Cold-water coral energy reserves and calcification in contrasting fjord environments.

The relationship between energy reserves of cold-water corals (CWCs) and their physiological performance remains largely unknown. In addition, it is poorly understood how the energy allocation to different metabolic processes might change with projected decreasing food supply to the deep sea in the future. This study explores the temporal and spatial variations of total energy reserves (proteins, carbohydrates and lipids) of the CWC Desmophyllum dianthus and their correlation with its calcification rate. We took advantage of distinct horizontal and vertical physico-chemical gradients in Comau Fjord (Chile) and examined the changes in energy reserves over one year in an in situ reciprocal transplantation experiment (20 m vs. 300 m and fjord head vs. mouth). Total energy reserves correlated positively with calcification rates. The fast-growing deep corals had higher and less variable energy reserves, while the slower-growing shallow corals showed pronounced seasonal changes in energy reserves. Novel deep corals (transplanted from shallow) were able to quickly increase both their calcification rates and energy reserves to similar levels as native deep corals. Our study shows the importance of energy reserves in sustaining CWC growth in spite of aragonite undersaturated conditions (deep corals) in the present, and potentially also future ocean.

Annexin A5 derived from matrix vesicles protects against osteoporotic bone loss via mineralization.

Matrix vesicles (MVs) have shown strong effects in diseases such as vascular ectopic calcification and pathological calcified osteoarthritis and in wound repair of the skeletal system due to their membranous vesicle characteristics and abundant calcium and phosphorus content. However, the role of MVs in the progression of osteoporosis is poorly understood. Here, we report that annexin A5, an important component of the matrix vesicle membrane, plays a vital role in bone matrix homeostasis in the deterioration of osteoporosis. We first identified annexin A5 from adherent MVs but not dissociative MVs of osteoblasts and found that it could be sharply decreased in the bone matrix during the occurrence of osteoporosis based on ovariectomized mice. We then confirmed its potential in mediating the mineralization of the precursor osteoblast lineage via its initial binding with collagen type I to achieve MV adhesion and the subsequent activation of cellular autophagy. Finally, we proved its protective role in resisting bone loss by applying it to osteoporotic mice. Taken together, these data revealed the importance of annexin A5, originating from adherent MVs of osteoblasts, in bone matrix remodeling of osteoporosis and provided a new strategy for the treatment and intervention of bone loss.

Epithelial-like transport of mineral distinguishes bone formation from other connective tissues.

We review unique properties of bone formation including current understanding of mechanisms of bone mineral transport. We focus on formation only; mechanism of bone degradation is a separate topic not considered. Bone matrix is compared to other connective tissues composed mainly of the same proteins, but without the specialized mechanism for continuous transport and deposition of mineral. Indeed other connective tissues add mechanisms to prevent mineral formation. We start with the epithelial-like surfaces that mediate transport of phosphate to be incorporated into hydroxyapatite in bone, or in its ancestral tissue, the tooth. These include several phosphate producing or phosphate transport-related proteins with special expression in large quantities in bone, particularly in the bone-surface osteoblasts. In all connective tissues including bone, the proteins that constitute the protein matrix are mainly type I collagen and γ-carboxylate-containing small proteins in similar molar quantities to collagen. Specialized proteins that regulate connective tissue structure and formation are surprisingly similar in mineralized and non-mineralized tissues. While serum calcium and phosphate are adequate to precipitate mineral, specialized mechanisms normally prevent mineral formation except in bone, where continuous transport and deposition of mineral occurs.

Bone-matrix mineralization dampens integrin-mediated mechanosignalling and metastatic progression in breast cancer.

In patients with breast cancer, lower bone mineral density increases the risk of bone metastasis. Although the relationship between bone-matrix mineralization and tumour-cell phenotype in breast cancer is not well understood, mineralization-induced rigidity is thought to drive metastatic progression via increased cell-adhesion forces. Here, by using collagen-based matrices with adjustable intrafibrillar mineralization, we show that, unexpectedly, matrix mineralization dampens integrin-mediated mechanosignalling and induces a less proliferative stem-cell-like phenotype in breast cancer cells. In mice with xenografted decellularized physiological bone matrices seeded with human breast tumour cells, the presence of bone mineral reduced tumour growth and upregulated a gene-expression signature that is associated with longer metastasis-free survival in patients with breast cancer. Our findings suggest that bone-matrix changes in osteogenic niches regulate metastatic progression in breast cancer and that in vitro models of bone metastasis should integrate organic and inorganic matrix components to mimic physiological and pathologic mineralization.

Differences in carbonate chemistry up-regulation of long-lived reef-building corals.

With climate projections questioning the future survival of stony corals and their dominance as tropical reef builders, it is critical to understand the adaptive capacity of corals to ongoing climate change. Biological mediation of the carbonate chemistry of the coral calcifying fluid is a fundamental component for assessing the response of corals to global threats. The Tara Pacific expedition (2016-2018) provided an opportunity to investigate calcification patterns in extant corals throughout the Pacific Ocean. Cores from colonies of the massive Porites and Diploastrea genera were collected from different environments to assess calcification parameters of long-lived reef-building corals. At the basin scale of the Pacific Ocean, we show that both genera systematically up-regulate their calcifying fluid pH and dissolved inorganic carbon to achieve efficient skeletal precipitation. However, while Porites corals increase the aragonite saturation state of the calcifying fluid (Ωcf) at higher temperatures to enhance their calcification capacity, Diploastrea show a steady homeostatic Ωcf across the Pacific temperature gradient. Thus, the extent to which Diploastrea responds to ocean warming and/or acidification is unclear, and it deserves further attention whether this is beneficial or detrimental to future survival of this coral genus.

Physiological Intracranial Calcifications in Children: A computed tomography-based study.

Objectives: This study aimed to examined the frequency of physiological intracranial calcifications (PICs) in paediatric population using computed tomography (CT). Methods: The brain CT scans of consecutive patients (age range: 0-15 years) who had visited Sultan Qaboos University Hospital, Muscat, Oman, from January 2017 to December 2020 were retrospectively assessed for the presence of PICs. The presence of calcifications was identified using 3 mm-thick axial images and coronal and sagittal reformats. Results: A total of 460 patients were examined, with a mean age of 6.54 ± 4.94 years. The frequency of PIC in boys and girls was 35.1% and 35.4%, respectively. PICs were most common in choroid plexus, observed in 35.2% (age range: 0.4-15 years, median: 12 years) of subjects, followed by the pineal gland in 21.1% (age range: 0.5-15 years, median: 12 years) and the habenular nucleus in 13.0% of subjects (age range: 2.9-15 years; median: 12 years). PICs were less common in falx cerebri, observed in 5.9% (age range: 2.8-15 years; median: 13 years) of subjects, and tentorium cerebelli, observed in 3.0% (age range: 7-15 years, median: 14 years) of subjects. PICs increased significantly with increase in age (P <0.001). Conclusion: Choroid plexus is the most frequent site of calcification. Choroid plexus and pineal gland calcifications may be present in infants younger than one year. Recognising PICs is clinically important for radiologists as they can be mistaken for haemorrhage or pathological entities such as neoplasms or metabolic diseases.

Testing hypotheses on the calcification in scleractinian corals using a spatio-temporal model that shows a high degree of robustness.

Calcification in photosynthetic scleractinian corals is a complicated process that involves many different biological, chemical, and physical sub-processes that happen within and around the coral tissue. Identifying and quantifying the role of separate processes in vivo or in vitro is difficult or not possible. A computational model can facilitate this research by simulating the sub-processes independently. This study presents a spatio-temporal model of the calcification physiology, which is based on processes that are considered essential for calcification: respiration, photosynthesis, Ca2＋-ATPase, carbonic anhydrase. The model is used to test different hypotheses considering ion transport across the calicoblastic cells and Light Enhanced Calcification (LEC). It is also used to quantify the effect of ocean acidification (OA) on the Extracellular Calcifying Medium (ECM) and ATP-consumption of Ca2＋-ATPase. It was able to reproduce the experimental data of three separate studies and finds that paracellular transport plays a minor role compared to transcellular transport. In the model, LEC results from increased Ca2＋-ATPase activity in combination with increased metabolism. Implementing OA increases the concentration of CO2 throughout the entire tissue, thereby increasing the availability of CO3- in the ECM. As a result, the model finds that calcification becomes more energy-demanding and the calcification rate increases.

Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

Matrix Vesicle-Mediated Mineralization and Osteocytic Regulation of Bone Mineralization.

Bone mineralization entails two mineralization phases: primary and secondary mineralization. Primary mineralization is achieved when matrix vesicles are secreted by osteoblasts, and thereafter, bone mineral density gradually increases during secondary mineralization. Nearby extracellular phosphate ions (PO43-) flow into the vesicles via membrane transporters and enzymes located on the vesicles' membranes, while calcium ions (Ca2+), abundant in the tissue fluid, are also transported into the vesicles. The accumulation of Ca2+ and PO43- in the matrix vesicles induces crystal nucleation and growth. The calcium phosphate crystals grow radially within the vesicle, penetrate the vesicle's membrane, and continue to grow outside the vesicle, ultimately forming mineralized nodules. The mineralized nodules then attach to collagen fibrils, mineralizing them from the contact sites (i.e., collagen mineralization). Afterward, the bone mineral density gradually increases during the secondary mineralization process. The mechanisms of this phenomenon remain unclear, but osteocytes may play a key role; it is assumed that osteocytes enable the transport of Ca2+ and PO43- through the canaliculi of the osteocyte network, as well as regulate the mineralization of the surrounding bone matrix via the Phex/SIBLINGs axis. Thus, bone mineralization is biologically regulated by osteoblasts and osteocytes.

pH variability at volcanic CO2 seeps regulates coral calcifying fluid chemistry.

Coral reefs are iconic ecosystems with immense ecological, economic and cultural value, but globally their carbonate-based skeletal construction is threatened by ocean acidification (OA). Identifying coral species that have specialised mechanisms to maintain high rates of calcification in the face of declining seawater pH is of paramount importance in predicting future species composition, and growth of coral reefs. Here, we studied multiple coral species from two distinct volcanic CO2 seeps in Papua New Guinea to assess their capacity to control their calcifying fluid (CF) chemistry. Several coral species living under conditions of low mean seawater pH, but with either low or high variability in seawater pH, were examined and compared with those living in 'normal' (non-seep) ambient seawater pH. We show that when mean seawater pH is low but highly variable, corals have a greater ability to maintain constant pHcf in their CF, but this characteristic was not linked with changes in abundance. Within less variable low pH seawater, corals with limited reductions in pHcf at the seep sites compared with controls tended to be more abundant at the seep site than at the control site. However, this finding was strongly influenced by a single species (Montipora foliosa), which was able to maintain complete pHcf homeostasis. Overall, although our findings indicate that there might be an association between ecological success and greater pHcf homeostasis, further research with additional species and at more sites with differing seawater pH regimes is required to solidify inferences regarding coral ecological success under future OA.

Cytoskeletal Protein 4.1G Is Essential for the Primary Ciliogenesis and Osteoblast Differentiation in Bone Formation.

The primary cilium is a hair-like immotile organelle with specific membrane receptors, including the receptor of Hedgehog signaling, smoothened. The cilium organized in preosteoblasts promotes differentiation of the cells into osteoblasts (osteoblast differentiation) by mediating Hedgehog signaling to achieve bone formation. Notably, 4.1G is a plasma membrane-associated cytoskeletal protein that plays essential roles in various tissues, including the peripheral nervous system, testis, and retina. However, its function in the bone remains unexplored. In this study, we identified 4.1G expression in the bone. We found that, in the 4.1G-knockout mice, calcium deposits and primary cilium formation were suppressed in the trabecular bone, which is preosteoblast-rich region of the newborn tibia, indicating that 4.1G is a prerequisite for osteoblast differentiation by organizing the primary cilia in preosteoblasts. Next, we found that the primary cilium was elongated in the differentiating mouse preosteoblast cell line MC3T3-E1, whereas the knockdown of 4.1G suppressed its elongation. Moreover, 4.1G-knockdown suppressed the induction of the cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation. These results demonstrate a new regulatory mechanism of 4.1G in bone formation that promotes the primary ciliogenesis in the differentiating preosteoblasts and induction of cilia-mediated osteoblast differentiation, resulting in bone formation at the newborn stage.

The deterioration of calcified cartilage integrity reflects the severity of osteoarthritis-A structural, molecular, and biochemical analysis.

The calcified cartilage zone (CCZ) is a thin interlayer between the hyaline articular cartilage and the subchondral bone and plays an important role in maintaining the joint homeostasis by providing biological and mechanical support from unmineralized cartilage to the underlying mineralized subchondral bone. The hallmark of CCZ characteristics in osteoarthritis (OA) is less well known. The aim of our study is to evaluate the structural, molecular, and biochemical composition of CCZ in tissues affected by primary knee OA and its relationship with disease severity. We collected osteochondral tissue samples stratified according to disease severity, from 16 knee OA patients who underwent knee replacement surgery. We also used meniscectomy-induced rat samples to confirm the pathophysiologic changes of human samples. We defined the characteristics of the calcified cartilage layer using a combination of morphological, biochemical, proteomic analyses on laser micro-dissected tissue. Our results demonstrated that the Calcium/Phosphate ratio is unchanged during the OA progression, but the calcium-binding protein and cadherin binding protein, as well as carbohydrate metabolism-related proteins, undergo significant changes. These changes were further accompanied by thinning of the CCZ, loss of collagen and proteoglycan content, the occurrence of the endochondral ossification, neovasculature, loss of the elastic module, loss of the collagen direction, and increase of the tortuosity indicating an altered structural and mechanical properties of the CCZ in OA. In conclusion, our results suggest that the calcified cartilage changes can reflect the disease progression.

Mineral tessellation in bone and the stenciling principle for extracellular matrix mineralization.

We review here the Stenciling Principle for extracellular matrix mineralization that describes a double-negative process (inhibition of inhibitors) that promotes mineralization in bone and other mineralized tissues, whereas the default condition of inhibition alone prevents mineralization elsewhere in soft connective tissues. The stenciling principle acts across multiple levels from the macroscale (skeleton/dentition vs soft connective tissues), to the microscale (for example, entheses, and the tooth attachment complex where the soft periodontal ligament is situated between mineralized tooth cementum and mineralized alveolar bone), and to the mesoscale (mineral tessellation). It relates to both small-molecule (e.g. pyrophosphate) and protein (e.g. osteopontin) inhibitors of mineralization, and promoters (enzymes, e.g. TNAP, PHEX) that degrade the inhibitors to permit and regulate mineralization. In this process, an organizational motif for bone mineral arises that we call crossfibrillar mineral tessellation where mineral formations - called tesselles - geometrically approximate prolate ellipsoids and traverse multiple collagen fibrils (laterally). Tesselle growth is directed by the structural anisotropy of collagen, being spatially restrained in the shorter transverse tesselle dimensions (averaging 1.6 × 0.8 × 0.8 µm, aspect ratio 2, length range 1.5-2.5 µm). Temporo-spatially, the tesselles abut in 3D (close ellipsoid packing) to fill the volume of lamellar bone extracellular matrix. Poorly mineralized interfacial gaps between adjacent tesselles remain discernable even in mature lamellar bone. Tessellation of a same, small basic unit to form larger structural assemblies results in numerous 3D interfaces, allows dissipation of critical stresses, and enables fail-safe cyclic deformations. Incomplete tessellation in osteomalacia/odontomalacia may explain why soft osteomalacic bones buckle and deform under loading.

Novel Elongator Protein 2 Inhibitors Mitigating Tumor Necrosis Factor-α Induced Osteogenic Differentiation Inhibition.

Tumor necrosis factor-α is a common cytokine that increases in inflammatory processes, slows the differentiation of bone formation, and induces osteodystrophy in the long-term inflammatory microenvironment. Our previous study confirmed that the Elongation protein 2 (ELP2) plays a significant role in osteogenesis and osteogenic differentiation, which is considered a drug discovery target in diseases related to bone formation and differentiation. In this study, we applied an in silico virtual screening method to select molecules that bind to the ELP2 protein from a chemical drug molecule library and obtained 95 candidates. Then, we included 11 candidates by observing the docking patterns and the noncovalent bonds. The binding affinity of the ELP2 protein with the candidate compounds was examined by SPR analysis, and 5 out of 11 compounds performed good binding affinity to the mouse ELP2 protein. After in vitro cell differentiation assay, candidates 2# and 5# were shown to reduce differentiation inhibition after tumor necrosis factor-α stimulation, allowing further optimization and development for potential clinical treatment of inflammation-mediated orthopedic diseases.

Dense intracellular ion pools in unicellular organisms.

Uptake and concentration of inorganic ions are part of the complex cellular processes required for cell homeostasis, as well as for mineral formation by organisms. These ion transport mechanisms include distinct cellular compartments and chemical phases that play various roles in the physiology of organisms. Here, the prominent cases of dense ion pools in unicellular organisms are briefly reviewed. The specific observations that were reported for different organisms are consolidated into a wide perspective that emphasizes general traits. It is suggested that the intracellular ion pools can be divided into three types: a high cytoplasmic concentration, a labile storage compartment that hosts dense ion-rich phases, and a mineral-forming compartment in which a stable long-lived structure is formed. Recently, many labile pools were identified in various organisms using advanced techniques, bringing many new questions about their possible roles in the formation of the stable mineralized structures.

Calycosin-7-O-ß-Glucoside Isolated from Astragalus membranaceus Promotes Osteogenesis and Mineralization in Human Mesenchymal Stem Cells.

Stem cells have received attention in various diseases, such as inflammatory, cancer, and bone diseases. Mesenchymal stem cells (MSCs) are multipotent stem cells that are critical for forming and repairing bone tissues. Herein, we isolated calycosin-7-O-ß-glucoside (Caly) from the roots of Astragalus membranaceus, which is one of the most famous medicinal herbs, and investigated the osteogenic activities of Caly in MSCs. Caly did not affect cytotoxicity against MSCs, whereas Caly enhanced cell migration during the osteogenesis of MSCs. Caly increased the expression and enzymatic activities of ALP and the formation of mineralized nodules during the osteogenesis of MSCs. The osteogenesis and bone-forming activities of Caly are mediated by bone morphogenetic protein 2 (BMP2), phospho-Smad1/5/8, Wnt3a, phospho-GSK3ß, and phospho-AKT, inducing the expression of runt-related transcription factor 2 (RUNX2). In addition, Caly-mediated osteogenesis and RUNX2 expression were attenuated by noggin and wortmannin. Moreover, the effects were validated in pre-osteoblasts committed to the osteoblast lineages from MSCs. Overall, our results provide novel evidence that Caly stimulates osteoblast lineage commitment of MSCs by triggering RUNX2 expression, suggesting Caly as a potential anabolic drug to prevent bone diseases.

Congenital Metabolic Bone Disorders as a Cause of Bone Fragility.

Bone fragility is a pathological condition caused by altered homeostasis of the mineralized bone mass with deterioration of the microarchitecture of the bone tissue, which results in a reduction of bone strength and an increased risk of fracture, even in the absence of high-impact trauma. The most common cause of bone fragility is primary osteoporosis in the elderly. However, bone fragility can manifest at any age, within the context of a wide spectrum of congenital rare bone metabolic diseases in which the inherited genetic defect alters correct bone modeling and remodeling at different points and aspects of bone synthesis and/or bone resorption, leading to defective bone tissue highly prone to long bone bowing, stress fractures and pseudofractures, and/or fragility fractures. To date, over 100 different Mendelian-inherited metabolic bone disorders have been identified and included in the OMIM database, associated with germinal heterozygote, compound heterozygote, or homozygote mutations, affecting over 80 different genes involved in the regulation of bone and mineral metabolism. This manuscript reviews clinical bone phenotypes, and the associated bone fragility in rare congenital metabolic bone disorders, following a disease taxonomic classification based on deranged bone metabolic activity.

Biomimicking Bone-Implant Interface Facilitates the Bioadaption of a New Degradable Magnesium Alloy to the Bone Tissue Microenvironment.

The most critical factor determining the success of biodegradable bone implants is the host tissue response, which greatly depends on their degradation behaviors. Here, a new magnesium-based implant, namely magnesium-silicon-calcium (Mg-0.2Si-1.0Ca) alloy, that coordinates its biodegradation along with the bone regenerative process via a self-assembled, multilayered bone-implant interface is designed. At first, its rapid biocorrosion contributes to a burst release of Mg2+ , leading to a pro-osteogenic immune microenvironment in bone. Meanwhile, with the simultaneous intervention of Ca and Si in the secondary phases of the new alloy, a hierarchical layered calcified matrix is rapidly formed at the degrading interface that favored the subsequent bone mineralization. In contrast, pure Mg or Mg-0.2Si alloy without the development of this interface at the beginning will unavoidably induce detrimental bone loss. Hence, it is believed this biomimicking interface justifies its bioadaptability in which it can modulate its degradation in vivo and accelerate bone mineralization.