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Osteoblast-derived extracellular vesicles (EVs) have demonstrated therapeutic utility for bone repair as transporters of key biomolecules capable of accelerating biomineralisation and tissue repair. The clinical translation of these biologically derived nanoparticles, however remains limited due to scalability, heterogeneity and standardisation issues. Herein we investigate the generation of nanovesicles (NVs) from mineralising osteoblasts by extrusion directly compared against natural EV counterparts from the same parental cells. Mineralising osteoblast-derived EVs (MO-EVs) were isolated via ultracentrifugation from cell culture media. The parental osteoblasts were then processed via serial extrusion to <200 nm. EVs and NVs were characterised by comparing their size, concentration and morphology. The presence of tetraspanin markers was detected by Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS). Osteoblasts viability and metabolic activity was assessed after both EV and NV-treatment before comparing their mineralising potency via alizarin red staining. EVs and NVs exhibited similar diameters of approximately 100 nm with a vesicular morphology. EVs were found to be richer in proteins and exhibited a significantly more negative ζ-potential compared to NVs. SP-IRIS analysis confirmed the presence of CD9, CD63 and CD81 in EVs. At both 1 and 10 μg/mL, EVs and NVs reduced the metabolic activity of osteoblasts, however, this was not associated with any cytotoxic effects. The biomineralisation study performed using osteoblasts showed that only EVs significantly increased mineral deposition (p < 0.05) compared to untreated control. In this study, we have established for the first time the biofabrication of cell-derived nanovesicles as a promising alternative to extracellular vesicles derived from mineralising osteoblasts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetes mellitus has been widely acknowledged to have a negative effect on the osteoblastic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). However, the underlying epigenetic mechanisms associated with this process remain to be elucidated. The goal of the present study was to investigate the effect of diabetes mellitus on the osteoblastic differentiation of BMSCs and assess the role of histone methylation in the observed phenomena. The osteoblastic differentiation ability of BMSCs was shown to be decreased in diabetes mellitus, as indicated by alkaline phosphatase activity and the mRNA levels of osteoblast-related genes. Furthermore, diabetes mellitus caused an increased expression of the histone methylase EZH2 and the levels of H3K27Me3 and decreased the expression of the histone demethylase KDM6B, as demonstrated by qRT-PCR and western blotting. Furthermore, immunofluorescence staining suggested that both EZH2 and H3K27Me3 were primarily localized in the nucleus. In addition, chromatin immunoprecipitation assays indicated an increased presence of H3K27Me3 on the promoter region of the BMP4 gene. In summary, in the present study, we demonstrated that the osteoblastic differentiation of BMSCs is dramatically reduced in diabetes mellitus. In addition, upregulation of EZH2 expression and downregulation of KDM6B expression may not be enough to eliminate transcriptional repression mediated by H3K27Me3 on the promoter region of the BMP4 gene during the osteoblastic differentiation of BMSCs in diabetes mellitus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: In clinical practice, alterations in the internal environment of type 2 diabetes can significantly affect bone quality. While the increased risk of fractures among diabetic patients is well-established, the precise mechanisms by which hyperglycemia influences bone quality remain largely unclear., Methods: Western blotting, immunohistochemistry (IHC), and micro-CT were used to examine ferroptosis-related protein expression and bone morphology changes in the bone tissues of type 2 diabetic mice. The CCK8 assay determined the optimal conditions for inducing ferroptosis in osteoblasts by high glucose and high fat (HGHF). Ferroptosis phenotypes in osteoblasts were analyzed using flow cytometry, Western blotting, and two-photon laser confocal microscopy. Transcriptomic sequencing of the control and HGHF groups, followed by bioinformatic analysis, identified and validated key genes. TIMP1 was knocked down in osteoblasts to assess its impact on ferroptosis, while TFRC expression was inhibited and activated to verify the role of TIMP1 in regulating ferroptosis through TFRC. The therapeutic effect of TIMP1 inhibition on osteoporosis was evaluated in a type 2 diabetic mouse model., Results: The expression of TIMP1 is increased in type 2 diabetic osteoporosis. In vitro, TIMP1 knockout inhibited ferroptosis in osteoblasts induced by high glucose and high fat (HGHF). However, overexpression of TFRC reversed the ferroptosis inhibition caused by TIMP1 knockout. Suppression of TIMP1 expression alleviated the progression of osteoporosis in type 2 diabetic mice. Mechanistic studies suggest that TIMP1 regulates HGHF-induced ferroptosis in osteoblasts through TFRC., Conclusion: This study demonstrates that TIMP1 expression is increased during type 2 diabetic osteoporosis and that TIMP1 promotes ferroptosis in osteoblasts by regulating TFRC. These findings suggest that TIMP1 is a promising novel therapeutic target for type 2 diabetic osteoporosis., Competing Interests: Declarations. Ethical approval and consent to participate: The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of The Second Hospital of Lanzhou University (D2023-117). This study did not involve human subjects. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Understanding the intricate cellular interactions involved in bone restoration is crucial for developing effective strategies to promote bone healing and mitigate conditions such as osteoporosis and fractures. Here, we provide compelling evidence supporting the anabolic effects of a pharmacological Pyk2 inhibitor (Pyk2-Inh) in promoting bone restoration. In vitro, Pyk2 signaling inhibition markedly enhances alkaline phosphatase (ALP) activity, a hallmark of osteoblast differentiation, through activation of canonical Wnt/β-catenin signaling. Notably, analysis of human mesenchymal stem cells through RNA-seq revealed a novel candidate, SCARA5, identified through Pyk2-Inh treatment. We demonstrate that Scara5 plays a crucial role in suppressing the differentiation from stromal cells into adipocytes, and accelerates lineage commitment to osteoblasts, establishing Scara5 as a negative regulator of bone formation. Additionally, Pyk2 inhibition significantly impedes osteoclast differentiation and bone resorption. In a co-culture system comprising osteoblasts and osteoclasts, Pyk2-Inh effectively suppressed osteoclast differentiation, accompanied by a substantial increase in the transcriptional expression of Tnfrsf11b and Csf1 in osteoblasts, highlighting a dual regulatory role in osteoblast-osteoclast crosstalk. In an ovariectomized mouse model of osteoporosis, oral administration of Pyk2-Inh significantly increased bone mass by simultaneously reducing bone resorption, promoting bone formation and decreasing bone marrow fat. These results suggest Pyk2 as a potential therapeutic target for both adipogenesis and osteogenesis in bone marrow. Our findings underscore the importance of Pyk2 signaling inhibition as a key regulator of bone remodeling, offering promising prospects for the development of novel osteoporosis therapies., Competing Interests: Declarations. Ethics approval and consent to participate: Experiments using mice were approved by the Institutional animal care and use committee of Hokkaido University, Japan and were performed in accordance with the committee’s guiding principle (approval no.: 21-0018). Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Naringenin (4,5,7-trihydroxyflavone, NAR) is an effective active ingredient in Rhizoma Drynariae , which has many biological functions, encompassing anti-inflammatory and -oxidant functions. Prior research has shown that NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes possessed a significant contribution to osteoporosis. However, the NAR impact on bone loss caused by microgravity remains unclear. Classical microgravity simulation methods were used to induce simulated microgravity (SMG) in mice and cells. Microcomputed tomography, immunohistochemical examination, and hematoxylin and eosin staining were implemented to ascertain alterations in bone microstructure and morphology in mice subsequent to NAR gavage. Cellular investigations were implemented encompassing quantitative real-time polymerase chain reaction, Western blotting, and immunofluorescence labeling to investigate the molecular mechanism behind NAR resistance to microgravity-induced bone loss. Our research has shown that NAR can significantly enhance the SMG-stimulated alterations in bone microstructure and morphology in mice, mainly by increasing the trabecular thickness, bone volume fraction, and trabecular number while increasing the bone trabecula number. Cell experiments also showed that SMG caused the activation of inflammatory corpuscles of NLRP3 and induced pyroptosis simultaneously, which can be confirmed by the upregulation of protein and mRNA expression levels such as those of NLRP3, cleaved caspase-1, gasdermin D, and apoptosis-associated speck-like protein. The occurrence of pyroptosis further led to the disorder of osteogenic differentiation, which showed that the osteopontin, Runt-related transcription factor 2, bone morphogenetic protein 2, and alkaline phosphatase expression levels were decreased. The intervention of NAR can activate the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway, reverse this phenomenon via controlling the reactive oxygen species generation in cells and correcting mitochondrial malfunction, weaken the pyroptosis of osteoblasts (OBs), and promote osteogenic differentiation. In summary, NAR could hinder the pyroptosis of OBs caused by SMG and promote osteogenic differentiation via activating the Nrf2/HO-1 pathway. This provides a unique view for inhibiting bone loss under weightlessness and confirms the NAR capacity in treating microgravity-stimulated bone loss, giving new ideas and methods for future space medicine development.

Staphylococcus aureus main internalization mechanism in osteoblasts relies on a tripartite interaction between bacterial fibronectin-binding proteins, extracellular matrix soluble fibronectin, and osteoblasts' β1 integrins. Caveolins, and particularly caveolin-1, have been shown to limit the plasma membrane microdomain mobility, and consequently reduce the uptake of S. aureus in keratinocytes. In this study, we aimed to deepen our understanding of the molecular mechanisms underlying S. aureus internalization in osteoblasts. Mechanistically, S. aureus internalization requires endosomal recycling of β1 integrins as well as downstream effectors such as Src, Rac1, and PAK1. Surprisingly, in β1 integrin deficient osteoblasts, S. aureus internalization is restored when Caveolin-1 is absent and requires αvβ3/5 integrins as backup fibronectin receptors. Altogether, our data support that β1 integrins regulate the level of detergent-resistant membrane at the plasma membrane in a an endosomal and Caveolin-1 dependent manner., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Objective: In this experiment, we screened key miRNAs involved in the dexamethasone-induced decrease in osteogenic capacity of mouse precursor osteoblasts MC3T3-E1 over and investigated their specific regulatory mechanisms., Methods: In this experiment, cell counting kit assay was utilized to act on MC3T3-E1 cells at 0, 5μM, 10μM, 15μM concentrations of dexamethasone for 24h, 48h and 72h to observe the changes in cell viability in order to select the appropriate dexamethasone concentration. Apoptosis and reactive oxygen species were detected by flow cytometry. The transcription of osteogenesis-related genes (Runx2, ALP, OCN, OPN, OPG, COL1A1) and protein expression levels (Runx2, ALP, OCN, OPN) were detected by Western Blot and qRT-PCR to validate the changes in cellular osteogenesis. The differentially expressed miRNAs related to MC3T3-E1 osteogenic differentiation after dexamethasone action were screened out. The expression levels of selected target miRNAs were verified in the experimental group and the control group by qRT-PCR. The miRNA inhibitor was transfected to knock down miRNA in dexamethasone-induced MC3T3-E1 injury. Alkaline phosphatase staining and flow cytometry were performed to detect apoptosis and reactive oxygen species changes. transcript and protein expression levels of osteogenesis-related genes in mouse MC3T3-E1 were detected by qRT-PCR and Western blot experiments. By miRNA target gene prediction, luciferase reporter gene experiments, qRT-PCR and Western blot experiments were used to verify whether the selected target miRNAs targeted the target gene., Results: First, it was determined that 10μM dexamethasone solution was effective in inducing a decrease in osteogenic function in mouse MC3T3-E1 by CCK8 experiments, which showed a significant decrease in alkaline phosphatase activity, a decrease in calcium nodules as shown by alizarin red staining, an increase in apoptosis and reactive oxygen species as detected by flow cytometry, as well as a decrease in the expression of osteogenesis-related genes and proteins. Five target miRNAs were identified: miR-706, miR-296-3p, miR-7011-5p, miR-145a-3p, and miR-149-3p. miR-145a-3p, which had the most pronounced and stable expression trend and was the most highly expressed miRNA, was chosen as the target of this experiment by qRT-PCR analysis. -145a-3p, as the subject of this experiment. Knockdown of miR-145a-3p in MC3T3-E1 cells after dexamethasone action significantly improved the expression of their impaired osteogenic indicators. It was shown that after knocking down the target miRNA, alkaline phosphatase staining was significantly increased compared with the dexamethasone-stimulated group and approached the level of the blank control group. Meanwhile, the expression of osteogenic function-related proteins and genes also increased in the dexamethasone-stimulated group after knocking down miR-145a-3p, and approached the level of the blank control group. A direct targeting relationship between miR-145a-3p and Runx2 was indeed confirmed by luciferase reporter gene assays, qRT-PCR and Western blot experiments., Conclusions: The results indicated that dexamethasone impaired the osteogenic differentiation ability of MC3T3-E1 cells by inducing the up-regulation of miR-145a-3p expression. MiR-145a-3p inhibited the osteogenic differentiation ability of MC3T3-E1 cells by targeting and suppressing the expression level of Runx2 protein. Inhibition of miR-145a-3p levels significantly improved the osteogenic differentiation ability of MC3T3-E1 cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Osteoporotic fragility fractures substantially impact aging societies, necessitating long-term care and increasing healthcare costs. Myokine irisin, secreted by skeletal muscle, influences bone metabolism; however, a comprehensive understanding of the mechanisms by which irisin affects bone metabolism is still lacking. Therefore, this study aimed to explore the effects of irisin on osteogenesis and osteoblast differentiation triggered by bone morphogenetic protein-2 (BMP-2). We used 4-week-old male ICR mice and implanted polyethylene glycol pellets containing recombinant human BMP-2 (rh-BMP-2) into the left dorsal muscle pouch. Mice received weekly intraperitoneal injections of either phosphate-buffered saline or recombinant irisin (re-irisin). Ectopic bone formation was evaluated 3 weeks post-surgery using micro-computed tomography (μ-CT) and histological analysis. In vitro experiments, C2C12 cells were treated with or without rh-BMP-2 and re-irisin, and we assessed osteoblast differentiation markers, e.g., runt-related transcription factor 2, alkaline phosphatase, osteocalcin, and osteopontin, using real-time reverse transcription-polymerase chain reaction. The μ-CT analyses showed that re-irisin significantly increased bone mineral content and bone volume of ectopic bones newly formed by rh-BMP-2. The gene expressions of the osteoblast markers were significantly increased by rh-BMP-2 and further upregulated by re-irisin. The treatment of cyclic AMP response element-binding protein (CREB) small interfering RNA attenuated these effects, suggesting that CREB signaling pathway was involved in rh-BMP-2/re-irisin-induced osteoblastic differentiation. This study demonstrates the potential of irisin to enhance osteogenesis through BMP signaling, offering insights for osteoporosis treatment and highlighting irisin as a promising therapeutic target for improving bone health and extending a healthy lifespan., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hydroxyapatite (HA) granules are frequently used in orthopedics and maxillofacial surgeries to fill bone defects and stimulate the regeneration process. Optimal HA granules should have high biocompatibility, high microporosity and/or mesoporosity, and high specific surface area (SSA), which are essential for their bioabsorbability, high bioactivity (ability to form apatite layer on their surfaces) and good osseointegration with the host tissue. Commercially available HA granules that are sintered at high temperatures (≥ 900 °C) are biocompatible but show low porosity and SSA (2-5 m 2 /g), reduced bioactivity, poor solubility and thereby, low bioabsorbability. HA granules of high microporosity and SSA can be produced by applying low sintering temperatures (below 900 °C). Nevertheless, although HA sintered at low temperatures shows significantly higher SSA (10-60 m 2 /g) and improved bioabsorbability, it also exhibits high ion reactivity and cytotoxicity under in vitro conditions. The latter is due to the presence of reaction by-products. Thus, the aim of this study was to fabricate novel biomaterials in the form of granules, composed of hydroxyapatite nanopowder sintered at a high temperature (1100 °C) and a biopolymer matrix: chitosan/agarose or chitosan/β-1,3-glucan (curdlan). It was hypothesized that appropriately selected ingredients would ensure high biocompatibility and microstructural properties comparable to HA sintered at low temperatures. Synthesized granules were subjected to the evaluation of their biological, microstructural, physicochemical, and mechanical properties. The obtained results showed that the developed nanocomposite granules were characterized by a lack of cytotoxicity towards both mouse preosteoblasts and normal human fetal osteoblasts, and supported cell adhesion to their surface. Moreover, produced biomaterials had the ability to induce precipitation of apatite crystals after immersion in simulated body fluid, which, combined with high biocompatibility, should ensure good osseointegration after implantation. Additionally, nanocomposite granules possessed microstructural parameters similar to HA sintered at a low temperature (porosity approx. 50%, SSA approx. 30 m²/g), Young's modulus (5-8 GPa) comparable to cancellous bone, and high fluid absorption capacity. Moreover, the nanocomposites were prone to biodegradation under the influence of enzymatic solution and in an acidic environment. Additionally, it was noted that the hydroxyapatite nanoparticles remaining after the physicochemical dissolution of the biomaterial were easily phagocytosed by mouse macrophages, mouse preosteoblasts, and normal human fetal osteoblasts (in vitro studies). The obtained materials show great potential as bone tissue implantation biomaterials with improved bioresorbability. The obtained materials show great potential as bone tissue implantation biomaterials with improved bioresorbability., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethics approval and consent to participate Informed consent was obtained from the volunteer to use his/her blood in the research and the study adhered to the tenets of the Declaration of Helsinki. Approval from the Bioethics Committee of the Medical University of Lublin for the use of human whole blood, plasma, and their components was obtained (no. KE-0254/187/10/2022, 06 Oct 2022)., (© 2024. The Author(s).)

Skeletal muscle and bone are the major organs for physical activity, in which there is a parallel correlation between muscle mass and bone density throughout a lifetime. Osteoporosis is a systemic bone metabolic disorder caused by reduced bone formation and increased bone resorption. Based on the metabolic symbiosis relationship between skeletal muscle and bone, we hypothesis that skeletal muscle secretory factors could play constructive roles in osteoporosis. Exosomes have been verified to transfer bioactive factors among cells. However, the role of skeletal muscle derived-exosomes (SM-Exos) in osteoporosis is still unclear. In this study, we performed neuromuscular electrical stimulation (NMES) intervention on denervated skeletal muscles and subsequently extracted exosomes (DN + ES-Exo) from the skeletal muscles, and then injected these DN + ES-Exo into sarco-osteoporotic rats through tail vein. In vitro studies, we cocultured SM-Exos from different states with differentiated MC3T3-E1 osteoblasts. In brief, our research findings demonstrate that SM-Exos could partially promote osteogenesis both in vivo and in vitro. Further, our findings indicate that skeletal muscle contraction induced by NMES can reverse the incidence of sarco-osteoporosis to a certain degree, and DN + ES-Exo contributes to the improvement in osteoporosis by facilitating osteoblast differentiation. Then, we revealed that NMES might regulate several miRNAs in skeletal muscle, the miRNAs that are encapsulated by SM-Exos might be involved in osteogenic differentiation in a network manner. All in all, this study confirmed the effect of NMES on sarco-osteoporosis and explored the role of SM-Exos in the improvement of osteoporosis, which provide an effective theoretical support for the physical therapy of clinical sarco-osteoporosis., Competing Interests: Declaration of competing interest This study was funded by the National Natural Science Foundation of China (No. 31871207) and supported by the Fundamental Research Funds for the Central Universities in UIBE., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hyperactive osteoclasts and hypoactive osteoblasts usually result in osteolytic conditions such as estrogen-deficiency bone loss. Few natural compounds that both attenuating bone resorption and enhancing bone formation could exert effects on this imbalance. 5-Deoxycajanin (5-D), an isoflavonoid extracted from Cajan leaf with estrogen-like properties, were found to have beneficial pharmacological effects on rebalancing the activities of osteoclasts and osteoblasts. This study revealed that 5-D at the same concentration could inhibit osteoclastogenesis of BMMs and promoted osteoblast differentiation of BMSCs. 5-D not only attenuated the fluorescent formation of RANKL-induced F-actin belts and NFATc1, but also activated ALP and RUNX2 expressions. As to downstream factor expressions, 5-D could block osteoclast-specific genes and proteins including NFATc1 and CTSK, while increased osteogenic genes and proteins including OPG and OCN, as confirmed by Real-time PCR and Western Blotting. Additionally, the network pharmacology and molecular docking identified the involvement of 5-D in the MIF and MAPK signaling pathways and the stable binding between 5-D and MAPK2K1. Further Western blot studies showed that 5-D decreased the phosphorylation of p38 and ERK in osteoclasts, but promoted these phosphorylations in osteoblasts. In a female C57BL/6J mouse model of estrogen deficiency-induced bone loss, 5-D demonstrated efficacy in enhancing BMD through attenuating osteoclast activities and promoting osteogenesis. These results underscore the potential application of 5-D on treating osteolysis resulting from hyperactive osteoclasts and hypoactive osteoblasts, shedding light on modulating osteoclast-osteoblast homeostasis., Competing Interests: Declaration of competing interest The authors declare no known competing financial interests or personal relationships that could influence this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Melatonin (MEL) has shown positive effects in anti-inflammatory and anti-oxidative stress research. This study investigates whether MEL can positively impact bone loss induced by valproic acid (VPA) in rats. The study examines changes in MC3T3-E1 cell viability and osteogenic potential, along with osteoclast differentiation in RAW264.7 cells in the presence of VPA using CCK-8, ALP staining, AR staining, and TRAP staining. In vitro experiments reveal that VPA-induced inhibition of osteogenic differentiation and promotion of osteoclastic differentiation are linked to increased inflammation and oxidative stress. Furthermore, MEL has demonstrated the ability to reduce oxidative stress and inflammation, boost osteogenic differentiation, and inhibit osteoclast differentiation. Animal experiments confirm that MEL significantly increases SOD2 expression and decreases TNF-α expression, leading to the restoration of impaired bone metabolism, enhanced bone strength, and higher bone mineral density. The combined experimental results strongly suggest that MEL can enhance osteogenic activity in the presence of VPA by reducing inflammation and oxidative stress, impeding osteoclast differentiation, and alleviating bone loss in VPA-treated rat models., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The accumulating evidence has made it clear that iron overload is a crucial mechanism in bone loss. Protocatechualdehyde (PCA) has also been used to prevent osteoporosis in recent years. Whether PCA can reverse the harmful effects of iron overload on bone mass in aged rats is still unknown. Therefore, this study aimed to assess the role of PCA in iron overload-induced bone loss in senile rats. In the aged rat model, we observed that iron overload affects bone metabolism and bone remodeling, manifested by bone loss and decreased bone mineral density. The administration of PCA effectively mitigated the detrimental effects caused by iron overload, and concomitant reduction in MDA serum levels and elevation of SOD were noted. In addition, PCA-treated rats were observed to have significantly increased bone mass and elevated expression of SIRT3，BMP2，SOD2 and reduced expression of TNF-α in bone tissue. We also observed that PCA was able to reduce oxidative stress and inflammation and restore the imbalance in bone metabolism. When MC3T3-E1 and RAW264.7 cells induced osteoblast and osteoclasts differentiation, PCA intervention could significantly recover the restriction of osteogenic differentiation and up-regulation of osteoclast differentiation treated by iron overload. Further, by detecting changes in ROS, SOD, MDA, expression of SIRT3 and mitochondrial membrane potentials, we confirm that the damage caused to cells by iron overload is associated with decreased SIRT3 activity, and that 3-TYP have similar effects on oxidative stress caused by FAC. In conclusion, PCA can resist iron overload-induced bone damage by improving SIRT3 activity, anti-inflammatory and anti-oxidative stress., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Osteoporosis (OP) is a metabolic bone disease characterized by progressive decline of bone mass and bone quality, leading to bone fragility and an increased risk of fracture. The osteogenic differentiation of bone mesenchymal stem cells (BMSCs) is crucial to maintain the balance of osteoblast and osteoclast. Bioinformatics prediction indicates that ZFP36 ring finger protein (ZFP36), an RNA-binding protein, is a potential target of OP. Herein, we sought to probe the regulatory role and mechanisms of ZFP36 in the progression of OP. Overexpression of ZFP36 enhanced osteoblast viability, differentiation and mineralization of human BMSCs (hBMSCs). RNA immunoprecipitation qPCR (RIP-qPCR) assays demonstrated that ZFP36 could inhibit the translation of JUN, which was also verified with dual luciferase reporter gene assay. Furthermore, administration with T-5224, a transcription factor c-Fos/activator protein (AP)-1 inhibitor, which specifically inhibits the DNA binding activity of c-Fos/JUN, abolished the effect of ZFP36 knockdown on the behaviors of hBMSCs, suggesting that ZFP36 might promotes osteogenic differentiation through regulating JUN. These findings provide insights into the progression and a potential therapeutic target of OP., Competing Interests: Declarations Competing interests The authors declare no competing interests. Conflict of interest The authors declare that there is no conflict interest., (© 2024. The Author(s).)

In vitro bone models are pivotal for understanding tissue behavior and cellular responses, particularly in unravelling certain pathologies' mechanisms and assessing the impact of new therapeutic interventions. A desirable in vitro bone model should incorporate primary human cells within a 3D environment that mimics the mechanical properties characteristics of osteoid and faithfully replicate all stages of osteogenic differentiation from osteoblasts to osteocytes. However, to date, no bio-printed model using primary osteoblasts has demonstrated the expression of osteocytic protein markers. This study aimed to develop bio-printed in vitro model that accurately captures the differentiation process of human primary osteoblasts into osteocytes. Given the considerable impact of hydrogel stiffness and relaxation behavior on osteoblast activity, we employed three distinct cross-linking solutions to fabricate hydrogels. These hydrogels were designed to exhibit either similar elastic behavior with different elastic moduli, or similar elastic moduli with varying relaxation behavior. These hydrogels, composed of gelatin (5% w/v), alginate (1%w/v) and fibrinogen (2%w/v), were designed to be compatible with micro-extrusion bioprinting and proliferative. The modulation of their biomechanical properties, including stiffness and viscoelastic behavior, was achieved by applying various concentrations of cross-linkers targeting both gelatin covalent bonding (transglutaminase) and alginate chains' ionic cross-linking (calcium). Among the conditions tested, the hydrogel with a low elastic modulus of 8 kPa and a viscoelastic behavior over time exhibited promising outcomes regarding osteoblast-to-osteocyte differentiation. The cessation of cell proliferation coincided with a significant increase in alkaline phosphatase activity, the development of dendrites, and the expression of the osteocyte marker PHEX. Within this hydrogel, cells actively influenced their environment, as evidenced by hydrogel contraction and the secretion of collagen I. This bio-printed model, demonstrating primary human osteoblasts expressing an osteocyte-specific protein, marks a significant achievement. We envision its substantial utility in advancing research on bone pathologies, including osteoporosis and bone tumors., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

Endowing titanium surfaces with multifunctional properties can reduce implant-related infections and enhance osseointegration. In this study, titanium dioxide nanotubes with strontium doping (STN) were first created on the titanium surface using anodic oxidation and hydrothermal synthesis techniques. Next, casein phosphopeptide (CCP) and an antimicrobial peptide (HHC36) were loaded into the STN with the aid of vacuum physical adsorption (STN-CP-H), giving the titanium surface a dual function of "antimicrobial-osteogenic". The surface of STN-CP-H has a suitable roughness and good hydrophilicity, which is conducive to osteoblasts. STN-CP-H had a 99 % antibacterial rate against S. aureus and E. coli and effectively prevented the growth of bacterial biofilm. Meanwhile, the antibacterial mechanism of STN-CP-H was initially explored with the help of transcriptome sequencing technology. STN-CP-H could greatly increase osteoblast adhesion, proliferation, and expression of osteogenic markers (alkaline phosphatase, runt-related transcription) when CCP and Sr worked together synergistically. In vivo, the STN-CP-H coating could effectively promote new osteogenesis around titanium implant bone and had no toxic effects on heart, liver, spleen, lung and kidney tissues. A potential anti-infection bone healing material, STN-CP-H bifunctional coating developed in this work efficiently inhibited bacterial infection of titanium implants and encouraged early osseointegration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Succinate receptor GPR91 is one of the G protein-coupled receptors (GPCRs) that interacts with various proteins to regulate diverse cellular functions such as cell morphology, apoptosis, and differentiation. In this study, we investigated whether the GPR91-mediated signaling pathway regulates mineralization in Porphyromonas gingivalis (P. gingivalis)-treated osteoblasts and to determine its potential role in osteoclast differentiation. Primary mouse osteoblasts from wild-type (WT) and GPR91 knockout (GPR91 -/- ) mice infected with P. gingivalis were used for in vitro experiments. The results showed that inhibition by 4C, a specific inhibitor, and GPR91 knockout promoted mineralization in P. gingivalis-infected osteoblasts. Surprisingly, GPR91 knockdown decreased the migration ability of osteoblasts. Moreover, compared with P. gingivalis-infected WT osteoblasts, GPR91 -/- osteoblasts exhibited decreased RANKL production, and conditioned media (CM) from bacteria-infected GPR91 -/- osteoblasts suppressed the formation of osteoclast precursors. Moreover, P. gingivalis mediated the role of GPR91 in osteoblast mineralization by activating the NF-κB pathway. These findings suggest that GPR91 activation reduces mineralization of P. gingivalis-infected osteoblasts and promotes osteoclastogenesis in macrophages. Therefore, targeting GPR91 may mitigate the loss of alveolar bone during bacterial infection., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethical approval Each author has read and passed the final version and reached an agreement to ensure all features of the work are accurate., (© 2024. The Author(s).)

Nanotherapeutic techniques are becoming increasingly important in the treatment of bone disorders owing to their targeted drug delivery. This study formulates zein nano coop composites containing chimeric antioxidants (ascorbic acid, luteolin, resveratrol, and coenzyme Q) (AZN) and evaluates its application in bone regeneration using osteoblasts and a Zebrafish osteoporosis model. In vitro experiments with human osteoblast-like MG63 cells showed enhancement of bone mineralization and regeneration. It further exhibited high biocompatibility in Zebrafish larvae, with increased calcium/phosphorus deposition and upregulation of osteogenic genes. The study has unequivocally demonstrated the potential of AZN in bone regeneration and fracture healing in both normal and osteoporosis models, underscoring the significance of this research. Further investigations using higher animal models are warranted to expand on these findings. The impact of this research seems far-reaching, with the possible development of new, effective, and safe treatment options for osteoporosis, addressing the limitations of the currently available treatments.

Osteoporosis is a degenerative bone disease highly prevalent in older women, causing high morbidity and mortality rates. Fourteen kinds of fucoidan were isolated from Sargassum fusiforme through acid (named as SFS), alkaline (SFJ) and water (SFW). SFW was passed through an anion exchange column to obtain SFW-0, SFW-0.5 and SFW-2. SFW-0.5 and SFW-2 were degraded to obtain different sulfate group contents SFW-x-M/S/O (x for 0.5 or 2). We further confirmed SFW-0.5-O was the most effective fraction of SFW. SFW-0.5-O may have alternating backbones of (Gal)n and (Fuc)n, and the main sulfation may be at C2/C3 of the Fuc/Gal residues. SFW-0.5-O inhibition of OC differentiation was associated with IRF-8 signaling; meanwhile, SFW-0.5-O promoted osteoblast differentiation and bone mineral nodule formation. SFW-0.5-O also effectively ameliorated osteoporosis symptom caused by estrogen deprivation in vivo. We uncovered that the fucoidan active fraction SFW-0.5-O demonstrated effective bone protection, may be exploited for osteoporosis therapy., (© 2024. The Author(s).)

Medication-related osteonecrosis of the jaw is a rare but severe complication with challenging treatment protocols. Cold atmospheric plasma (CAP) has shown promising effects in wound healing, cell proliferation and viability. This study investigated the effect of CAP on osteoblasts in a culture medium containing bisphosphonates. Pilot study was designed to determine the optimal setting of CAP. MG-63 cells were exposed to zoledronic acid at a concentration of 10 micromolar for 72 h. Study groups with the best MTT assay results, were chosen for assessing OPG and RANKL genes by RT-PCR. Cell viability was significantly higher in groups 9 kV.1 mm.90 s, 10 kV.1 mm.60 s, and 12 kV.1 mm.60 s (P < 0.05). Expression of RANKL in these groups was significantly lower than the positive control group (medium culture without zoledronic and plasma treatment) and higher than the BP group (culture with zoledronic without plasma treatment), except for the 12 kV.1 mm.60 s group, which did not differ significantly from the positive control group (P > 0.05). OPG decreased in all test groups compared to BP (p < 0.05), except for the 12 kV.1 mm.60 s group, which did not significantly differ from BP (P > 0.05). RANKL/OPG ratio in groups 9 kV.1 mm.90 s and 12 kV.1 mm.60 s significantly increased compared to BP (P < 0.05).CAP treatment may enhance the viability of osteoblasts exposed to bisphosphonates, increase their activity levels, enhance osteoclast activity, and improve bone turnover rates., (© 2024. The Author(s).)

Osteoporosis is a major degenerative metabolic bone disease that threatens the life and health of postmenopausal women. Owing to limitations in detection methods and prevention strategy awareness, the purpose of osteoporosis treatment is more to delay further deterioration rather than to fundamentally correct bone mass. We aimed to clarify the pathogenesis of postmenopausal osteoporosis and optimize treatment plans. Our experiments were based on previous findings that oxidative stress mediates bone metabolism imbalance after oestrogen deficiency. Through energy metabolism-targeted metabolomics, we revealed that purine metabolism disorder is the main mechanism involved in inducing oxidative damage in bone tissue, which was verified via the use of machine-learning data from human databases. Xanthine and xanthine oxidase were used to treat osteoblasts to construct a purine metabolism disorder model. The activity and differentiation ability of osteoblasts decreased after X/XO treatment. Transcriptomic sequencing indicated that autophagic flux damage was involved in purine metabolism-induced oxidative stress in osteoblasts. Additionally, we performed serum metabolomics combined with network pharmacology to determine the pharmacological mechanism of metformin in the treatment of postmenopausal osteoporosis. HPRT1 was the potential target filtered from the hub genes, and FoxO1 signalling was the key pathway mediating the effect of metformin in osteoblasts. We also revealed that SIRT3-mediated deacetylation promoted the nuclear localization of FoxO1 to increase the expression of HPRT1. HPRT1 upregulation promoted purine anabolism and prevented the accumulation of ROS caused by purine catabolism to reverse oxidative damage in osteoblasts. We propose that purine metabolism disorder-induced oxidative stress is important for the pathogenesis of postmenopausal osteoporosis. The therapeutic mechanism of metformin should be confirmed through subsequent drug optimization and development studies to improve bone health in postmenopausal women., (© 2024. The Author(s).)

We investigated the regulation of histone deacetylases (HDACs) by miR-2861 in the osteoblastic differentiation of human mesenchymal stem cells (MSCs) and miR-2861 binding site by CRISPR activation (CRISPRa). Transfection of miR-2861 into human MSCs was performed and the effect on osteoblast differentiation was analyzed. Using catalytically inactive Cas12a, the CRISPRa system induced targeted overexpression of endogenous miRNA and repressed the luciferase activities of reporters that contained functional miRNA target sites. The delivery of miR-2861 into MSCs enhanced osteoblast differentiation by decreased expressions of the HDAC1, 4 and 5 genes. The mechanism of HDAC5 repression by miR-2861 in humans has not been fully elucidated. To this end, the HDAC5 mRNA sequence was analyzed and a putative primate-specific miR-2861 binding site was identified in the 3' untranslated region (3'-UTR). CRISPRa was applied to validate the putative binding site and an increase in endogenous miR-2861 was found to repress the expression of a reporter that contained the novel miR-2861 binding site. The delivery of miR-2861 to human MSCs enhanced osteoblast differentiation. In the 3'-UTR, the HDAC5 repression was mediated by the miR-2861 binding site, and miR-2861 promoted osteoblast differentiation via the inhibition of HDAC5 through a primate-specific miRNA binding site. Therefore, miRNAmiR-2861 with the CRISPRa methods might be a good biomaterial for osteogenesis augmentation., (© 2024. The Author(s).)

The differentiation and maturation of osteoblasts are essential for bone formation. Zn 2+ plays a crucial role in cell differentiation and is involved in osteogenic differentiation. The concentration and distribution of Zn 2+ in the nucleus and cytoplasm indicate the differentiation states of osteoblasts. However, there is an absence of a real-time method for monitoring the dynamic fluctuations of endogenous Zn 2+ within the nucleus. Here, a novel Zn 2+ fluorescent probe ( NTAD-N1 ) with nuclear membrane permeability was designed and developed, allowing for distribution throughout the entire cell, including the nucleus. The NTAD-N1 probe successfully showed the dynamic distribution and concentration changes of Zn 2+ in the nucleus and cytoplasm of preosteoblast MC3T3-E1 during the 21-day differentiation period. The results showed that free Zn 2+ increased significantly during differentiation of osteoblasts (2-21 days). Importantly, after 4 days of differentiation, osteoblasts are mainly distributed in the nucleus, which is confirmed by metallothionein expression. Subsequently, the level of free Zn 2+ in the cytoplasm remained at a high level, which promoted the increase in alkaline phosphatase activity and inhibited the activity of cis-aconitase in the tricarboxylic acid cycle, resulting in the accumulation of citric acid. This series of events promotes the formation of mineralized nodules. In the process of osteoblast differentiation, the detection time of Zn 2+ (≤7 days) is ahead of the late marker of alkaline phosphatase (14 days) and mineralized nodules (14-21 days). This indicates that Zn 2+ can be used as a biomarker and an intervention point for early differentiation of osteoblasts.

The present medical treatments of osteoporosis come with adverse effects. It leads to the exploration of natural products as safer alternative medical prevention and treatment. The sea cucumber, Holothuria scabra, has commercial significance in Asian countries with rising awareness of its properties as a functional food. This study aims to investigate the effects of the inner wall (IW) extract isolated from H. scabra on extracellular matrix maturation, mineralization, and osteogenic signaling pathways on MC3T3-E1 preosteoblasts. The IW showed the expression of several growth factors. Molecular docking revealed that H. scabra BMP2/4 binds specifically to mammal BMP2 type I receptor (BMPR-IA). After osteogenic induction, the viability of cells treated with IW extract was assessed and designated with treatment of 0.1, 0.5, 1, and 5 µg/ml of IW extract for 21 consecutive days. On days 14 and 21, treatments with IW extract at 1 and 5 µg/ml showed increased alkaline phosphatase (ALP) activity and calcium deposit levels in a dose-dependent manner compared to the control group. Moreover, the transcriptomic analysis of total RNA of cells treated with 5 µg/ml of IW extract exhibited upregulation of TGF-β, PI3K/Akt, MAPK, Wnt and PTH signaling pathways at days 14. This study suggests that IW extract from H. scabra exhibits the potential to enhance osteogenic differentiation and mineralization of MC3T3-E1 preosteoblasts through TGF-β, PI3K/Akt, MAPK, Wnt and PTH signaling pathways. Further investigation into the molecular mechanisms underlying the effect of IW extract on osteogenesis is crucial to support its application as a naturally derived supplement for prevention or treatment of osteoporosis., (© 2024. The Author(s).)

Synthesis of mechanically stable porous scaffolds with an architecture analogous to cancellous bone tissue poses significant challenges to bioactive glass (BG) based scaffolds. This is primarily due to densification and crystallization of the BG's during heat treatment. This study presents a modified BG series (42SiO 2 -xTiO 2 -24Na 2 O-21CaO-13P 2 O 5 , where x = 8 and 16 TiO 2 ). TiO 2 replaced the SiO 2 concentration in the glass and was incorporated due to its biocompatibility and influence on glass structure. Material characterization determined that TiO 2 did not induce crystallization within the glass but did increase the glass transition temperature (T g ) from 520°C to 600°C thereby indicating a more stable network connectivity. Scaffolds were synthesized using the foam replication method, resulting in scaffolds with a pore size of approximately 500 μm with the BG-4 composition (30SiO 2 -12TiO 2 -24Na 2 O-21CaO-13P 2 O 5 ) retaining its amorphous character post-heat treatment. Scaffold ion release was monitored over 5-60 days in simulated body fluid (SBF). Si 4+ release was found to decrease, while Ca 2+ levels increased in SBF as TiO 2 replaced SiO 2 within the glass series. Cytocompatibility studies revealed that MC3T3 Osteoblast cells proliferated on the BG-4 scaffold surface and at its interface within culture media, and cell numbers were not significantly reduced., (© 2024 Wiley Periodicals LLC.)

The identification of both autophagy-related material degradation and unconventional secretion has paved the way for significant breakthroughs linking autophagy to a plethora of physiological processes and disease conditions. However, the mechanisms that coordinate these two pathways remain elusive. Here, we demonstrate that a switch from the lysosomal degradation to a secretory autophagy pathway is governed by protein tyrosine phosphatase 1B (PTP1B, encoded by PTPN1). Dephosphorylation at two tyrosine residues of syntaxin17 (STX17) by PTP1B reduces autophagosome-lysosome fusion while switching the cells to a secretory autophagy pathway. Both PTP1B overexpression and tumour-derived extracellular vesicles (EVs) can activate the secretory autophagy pathway in osteoblasts. Moreover, we demonstrate that osteoblastic LC3+ EVs, generated via the secretory autophagy pathway, are the primary contributor to tumour-associated bone remodelling in prostate cancer. Depletion of tumour-derived EVs secretion or genetic ablation of osteoblastic PTP1B rescues aberrant bone remodelling and lesions, highlighting the relevance between LC3+ EVs and the formation of bone metastatic niche. Our results reveal the significance of tumour-regulated PTP1B in the fate decision of autophagosomes, and propose a role ofLC3+ EVs in shaping the bone metastatic niche., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Objective: Bone incision leads to interrupted and sluggish blood flow in the process of distraction osteogenesis (DO), creating a hypoxia (0-2% oxygen tension) at the center of the bone callus. This hypoxia is critical in the coupling of osteogenesis and angiogenesis during DO. This study aimed to investigate the effect of Uniaxial Static Strain (USS) on osteogenesis in osteoblasts under hypoxic conditions, with a focus on the expression of osteogenic markers and angiogenic factors., Methods: The USS was made by a multi-unit tension compression device.Osteoblasts were subjected to 10% USS made under hypoxic conditions to mimic the process of DO in vitro. The cell proliferation, alkaline phosphatase (ALP) activity, mineralized nodule formation, and expression of osteogenic and angiogenic markers were evaluated by using a CCK-8 assay, alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red S staining, qRT-PCR, Western blotting and ELISA., Results: Hypoxia inhibited osteoblast cell proliferation, ALP activity, mineralized nodule formation, and the expression of runt-related transcription factor 2 (Runx- 2), osteopontin(OPN), osteocalcin (OCN), collagen type I (Col1a1). Conversely, hypoxia upregulated the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF), which are associated with angiogenesis. However, the application of USS enhanced osteoblasts' osteogenic capacity and upregulated angiogenic factors under hypoxic conditions., Conclusion: USS can enhance osteogenesis in osteoblasts under hypoxic conditions. Moreover, it may stimulate angiogenesis by promoting the expression of VEGF, which further contributes to bone formation. This finding provides important implications for understanding the mechanisms involved in bone regeneration and may have clinical applications in optimizing the effectiveness of DO techniques., (© 2024. The Author(s).)

Objectives: To investigate the effect of tranexamic acid (TXA) through in vitro culture of primary human osteoblasts (HOB) and in vivo using an operative rat femur fracture model. It was hypothesized that there would not be any effect on fracture healing in both studies., Methods: Primary HOBs were exposed to varying concentrations of TXA over different time periods. Cells were assessed for viability, metabolism, and mineralization. For the in vivo model, fractures were created in the femora of adult rats, exposed to either TXA or saline, and then assessed for healing at different time points. A modified radiographic union score for tibia was used to evaluate radiographs, callus mineralization was assessed with microcomputed tomography, and biomechanical tests were performed., Results: Overall, HOB viability and metabolism decreased as TXA concentration and exposure time increased. However, at concentrations below 56.44 mg/mL, HOB viability was not affected. Similarly, mineralization also decreased as TXA concentration and exposure time increased. In both groups, in vivo results demonstrated increasing radiographic healing, callus mineralization, and biomechanical strength as a function of time. There was a trend for increased healing in the TXA group at 6 weeks after fracture; however, the difference compared with untreated animals was not statistically significant., Conclusions: Although a degradation of HOB viability and metabolism occurred with increased TXA concentrations and exposure times, clinically relevant concentrations do not adversely affect HOB viability, metabolism, or mineralization. In addition, there were no noticeable adverse effects of TXA administration in the in vivo model., Competing Interests: The authors report no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Bone provides structural support, enables movement, protects internal organs, regulates calcium and phosphorus levels, and contains bone marrow essential for hematopoiesis. Osteoblasts are specialized cells responsible for bone formation through the secretion of extracellular matrix components. Transmembrane protein 175 (TMEM175), which functions as an endosomal/lysosomal K + channel and a lysosomal H + channel, regulates lysosomal function and autophagy. Despite the recognized importance of lysosomes and autophagy in osteoblast differentiation, the specific role of TMEM175 in osteoblast differentiation has not been revealed. In this study, we investigated whether TMEM175 is associated with human bone mineral density and fracture and examined the role of TMEM175 in osteoblast differentiation. In analyses of single nucleotide polymorphisms of pore ion channel genes using the mouse2human database, a significant correlation between TMEM175 single nucleotide polymorphisms and human bone mineral density and fracture was identified. TMEM175 expression levels were found to increase during osteoblast differentiation from bone chip-derived mesenchymal stem cells (BMSCs). Knockdown of TMEM175 in BMSCs suppressed osteoblast differentiation, as evidenced by decreased matrix mineralization and lower expression levels of osteoblast marker genes. Further analysis indicated that TMEM175 deficiency leads to lysosomal dysfunction and partially impairs autophagic clearance during osteoblast differentiation. Moreover, the TMEM175 inhibitor 4-aminopyridine decreased osteoblast differentiation of BMSCs. Taken together, this study reveals that TMEM175 plays an important role in osteoblast differentiation by regulating lysosomal function and autophagic clearance., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cellular therapy using adipose tissue-derived mesenchymal stromal cells (at-MSCs) has garnered attention for the treatment of bone defects. Therefore, preconditioning strategies to enhance the osteogenic potential of at-MSCs could optimize cell therapy outcomes, and photobiomodulation (PBM) therapy has emerged as an effective, noninvasive, and low-cost alternative. This study explored the impacts of PBM on at-MSCs differentiation and the subsequent repair of bone defects treated with cell injection. Rat at-MSCs were cultured and irradiated (at-MSCs PBM ) following the PBM protocol (660 nm; 20 mW; 0.714 W/cm 2 ; 0.14 J; 5 J/cm 2 ). Cellular differentiation was assessed based on the expression of gene and protein markers. Reactive oxygen species (ROS) were detected using fluorescence. At-MSCs PBM were injected into 5-mm calvarial lesions, and bone formation was analyzed using micro-CT and histological evaluations. At-MSCs were used as control. Data were analyzed using the ANOVA or t-test. At-MSCs PBM exhibited high levels of gene and protein runt-related transcription factor-2 (Runx2) and alkaline phosphatase (Alp) expression. PBM increased ALP activity and significantly reduced ROS levels. In addition, PBM increased the expression of Wnt pathway-associated genes. In vivo, there was an increase in the morphometric parameters, including bone volume, percentage of bone volume, bone surface area, and trabecular number, in at-MSCs PBM -treated defects compared with those in the control. These findings suggest that PBM enhances the osteogenic potential of at-MSCs, thereby supporting the advancement of improved cellular therapies for bone regeneration., Competing Interests: Declaration of competing interest Emanuela Prado Ferraz reports financial support was provided by State of Sao Paulo Research Foundation. Natalia Pieretti Bueno reports financial support was provided by National Council for Scientific and Technological Development. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Osteoporosis (OP) is a common cause of morbidity and mortality in older individuals. The importance of Wnt3a in osteogenic activity and bone tissue homeostasis is well known. Here, we explored the possible molecular mechanism by which Wnt3a mediates the LRP6/mTORC1/β-catenin axis to regulate osteoblast differentiation in OP., Methods: OP-related key genes were identified through a bioinformatics analysis. A ROS17/2.8 cell differentiation system for rat osteogenic progenitors and a rat model of senile OP were constructed for in vitro and in vivo mechanism verification., Results: Bioinformatics analysis revealed that LRP6 was poorly expressed in OP and may play a key role in the occurrence of OP by affecting osteoblast differentiation. LRP6 knockdown inhibited osteoblast differentiation in an in vitro model. In addition, Wnt3a promoted osteoblast differentiation by inducing LRP6 phosphorylation. Moreover, LRP6 promoted mTORC1 expression, which indirectly promoted β-catenin expression, thus promoting osteoblast differentiation. Finally, an in vivo assay revealed that LRP6 inhibition improved OP., Conclusion: Our study provides evidence that Wnt3a induces phosphorylation of LRP6 to activate the mTORC1/β-catenin axis, thus promoting osteoblast differentiation and ultimately improving OP in aged rats., (Copyright © 2024. Published by Elsevier Inc.)

Recently, exosomes that are derived from bone marrow mesenchymal stem cells (BMSCs) have garnered considerable interest due to their significant roles in the processes of bone regeneration and repair. Among the various molecular components present within these exosomes, miR-668-3p has emerged as a pivotal microRNA that may be instrumental in modulating the function and proliferation of osteoblasts, the cells responsible for bone formation. The primary objective of this research was to examine the enhancing effects of BMSC-derived exosomes that are enriched with miR-668-3p on the advancement of osteoblasts in the context of osteonecrosis of the femoral head. Furthermore, the study aimed to analyze how the expression of specific exosomal proteins, namely CD63 and CD9, influences this biological process. To conduct the investigation, BMSCs were isolated from healthy rat models, followed by the extraction of their secreted exosomes. The subsequent phase of the study involved assessing the proliferation and differentiation of osteoblasts by introducing the exosomes enriched with miR-668-3p into an experimental setup representing osteonecrosis of the femoral head. The findings revealed that exosomes derived from BMSCs, which contained miR-668-3p, significantly enhanced the proliferation of osteoblasts as well as the expression of key osteogenic marker genes. Notably, the levels of CD63 and CD9 proteins were markedly increased in the treated groups, indicating that the mechanisms underlying this promotion might involve cell adhesion and the endocytic uptake of exosomes., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Adipocyte enhancer-binding protein 1 (AEBP1) is closely implicated in osteoblastic differentiation and bone fracture; this research aimed to investigate the effect of AEBP1 on restoring osteoblastic differentiation under dexamethasone (Dex) treatment, and its interaction with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Pre-osteoblastic MC3T3-E1 cells were cultured in osteogenic medium and treated by Dex to mimic steroid-induced osteonecrosis cellular model. They were then further transfected with control or AEBP1-overexpressed lentiviral vectors. Finally, cells were treated with the PI3K inhibitor LY294002, with or without AEBP1-overexpressed lentiviral vectors. AEBP1 expression showed a downward trend in MC3T3-E1 cells under Dex treatment in a dose-dependent manner. AEBP1-overexpressed lentiviral vectors increased relative cell viability, alkaline phosphatase (ALP) staining, Alizarin red staining and osteoblastic differentiation markers including osteocalcin (OCN), osteopontin (OPN), collagen type I alpha 1 (COL1A1), runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), but decreased cell apoptosis rate in MC3T3-E1 cells under Dex treatment; besides, AEBP1-overexpressed lentiviral vectors positively regulated p-PI3K and p-AKT expressions. Furthermore, LY294002 treatment decreased relative cell viability, Alizarin red staining, osteoblastic differentiation markers including OCN, OPN, RUNX2 and BMP, increased cell apoptosis rate and did not affect ALP staining in MC3T3-E1 cells under Dex treatment; meanwhile, LY294002 treatment weakened the effect of AEBP1 overexpression vectors on the above cell functions. AEBP1 restores osteoblastic differentiation under Dex treatment by activating the PI3K/AKT pathway., (© 2024 John Wiley & Sons Australia, Ltd.)

Background: The incidence of osteoarthritis (OA) is increasing, yet its pathogenesis remains largely unknown. Recent studies suggest that abnormal subchondral bone remodeling plays a crucial role in OA development, highlighting a gap in clinical treatments targeting this aspect. Soybean Isoflavone (SI) has shown potential in treating OA, although its mechanisms are not fully understood., Methods: This research investigated the effects of SI on subchondral bone remodeling in an OA rat model, assessing joint damage, OARSI scores, and type H vessel formation (CD31hiEmcnhi expression). Additionally, the expression of ALP, OCN, BMP, and TSC1 was evaluated to determine involvement of the mTORC1 pathway. In vitro studies on IL-1β-induced osteoblasts further examined the impact of SI on TSC1/mTORC1 signaling and related markers., Results: SI treatment reduced joint damage and OARSI scores in the rat OA model, significantly decreasing CD31hiEmcnhi expression, indicating a reduction in type H vessel formation. SI also downregulated ALP, OCN, and BMP expression while upregulating TSC1, suggesting inhibition of the mTORC1 signaling pathway and VEGF release. In vitro, SI increased TSC1 expression and decreased mTORC1 signaling, VEGF, ALP, OCN, and BMP levels in IL-1β-induced osteoblasts., Conclusion: SI targets the TSC1/mTORC1 signaling pathway to suppress osteoblast activation and VEGF release, inhibiting type H vessel formation and slowing abnormal subchondral bone remodeling. These findings provide a novel therapeutic approach for OA by focusing on subchondral bone remodeling mechanisms.

Bone cells produce fibroblast growth factor 23 (FGF23), a hormone regulating renal phosphate and vitamin D homeostasis, and a paracrine factor produced in further tissues. Chronic kidney disease and cardiovascular disorders are associated with early elevations of plasma FGF23 levels associated with clinical outcomes. FGF23 production is dependent on many conditions including inflammation. Prostaglandin E 2 (PGE 2 ) is a major eicosanoid with a broad role in pain, inflammation, and fever. Moreover, it regulates renal blood flow, renin secretion, natriuresis as well as bone formation through prostaglandin E receptor 2 (EP2). Here, we studied the role of PGE 2 and its signaling for the production of FGF23. Osteoblast-like UMR-106 cells were exposed to EP receptor agonists, antagonists or RNAi. Wild type and EP2 knockout mice were treated with stable EP2 agonist misoprostol. Fgf23 or Nurr1 gene expression was determined by quantitative real-time PCR, hormone and further blood parameters by enzyme-linked immunosorbent assay and colorimetric methods. PGE 2 and EP2 agonists misoprostol and butaprost enhanced FGF23 production in UMR-106 cells, effects mediated by EP2 and transcription factor Nurr1. A single dose of misoprostol up-regulated bone Fgf23 expression and FGF23 serum levels in wild type mice with subtle effects on parameters of mineral metabolism only. Compared to wild type mice, the FGF23 effect of misoprostol was significantly lower in EP2-deficient mice. To conclude, PGE 2 signaling through EP2 and Nurr1 induces FGF23 production. Given the broad physiological and pathophysiological implications of PGE 2 signaling, this effect is likely of clinical relevance., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Martina Feger and Michael Föller report financial support was provided by German Research Foundation. Michael Föller reports a relationship with Kyowa Kirin Co Ltd that includes: speaking and lecture fees. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

In the process of bone metabolism and bone remodeling, bone marrow mesenchymal stem cells (BM-MSCs) differentiate into osteoblasts (OBs) under certain conditions to enable the formation of new bone, and normal bone reconstruction and pathological bone alteration are closely related to the differentiation and proliferation functions of OBs. Osteogenic differentiation of BM-MSCs involves multiple signaling pathways, which function individually but interconnect intricately to form a complex signaling regulatory network. Natural compounds have fewer adverse effects than chemically synthesized drugs, optimize bone health, and are more suitable for long-term use. In this paper, we focus on OBs, summarize the current research progress of signaling pathways related to OBs differentiation, and review the molecular mechanisms by which chemically synthesized drugs with potential anti-osteoporosis properties regulate OBs-mediated bone formation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The pathogenesis of osteoporosis is driven by several mechanisms including the imbalance between osteoblastic bone formation and osteoclastic bone resorption. Currently, the role of Niacin (NA), also known as vitamin B3, in the regulation of osteoblastic differentiation is not fully understood. Data from the NHANES database were employed to investigate the association of NA intake with the prevalence of osteoporosis. Alterations in mRNA and protein levels of genes and proteins involved in osteogenic differentiation were evaluated via techniques including qRT-PCR, protein immunoblotting, Alkaline Phosphatase (ALP) activity analysis, ALP staining, and Alizarin Red staining. Changes in the mouse skeletal system were investigated by organizational analysis and Micro-CT. The results indicated that NA promoted osteogenic differentiation. Co-immunoprecipitation and chromatin immunoprecipitation were performed to explore the underlying mechanisms. It was observed that NA promoted AREG expression by deacetylating C/EBPβ via SIRT2, thereby activating the PI3K-AKT signaling pathway. It also enhanced the activity of the pivotal glycolytic enzyme, PFKFB3. This cascade amplified osteoblast glycolysis, facilitating osteoblast differentiation. These findings demonstrate that NA modulates glucose metabolism and influences osteogenic differentiation via the SIRT2-C/EBPβ-AREG pathway, suggesting that NA may be a potential therapeutic agent for the management of osteoporosis, and AREG could be a plausible target., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Dental and orthopedic implants have become routine medical technologies for tooth replacement and bone fixation. Despite significant progress in implantology, achieving sufficient osseointegration remains a challenge, often leading to implant failure over the long term. Nanotechnology offers the potential to mimic the natural patterns of living tissues, providing a promising platform for tissue engineering and implant surface design. Among the various methods for developing nanostructures, High-Regular Laser-Induced Periodic Surface Structures (HR-LIPSS) techniques stand out for their ability to fabricate highly ordered nanostructures with excellent long-range repeatability and production efficiency. In this study, we utilized an innovative technical approach to generate traditional laser-induced superficial LIPSS nanostructures, followed by detailed surface analysis using classical microscopy and physicochemical methods. Our findings demonstrate for the first time that nanostructured LIPSS surfaces can significantly enhance cell adhesion and proliferation while providing an optimal environment for cell metabolism. Given the high reproducibility, low cost, and potential of HR-LIPSS techniques to support cell growth and differentiation, this novel technology has the potential to impact both the industrial development of new implants and clinical outcomes after implantation., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

As obesity rates continue to rise, the prevalence of metabolic dysfunction and alcohol-associated steatotic liver disease (MetALD), a new term for Nonalcoholic Fatty Liver Disease (NAFLD), also increases. In an aging population, it is crucial to understand the interplay between metabolic disorders, such as MetALD, and bone health. This understanding becomes particularly significant in the context of implant osseointegration. This study introduces an in vitro model simulating high lipogenesis through the use of human Mesenchymal Stroma Cells-derived adipocytes, 3D intrahepatic cholangiocyte organoids (ICO), and Huh7 hepatocytes, to evaluate the endocrine influence on osteoblasts interacting with titanium. We observed a significant increase in intracellular fat accumulation in all three cell types, along with a corresponding elevation in metabolic gene expression compared to the control groups. Notably, osteoblasts undergoing mineralization in this high-lipogenesis environment also displayed lipid vesicle accumulation. The study further revealed that titanium surfaces modulate osteogenic gene expression and impact cell cycle progression, cell survival, and extracellular matrix remodeling under lipogenic conditions. These findings provide new insights into the challenges of implant integration in patients with obesity and MetALD, offering a deeper understanding of the metabolic influences on bone regeneration and implant success., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Inspired by the formation of natural abalone shells (AS) similar to calcium salt deposition in human orthodontics, AS is used as an emulsifier in the scaffold to solve the problem of coexistence of natural and synthetic polymers and promote new bone formation. In this study, AS-stabilized and reinforced carboxymethyl chitosan/collagen/PLGA porous bionic composite scaffolds (AS/CMCS/Col/PLGA) were fabricated through the emulsion polymerization and bionic hybrid technology. As the addition of AS increased from 0.75 to 3.0 wt%, homogeneous distribution of flower-like particles could be observed on the inner surface of the scaffold, and its mechanical properties were improved. Particularly, 3.0 wt% AS-doped scaffolds (S3 and C + S3) exhibited excellent inorganic mineral deposition and osteoblast proliferation and differentiation abilities in vitro. In a SD rat calvarial defect model, they effectively promoted new bone formation in the defect and accelerated expression of osteogenic-angiogenic related proteins (COLI, OCN, VEGF). By virtue of its combined merits including good mechanical properties, inducing mineralization crystallization and facilitating osteogenesis, the 3.0 wt% AS-doped scaffold promises to be employed as a novel bone repair material for bone tissue regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Electrical stimulation (ES) plays an important role in regulating cell osteoblast differentiation. As a noninvasive rehabilitation therapy method, Es has a unique role in postoperative recovery. Bone morphogenetic protein-2 (BMP-2) is the most commonly used bioactive molecule in in situ tissue engineering scaffolds, and it plays an important regulatory role in the whole process of bone injury repair. In this study, the osteogenic regulation of MC-3T3-E1 cells was studied by combining pulsed electrical stimulation (PES) and different concentrations of BMP-2. The results showed that PES and BMP-2 could synergically promote the proliferation of MC-3T3-E1 cells. The qPCR results of osteoblast-related genes showed that PES was synergistic with BMP-2 to promote osteoblast differentiation mainly through the regulation of the Smad/BMP and insulin like growth factor 1 (IGF1) signaling pathways. The expression level of alkaline phosphatase (ALP) and alizarin red staining further demonstrated the synergistic effect of PES and BMP-2 on promoting osteogenic differentiation and mineralization of cells. PES and BMP-2 could also synergically promote cell proliferation, expression of collagen I (COL-I) and ALP, and cell mineralization on the 3D-printed polylactic acid scaffold. These results suggest that the use of PES can enhance the osteogenic effect of in situ bone repair scaffolds containing BMP-2, reduce the dose of BMP-2 alone, and reduce the possible side effects of high-dose BMP-2 in vivo., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This work investigated the range of substitution of two biologically relevant ions, namely Mn 2+ and Co 2+ , into the structure of β-tricalcium phosphate, as well as their influence on bone cells response. To this aim, β-TCP was synthesized by solid state reaction in the presence of increasing amount of the substituent ions. The results of the X-ray diffraction analysis reveal that just limited amounts of these ions can enter into the β-TCP structure: 15 at% and 20 at% for cobalt and manganese, respectively. Substitution provokes aggregation of the micrometric particles and reduction of the lattice constants. In particular, the dimension of the c-parameter exhibits a discontinuity at about 10 at% for both cations, although with different trend. Moreover, Rietveld refinement demonstrates a clear preference of both manganese and cobalt for the octahedral site (V). The influence of these ions on cell response was tested on osteoblast, osteoclast and endothelial cells. The results indicate that the presence of manganese promotes a good osteoblast viability, significantly enhances the expression of osteoblast key genes and the angiogenic process of endothelial cells, while inhibiting osteoclast resorption. At variance, osteoblast viability appears reduced in the presence of Co samples, on which osteoblast genes reach higher expression than on β-TCP just in a few cases. On the other hand, the results clearly show that cobalt significantly stimulates the angiogenic process and inhibits osteoclast resorption., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Population aging, reduced economic capacity, and neglecting the treatments for oral pathologies, are the main causal factors for about 3 billion individuals who are suffering from partial/total edentulism or alveolar bone resorption: thus, the demand for dental implants is increasingly growing. To achieve a good prognosis for implant-supported restorations, adequate peri-implant bone volume is mandatory. The Guided Bone Regeneration (GBR) technique is one of the most applied methods for alveolar bone reconstruction and treatment of peri-implant bone deficiencies. This technique involves the use of different types of membranes in association with some bone substitutes (autologous, homologous, or heterologous). However, time for bone regeneration is often too long and the bone quality is not simply predictable. This study aims at engineering and evaluating the efficacy of modified barrier membranes, enhancing their bioactivity for improved alveolar bone tissue regeneration. We investigated membranes functionalized with chitosan (CS) and chitosan combined with the peptide GBMP1α (CS + GBMP1α), to improve bone growth. OsseoGuard® membranes, derived from bovine Achilles tendon type I collagen crosslinked with formaldehyde, were modified using CS and CS + GBMP1α. The functionalization, carried out with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide and sulfo-N-Hydroxysuccinimide (EDC/sulfo-NHS), was assessed through FT-IR and XPS analyses. Biological assays were performed by directly seeding human osteoblasts onto the materials to assess cell proliferation, mineralization, gene expression of Secreted Phosphoprotein 1 (SPP1) and Runt-Related Transcription Factor 2 (Runx2), and antibacterial properties. Both CS and CS + GBMP1α functionalizations significantly enhanced human osteoblast proliferation, mineralization, gene expression, and antibacterial activity compared to commercial membranes. The CS + GBMP1α functionalization exhibited superior outcomes in all biological assays. Mechanical tests showed no significant alterations of membrane biomechanical properties post-functionalization. The engineered membranes, especially those functionalized with CS + GBMP1α, are suitable for GBR applications thanks to their ability to enhance osteoblast activity and promote bone tissue regeneration. These findings suggest a potential advancement in the treatment of oral cavity problems requiring bone regeneration., (© 2024 Wiley Periodicals LLC.)

Skeletal tissue development and regeneration in mammals are intricate, multistep, and highly regulated processes. Various signaling pathways have been implicated in the regulation of these processes, including redox. Redox signaling is the signal transduction by electron transfer reactions involving free radicals or related species. Redox homeostasis is essential to cell metabolic states, as the ROS not only regulates cell biological processes but also mediates physiological processes. Following a bone fracture, redox signaling is also triggered to regulate bone healing and regeneration by targeting resident stromal cells, osteoblasts, osteoclasts and endothelial cells. This review will focus on how the redox signaling impact the bone development and bone regeneration.

Scaffolds with multiphasic structures are considered to be superior for guided tissue regeneration. Two types of tilapia skin collagen gradient membranes (stepped gradient and linear gradient) with multiphasic structures were prepared by controlling the collagen concentrations and the freezing rates. The results revealed that collagen gradient membranes were more capable of guiding tissue regeneration compared to homogeneous membranes. These two gradient membranes featured a dense outer layer and a loose inner layer, with good mechanical properties as indicated by tensile strengths of more than 250 Kpa and porosities exceeding 85 %. Additionally, these membranes also showed good hydrophilicity and water absorption, with an inner layer contact angle of less than 91° and a water absorption ratio greater than 40 times. Furthermore, the multiphasic scaffolds were proved to be biocompatible by the acute toxicity assay, the intradermal irritation test and so on. Gradient membranes could effectively promote the adhesion and proliferation of fibroblasts and osteoblasts, through elevating the TGF-β/Smad signaling pathway by TGF-β and Smads, and activating the Wnt/β-catenin signaling pathway by LRP5 and β-catenin, similar to homogenous membranes. Therefore, collagen gradient membranes from tilapia skin show important application value in guiding tissue regeneration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Osteopenia and osteoporosis are among the most common metabolic bone diseases and represent major public health problems, with sufferers having an increased fracture risk. Diabetes is one of the most common diseases contributing to osteopenia and osteoporosis. However, the mechanisms underlying diabetes-induced osteopenia and osteoporosis remain unclear. Bone reconstruction, including bone formation and absorption, is a dynamic process. Large-conductance Ca 2+ -activated K + channels (BK channels) regulate the function of bone marrow-derived mesenchymal stem cells, osteoblasts, and osteoclasts. Our previous studies revealed the relationship between BK channels and the function of osteoblasts via various pathways under physiological conditions. In this study, we reported a decrease in the expression of BK channels in mice with diabetes-induced osteopenia. BK deficiency enhanced mitochondrial Ca 2+ and activated classical PINK1 (PTEN induced putative kinase 1)-PRKN/Parkin (parkin RBR E3 ubiquitin protein ligase)-dependent mitophagy, whereas the upregulation of BK channels inhibited mitophagy in osteoblasts. Moreover, SLC25A5/ANT2 (solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 5), a critical inner mitochondrial membrane protein participating in PINK1-PRKN-dependent mitophagy, was also regulated by BK channels. Overall, these data identified a novel role of BK channels in regulating mitophagy in osteoblasts, which might be a potential target for diabetes-induced bone diseases. Abbreviations : AGE, advanced glycation end products; Baf A1, bafilomycin A 1 ; BK channels, big-conductance Ca 2+ -activated K + channels; BMSCs, bone marrow-derived mesenchymal stem cells; BSA, bovine serum albumin; FBG, fasting blood glucose; IMM, inner mitochondrial membrane; ITPR1, inositol 1,4,5-trisphosphate receptor 1; MAM, mitochondria-associated ER membrane; OMM, outer mitochondrial membrane; PINK1, PTEN induced putative kinase 1; PPID/CyP-D, peptidylprolyl isomerase D (cyclophilin D); PRKN/PARK2, parkin RBR E3 ubiquitin protein ligase; ROS, reactive oxygen species; SLC25A5/ANT2, solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 5; STZ, streptozotocin.

Successful bone regeneration requires close cooperation between bone marrow mesenchymal stem cells (BMSCs) and macrophages, but the low osteogenic differentiation efficiency of stem cells and the excessive inflammatory response of immune cells hinder the development of bone repair. It is necessary to develop a strategy that simultaneously regulates the osteogenic differentiation of BMSCs and the anti-inflammatory polarization of macrophages for accelerating the bone regeneration. Herein, calcium-chlorogenic acid nanoparticles (Ca-CGA NPs) are synthesized by combining the small molecules of chlorogenic acid (CGA) with Ca 2+ . Ca-CGA NPs internalized by cells can be dissolved to release free CGA and Ca 2+ under low pH conditions in lysosomes. In vitro results demonstrate that Ca-CGA NPs can not only enhance the osteogenic differentiation of BMSCs but also promote the phenotype transformation of macrophages from M1 to M2. Furthermore, in vivo experiments confirm that Ca-CGA NPs treatment facilitates the recovery of rat skull defect model through both osteoinduction and immunomodulation. This study develops a new Ca-CGA NPs-based strategy to induce the differentiation of BMSCs into osteoblasts and the polarization of macrophages into M2 phenotype, which is promising for accelerating bone repair., (© 2024 Wiley‐VCH GmbH.)

Osteoporosis and resulting fractures affect a significant group of people in the world. It has been shown in many studies that selenium has positive effects on bone metabolism. Based on this information, the aim of this study is to investigate whether bone differentiation will start in a shorter time by applying selenomethionine (SeMet) to hFOB cells.First, hFOB 1.19 cells were cultured. Safe doses of SeMet were determined by MTT and LDH tests. Ossification levels were determined by alizarin red staining and measurement of alkaline phosphatase enzyme levels. The results were analyzed with statistical tests.It was observed that SeMet increased cell viability at concentrations of 10, 25, 50, 100, and 200 µM in 24 h. At these concentrations, cell viability increased above the control, the viabilities were as follows: 109.4%, 104.9%, 104.3%, 103.15%, and 100.27%. High doses of SeMet significantly reduce cell viability. According to Alizarin red staining, SeMet increases the amount of calcium deposits in hFOB cells in a dose-dependent manner. In the experimental groups, the highest ALP enzyme was determined in the 7-day SeMet application. The most effective dose was measured as 15 µM.It was determined that SeMet, which is found as a trace element in living things in nature, increases the viability of hFOB cells, which are osteoblast cell precursors, and increases osteoblastic differentiation and osteoblastic activity in these cells. Our results are at a level that sheds light on an important problem in public health., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Mutations in genes related to cholesterol metabolism, or maternal diet and health status, affect craniofacial bone formation. However, the precise role of intracellular cholesterol metabolism in craniofacial bone development remains unclear., Objective: The aim of this study is to determine how cholesterol metabolism aberrations affect craniofacial bone development., Methods: Mice with a deficiency in Sc5d, which encodes an enzyme involved in cholesterol synthesis, were analyzed with histology, micro computed tomography (microCT), and cellular and molecular biological methods., Results: Sc5d null mice exhibited mandible hypoplasia resulting from defects in osteoblast differentiation. The activation of the hedgehog and WNT/β-catenin signaling pathways, which induce expression of osteogenic genes Col1a1 and Spp1, was compromised in the mandible of Sc5d null mice due to a failure in the formation of the primary cilium, a cell surface structure that senses extracellular cues. Treatments with an inducer of hedgehog or WNT/β-catenin signaling or with simvastatin, a drug that restores abnormal cholesterol production, partially rescued the defects in osteoblast differentiation seen in Sc5d mutant cells., Conclusion: Our results indicate that loss of Sc5d results in mandibular hypoplasia through defective primary cilia-mediated hedgehog and WNT/β-catenin signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Metals in living organisms and environments are essential for key biological functions such as enzymatic activity, and DNA and RNA synthesis. This means that disruption of metal ion homeostasis and exchange between cells can lead to diseases. EVs are believed to play an essential role in transporting metals between cells, but the mechanism of metal packaging and exchange remains to be elucidated. Here, we established the elemental composition of EVs at the nanoscale and single-vesicle level and showed that the metal content depends on the cell type and culture microenvironment. We also demonstrated that EVs participate in the exchange of metal elements between cells. Specifically, we used two classes of EVs derived from papaya fermented fluid (PaEVs), and decidual mesenchymal stem/stromal cells (DEVs). To show that EVs transfer metal elements to cells, we treated human osteoblast-like cells (MG63) and bone marrow mesenchymal stem cells (BMMSCs) with both classes of EVs. We found that both classes of EVs contained various metal elements, such as Ca, P, Mg, Fe, Na, Zn, and K, originating from their parent cells, but their relative concentrations did not mirror the ones found in the parent cells. Single-particle analysis of P, Ca, and Fe in DEVs and PaEVs revealed varying element masses. Assuming spherical geometry, the mean mass of P was converted to a mean size of 62 nm in DEVs and 24 nm in PaEVs, while the mean sizes of Ca and Fe in DEVs were smaller, converting to 20 nm and 30 nm respectively. When EVs interacted with BMMSCs and MG63, DEVs increased Ca, P, and Fe concentrations in BMMSCs and increased Fe concentration in MG63, while PaEVs increased Ca concentrations in BMMSCs and had no effect on MG63. The EV cargo, including proteins, nucleic acids, and lipids, differs from their origin in composition, and this variation extends to the element composition of EVs in our study. This fundamental understanding of EV-mediated metal exchange between cells could offer a new way of assessing EV functionality by measuring their elemental composition. Additionally, it will contribute novel insights into the mechanisms underlying EV production and their biological activity.