IL-1ß contributes to the secretion of sclerostin by osteocytes and targeting sclerostin promotes spinal fusion at early stages.

BACKGROUND: Despite extensive research, there is still a need for safe and effective agents to promote spinal fusion. Interleukin (IL)-1ß is an important factor which influences the bone repair and remodelling. The purpose of our study was to determine the effect of IL-1ß on sclerostin in osteocytes and to explore whether inhibiting the secretion of sclerostin from osteocytes can promote spinal fusion at early stages. METHODS: Small-interfering RNA was used to suppress the secretion of sclerostin in Ocy454 cells. MC3T3-E1 cells were cocultured with Ocy454 cells. Osteogenic differentiation and mineralisation of MC3T3-E1 cells were evaluated in vitro. SOST knock-out rat generated using the CRISPR-Cas9 system and rat spinal fusion model was used in vivo. The degree of spinal fusion was assessed by manual palpation, radiographic analysis and histological analysis at 2 and 4 weeks. RESULTS: We found that IL-1ß level had a positive association with sclerostin level in vivo. IL-1ß promoted the expression and secretion of sclerostin in Ocy454 cells in vitro. Inhibition of IL-1ß-induced secretion of sclerostin from Ocy454 cells could promote the osteogenic differentiation and mineralisation of cocultured MC3T3-E1 cells in vitro. The extent of spinal graft fusion was greater in SOST-knockout rats than in wild-type rats at 2 and 4 weeks. CONCLUSIONS: The results demonstrate that IL-1ß contributes to a rise in the level of sclerostin at early stages of bone healing. Suppressing sclerostin may be an important therapeutic target capable of promoting spinal fusion at early stages.

Mechanical loading modulates phosphate related genes in rat bone.

Mechanical loading determines bone mass and bone structure, which involves many biochemical signal molecules. Of these molecules, Mepe and Fgf23 are involved in bone mineralization and phosphate homeostasis. Thus, we aimed to explore whether mechanical loading of bone affects factors of phosphate homeostasis. We studied the effect of mechanical loading of bone on the expression of Fgf23, Mepe, Dmp1, Phex, Cyp27b1, and Vdr. Twelve-week old female rats received a 4-point bending load on the right tibia, whereas control rats were not loaded. RT-qPCR was performed on tibia mRNA at 4, 5, 6, 7 or 8 hours after mechanical loading for detection of Mepe, Dmp1, Fgf23, Phex, Cyp27b1, and Vdr. Immunohistochemistry was performed to visualise FGF23 protein in tibiae. Serum FGF23, phosphate and calcium levels were measured in all rats. Four-point bending resulted in a reduction of tibia Fgf23 gene expression by 64% (p = 0.002) and a reduction of serum FGF23 by 30% (p<0.001), six hours after loading. Eight hours after loading, Dmp1 and Mepe gene expression increased by 151% (p = 0.007) and 100% (p = 0.007). Mechanical loading did not change Phex, Cyp27b1, and Vdr gene expression at any time-point. We conclude that mechanical loading appears to provoke both a paracrine as well as an endocrine response in bone by modulating factors that regulate bone mineralization and phosphate homeostasis.

Wichterle hydron for breast augmentation - case reports and brief review.

During the period of 1960s and 1970s, a new alloplastic material - Wichterle gel - was introduced in the field of plastic surgery. In 1961, a Czech scientist, prof. Otto Wichterle, had developed, along with his research team, a hydrophilic gel made of polymers, which fulfilled the high standards for prosthetic materials due to its hydrophilic, chemical, thermal and shape stability that provided a better tolerance in the body compared with other hydrophobic gels. Plastic surgeons had started to use the gel for breast augmentations and reconstructions. Success of the gel had been reinforced due to its easy preoperative preparation. The material had been implanted during general anaesthesia via submammary approach over the muscle fixed with a stitch to the fascia. Fixing corset bandage was applied after the surgery. The implanted material had proved to be suitable for postoperative processes with a minimum of complications. In the later postoperative period, however, serious complications occurred - mainly infections and calcifications. Long-term results are presented by case reports. Today, this material is no longer used and it is replaced by more modern implants.

Feasibility and intra-and interobserver reproducibility of quantitative susceptibility mapping with radiomic features for intracranial dissecting intramural hematomas and atherosclerotic calcifications.

Quantitative susceptibility mapping (QSM) for 61 patients with dissecting intramural hematomas (n = 36) or atherosclerotic calcifications (n = 25) in intracranial vertebral arteries were collected to assess intra- and interobserver reproducibility in a 3.0-T MR system between January 2015 and December 2017. Two independent observers each segmented regions of interest for lesions twice. The reproducibility was evaluated using intra-class correlation coefficients (ICC) and within-subject coefficients of variation (wCV) for means and concordance correlation coefficients (CCC) and ICC for radiomic features (CCC and ICC > 0.85) were used. Mean QSM values were 0.277 ± 0.092 ppm for dissecting intramural hematomas and - 0.208 ± 0.078 ppm for atherosclerotic calcifications. ICCs and wCVs were 0.885-0.969 and 6.5-13.7% in atherosclerotic calcifications and 0.712-0.865 and 12.4-18.7% in dissecting intramural hematomas, respectively. A total of 9 and 19 reproducible radiomic features were observed in dissecting intramural hematomas and atherosclerotic calcifications, respectively. QSM measurements in dissecting intramural hematomas and atherosclerotic calcifications were feasible and reproducible between intra- and interobserver comparisons, and some reproducible radiomic features were demonstrated.

FBXO11 regulates bone development.

FBXO11 is the substrate-recognition component of a ubiquitin ligase complex called SKP1-cullin-F-boxes. The role of FBXO11 in bone development is unexplored. In this study, we reported a novel mechanism of how bone development is regulated by FBXO11. FBXO11 gene knockdown by lentiviral transduction in mouse pre-osteoblast MC3T3-E1 cells leads to reduced osteogenic differentiation, while overexpressing FBXO11 accelerates their osteogenic differentiation in vitro. Furthermore, we generated two osteoblastic-specific FBXO11 conditional knockout mouse models, Col1a1-ERT2-FBXO11KO and Bglap2-FBXO11KO mice. In both conditional FBXO11KO mouse models, we found FBXO11 deficiency inhibits normal bone growth, in which the osteogenic activity in FBXO11cKO mice is reduced, while osteoclastic activity is not significantly changed. Mechanistically, we found FBXO11 deficiency leads to Snail1 protein accumulation in osteoblasts, leading to suppression of osteogenic activity and inhibition of bone matrix mineralization. FBXO11 knockdown in MC3T3-E1 cells reduced Snail1 protein ubiquitination and increased Snail1 protein accumulation in the cells, which eventually inhibited osteogenic differentiation. In conclusion, FBXO11 deficiency in osteoblasts inhibits bone formation through Snail1 accumulation, inhibiting osteogenic activity and bone mineralization.

Activated FGFR3 suppresses bone regeneration and bone mineralization in an ovariectomized mouse model.

BACKGROUND: Postmenopausal osteoporosis is a widespread health concern due to its prevalence among older adults and an associated high risk of fracture. The downregulation of bone regeneration delays fracture healing. Activated fibroblast growth factor receptor 3 (FGFR3) accelerates bone regeneration at juvenile age and downregulates bone mineralization at all ages. However, the impact of FGFR3 signaling on bone regeneration and bone mineralization post-menopause is still unknown. This study aimed to evaluate the impact of FGFR3 signaling on bone regeneration and bone mineralization during menopause by developing a distraction osteogenesis (DO) mouse model after ovariectomy (OVX) using transgenic mice with activated FGFR3 driven by Col2a1 promoter (Fgfr3 mice). METHODS: The OVX or sham operations were performed in 8-week-old female Fgfr3 and wild-type mice. After 8 weeks of OVX surgery, DO surgery in the lower limb was performed. The 5-day-latency period followed by performing distraction for 9 days. Bone mineral density (BMD) and bone regeneration was assessed by micro-computed tomography (micro-CT) scan and soft X-ray. Bone volume in the distraction area was also evaluated by histological analysis after 7 days at the end of distraction. Osteogenic differentiation and mineralization of bone marrow-derived mesenchymal stem cells (BMSCs) derived from each mouse after 8 weeks of the OVX or sham operations were also evaluated with and without an inhibitor for FGFR3 signaling (meclozine). RESULTS: BMD decreased after OVX in both groups, and it further deteriorated in Fgfr3 mice. Poor callus formation after DO was also observed in both groups with OVX, and the amount of regenerated bone was further decreased in Fgfr3 mice. Similarly, histological analysis revealed that Fgfr3 OVX mice showed lower bone volume. Osteogenic differentiation and mineralization of BMSCs were also deteriorated in Fgfr3 OVX mice. An inhibitor for FGFR3 signaling dramatically reversed the inhibitory effect of OVX and FGFR3 signaling on BMSC mineralization. CONCLUSION: Upregulated FGFR3 decreased newly regenerated bone after DO and BMD in OVX mice. FGFR3 signaling can be a potential therapeutic target in patients with postmenopausal osteoporosis.

Zebrafish Models to Study Ectopic Calcification and Calcium-Associated Pathologies.

Ectopic calcification refers to the pathological accumulation of calcium ions in soft tissues and is often the result of a dysregulated action or disrupted function of proteins involved in extracellular matrix mineralization. While the mouse has traditionally been the go-to model organism for the study of pathologies associated with abnormal calcium deposition, many mouse mutants often have exacerbated phenotypes and die prematurely, limiting the understanding of the disease and the development of effective therapies. Since the mechanisms underlying ectopic calcification share some analogy with those of bone formation, the zebrafish (Danio rerio)-a well-established model for studying osteogenesis and mineralogenesis-has recently gained momentum as a model to study ectopic calcification disorders. In this review, we outline the mechanisms of ectopic mineralization in zebrafish, provide insights into zebrafish mutants that share phenotypic similarities with human pathological mineralization disorders, list the compounds capable of rescuing mutant phenotypes, and describe current methods to induce and characterize ectopic calcification in zebrafish.

Collagen/κ-Carrageenan-Based Scaffolds as Biomimetic Constructs for In Vitro Bone Mineralization Studies.

Tissue engineering offers attractive strategies to develop three-dimensional scaffolds mimicking the complex hierarchical structure of the native bone. The bone is formed by cells incorporated in a molecularly organized extracellular matrix made of an inorganic phase, called biological apatite, and an organic phase mainly made of collagen and noncollagenous macromolecules. Although many strategies have been developed to replicate the complexity of bone at the nanoscale in vitro, a critical challenge has been to control the orchestrated process of mineralization promoted by bone cells in vivo and replicate the anatomical and biological properties of native bone. In this study, we used type I collagen to fabricate mineralized scaffolds mimicking the microenvironment of the native bone. The sulfated polysaccharide κ-carrageenan was added to the scaffolds to fulfill the role of noncollagenous macromolecules in the organization and mineralization of the bone matrix and cell adhesion. Scanning electron microscopy images of the surface of the collagen/κ-carrageenan scaffolds showed the presence of a dense and uniform network of intertwined fibrils, while images of the scaffolds' lateral sides showed the presence of collagen fibrils with a parallel alignment, which is characteristic of dense connective tissues. MC3T3-E1 osteoblasts were cultured in the collagen scaffolds and were viable after up to 7 days of culture, both in the absence and in the presence of κ-carrageenan. The presence of κ-carrageenan in the collagen scaffolds stimulated the maturation of the cells to a mineralizing phenotype, as suggested by the increased expression of key genes related to bone mineralization, including alkaline phosphatase (Alp), bone sialoprotein (Bsp), osteocalcin (Oc), and osteopontin (Opn), as well as the ability to mineralize the extracellular matrix after 14 and 21 days of culture. Taken together, the results described in this study shed light on the potential use of collagen/κ-carrageenan scaffolds to study the role of the structural organization of bone-mimetic synthetic matrices in cell function.

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.

Adsorptive exchange of coccolith biominerals facilitates viral infection.

Marine coccolithophores are globally distributed, unicellular phytoplankton that produce nanopatterned, calcite biominerals (coccoliths). These biominerals are synthesized internally, deposited into an extracellular coccosphere, and routinely released into the external medium, where they profoundly affect the global carbon cycle. The cellular costs and benefits of calcification remain unresolved. Here, we show observational and experimental evidence, supported by biophysical modeling, that free coccoliths are highly adsorptive biominerals that readily interact with cells to form chimeric coccospheres and with viruses to form "viroliths," which facilitate infection. Adsorption to cells is mediated by organic matter associated with the coccolith base plate and varies with biomineral morphology. Biomineral hitchhiking increases host-virus encounters by nearly an order of magnitude and can be the dominant mode of infection under stormy conditions, fundamentally altering how we view biomineral-cell-virus interactions in the environment.

Dynamic network biomarker identifies cdkn1a-mediated bone mineralization in the triggering phase of osteoporosis.

The identification of predictive markers to determine the triggering phase prior to the onset of osteoporosis is essential to mitigate further irrevocable deterioration. To determine the early warning signs before osteoporosis, we used the dynamic network biomarker (DNB) approach to analyze time-series gene expression data in a zebrafish osteoporosis model, which revealed that cyclin-dependent kinase inhibitor 1 A (cdkn1a) is a core DNB. We found that cdkn1a negatively regulates osteogenesis, as evidenced by loss-of-function and gain-of-function studies. Specifically, CRISPR/Cas9-mediated cdkn1a knockout in zebrafish significantly altered skeletal development and increased bone mineralization, whereas inducible cdkn1a expression significantly contributed to osteoclast differentiation. We also found several mechanistic clues that cdkn1a participates in osteoclast differentiation by regulating its upstream signaling cascades. To summarize, in this study, we provided new insights into the dynamic nature of osteoporosis and identified cdkn1a as an early-warning signal of osteoporosis onset.

An advanced Ca/Zn hybrid battery enabled by the dendrite-free zinc anode and a reversible calcification/decalcification NASICON cathode.

The proposal of hybrid ion batteries, which can integrate the advantages of the single ion battery, opens up a new route for developing high-performance secondary batteries. Herein, we successfully constructed an aqueous hybrid battery comprised of polyanionic-type cathode material (Na3V2(PO4)3, NVP), Zn metal anode, and aqueous Ca2+/Zn2+ hybrid electrolyte. This exciting combination gives full play to not only the excellent diffusion dynamics of Ca2+ in the NASICON (sodium super ion conductors) structure but also the electrostatic shielding effect of Ca2+ with low reduction potential that inhibits the formation of zinc dendrites. As results, the NVP//Zn Zn/Ca hybrid battery delivers favorable specific capacity with outstanding rate performance (85.3 mAh g-1 capacity at 1 C, 60.5 mAh g-1 capacity at 20 C), and excellent cycle stability (74 % capacity retention after 1300 cycles).

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization.

Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (Pi) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 ± 19 nm) and by Atomic Force Microcopy (AFM) (180 ± 85 nm). The MVs had a specific activity for TNAP of 9.2 ± 4.6 U·mg-1 confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by 31P NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69-72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8-13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3-8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm-1 band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3-8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen.

The Inhibition of the Inducible Nitric Oxide Synthase Enhances the DPSC Mineralization under LPS-Induced Inflammation.

Nitric oxide (NO) is a key messenger in physiological and pathological processes in mammals. An excessive NO production is associated with pathological conditions underlying the inflammation response as a trigger. Among others, dental pulp inflammation results from the invasion of dentin by pathogenic bacteria. Vital functions of pulp mesenchymal stem cells (DPSCs, dental pulp stem cells), such as mineralization, might be affected by the inducible NOS (iNOS) upregulation. In this context, the iNOS selective inhibition can be considered an innovative therapeutic strategy to counteract inflammation and to promote the regeneration of the dentin-pulp complex. The present work aims at evaluating two acetamidines structurally related to the selective iNOS inhibitor 1400W, namely CM544 and FAB1020, in a model of LPS-stimulated primary DPSCs. Our data reveal that CM544 and even more FAB1020 are promising anti-inflammatory compounds, decreasing IL-6 secretion by enhancing CD73 expression-levels, a protein involved in innate immunity processes and thus confirming an immunomodulatory role of DPSCs. In parallel, cell mineralization potential is retained in the presence of compounds as well as VEGF secretion, and thus their angiogenetic potential. Data presented lay the ground for further investigation on the anti-inflammatory potential of acetamidines selectively targeting iNOS in a clinical context.

Osteoblast Demineralization Induced by Oxidized High-Density Lipoprotein via the Inflammatory Pathway Is Suppressed by Adiponectin.

Low mineralization activity by human osteoblast cells (HOBs) indicates abnormal bone remodeling that potentially leads to osteoporosis. Oxidation, the most prominent form of high-density lipoprotein (HDL) modification, is suggested to affect bone mineralization through the inflammatory pathway. Adiponectin, which possesses anti-inflammatory activity, is postulated to have the ability to suppress the detrimental effects of oxidized HDL (oxHDL). This study aimed to investigate the effects of HDL before and after oxidation on markers of mineralization and inflammation. The protective effects of adiponectin on demineralization and inflammation induced by oxHDL were also investigated. OxHDL at 100 µg/mL protein had the highest inhibitory effect on mineralization, followed by lower calcium incorporation. OxHDL also had significantly lower expression of a mineralization marker (COL1A2) and higher expression of inflammatory markers (IL-6, TNF-α, and RELA proto-oncogene, NF-κß (p65)) compared to the unstimulated control group. These findings suggest that oxHDL reduces the mineralization activity of HOBs by increasing the expression of inflammatory markers. Interestingly, co-incubation of adiponectin and oxHDL in HOBs resulted in higher expression of mineralization markers (ALPL, COL1A2, BGLAP, and RUNX2) and significantly reduced all targeted inflammatory markers compared to the oxHDL groups. On the contrary, HDL increased the expression of mineralization markers (COL1A2 and STAT-3) and exhibited lower expression of inflammatory cytokines (IL-6 and TNF-α), proving the protective effect of HDL beyond the reverse cholesterol transport activity.

Structure-from-motion photogrammetry demonstrates variability in coral growth within colonies and across habitats.

Coral growth is an important metric of coral health and underpins reef-scale functional attributes such as structural complexity and calcium carbonate production. There persists, however, a paucity of growth data for most reef-building regions, especially for coral species whose skeletal architecture prevents the use of traditional methods such as coring and Alizarin staining. We used structure-from-motion photogrammetry to quantify a range of colony-scale growth metrics for six coral species in the Mexican Caribbean and present a newly developed workflow to measure colony volume change over time. Our results provide the first growth metrics for two species that are now major space occupiers on Caribbean reefs, Agaricia agaricites and Agaricia tenuifolia. We also document higher linear extension, volume increase and calcification rates within back reef compared to fore reef environments for four other common species: Orbicella faveolata, Porites astreoides, Siderastrea siderea and Pseudodiploria strigosa. Linear extension rates in our study were lower than those obtained via computed tomography (CT) scans of coral cores from the same sites, as the photogrammetry method averages growth in all dimensions, while the CT method depicts growth only along the main growth axis (upwards). The comparison of direct volume change versus potential volume increase calculated from linear extension emphasizes the importance of assessing whole colony growth to improve calcification estimates. The method presented here provides an approach that can generate accurate calcification estimates alongside a range of other whole-colony growth metrics in a non-invasive way.

Bone surface mimicked PDMS membranes stimulate osteoblasts and calcification of bone matrix.

Cellular microenvironments play a crucial role in cell behavior. In addition to the biochemical cues present in the microenvironments, biophysical and biomechanical properties on surfaces have an impact on cellular functionality and eventually cellular fate. Effects of surface topography on cell behavior are being studied extensively in the literature. However, these studies often try to replicate topographical features of tissue surfaces by using techniques such as chemical etching, photolithography, and electrospinning, which may result in the loss of crucial micro- and nano- features on the tissue surfaces such as bone. This study investigates the topographical effects of bone surface by transferring its surface features onto polydimethylsiloxane (PDMS) membranes using soft lithography from a bovine femur. Our results have shown that major features on bone surfaces were successfully transferred onto PDMS using soft lithography. Osteoblast proliferation and calcification of bone matrix have significantly increased along with osteoblast-specific differentiation and maturation markers such as osteocalcin (OSC), osterix (OSX), collagen type I alpha 1 chain (COL1A1), and alkaline phosphatase (ALP) on bone surface mimicked (BSM) PDMS membranes in addition to a unidirectional alignment of osteoblast cells compared to plain PDMS surfaces. This presented bone surface mimicking method can provide a versatile native-like platform for further investigation of intracellular pathways regarding osteoblast growth and differentiation.

Cellular adaptations leading to coral fragment attachment on artificial substrates in Acropora millepora (Am-CAM).

Reproductive propagation by asexual fragmentation in the reef-building coral Acropora millepora depends on (1) successful attachment to the reef substrate through modification of soft tissues and (2) a permanent bond with skeletal encrustation. Despite decades of research examining asexual propagation in corals, the initial response, cellular reorganisation, and development leading to fragment substrate attachment via a newly formed skeleton has not been documented in its entirety. Here, we establish the first "coral attachment model" for this species ("Am-CAM") by developing novel methods that allow correlation of fluorescence and electron microscopy image data with in vivo microscopic time-lapse imagery. This multi-scale imaging approach identified three distinct phases involved in asexual propagation: (1) the contact response of the coral fragment when contact with the substrate, followed by (2) fragment stabilisation through anchoring by the soft tissue, and (3) formation of a "lappet-like appendage" structure leading to substrate bonding of the tissue for encrustation through the onset of skeletal calcification. In developing Am-CAM, we provide new biological insights that can enable reef researchers, managers and coral restoration practitioners to begin evaluating attachment effectiveness, which is needed to optimise species-substrate compatibility and achieve effective outplanting.

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.

The combined cartilage growth - calcification patterns in the wing-fins of Rajidae (Chondrichthyes): A divergent model from endochondral ossification of tetrapods.

The relationship between cartilage growth - mineralization patterns were studied in adult Rajidae with X-ray morphology/morphometry, undecalcified resin-embedded, heat-deproteinated histology and scanning electron microscopy. Morphometry of the wing-fins, nine central rays of the youngest and oldest specimens documented a significant decrement of radials mean length between inner, middle and outer zones, but without a regular progression along the ray. This suggests that single radial length growth is regulated in such a way to align inter-radial joints parallel to the wing metapterygia curvature. Trans-illumination and heat-deproteination techniques showed polygonal and cylindrical morphotypes of tesserae, whose aligned pattern ranged from mono-columnar, bi-columnar, and multi-columnar up to the crustal-like layout. Histology of tessellated cartilage allowed to identify of zones of the incoming mineral deposition characterized by enhanced duplication rate of chondrocytes with the formation of isogenic groups, whose morphology and topography suggested a relationship with the impending formation of the radials calcified column. The morphotype and layout of radial tesserae were related to mechanical demands (stiffening) and the size/mass of the radial cartilage body. The cartilage calcification pattern of the batoids model shares several morphological features with tetrapods' endochondral ossification, that is, (chondrocytes' high duplication rate, alignment in rows, increased volume of chondrocyte lacunae), but without the typical geometry of the metaphyseal growth plates. RESEARCH HIGHLIGHTS: 1. The wing-fins system consists of stiff radials, mobile inter-radial joints and a flat inter-radial membrane adapted to the mechanical demand of wing wave movement. 2. Growth occurs by forming a mixed calcified-uncalcified cartilage texture, developing intrinsic tensional stresses documented by morphoanatomical data.