Potential benefits of advanced chelate-based trace minerals in improving bone mineralization, antioxidant status, immunity, and gene expression modulation in heat-stressed broilers.

Organic sources of trace minerals (TM) in broiler diets are more bioavailable and stable than inorganic sources, making them particularly beneficial during challenging periods such as heat stress (HS) conditions. A 42-d study investigated the effects of using advanced chelate technology-based TM (ACTM) or adding varying amounts of ACTM to broiler diets during HS conditions. The study involved 672 male broiler chickens in 7 treatment groups, including a thermoneutral control (TNC) group and six HS treatments. There were 8 replicate pens per treatment and 12 birds per replicate. The six HS treatments included birds exposed to a cyclic HS environment (34°C) for 8 h and were as follows: HSC, which consisted of the same basal diet with the recommended ITM levels; ACTM50 and ACTM100, which replaced the basal diet with 50% and 100% ACTM instead of ITM; ITM+ACTM12.5 and ITM+ACTM25, which involved adding extra ACTM to the ITM basal diet at 12.5% and 25%, respectively; and ITM125, which used 125% of the recommended levels of ITM in the basal diet. Compared with the HSC treatment, the TNC, ACTM100, and ITM+ACTM25 treatments resulted in increased (P < 0.05) body weight; tibia weight; tibia ash, phosphorus, iron, and manganese contents; secondary antibody titers; and serum TAC and SOD values but decreased (P < 0.05) serum MDA concentrations and the expression levels of the hepatic genes IL-1ß, IL-6, and INF-γ. The TNC and ACTM100 groups also showed greater (P < 0.05) feed efficiency, tibia length, tibia zinc content, and hepatic SOD1 expression but exhibited reduced (P < 0.05) hepatic NF-kB expression. Significant increases (P < 0.05) in primary anti-NDV titers, serum GPx1 activity, and Nrf2 and GPx1 gene expression levels were also detected in the ACTM100, ITM+ACTM12.5, and ITM+ACTM25 groups. In conclusion, the findings suggest that replacing ITM with ACTM or adding ACTM to ITM diets, especially at a 25% higher dose, can effectively protect broilers from heat stress by promoting growth, reducing inflammation, and increasing the expression of antioxidant proteins.

The effect of citric acid on mineralisation and vascular endothelial growth factor secretion from apical papilla stem cells.

OBJECTIVE: To investigate the influence of citric acid on the osteogenic and angiogenic potential of stem cells from apical papillae (SCAPs). MATERIALS AND METHODS: Stem cells from apical papillae were isolated from freshly extracted third permanent molars. These cells were treated with 20 and 100 µM citric acid. Alizarin red staining was used to evaluate mineral deposition. The secreted levels of vascular endothelial growth factor (VEGF) were assessed by ELISA on days 18, 24 and 28. Immunofluorescence analysis was performed to assess the expression of surface markers after exposure to 20 and 100 µM citric acid. RESULTS: Different mineralisation patterns were observed. Supplemented with citric acid, media showed more diffuse and less dense crystals. On day 18, most VEGF was secreted from the cells with no added citric acid. On day 24, there was a significant increase (p < 0.05) in the levels of VEGF secreted from cells treated with 20 µM citric acid. On day 28, cells from the control group did not secrete VEGF. There was a reduction in the levels of VEGF secreted by cells treated with 20 µM citric acid and a significant increase in the cells exposed to 100 µM citric acid (p < 0.05). CONCLUSION: Citric acid can promote the differentiation of SCAPs and angiogenesis.

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model.

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Physiological resilience of intertidal chitons in a persistent upwelling coastal region.

Current climate projections for mid-latitude regions globally indicate an intensification of wind-driven coastal upwelling due to warming conditions. The dynamics of mid-latitude coastal upwelling are marked by environmental variability across temporal scales, which affect key physiological processes in marine calcifying organisms and can impact their large-scale distribution patterns. In this context, marine invertebrates often exhibit phenotypic plasticity, enabling them to adapt to environmental change. In this study, we examined the physiological performance (i.e., metabolism, Thermal Performance Curves, and biomass and calcification rates) of individuals of the intertidal mollusk Chiton granosus, a chiton found from northern Peru to Cape Horn (5° to 55°S). Our spatial study design indicated a pattern of contrasting conditions among locations. The Talcaruca site, characterized by persistent upwelling and serving as a biogeographic break, exhibited lower pH and carbonate saturation states, along with higher pCO2, compared to the sites located to the north and south of this location (Huasco and Los Molles, respectively). In agreement with the spatial pattern in carbonate system parameters, long-term temperature records showed lower temperatures that changed faster over synoptic scales (1-15 days) at Talcaruca, in contrast to the more stable conditions at the sites outside the break. Physiological performance traits from individuals from the Talcaruca population exhibited higher values and more significant variability, along with significantly broader and greater warming tolerance than chitons from the Huasco and Los Molles populations. Moreover, marked changes in local abundance patterns over three years suggested population-level responses to the challenging environmental conditions at the biogeographic break. Thus, C. granosus from the Talcaruca upwelling zone represents a local population with wide tolerance ranges that may be capable of withstanding future upwelling intensification on the Southern Eastern Pacific coast and likely serving as a source of propagules for less adapted populations.

Biology of bone mineralization and ectopic calcifications: the same actors for different plays.

Bone has several crucial functions. It is essential for locomotion and allows our body to stand erect against gravity. A mismatch between the mechanical stresses applied to it and its mechanical resistance leads to fractures. Bone also has numerous endocrine functions. It acts as a reservoir for minerals such as calcium and phosphorus, making it the target of calciotropic hormones that mobilize these minerals, particularly calcium, according to the body's needs. Additionally, bone secretes hormones, notably fibroblast growth factor 23 (FGF23), which regulates urinary excretion of phosphate and the bioavailability of active vitamin D. Bone mineralization is the process that facilitates the organized deposition of minerals in the bone matrix, providing rigidity and appropriate mechanical resistance. This process is compromised in genetically related bone mineralization disorders, such as those causing hypophosphatemia or hypophosphatasia. Conversely, calcification can be pathological, affecting soft tissues like the blood vessels, as seen in generalized arterial calcification of infancy (GACI) or arterial calcification due to CD73 deficiency (ACDC). The aim of this article is to first present the composition and structure of the mineralized bone matrix, to review the current understanding of the molecular mechanisms of mineralization, and finally to discuss the conditions associated with ectopic calcification and the underlying mechanisms.

Increased bone mass but delayed mineralization: in vivo and in vitro study for zoledronate in bone regeneration.

BACKGROUND: Bisphosphonates (BPs) are widely used to inhibit excessive osteoclast activity. However, the potential to compromise bone defect healing has limited their broader application. To better understand the influence of BPs on bone regeneration, we established a bone grafting model with Zoledronate administration, aiming to deepen the understanding of bone remodeling and mineralization processes. METHODS: A bone grafting model was established in the distal femurs of male Sprague-Dawley rats. The experimental group received systemic administration of Zoledronate (ZOL, 0.2 mg/kg, administered twice). Histological analysis and immunohistochemistry (IHC) were employed to assess osteoblastic and macrophage activity, tartrate-resistant acid phosphatase (TRAP) staining was used to evaluate osteoclastogenesis. Mineralization was assessed through Micro-CT analysis, Raman spectroscopy, and back-scatter scanning electron microscopy (BSE-SEM). Additionally, the in vitro effects of ZOL on osteoblast and osteoclast activity were investigated to further elucidate its impact on bone regeneration. RESULTS: In vivo, the ZOL group showed increased bone mass, as observed in histological and radiological assessments. However, Micro-CT, Raman spectroscopy, and BSE-SEM detection revealed lower mineralization levels in ZOL group's regenerated bone. Acid-etched SEM analysis showed abnormal osteocyte characteristics in ZOL-group's regenerated bone. Simultaneously, elevated osteopontin (OPN), F4/80 expression along with reduced TRAP expressing was found in the grafting region of ZOL group. In vitro, ZOL did not negatively impact osteogenetic activity (ALP, BMP4, OCN expression) at the tested concentrations (0.02-0.5 g/ml) but significantly impaired mineralization and inhibited osteoclast formation, even at the lowest concentration. CONCLUSIONS: This study highlights a less recognized negative effect of ZOL on bone mineralization during bone regeneration. More research is needed to elucidate the underlying mechanism.

Genome of Halimeda opuntia reveals differentiation of subgenomes and molecular bases of multinucleation and calcification in algae.

Algae mostly occur either as unicellular (microalgae) or multicellular (macroalgae) species, both being uninucleate. There are important exceptions, however, as some unicellular algae are multinucleate and macroscopic, some of which inhabit tropical seas and contribute to biocalcification and coral reef robustness. The evolutionary mechanisms and ecological significance of multinucleation and associated traits (e.g., rapid wound healing) are poorly understood. Here, we report the genome of Halimeda opuntia, a giant multinucleate unicellular chlorophyte characterized by interutricular calcification. We achieve a high-quality genome assembly that shows segregation into four subgenomes, with evidence for polyploidization concomitant with historical sea level and climate changes. We further find myosin VIII missing in H. opuntia and three other unicellular multinucleate chlorophytes, suggesting a potential mechanism that may underpin multinucleation. Genome analysis provides clues about how the unicellular alga could survive fragmentation and regenerate, as well as potential signatures for extracellular calcification and the coupling of calcification with photosynthesis. In addition, proteomic alkalinity shifts were found to potentially confer plasticity of H. opuntia to ocean acidification (OA). Our study provides crucial genetic information necessary for understanding multinucleation, cell regeneration, plasticity to OA, and different modes of calcification in algae and other organisms, which has important implications in reef conservation and bioengineering.

Widespread scope for coral adaptation under combined ocean warming and acidification.

Reef-building coral populations are at serious risk of collapse due to the combined effects of ocean warming and acidification. Nonetheless, many corals show potential to adapt to the changing ocean conditions. Here we examine the broad sense heritability (H2) of coral calcification rates across an ecologically and phylogenetically diverse sampling of eight of the primary reef-building corals across the Indo-Pacific. We show that all eight species exhibit relatively high heritability of calcification rates under combined warming and acidification (0.23-0.56). Furthermore, tolerance to each factor is positively correlated and the two factors do not interact in most of the species, contrary to the idea of trade-offs between temperature and pH sensitivity, and all eight species can co-evolve tolerance to elevated temperature and reduced pH. Using these values together with historical data, we estimate potential increases in thermal tolerance of 1.0-1.7°C over the next 50 years, depending on species. None of these species are probably capable of keeping up with a high global change scenario and climate change mitigation is essential if reefs are to persist. Such estimates are critical for our understanding of how corals may respond to global change, accurately parametrizing modelled responses, and predicting rapid evolution.

Stanniocalcin 1 and 1,25-dihydroxyvitamin D3 cooperatively regulate bone mineralization by osteoblasts.

Stanniocalcin 1 (STC1) is a calcium- and phosphate-regulating hormone that is expressed in all tissues, including bone tissues, and is involved in calcium and phosphate homeostasis. Previously, STC1 expression was found to be increased by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] administration in renal proximal tubular cells. In this study, we investigated whether STC1 directly regulates osteoblast differentiation or reciprocally controls the effects of 1,25(OH)2D3 on osteoblasts to contribute to bone homeostasis. We found that STC1 inhibited osteoblast differentiation in vitro and bone morphogenetic protein 2 (BMP2)-induced ectopic bone formation in vivo. Moreover, 1,25(OH)2D3 increased STC1 expression through direct binding to the Stc1 promoter of the vitamin D receptor (VDR). STC1 activated the 1,25(OH)2D3-VDR signaling pathway through the upregulation of VDR expression mediated by the inhibition of Akt phosphorylation in osteoblasts. STC1 further increased the effects of 1,25(OH)2D3 on receptor activator of nuclear factor-κB ligand (RANKL) secretion and inhibited osteoblast differentiation by exhibiting a positive correlation with 1,25(OH)2D3. The long-bone phenotype of transgenic mice overexpressing STC1 specifically in osteoblasts was not significantly different from that of wild-type mice. However, compared with that in the wild-type mice, 1,25(OH)2D3 administration significantly decreased bone mass in the STC1 transgenic mice. Collectively, these results suggest that STC1 negatively regulates osteoblast differentiation and bone formation; however, the inhibitory effect of STC1 on osteoblasts is transient and can be reversed under normal conditions. Nevertheless, the synergistic effect of STC1 and 1,25(OH)2D3 through 1,25(OH)2D3 administration may reduce bone mass by inhibiting osteoblast differentiation.

Modulation of osteoblastogenesis by NRF2: NRF2 activation suppresses osteogenic differentiation and enhances mineralization in human bone marrow-derived mesenchymal stromal cells.

Mesenchymal stromal stem cells (MSCs) or skeletal stem cells (SSCs) play a major role in tissue repair due to their robust ability to differentiate into osteoblasts, chondrocytes, and adipocytes. Complex cell signaling cascades tightly regulate this differentiation. In osteogenic differentiation, Runt-related transcription factor 2 (RUNX2) and ALP activity are essential. Furthermore, during the latter stages of osteogenic differentiation, mineral formation mediated by the osteoblast occurs with the secretion of a collagenous extracellular matrix and calcium deposition. Activation of nuclear factor erythroid 2-related factor 2 (NRF2), an important transcription factor against oxidative stress, inhibits osteogenic differentiation and mineralization via modulation of RUNX2 function; however, the exact role of NRF2 in osteoblastogenesis remains unclear. Here, we demonstrate that NRF2 activation in human bone marrow-derived stromal cells (HBMSCs) suppressed osteogenic differentiation. NRF2 activation increased the expression of STRO-1 and KITLG (stem cell markers), indicating NRF2 protects HBMSCs stemness against osteogenic differentiation. In contrast, NRF2 activation enhanced mineralization, which is typically linked to osteogenic differentiation. We determined that these divergent results were due in part to the modulation of cellular calcium flux genes by NRF2 activation. The current findings demonstrate a dual role for NRF2 as a HBMSC maintenance factor as well as a central factor in mineralization, with implications therein for elucidation of bone formation and cellular Ca2+ kinetics, dystrophic calcification and, potentially, application in the modulation of bone formation.

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.

Effects of boric acid combined with injectable platelet rich fibrin on the mineralized nodule formation and the viability of human dental pulp stem cells.

BACKGROUND: The present study aimed to evaluate the viability of human dental pulp stem cells (hDPSCs) exposed to boric acid (BA) and injectable platelet-rich fibrin (I-PRF). MATERIALS AND METHODS: hDPSCs were isolated from impacted third molars. Nine milliliters of whole blood was transferred to I-PRF tubes and centrifuged at 700 rpm for 3 minutes. A BA solution was prepared by dissolving BA in a 0.1 g/ml stock solution. The cells were divided into four groups: control, I-PRF, BA, and BA + I-PRF. Cell viability was evaluated using flow cytometry. Mineralized calcium nodules were observed using Alizarin Red staining. The data were analyzed using two-way analysis of variance and Tukey's HSD test (p<0.05). RESULTS: The highest percentage of viable cells was in the I-PRF group, and the lowest percentage of viable cells was in the BA group at all times. Larger calcium nodules were observed in the BA group compared to the other groups. CONCLUSION: The use of I-PRF with or without BA had a positive effect on cell viability. BA and I-PRF affected the formation of mineralized calcium nodules. I-PRF and BA may be used in combination because these substances minimally reduce cell viability and promote mineralized nodule formation.

Zinc phosphate glass microspheres promoted mineralization and expression of BMP2 in MC3T3-E1 cells.

Degradable phosphate glasses have shown favorable properties for tissue engineering. By changing the composition of the glasses, the degradation rate, and ion release are controllable. Zinc oxide can function as a glass network modifier and has been shown to play a positive role in bone formation. Also, phosphate glasses can easily be processed into microspheres, which can be used as microcarriers. This study aims to develop zinc phosphate glasses microspheres and explore the optimized size and composition for applications in bone tissue engineering. Zinc-titanium-calcium-sodium phosphate glasses with 0, 1, 3, 5, or 10 mol % zinc oxide were prepared and processed into microspheres. The smaller microspheres ranged in size from 50 to 106 µm, while the larger ones ranged from 106 to 150 µm. The characteristics of glasses were examined. The osteoblastic cell line MC3T3-E1 was cultured on the surface of microspheres and the cell viability was examined. To evaluate osteogenic differentiation, Alizarin Red S staining, quantitative reverse transcription polymerase chain reaction, and western blot analysis were performed after 14 days. Different sizes of zinc phosphate glass microspheres were successfully made. The glass microspheres with <10 mol % zinc oxide were able to support the adhesion and proliferation of MC3T3-E1 cell lines. The relative gene expression of BMP2 was significantly upregulated in the smaller glass microspheres containing 3 mol % zinc oxide (26-fold, p < .001) and both sizes of microspheres containing 5 mol % zinc oxide (smaller: 27-fold, p < .001; larger: 35-fold, p < .001). Additionally, cluster formation was observed in glass microspheres after 14 days, and the mineralization of MC3T3-E1 cell lines was promoted. Based on these findings, the glass microspheres containing 3-5 mol % of zinc oxide can promote osteogenic differentiation for MC3T3-E1 cells.

A persistent mineralization process in alveolar bone throughout the postnatal growth stage in rats.

OBJECTIVE: Alveolar bone quality is essential for the maxillofacial integrity and function, and depends on alveolar bone mineralization. This study aims to investigate the in vivo changes in alveolar bone mineralization, from the perspective of mineral deposition and crystal transition in postnatal rats. DESIGN: Nine postnatal time points of Wistar rats, ranging from day 1 to 56, were set to obtain the maxillary alveolar bone samples. Each time point consisted of ninety rats, with 45 females and 45 males. Macromorphology of alveolar bone was reconducted by Micro-Computed Tomography and the mineral content was quantified via Thermogravimetric analysis, Scanning Electron Microscope, High-Resolution Transmission Electron Microscopy and vibrational spectroscopy. Furthermore, the crystallinity and composition were characterized by vibrational spectroscopy, X-ray Diffraction, X-ray Photoelectron Spectroscopy and Selected Area Electron Diffraction. RESULTS: The progressive increase of mineral deposition was accompanied by substantial growth in alveolar bone mass and volume in postnatal rats. Whereas the mineral percentage initially decreased and then increased, reaching a nadir on postnatal day 14 (P14) when tooth eruption was first observed. Besides, localized mineralization was initiated by the formation of amorphous precursors and then converted into mineral crystals, while there was no statistically significant change in the average crystallinity of the bone during growth. CONCLUSION: Mineralization of alveolar bone is ongoing throughout the early growth in postnatal rats. Mineral deposition increases with age, whereas the crystallinity remains stable within a certain range. Besides, the mineral percentage reaches its lowest point on P14, which may be attributed to tooth eruption.

Development of the mineralisation of individual bones and bone regions in replacement gilts according to dietary calcium and phosphorus.

Skeleton bones, distinguished by trabecular and cortical bone tissue content, exhibit varied growth and composition, in response to modified dietary calcium and phosphorus levels. The study investigated how gilts adapt their individual bone and bone region mineralisation kinetics in response to changing intake of Ca and P. A total of 24 gilts were fed according to a two-phase (Depletion (D) 60-95 and Repletion (R) 95-140 kg BW, respectively). During the D phase, gilts were fed either 60% (D60) or 100% (D100) of the estimated P requirement. Subsequently, during the R phase, half of the gilts from each D diet were fed either 100% (R100) or 160% (R160) of the estimated P requirement according to a 2 × 2 factorial arrangement. Bone mineral content (BMC) was assessed in the whole body, individual bones (femur and lumbar spine L2-L4), and bone regions (head, front legs, trunk, pelvis, femur, and hind legs) every 2 weeks using dual-energy X-ray absorptiometry (DXA). At 95 kg BW, gilts fed D60 showed reduced BMC and BMC/BW ratio in all studied sites compared to those fed D100 (P < 0.001). During the depletion phase, the allometric BW-dependent regressions slopes for BMC of D100 gilts remained close to 1 for all sites and did not differ from each other. In contrast, the slopes were lower in D60 gilts (P < 0.05), with an 18% reduction in the whole body, except for the front and hind legs, femur, and pelvis, which exhibited higher reductions (P < 0.05). At 140 kg BW, BMC and BMC/BW ratio of all studied sites were similar in gilts previously fed D60 and D100, but higher in R160 than in R100 gilts (P < 0.05), except for front and hind legs. During the repletion phase, the allometric BW dependent regressions slopes for BMC were lower (P < 0.05) in R100 than in R160 gilts (for whole body -10%; P < 0.01) except for front and hind legs, femur, and pelvis. In conclusion, bone demineralisation and recovery followed similar trends for all measured body sites. However, the lumbar spine region was most sensitive whereas the hind legs were least sensitive. These data suggest that using bone regions such as the head and forelegs that can be collected easily at the slaughterhouse may be a viable alternative to whole body DXA measurement.

Biocalcification in porcelaneous foraminifera.

Living organisms control the formation of mineral skeletons and other structures through biomineralization. Major phylogenetic groups usually consistently follow a single biomineralization pathway. Foraminifera, which are very efficient marine calcifiers, making a substantial contribution to global carbonate production and global carbon sequestration, are regarded as an exception. This phylum has been commonly thought to follow two contrasting models of either in situ 'mineralization of extracellular matrix' attributed to hyaline rotaliid shells, or 'mineralization within intracellular vesicles' attributed to porcelaneous miliolid shells. Our previous results on rotaliids along with those on miliolids in this paper question such a wide divergence of biomineralization pathways within the same phylum of Foraminifera. We have found under a high-resolution scanning electron microscopy (SEM) that precipitation of high-Mg calcitic mesocrystals in porcelaneous shells takes place in situ and form a dense, chaotic meshwork of needle-like crystallites. We have not observed calcified needles that already precipitated in the transported vesicles, what challenges the previous model of miliolid mineralization. Hence, Foraminifera probably utilize less divergent calcification pathways, following the recently discovered biomineralization principles. Mesocrystalline chamber walls in both models are therefore most likely created by intravesicular accumulation of pre-formed liquid amorphous mineral phase deposited and crystallized within the extracellular organic matrix enclosed in a biologically controlled privileged space by active pseudopodial structures. Both calcification pathways evolved independently in the Paleozoic and are well conserved in two clades that represent different chamber formation modes.

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.

Mitigating lipopolysaccharide-induced impairment in human dental pulp stem cells with tideglusib-doped nanoparticles: Enhancing osteogenic differentiation and mineralization.

OBJECTIVE: Drug-loaded non-resorbable polymeric nanoparticles (NPs) are proposed as an adjunctive treatment for pulp regenerative strategies. The present in vitro investigation aimed to evaluate the effectiveness of tideglusib-doped nanoparticles (TDg-NPs) in mitigating the adverse effects of bacterial lipopolysaccharide endotoxin (LPS) on the viability, morphology, migration, differentiation and mineralization potential of human dental pulp stem cells (hDPSCs). METHODS: Cell viability, proliferation, and differentiation were assessed using a MTT assay, cell migration evaluation, cell cytoskeleton staining analysis, Alizarin Red S staining and expression of the odontogenic related genes by a real-time quantitative polymerase chain reaction (RT-qPCR) were also performed. Cells were tested both with and without stimulation with LPS at various time points. One-way ANOVA and Tukey's test were employed for statistical analysis (p < 0.05). RESULTS: Adequate cell viability was encountered in all groups and at every tested time point (24, 48, 72 and 168 h), without differences among the groups (p > 0.05). The analysis of cell cytoskeleton showed nuclear alteration in cultures with undoped NPs after LPS stimulation. These cells exhibited an in blue diffuse and multifocal appearance. Some nuclei looked fragmented and condensed. hDPSCs after LPS stimulation but in the presence of TDg-NPs exhibited less nuclei changes. LPS induced down-regulation of Alkaline phosphatase, Osteonectin and Collagen1 gene markers, after 21d. LPS half-reduced the cells production of calcium deposits in all groups (p < 0.05), except in the group with TDg-NPs (decrease about 10 %). SIGNIFICANCE: LPS induced lower mineral deposition and cytoskeletal disorganization in hDPSCs. These effects were counteracted by TDg-NPs, enhancing osteogenic differentiation and mineralization.

LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response.

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells.

ß-glycerophosphate, not low magnitude fluid shear stress, increases osteocytogenesis in the osteoblast-to-osteocyte cell line IDG-SW3.

AIM: As osteoblasts deposit a mineralized collagen network, a subpopulation of these cells differentiates into osteocytes. Biochemical and mechanical stimuli, particularly fluid shear stress (FSS), are thought to regulate this, but their relative influence remains unclear. Here, we assess both biochemical and mechanical stimuli on long-term bone formation and osteocytogenesis using the osteoblast-osteocyte cell line IDG-SW3. METHODS: Due to the relative novelty and uncommon culture conditions of IDG-SW3 versus other osteoblast-lineage cell lines, effects of temperature and media formulation on matrix deposition and osteocytogenesis were initially characterized. Subsequently, the relative influence of biochemical (ß-glycerophosphate (ßGP) and ascorbic acid 2-phosphate (AA2P)) and mechanical stimulation on osteocytogenesis was compared, with intermittent application of low magnitude FSS generated by see-saw rocker. RESULTS: ßGP and AA2P supplementation were required for mineralization and osteocytogenesis, with 33°C cultures retaining a more osteoblastic phenotype and 37°C cultures undergoing significantly higher osteocytogenesis. ßGP concentration positively correlated with calcium deposition, whilst AA2P stimulated alkaline phosphatase (ALP) activity and collagen deposition. We demonstrate that increasing ßGP concentration also significantly enhances osteocytogenesis as quantified by the expression of green fluorescent protein linked to Dmp1. Intermittent FSS (~0.06 Pa) rocker had no effect on osteocytogenesis and matrix deposition. CONCLUSIONS: This work demonstrates the suitability and ease with which IDG-SW3 can be utilized in osteocytogenesis studies. IDG-SW3 mineralization was only mediated through biochemical stimuli with no detectable effect of low magnitude FSS. Osteocytogenesis of IDG-SW3 primarily occurred in mineralized areas, further demonstrating the role mineralization of the bone extracellular matrix has in osteocyte differentiation.