen, MAPK, insulin resistance, PPAR and AMPK signalling pathways. It also decreased the expression of genes typical of osteoclasts. The results of our study uncover a previously unknown function of EC in controlling bone formation and emphasize the potential of EC as a therapeutic target. The osteoprotective effect of EC indicates its potential as a cost-effective strategy for treating GIOP.

Cadmium impacts on calcium mineralization of zebrafish skeletal development and behavioral impairment.

Cadmium (Cd) poses significant risks to aquatic organisms due to its toxicity and ability to disrupt the cellular processes. Given the similar atomic radius of Cd and calcium (Ca), Cd may potentially affect the Ca homeostasis, which can lead to impaired mineralization of skeletal structures and behavioral abnormalities. The formation of the spinal skeleton involves Ca transport and mineralization. In this study, we conducted an in-depth investigation on the effects of Cd at environmental concentrations on zebrafish (Danio rerio) skeletal development and the underlying molecular mechanisms. As the concentration of Cd increased, the accumulation of Cd in zebrafish larvae also rose, while the Ca content decreased significantly by 3.0 %-57.3 %, and vertebral deformities were observed. Transcriptomics analysis revealed that sixteen genes involved in metal absorption were affected. Exposure to 2 µg/L Cd significantly upregulated the expression of these genes, whereas exposure to 10 µg/L resulted in their downregulation. Consequently, exposure of zebrafish larvae to 10 µg/L of Cd inhibited the body segmentation growth and skeletal mineralization development by 29.1 %-56.7 %. This inhibition was evidenced by the downregulation of mineral absorption genes and decreased Ca accumulation. The findings of this study suggested that the inhibition of skeletal mineralization was likely attributed to the disruption of mineral absorption, thus providing novel insights into the mechanisms by which metal pollutants inhibit the skeletal development of fish.

Fabrication of alginate composite hydrogel encapsulated retinoic acid and nano Se doped biphasic CaP to augment in situ mineralization and osteoimmunomodulation for bone regeneration.

BACKGROUND: Bone tissue engineering endows alternates to support bone defects/injuries that are circumscribed to undergo orchestrated process of remodeling on its own. In this regard, hydrogels have emerged as a promising platform that can confront irregular defects and encourage in situ bone repair. METHODS: In this study, we aimed to develop a new approach for bone tissue regeneration by developing an alginate based composite hydrogel incorporating selenium doped biphasic calcium phosphate nanoparticles, and retinoic acid. The fabricated hydrogel was physiochemically evaluated for morphological, bonding, and mechanical behavior. Additionally, the biological response of the fabricated hydrogel was evaluated on MC3T3-E1 pre-osteoblast cells. RESULTS: The developed composite hydrogel confers excellent biocompatibility, and osteoconductivity owing to the presence of alginate, and biphasic calcium phosphate, while selenium presents pro osteogenic, antioxidative, and immunomodulatory properties. The hydrogels exhibited highly porous microstructure, superior mechanical attributes, with enhanced calcification, and biomineralization abilities in vitro. SIGNIFICANCE: By combining the osteoconductive properties of biphasic calcium phosphate with multifaceted benefits of selenium and retinoic acid, the fabricated composite hydrogel offers a potential transformation in the landscape of bone defect treatment. This strategy could direct a versatile and effective approach to tackle complex bone injuries/defects and present potential for clinical translation.

Dual sgRNA-directed tyrosinases knockout using CRISPR/Cas9 technology in Pacific oyster (Crassostrea gigas) reveals their roles in early shell calcification.

Biomineralization processes in bivalves, particularly the initial production of molecular components (such as matrix deposition and calcification) in the early stages of shell development are highly complex and well-organized. This study investigated the temporal dynamics of organic matrix and calcium carbonate (CaCO3) deposition in Pacific oysters (Crassostrea gigas) across various development stages. The shell-field initiated matrix secretion during the gastrula stage. Subsequent larval development triggered central shell-field calcification, accompanied by expansion of the calcium ring from its interior to the periphery. Notably, the expression patterns of CgTyrp-2 and CgTyr closely correlated with matrix deposition and calcification during early developmental stages, with peak expression occurring in oyster's gastrula and D-veliger stages. Subsequently, the CRISPR/Cas9 system was utilized to knock out CgTyrp-2 and CgTyr with more distinct phenotypic alterations observed when both genes were concurrently knocked out. The relative gene expression was analyzed post-knockout, indicating that the knockout of CgTyr or CgTyrp-2 led to reduced expression of CgChs1, along with increased expression of CgChit4. Furthermore, when dual-sgRNAs were employed to knockout CgTyrp-2, a large deletion (2 kb) within the CgTyrp-2 gene was identified. In summary, early shell formation in C. gigas is the result of a complex interplay of multiple molecular components with CgTyrp-2 and CgTyr playing key roles in regulating CaCO3 deposition.

Elevated heterotrophic capacity as a strategy for Mediterranean corals to cope with low pH at CO2 vents.

The global increase in anthropogenic CO2 is leading to ocean warming and acidification, which is threatening corals. In Ischia, Italy, two species of Mediterranean scleractinian corals-the symbiotic Cladocora caespitosa and the asymbiotic Astroides calycularis-were collected from ambient pH sites (average pHT = 8.05) and adjacent CO2 vent sites (average pHT = 7.8) to evaluate their response to ocean acidification. Coral colonies from both sites were reared in a laboratory setting for six months at present day pH (pHT ~ 8.08) or low pH (pHT ~7.72). Previous work showed that these corals were tolerant of low pH and maintained positive calcification rates throughout the experiment. We hypothesized that these corals cope with low pH by increasing their heterotrophic capacity (i.e., feeding and/or proportion of heterotrophically derived compounds incorporated in their tissues), irrespective of site of origin, which was quantified indirectly by measuring δ13C, δ15N, and sterols. To further characterize coral health, we quantified energy reserves by measuring biomass, total lipids, and lipid classes. Additional analysis for C. caespitosa included carbohydrates (an energy reserve) and chlorophyll a (an indicator of photosynthetic capacity). Isotopic evidence shows that ambient-sourced Mediterranean corals, of both species, decreased heterotrophy in response to six months of low pH. Despite maintaining energy reserves, lower net photosynthesis (C. caespitosa) and a trend of declining calcification (A. calycularis) suggest a long-term cost to low heterotrophy under ocean acidification conditions. Conversely, vent-sourced corals maintained moderate (C. caespitosa) or high (A. calycularis) heterotrophic capacity and increased photosynthesis rates (C. caespitosa) in response to six months at low pH, allowing them to sustain themselves physiologically. Provided there is sufficient zooplankton and/or organic matter to meet their heterotrophic needs, vent-sourced corals are more likely to persist this century and potentially be a source for new corals in the Mediterranean.

Numerical and graphical simulation of the non-linear fractional dynamical system of bone mineralization.

The objective of the present study was to improve our understanding of the complex biological process of bone mineralization by performing mathematical modeling with the Caputo-Fabrizio fractional operator. To obtain a better understanding of Komarova's bone mineralization process, we have thoroughly examined the boundedness, existence, and uniqueness of solutions and stability analysis within this framework. To determine how model parameters affect the behavior of the system, sensitivity analysis was carried out. Furthermore, the fractional Adams-Bashforth method has been used to carry out numerical and graphical simulations. Our work is significant owing to its comparison of fractional- and integer-order models, which provides novel insight into the effectiveness of fractional operators in representing the complex dynamics of bone mineralization.

The Na+/Ca2+ exchanger NCX3 mediates Ca2+ entry into matrix vesicles to facilitate initial steps of mineralization in osteoblasts.

Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na+/Ca2+ exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca2+ transporter responsible for Ca2+ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca2+ deposition due to reduced Ca2+ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3-/-) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, µCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3-/- mice, thus supporting the critical role of NCX3 in facilitating Ca2+ uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.

Effect of bone and analytical method on assessment of bone mineralization in response to dietary phosphorus, phytase, and vitamin D in finishing pigs.

A total of 882 pigs (PIC TR4 × [Fast LW × PIC L02]; initially 33.2 ±â€…0.31 kg) were used in a 112-d study to evaluate the effects of different bones and analytical methods on the assessment of bone mineralization response to changes in dietary P, phytase, and vitamin D in growing pigs. Pens of pigs (20 pigs per pen) were randomized to one of five dietary treatments with nine pens per treatment. Dietary treatments were designed to create differences in bone mineralization and included: 1) P at 80% of NRC (2012) standardized total tract digestible (STTD) P requirement, 2) NRC STTD P with no phytase, 3) NRC STTD P with phytase providing an assumed release of 0.14% STTD P from 2,000 FYT/kg, 4) high STTD P (128% of the NRC P) using monocalcium phosphate and phytase, and 5) diet 4 with additional vitamin D3 from 25(OH)D3. On day 112, one pig per pen was euthanized for bone, blood, and urine analysis. Additionally, 11 pigs identified as having poor body condition which indicated a history of low feed intake (unhealthy) were sampled. There were no differences between treatments for final body weight, average daily gain, average daily feed intake, gain to feed, or bone ash measurements (treatment × bone interaction) regardless of bone ash method. The response to treatment for bone density and bone mineral content was dependent upon the bone sampled (density interaction, P = 0.053; mineral interaction, P = 0.078). For 10th rib bone density, pigs fed high levels of P had increased (P < 0.05) bone density compared with pigs fed NRC levels with phytase, with pigs fed deficient P, NRC levels of P with no phytase, and high STTD P with extra 25(OH)D3 intermediate, with no differences for metacarpals, fibulas, or 2nd ribs. Pigs fed extra vitamin D from 25(OH)D3 had increased (P < 0.05) 10th rib bone mineral content compared with pigs fed deficient P and NRC levels of P with phytase, with pigs fed industry P and vitamin D, and NRC P with monocalcium intermediate. Healthy pigs had greater (P < 0.05) serum Ca, P, vitamin D concentrations, and defatted bone ash than those unhealthy, with no difference between the two health statuses for non-defatted bone ash. In summary, differences between bone ash procedures were more apparent than differences between diets. Differences in bone density and mineral content in response to dietary P and vitamin D were most apparent with 10th ribs.

Lameness is defined as impaired movement or deviation from normal gait. The evaluation of bone mineralization can be an important component of a diagnostic investigation of lameness. Lameness in growing pigs can cause an increase in morbidity and mortality, which leads to economic losses and animal welfare concerns for producers. Calcium and P are the primary minerals in skeletal tissue and their deficiency is considered to be one of the causes of lameness. To evaluate bone mineralization, it is important to know the differences between methodologies used to determine bone ash and the expected differences between the bones analyzed. Furthermore, there has been limited data comparing bone mineralization and serum Ca and P concentrations between healthy pigs and those exhibiting clinical signs of illness (unhealthy). By removing the lipid in the bone (defatting) before the bone is ashed, variation across bones is decreased compared with not removing lipid before ashing (non-defatted). The reduction in variation across bones allows for more differences to be detected among dietary treatments and health statuses of pigs. The 10th rib is more sensitive to detect dietary differences using bone density than metacarpals, fibulas, and 2nd ribs. When comparing healthy vs. unhealthy pigs exhibiting clinical signs of illness, healthy pigs have increased defatted percentage bone ash and serum Ca, P, and vitamin D.

Enzyme-Activated Biomimetic Vesicles Confining Mineralization for Bone Maturation.

Inspired by the crucial role of matrix vesicles (MVs), a series of biomimetic vesicles (BVs) fabricated by calcium glycerophosphate (CaGP) modified polyurethane were designed to mediate the mineralization through in situ enzyme activation for bone therapy. In this study, alkaline phosphatase (ALP) was harbored in the porous BVs by adsorption (Ad-BVs) or entrapment (En-BVs). High encapsulation of ALP on En-BVs was effectively self-activating by calcium ions of CaGP-modified PU that specifically hydrolyzed the organophosphorus (CaGP) to inorganic phosphate, thus promoting the formation of the highly oriented bone-like apatite in vitro. Enzyme-catalyzed kinetics confirms the regulation of apatite crystallization by the synergistic action of self-activated ALP and the confined microcompartments of BVs. This leads to a supersaturated microenvironment, with the En-BVs group exhibiting inorganic phosphate (Pi) levels 4.19 times higher and Ca2+ levels 3.67 times higher than those of simulated body fluid (SBF). Of note, the En-BVs group exhibited excellent osteo-inducing differentiation of BMSCs in vitro and the highest maturity with reduced bone loss in rat femoral defect in vivo. This innovative strategy of biomimetic vesicles is expected to provide valuable insights into the enzyme-activated field of bone therapy.

Rubble persistence under ocean acidification threatened by accelerated bioerosion and lower-density coral skeletons.

As the balance between erosional and constructive processes on coral reefs tilts in favor of framework loss under human-induced local and global change, many reef habitats worldwide degrade and flatten. The resultant generation of coral rubble and the beds they form can have lasting effects on reef communities and structural complexity, threatening the continuity of reef ecological functions and the services they provide. To comprehensively capture changing framework processes and predict their evolution in the context of climate change, heavily colonized rubble fragments were exposed to ocean acidification (OA) conditions for 55 days. Controlled diurnal pH oscillations were incorporated in the treatments to account for the known impact of diel carbonate chemistry fluctuations on calcification and dissolution response to OA. Scenarios included contemporary pH (8.05 ± 0.025 diel fluctuation), elevated OA (7.90 ± 0.025), and high OA (7.70 ± 0.025). We used a multifaceted approach, combining chemical flux analyses, mass alteration measurements, and computed tomography scanning images to measure total and chemical bioerosion, as well as chemically driven secondary calcification. Rates of net carbonate loss measured in the contemporary conditions (1.36 kg m-2 year-1) were high compared to literature and increased in OA scenarios (elevated: 1.84 kg m-2 year-1 and high: 1.59 kg m-2 year-1). The acceleration of these rates was driven by enhanced chemical dissolution and reduced secondary calcification. Further analysis revealed that the extent of these changes was contingent on the density of the coral skeleton, in which the micro- and macroborer communities reside. Findings indicated that increased mechanical bioerosion rates occurred in rubble with lower skeletal density, which is of note considering that corals form lower-density skeletons under OA. These direct and indirect effects of OA on chemical and mechanical framework-altering processes will influence the permanence of this crucial habitat, carrying implications for biodiversity and reef ecosystem function.

Efficient carbon recycling between calcification and photosynthesis in red coralline algae.

Red coralline algae create abundant, spatially vast, reef ecosystems throughout our coastal oceans with significant ecosystem service provision, but our understanding of their basic physiology is lacking. In particular, the balance and linkages between carbon-producing and carbon-sequestering processes remain poorly constrained, with significant implications for understanding their role in carbon sequestration and storage. Using dual radioisotope tracing, we provide evidence for coupling between photosynthesis (which requires CO2) and calcification (which releases CO2) in the red coralline alga Boreolithothamnion soriferum (previously Lithothamnion soriferum)-a marine ecosystem engineer widely distributed across Atlantic mid-high latitudes. Of the sequestered HCO3 -, 38 ± 22% was deposited as carbonate skeleton while 39 ± 14% was incorporated into organic matter via photosynthesis. Only 38 ± 2% of the sequestered HCO3 - was transformed into CO2, and almost 40% of that was internally recycled as photosynthetic substrate, reducing the net release of carbon to 23 ± 3% of the total uptake. The calcification rate was strongly dependent on photosynthetic substrate production, supporting the presence of photosynthetically enhanced calcification. The efficient carbon-recycling physiology reported here suggests that calcifying algae may not contribute as much to marine CO2 release as is currently assumed, supporting a reassessment of their role in blue carbon accounting.

Correlative geochemical imaging of Desmophyllum dianthus reveals biomineralisation strategy as a key coral vital effect.

The chemical and isotopic composition of stony coral skeletons form an important archive of past climate. However, these reconstructions are largely based on empirical relationships often complicated by "vital effects" arising from uncertain physiological processes of the coral holobiont. The skeletons of deep-sea corals, such as Desmophyllum dianthus, are characterised by micron-scale or larger geochemical heterogeneity associated with: (1) centres of calcification (COCs) where nucleation of new skeleton begins, and (2) fibres that thicken the skeleton. These features are difficult to sample cleanly using traditional techniques, resulting in uncertainty surrounding both the causes of geochemical differences and their influence on environmental signals. Here we combine optical, and in-situ chemical and isotopic, imaging tools across a range of spatial resolutions (~ 100 nm to 10 s of µm) in a correlative multimodal imaging (CMI) approach to isolate the microstructural geochemistry of each component. This reveals COCs are characterised by higher organic content, Mg, Li and Sr and lower U, B and δ11B compared to fibres, reflecting the contrasting biomineralisation mechanisms employed to construct each feature. CMI is rarely applied in Environmental/Earth Sciences, but here we illustrate the power of this approach to unpick the "vital effects" in D. dianthus, and by extension, other scleractinian corals.

17ß-Estradiol (E2) Activates Matrix Mineralization through Genomic/Nongenomic Pathways in MC3T3-E1 Cells.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17ß-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERß antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

Multiscale effects of the calcimimetic drug, etelcalcetide on bone health of rats with secondary hyperparathyroidism induced by chronic kidney disease.

Chronic kidney disease-induced secondary hyperparathyroidism (CKD-SHPT) heightens fracture risk through impaired mineral homeostasis and elevated levels of uremic toxins (UTs), which in turn enhance bone remodeling. Etelcalcetide (Etel), a calcium-sensing receptor (CaSR) agonist, suppresses parathyroid hormone (PTH) in hyperparathyroidism to reduce excessive bone resorption, leading to increased bone mass. However, Etel's effect on bone quality, chemical composition, and strength is not well understood. To address these gaps, we established a CKD-SHPT rat model and administered Etel at a human-equivalent dose concurrently with disease induction. The effects on bone and mineral homeostasis were compared with a CKD-SHPT (vehicle-treated group) and a control group (rats without SHPT). Compared with vehicle-treated CKD-SHPT rats, Etel treatment improved renal function, reduced circulating UT levels, improved mineral homeostasis parameters, decreased PTH levels, and prevented mineralization defects. The upregulation of mineralization-promoting genes by Etel in CKD-SHPT rats might explain its ability to prevent mineralization defects. Etel preserved both trabecular and cortical bones with attendant suppression of osteoclast function, besides increasing mineralization. Etel maintained the number of viable osteocytes to the control level, which could also contribute to its beneficial effects on bone. CKD-SHPT rats displayed increased carbonate substitution of matrix and mineral, decreased crystallinity, mineral-to-matrix ratio, and collagen maturity, and these changes were mitigated by Etel. Further, Etel treatment prevented CKD-SHPT-induced deterioration in bone strength and mechanical behavior. Based on these findings, we conclude that in CKD-SHPT rats, Etel has multiscale beneficial effects on bone that involve remodeling suppression, mineralization gene upregulation, and preservation of osteocytes.

Exposure to BPA and BPS during pregnancy disrupts the bone mineralization in the offspring.

Exposure to plastic-derived estrogen-mimicking endocrine-disrupting bisphenols can have a long-lasting effect on bone health. However, gestational exposure to bisphenol A (BPA) and its analogue, bisphenol S (BPS), on offspring's bone mineralization is unclear. The effects of in-utero bisphenol exposure were examined on the offspring's bone parameters. BPA and BPS (0.0, 0.4 µg/kg bw) were administered to pregnant Wistar rats via oral gavage from gestational day 4-21. Maternal exposure to BPA and BPS increased bone mineral content and density in the offspring aged 30 and 90 days (P < 0.05). Plasma analysis revealed that alkaline phosphatase, and Gla-type osteocalcin were significantly elevated in the BPS-exposed offspring (P < 0.05). The expression of BMP1, BMP4, and their signaling mediators SMAD1 mRNAs were decreased in BPS-exposed osteoblast SaOS-2 cells (P < 0.05). The expression of extracellular matrix proteins such as ALPL, COL1A1, DMP1, and FN1 were downregulated (P < 0.05). Bisphenol co-incubation with noggin decreased TGF-ß1 expression, indicating its involvement in bone mineralization. Altered mineralization could be due to dysregulated expression of bone morphogenetic proteins and signalling mediators in the osteoblast cells. Thus, bisphenol exposure during gestation altered growth and bone mineralization in the offspring, possibly by modulating the expression of Smad-dependent BMP/TGF-ß1 signalling mediators.

Harnessing oriented arrangement of collagen fibers by 3D printing for enhancing mechanical and osteogenic properties of mineralized collagen scaffolds.

As the structural basis of connective and load-bearing tissues, collagen fibers with orientation play an important role in the mechanical properties and physiological and biochemical functions of the tissues, but viable methods for preparing scaffolds with highly oriented collagenous structure still need to be further studied. In this study, pure collagen was used as printing ink to 3D printing. Harnessing oriented collagen fiber structure by 3D printing for promoting mechanical and osteogenic properties of scaffolds. The scaffolds with different printed angles and thicknesses were prepared to fit the bone defect site and realize personalized customization. The orientation assembly of collagen fibers was promoted by shear force action of 3D printing, the regular arrangement of collagen fibers and stabilization of fiber structure were promoted by pH adjustment and glutaraldehyde cross-linking, and the collagen fibers were mineralized by cyclic mineralization method. The microscopic morphology of fiber arrangement in the scaffolds were investigated by scanning electron microscopy. Results demonstrated that collagen fibers were changed from non-oriented to oriented after 3D printing. And the tensile modulus of the scaffolds with oriented collagen fibers was nine times higher than that of the scaffolds with non-oriented fibers. Moreover, the effects of oriented collagen fibers on the proliferation, differentiation and mineralization of MC3T3-E1 cells were studied by CCK-8 assay, live/dead cell staining, alkaline phosphatase activity test, and Alizarin red staining. The results indicated that cell proliferation, differentiation and mineralization were significantly promoted by oriented collagen fibers, and the cells proliferated directionally in the direction of the fibers. Taken together, mineralized collagen fiber scaffolds with oriented collagen fibers have great potential in bone tissue engineering applications.