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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.)

Osteocytes' response to dynamic loading plays a crucial role in regulating the bone mass but quickly becomes saturated such that downstream induction of bone formation plateaus. The underlying mechanisms that downregulate osteocytes' sensitivity and overall response to loading remain unknown. In other cell types, purinergic signaling through the P2Y2 receptor has the potential to downregulate the sensitivity to loading by modifying cell stiffness through actin polymerization and cytoskeleton organization. Herein, we examined the role of P2Y2 activation in regulating osteocytes' mechanotransduction using a P2Y2 knockout cell line alongside conditional knockout mice. Our findings demonstrate that the absence of P2Y2 expression in MLO-Y4 cells prevents actin polymerization while increasing the sensitivity to fluid flow-induced shear stress. Deleting osteocytes' P2Y2 expression in conditional-knockout mice enabled bone formation to increase when increasing the duration of exercise. Overall, P2Y2 activation under loading produces a negative feedback loop, limiting osteocytes' response to continuous loading by shifting the sensitivity to mechanical strain through actin stress fiber formation., (© 2024 Chougule et al.)

The lacunocanalicular network (LCN) is an intricate arrangement of cavities (lacunae) and channels (canaliculi), which permeates the mineralized bone matrix. In its porosity, the LCN accommodates the cell network of osteocytes. These two nested networks are attributed a variety of essential functions including transport, signaling, and mechanosensitivity due to load-induced fluid flow through the LCN. For a more quantitative assessment of the networks' function, the three-dimensional architecture has to be known. For this reason, we aimed (i) to quantitatively characterize spatial heterogeneities of the LCN in whole mouse tibial cross-sections of BALB/c mice and (ii) to analyze differences in LCN architecture by comparison with another commonly used inbred mouse strain, the C57BL/6 mouse. Both tibiae of five BALB/c mice (female, 26-week-old) were stained using rhodamine 6G and whole tibiae cross-sections were imaged using confocal laser scanning microscopy. Using image analysis, the LCN was quantified in terms of density and connectivity and lacunar parameters, such as lacunar degree, volume, and shape. In the same tibial cross-sections, the calcium content was measured using quantitative backscattered electron imaging (qBEI). A structural analysis of the LCN properties showed that spatially denser parts of the LCN are mainly due to a higher density of branching points in the network. While a high intra-individual variability of network density was detected within the cortex, the inter-individual variability between different mice was low. In comparison to C57BL/6J mice, BALB/c mice showed a distinct lower canalicular density. This reduced network was already detectable on a local network level with fewer canaliculi emanating from lacunae. Spatial correlation with qBEI images demonstrated that bone modeling resulted in disruptions in the network architecture. The spatial heterogeneity and differences in density of the LCN likely affects the fluid flow within the network and therefore bone's mechanoresponse to loading., (© 2024. The Author(s).)

We and others have shown that application of high-level mechanical loading promotes the formation of transient plasma membrane disruptions (PMD) which initiate mechanotransduction. We hypothesized that increasing osteocyte cell membrane fragility, by disrupting the cytoskeleton-associated protein β2-spectrin (Sptbn1), could alter osteocytic responses and bone adaptation to loading in a PMD-related fashion. In MLO-Y4 cells, treatment with the spectrin-disrupting agent diamide or knockdown of Sptbn1 via siRNA increased the number of PMD formed by fluid shear stress. Primary osteocytes from an osteocyte-targeted DMP1-Cre Sptbn1 conditional knockout (CKO) model mimicked trends seen with diamide and siRNA treatment and suggested the creation of larger PMD, which repaired more slowly, for a given level of stimulus. Post-wounding cell survival was impaired in all three models, and calcium signaling responses from the wounded osteocyte were mildly altered in Sptbn1 CKO cultures. Although Sptbn1 CKO mice did not demonstrate an altered skeletal phenotype as compared to WT littermates under baseline conditions, they showed a blunted increase in cortical thickness when subjected to an osteogenic tibial loading protocol as well as evidence of increased osteocyte death (increased lacunar vacancy) in the loaded limb after 2 weeks of loading. The impaired post-wounding cell viability and impaired bone adaptation seen with Sptbn1 disruption support the existence of an important role for Sptbn1, and PMD formation, in osteocyte mechanotransduction and bone adaptation to mechanical loading., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Basic, translational and clinical research over the past few decades has provided new understanding on the mechanisms by which activation of the receptor of parathyroid hormone (parathyroid hormone 1 receptor (PTH1R)) regulates bone physiology and pathophysiology. A fundamental change in the field emerged upon the recognition that osteocytes, which are permanent residents of bone and the most abundant cells in bone, are targets of the actions of natural and synthetic ligands of PTH1R (parathyroid hormone and abaloparatide, respectively), and that these cells drive essential actions related to bone remodelling. Among the numerous genes regulated by PTH1R in osteocytes, SOST (which encodes sclerostin, the WNT signalling antagonist and inhibitor of bone formation) has a critical role in bone homeostasis and changes in its expression are associated with several bone pathologies. The bone fragility syndrome induced by diabetes mellitus is accompanied by increased osteocyte apoptosis and changes in the expression of osteocytic genes, including SOST. This Review will discuss advances in our knowledge of the role of osteocytes in PTH1R signalling and the new opportunities to restore bone health in diabetes mellitus by targeting the osteocytic PTH1R-sclerostin axis., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Different gravity fields have important effects on the structural morphology of bone. The fluid flow caused by loadings in the bone lacunar-canalicular system (LCS), converts mechanical signals into biological signals and regulates bone reconstruction by affecting effector cells, which ensures the efficient transport of signaling molecules, nutrients, and waste products. In this study, the fluid flow and mass transfer effects of bone lacunar-canalicular system at multi-scale were firstly investigated, and a three-dimensional axisymmetric fluid-solid coupled finite element model of the LCS within three continuous osteocytes was established. The changes in fluid pressure field, flow velocity field, and fluid shear force variation on the surface of osteocytes within the LCS were studied comparatively under different gravitational fields (0 G, 1 G, 5 G), frequencies (1 Hz, 1.5 Hz, 2 Hz) and forms of cyclic compressive loading. The results showed that different frequencies represented different exercise intensities, suggesting that high-intensity exercise may accelerate the fluid flow rate within the LCS and enhance osteocytes activity. Hypergravity enhanced the transport of solute molecules, nutrients, and signaling molecules within the LCS. Conversely, the mass transfer in the LCS may be inhibited under microgravity, which may cause bone loss and eventually lead to the onset of osteoporosis. This investigation provides theoretical guidance for rehabilitative training against osteoporosis.

Glucocorticoid-induced osteoporosis serves as a primary cause for secondary osteoporosis and fragility fractures, representing the most prevalent adverse reaction associated with prolonged glucocorticoid use. In this study, to elucidate the impact and underlying mechanisms of fluid shear stress (FSS)-mediated Piezo1 on dexamethasone (Dex)-induced apoptosis, we respectively applied Dex treatment for 6 h, FSS at 9 dyne/cm 2 for 30 min, Yoda1 treatment for 2 h, and Piezo1 siRNA transfection to intervene in MLO-Y4 osteocytes. Western blot analysis was used to assess the expression of Cleaved Caspase-3, Bax, Bcl-2, and proteins associated with the PI3K/Akt pathway. Additionally, qRT-PCR was utilized to quantify the mRNA expression levels of these molecules. Hoechst 33258 staining and flow cytometry were utilized to evaluate the apoptosis levels. The results indicate that FSS at 9 dyne/cm 2 for 30 min significantly upregulates Piezo1 in osteocytes. Following Dex-induced apoptosis, the phosphorylation levels of PI3K and Akt are markedly suppressed. FSS-mediated Piezo1 exerts a protective effect against Dex-induced apoptosis by activating the PI3K/Akt pathway. Additionally, downregulating the expression of Piezo1 in osteocytes using siRNA exacerbates Dex-induced apoptosis. To further demonstrate the role of the PI3K/Akt signaling pathway, after intervention with the PI3K pathway inhibitor, the activation of the PI3K/Akt pathway by FSS-mediated Piezo1 in osteocytes was significantly inhibited, reversing the anti-apoptotic effect. This study indicates that under FSS, Piezo1 in MLO-Y4 osteocytes is significantly upregulated, providing protection against Dex-induced apoptosis through the activation of the PI3K/Akt pathway., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yayi Xia reports financial support was provided by National Natural Science Foundation of China and Natural Science Foundation of Gansu Province. 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 Inc. All rights reserved.)

Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1-34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11 + cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts., (© 2024. The Author(s).)

Bone is a three-dimensional (3D) highly dynamic tissue under constant remodeling. Commonly used tools to investigate bone biology require sample digestion for biomolecule extraction or provide only two-dimensional (2D) spatial information. There is a need for 3D tools to investigate spatially preserved biomarker expression in osteocytes. In this work, we present a new method, WISH-BONE, to label osteocyte messenger RNA (mRNA) and protein in whole-mount mouse bone. For mRNA labeling, we used hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) to label genes of interest in osteocytes. For protein labeling, samples were preserved using an epoxy-based solution that protects tissue structure and biomolecular components. Then an enzymatic matrix permeabilization step was performed to enable antibody penetration. Immunostaining was used to label various proteins involved in bone homeostasis. We also demonstrate the use of customized fluorescent nanobodies to target and label proteins in the cortical bone (CB). However, the relatively dim signal observed from nanobodies' staining limited detection. mRNA and protein labeling were performed in separate samples. In this study, we share protocols, highlight opportunities, and identify the challenges of this novel 3D labeling method. They are the first protocols for whole-mount osteocyte 3D labeling of mRNA and protein in mature mouse bones. WISH-BONE will allow the investigation of molecular signaling in bone cells in their 3D environment and could be applied to various bone-related fields of research., (© 2024 Federation of American Societies for Experimental Biology.)

Cortical bone loss is intricately associated with ageing and coincides with iron accumulation. The precise role of ferroptosis, characterized by iron overload and lipid peroxidation, in senescent osteocytes remains elusive. We found that ferroptosis was a crucial mode of osteocyte death in cortical bone during ageing. Using a single-cell transcriptome analysis, we identified activating transcription factor 3 (ATF3) as a critical driver of osteocyte ferroptosis. Elevated ATF3 expression in senescent osteocytes promotes iron uptake by upregulating transferrin receptor 1 while simultaneously inhibiting solute carrier family 7-member 11-mediated cystine import. This process leads to an iron overload and lipid peroxidation, culminating in ferroptosis. Importantly, ATF3 inhibition in aged mice effectively alleviated ferroptosis in the cortical bone and mitigated cortical bone mass loss. Taken together, our findings establish a pivotal role of ferroptosis in cortical bone loss in older adults, providing promising prevention and treatment strategies for osteoporosis and fractures., (© 2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

The bone turnover capability influences the acquisition and maintenance of osseointegration. The architectures of osteocyte three-dimensional (3D) networks determine the direction and activity of bone turnover through osteocyte intercellular crosstalk, which exchanges prostaglandins through gap junctions in response to mechanical loading. Titanium nanosurfaces with anisotropically patterned dense nanospikes promote the development of osteocyte lacunar-canalicular networks. We investigated the effects of titanium nanosurfaces on intercellular network development and regulatory capabilities of bone turnover in osteocytes under cyclic compressive loading. MLO-Y4 mouse osteocyte-like cell lines embedded in type I collagen 3D gels on titanium nanosurfaces promoted the formation of intercellular networks and gap junctions even under static culture conditions, in contrast to the poor intercellular connectivity in machined titanium surfaces. The osteocyte 3D network on the titanium nanosurfaces further enhanced gap junction formation after additional culturing under cyclic compressive loading simulating masticatory loading, beyond the degree observed on machined titanium surfaces. A prostaglandin synthesis inhibitor cancelled the dual effects of titanium nanosurfaces and cyclic compressive loading on the upregulation of gap junction-related genes in the osteocyte 3D culture. Supernatants from osteocyte monolayer culture on titanium nanosurfaces promoted osteocyte maturation and intercellular connections with gap junctions. With cyclic loading, titanium nanosurfaces induced expression of the regulatory factors of bone turnover in osteocyte 3D cultures, toward higher osteoblast activation than that observed on machined surfaces. Titanium nanosurfaces with anisotropically patterned dense nanospikes promoted intercellular 3D network development and regulatory function toward osteoblast activation in osteocytes activated by cyclic compressive loading, through intercellular crosstalk by prostaglandin., 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 Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: The skeleton is one of the largest organs in the body, wherein metabolism is integrated with systemic energy metabolism. However, the bioenergetic programming of osteocytes, the most abundant bone cells coordinating bone metabolism, is not well defined. Here, using a mouse model with partial penetration of an osteocyte-specific PPARG deletion, we demonstrate that PPARG controls osteocyte bioenergetics and their contribution to systemic energy metabolism independently of circulating sclerostin levels, which were previously correlated with metabolic status of extramedullary fat depots., Methods: In vivo and in vitro models of osteocyte-specific PPARG deletion, i.e. Dmp1 Cre Pparγ flfl male and female mice (γOT KO ) and MLO-Y4 osteocyte-like cells with either siRNA-silenced or CRISPR/Cas9-edited Pparγ. As applicable, the models were analyzed for levels of energy metabolism, glucose metabolism, and metabolic profile of extramedullary adipose tissue, as well as the osteocyte transcriptome, mitochondrial function, bioenergetics, insulin signaling, and oxidative stress., Results: Circulating sclerostin levels of γOT KO male and female mice were not different from control mice. Male γOT KO mice exhibited a high energy phenotype characterized by increased respiration, heat production, locomotion and food intake. This high energy phenotype in males did not correlate with "beiging" of peripheral adipose depots. However, both sexes showed a trend for reduced fat mass and apparent insulin resistance without changes in glucose tolerance, which correlated with decreased osteocytic responsiveness to insulin measured by AKT activation. The transcriptome of osteocytes isolated from γOT KO males suggested profound changes in cellular metabolism, fuel transport, mitochondria dysfunction, insulin signaling and increased oxidative stress. In MLO-Y4 osteocytes, PPARG deficiency correlated with highly active mitochondria, increased ATP production, and accumulation of reactive oxygen species (ROS)., Conclusions: PPARG in male osteocytes acts as a molecular break on mitochondrial function, and protection against oxidative stress and ROS accumulation. It also regulates osteocyte insulin signaling and fuel usage to produce energy. These data provide insight into the connection between osteocyte bioenergetics and their sex-specific contribution to the balance of systemic energy metabolism. These findings support the concept that the skeleton controls systemic energy expenditure via osteocyte metabolism., 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 GmbH.. All rights reserved.)

In response to mechanical loading of bone, osteocytes produce nitric oxide (NO•) and decrease sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO• production and sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca 2+ ) influx. Increased intracellular Ca 2+ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO• as a regulator of sclerostin degradation downstream of mechano-activated CaMKII. Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO• donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO• production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NO•, and that NO• is necessary and sufficient for sclerostin protein control. Further, selective inhibition of elements within this sclerostin-controlling mechano-transduction pathway indicated that NO• production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO• production following FSS, indicating that CaMKII is needed for NO• production. However, NO• donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO• may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NO•, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:JPS and CWW hold a patent (US 2017/0276666) related to the fluid shear stress device used in this manuscript., (Copyright © 2024. Published by Elsevier Inc.)

Background: Glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH) is a progressive bone disorder which frequently results in femoral head collapse and hip joint dysfunction. Sclerostin (SOST) is principally secreted by osteocytes in bone and plays an important role in bone homeostasis and homeostasis of skeletal integrity. Our previous study reported that short-term use of glucocorticoid increased serum sclerostin levels. Here this study is aimed to identify whether sclerostin played an essential role in the occurrence and development of GA-ONFH., Methods: Glucocorticoid-induced osteonecrosis of femoral head (ARCO stage II) samples were collected and sclerostin staining was conducted. Osteocyte cell line Ocy454, MC3T3-E1 and endothelial cells was used. MC3T3-E1 or endothelial cells were co-cultured with Ocy454 or SOST-silencing Ocy454 in presence of dexamethasone to mimic the crosstalk of various cells in the bone niche. GA-ONFH rat model and SOST knockout model was built to better understand the phenomenon in vivo., Results: Sclerostin was highly concentrated in osteonecrosis patient sample in the necrotic area. Co-culture with osteocytes aggravated the inhibition of dexamethasone on MC3T3-E1 and endothelial cells. Sclerostin derived from osteocytes impaired osteogenesis and angiogenesis via inhibiting the Wnt pathway. In GA-ONFH rat model, SOST knockout ameliorated the incidence of osteonecrosis and improved bone metabolism compared with the wild type group through histological, immunohistochemical and bone metabolic analyses., Conclusion: Sclerostin contribute to pathologic process of GA-ONFH by impairing osteogenesis and angiogenesis., (© 2024. The Author(s).)

Osteocytes are the main mechanosensory cells and the primary regulators of bone metabolic homeostasis. Here, we present a protocol for evaluating the effects of the large gradient high magnetic field (LG-HMF) on osteocyte function. We describe steps for establishing a corresponding cell culture system in the LG-HMF generated by a superconducting magnet. We then detail procedures for using this cell culture system to study the effects of magnetic forces on the structure and function of murine long bone osteocyte Y4 cells. For complete details on the use and execution of this protocol, please refer to Zhang et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Macrophages and osteocytes are important regulators of inflammation, osteogenesis and osteoclastogenesis. However, their interactions under adverse conditions, such as biomaterial-associated infection (BAI) are not fully understood. We aimed to elucidate how factors released from macrophages modulate osteocyte responses in an in vitro indirect 3D co-culture model. Human monocyte-derived macrophages were cultured on etched titanium disks and activated with either IL-4 cytokine (anti-inflammatory M2 phenotype) or Staphylococcus aureus secreted virulence factors to simulate BAI (pro-inflammatory M1 phenotype). Primary osteocytes in collagen gels were then stimulated with conditioned media (CM) from these macrophages. The osteocyte response was analyzed by gene expression, protein secretion, and immunostaining. M1 phenotype macrophages were confirmed by IL-1β and TNF-α secretion, and M2 macrophages by ARG-1 and MRC-1.Osteocytes receiving M1 CM revealed bone inhibitory effects, denoted by reduced secretion of bone formation osteocalcin (BGLAP) and increased secretion of the bone inhibitory sclerostin (SOST). These osteocytes also downregulated the pro-mineralization gene PHEX and upregulated the anti-mineralization gene MEPE. Additionally, exhibited pro-osteoclastic potential by upregulating pro-osteoclastic gene RANKL expression. Nonetheless, M1-stimulated osteocytes expressed a higher level of the potent pro-osteogenic factor BMP-2 in parallel with the downregulation of the bone inhibitor genes DKK1 and SOST, suggesting a compensatory feedback mechanisms. Conversely, M2-stimulated osteocytes mainly upregulated anti-osteoclastic gene OPG expression, suggesting an anti-catabolic effect. Altogether, our findings demonstrate a strong communication between M1 macrophages and osteocytes under M1 (BAI)-simulated conditions, suggesting that the BAI adverse effects on osteoblastic and osteoclastic processes in vitro are partly mediated via this communication. STATEMENT OF SIGNIFICANCE: Biomaterial-associated infections are major challenges and the underlying mechanisms in the cellular interactions are missing, especially among the major cells from the inflammatory side (macrophages as the key cell in bacterial clearance) and the regenerative side (osteocyte as main regulator of bone). We evaluated the effect of macrophage polarization driven by the stimulation with bacterial virulence factors on the osteocyte function using an indirect co-culture model, hence mimicking the scenario of a biomaterial-associated infection. The results suggest that at least part of the adverse effects of biomaterial associated infection on osteoblastic and osteoclastic processes in vitro are mediated via macrophage-to-osteocyte communication., Competing Interests: Declaration competing of 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 Author(s). Published by Elsevier Ltd.. All rights reserved.)

Osteogenesis imperfecta (OI) is a group of severe genetic bone disorders characterized by congenital low bone mass, deformity, and frequent fractures. Type XV OI is a moderate to severe form of skeletal dysplasia caused by WNT1 variants. In this cohort study from southern China, we summarized the clinical phenotypes of patients with WNT1 variants and found that the proportion of type XV patients was around 10.3% (25 out of 243) with a diverse spectrum of phenotypes. Functional assays indicated that variants of WNT1 significantly impaired its secretion and effective activity, leading to moderate to severe clinical manifestations, porous bone structure, and enhanced osteoclastic activities. Analysis of proteomic data from human skeleton indicated that the expression of SOST (sclerostin) was dramatically reduced in type XV patients compared to patients with COL1A1 quantitative variants. Single-cell transcriptome data generated from human tibia samples of patients diagnosed with type XV OI and leg-length discrepancy, respectively, revealed aberrant differentiation trajectories of skeletal progenitors and impaired maturation of osteocytes with loss of WNT1, resulting in excessive CXCL12+ progenitors, fewer mature osteocytes, and the existence of abnormal cell populations with adipogenic characteristics. The integration of multi-omics data from human skeleton delineates how WNT1 regulates the differentiation and maturation of skeletal progenitors, which will provide a new direction for the treatment strategy of type XV OI and relative low bone mass diseases such as early onset osteoporosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Osteoarthritis is a highly prevalent progressive joint disease that still requires an optimal therapeutic approach. Intermittent fasting is an attractive dieting strategy for improving health. Here this study shows that intermittent fasting potently relieves medial meniscus (DMM)- or natural aging-induced osteoarthritic phenotypes. Osteocytes, the most abundant bone cells, secrete excess neuropeptide Y (NPY) during osteoarthritis, and this alteration can be altered by intermittent fasting. Both NPY and the NPY-abundant culture medium of osteocytes (OCY-CM) from osteoarthritic mice possess pro-inflammatory, pro-osteoclastic, and pro-neurite outgrowth effects, while OCY-CM from the intermittent fasting-treated osteoarthritic mice fails to induce significant stimulatory effects on inflammation, osteoclast formation, and neurite outgrowth. Depletion of osteocyte NPY significantly attenuates DMM-induced osteoarthritis and abolishes the benefits of intermittent fasting on osteoarthritis. This study suggests that osteocyte NPY is a key contributing factor in the pathogenesis of osteoarthritis and intermittent fasting represents a promising nonpharmacological antiosteoarthritis method by targeting osteocyte NPY., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Blood vessel growth and osteogenesis in the skeletal system are coupled; however, fundamental aspects of vascular function in osteoblast-to-osteocyte transition remain unclear. Our study demonstrates that vascular smooth muscle cells (VSMCs), but not endothelial cells, are sufficient to drive bone marrow mesenchymal stromal cell-derived osteoblast-to-osteocyte transition via β-catenin signaling and exosome-mediated communication. We found that VSMC-derived exosomes are loaded with transcripts encoding proteins associated with the osteocyte phenotype and members of the WNT/β-catenin signaling pathway. In contrast, endothelial cell-derived exosomes facilitated mature osteoblast differentiation by reprogramming the TGFB1 gene family and osteogenic transcription factors osterix (SP7) and RUNX2. Notably, VSMCs express significant levels of tetraspanins (CD9, CD63, and CD81) and drive the intracellular trafficking of exosomes with a lower membrane zeta potential than those from other cells. Additionally, the high ATP content within these exosomes supports mineralization mechanisms, as ATP is a substrate for alkaline phosphatase. Osteocyte function was further validated by RNA sequencing, revealing activity in genes related to intermittent mineralization and sonic hedgehog signaling, alongside a significant increase in TNFSF11 levels. Our findings unveil a novel role of VSMCs in promoting osteoblast-to-osteocyte transition, thus offering new insights into bone biology and homeostasis, as well as in bone-related diseases. Clinically, these insights could pave the way for innovative therapeutic strategies targeting VSMC-derived exosome pathways to treat bone-related disorders such as osteoporosis. By manipulating these signaling pathways, it may be possible to enhance bone regeneration and improve skeletal health in patients with compromised bone structure and function., 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.)

We and others have seen that osteocytes sense high-impact osteogenic mechanical loading via transient plasma membrane disruptions (PMDs) which initiate downstream mechanotransduction. However, a PMD must be repaired for the cell to survive this wounding event. Previous work suggested that the protein Prkd1 (also known as PKCμ) may be a critical component of this PMD repair process, but the specific role of Prkd1 in osteocyte mechanobiology had not yet been tested. We treated MLO-Y4 osteocytes with Prkd1 inhibitors (Go6976, kbNB 142-70, staurosporine) and generated an osteocyte-targeted (Dmp1-Cre) Prkd1 conditional knockout (CKO) mouse. PMD repair rate was measured via laser wounding and FM1-43 dye uptake, PMD formation and post-wounding survival were assessed via fluid flow shear stress (50 dyn/cm 2 ), and in vitro osteocyte mechanotransduction was assessed via measurement of calcium signaling. To test the role of osteocyte Prkd1 in vivo, Prkd1 CKO and their wildtype (WT) littermates were subjected to 2 weeks of unilateral axial tibial loading and loading-induced changes in cortical bone mineral density, geometry, and formation were measured. Prkd1 inhibition or genetic deletion slowed osteocyte PMD repair rate and impaired post-wounding cell survival. These effects could largely be rescued by treating osteocytes with the FDA-approved synthetic copolymer Poloxamer 188 (P188), which was previously shown to facilitate membrane resealing and improve efficiency in the repair rate of PMD in skeletal muscle myocytes. In vivo, while both WT and Prkd1 CKO mice demonstrated anabolic responses to tibial loading, the magnitude of loading-induced increases in tibial BMD, cortical thickness, and periosteal mineralizing surface were blunted in Prkd1 CKO as compared to WT mice. Prkd1 CKO mice also tended to show a smaller relative difference in the number of osteocyte PMD in loaded limbs and showed greater lacunar vacancy, suggestive of impaired post-wounding osteocyte survival. While P188 treatment rescued loading-induced increases in BMD in the Prkd1 CKO mice, it surprisingly further suppressed loading-induced increases in cortical bone thickness and cortical bone formation. Taken together, these data suggest that Prkd1 may play a pivotal role in the regulation and repair of the PMD response in osteocytes and support the idea that PMD repair processes can be pharmacologically targeted to modulate downstream responses, but suggest limited utility of PMD repair-promoting P188 in improving bone anabolic responses to loading., Competing Interests: Declaration of competing interest The authors state that they have no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Osteocytes are the major actors in bone mechanobiology. Within bone matrix, they are trapped close together in a submicrometric interconnected network: the lacunocanalicular network (LCN). The interstitial fluid circulating within the LCN transmits the mechanical information to the osteocytes that convert it into a biochemical signal. Understanding the interstitial fluid dynamics is necessary to better understand the bone mechanobiology. Due to the submicrometric dimensions of the LCN, making it difficult to experimentally investigate fluid dynamics, numerical models appear as a relevant tool for such investigation. To develop such models, there is a need for geometrical and morphological data on the human LCN. This study aims at providing morphological data on the human LCN from measurement of 27 human femoral diaphysis bone samples using synchrotron radiation nano-computed tomography with an isotropic voxel size of 100 nm. Except from the canalicular diameter, the canalicular morphological parameters presented a high variability within one sample. Some differences in terms of both lacunar and canalicular morphology were observed between the male and female populations. But it has to be highlighted that all the canaliculi cannot be detected with a voxel size of 100 nm. Hence, in the current study, only a specific population of large canaliculi that could be characterize. Still, to the authors knowledge, this is the first time such a data set was introduced to the community. Further processing will be achieved in order to provide new insight on the LCN permeability., 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 Author(s). Published by Elsevier Inc. All rights reserved.)

Osteocytes engage in bone resorption and mineralization surrounding their expansive lacunar-canalicular system (LCS) through peri-LCS turnover. However, fundamental questions persist about where, when, and how often osteocytes engage in peri-LCS turnover and how these processes change with aging. Furthermore, whether peri-LCS turnover is associated with natural variation in cortical tissue strain remains unexplored. To address these questions, we utilized confocal scanning microscopy, immunohistochemistry, and scanning electron microscopy to characterize osteocyte peri-LCS turnover in the cortical (mid-diaphysis) and cancellous (metaphysis) regions of femurs from young adult (5 mo) and early-old-age (22 mo) female C57BL/6JN mice. LCS bone mineralization was measured by the presence of perilacunar fluorochrome labels. LCS bone resorption was measured by immunohistochemical marker of bone resorption. The dynamics of peri-LCS turnover were estimated from serial fluorochrome labeling, where each mouse was administered two labels between 2 and 16 days before euthanasia. Osteocyte participation in mineralizing their surroundings is highly abundant in both cortical and cancellous bone of young adult mice but significantly decreases with aging. LCS bone resorption also decreases with aging. Aging has a greater impact on peri-LCS turnover dynamics in cancellous bone than in cortical bone. Lacunae with recent peri-LCS turnover are larger in both age groups. While peri-LCS turnover is associated with variation in tissue strain between cortical quadrants and intracortical location for 22 mo mice, these associations were not seen for 5 mo mice. The impact of aging on decreasing peri-LCS turnover may have significant implications for bone quality and mechanosensation., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: This study aimed to elucidate whether the administration of parathyroid hormone (PTH) results in remodeling- or modeling-based bone formation in different regions of the murine femora, and whether the PTH-driven bone formation would facilitate osteoblastic differentiation into osteocytes., Methods: Six-week-old male C57BL/6J mice were employed to examine the distribution of alkaline phosphatase (ALP), PHOSPHO1, podoplanin, and calcein labeling in two distinct long bone regions: the metaphyseal trabeculae close to the chondro-osseous junction (COJ) and those distant from the COJ in three mouse groups, a control group receiving a vehicle (sham group) and groups receiving hPTH (1-34) twice a day (PTH BID group) or four times a day (PTH QID group) for two weeks., Results: The sham group showed PHOSPHO1-reactive mature osteoblasts localized primarily at the COJ, whereas the PTH BID/QID groups exhibited extended lines of PHOSPHO1-reactive osteoblasts even in regions distant from the COJ. The PTH QID group displayed fragmented calcein labeling in trabeculae close to the COJ, whereas continuous labeling was observed in trabeculae distant from the COJ. Osteoblasts tended to express podoplanin and PHOSPHO1 independently in the close and distant regions of the sham group, while osteoblasts in the PTH-administered groups showed immunoreactivity of podoplanin and PHOSPHO1 together in the close and distant regions., Conclusions: Administration of PTH may accelerate remodeling-based bone formation in regions close to the COJ while predominantly inducing modeling-based bone formation in distant regions. PTH appeared to simultaneously facilitate osteoblastic bone mineralization and differentiation into osteocytes in both remodeling- and modeling-based 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 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Background: Chondrocyte viability, apoptosis, and migration are closely related to cartilage injury in osteoarthritis (OA) joints. Exosomes are identified as potential therapeutic agents for OA., Objective: This study aimed to investigate the role of exosomes derived from osteocytes in OA, particularly focusing on their effects on cartilage repair and molecular mechanisms., Methods: An injury cell model was established by treating chondrocytes with IL-1β. Cartilage repair was evaluated using cell counting kit-8, flow cytometry, scratch test, and Western Blot. Molecular mechanisms were analyzed using quantitative real-time PCR, bioinformatic analysis, and Western Blot. An OA mouse model was established to explore the role of exosomal DLX2 in vivo ., Results: Osteocyte-released exosomes promoted cell viability and migration, and inhibited apoptosis and extracellular matrix (ECM) deposition. Moreover, exosomes upregulated DLX2 expression, and knockdown of DLX2 activated the Wnt pathway. Additionally, exosomes attenuated OA in mice by transmitting DLX2., Conclusion: Osteocyte-derived exosomal DLX2 alleviated IL-1β-induced cartilage repair and inactivated the Wnt pathway, thereby alleviating OA progression. The findings suggested that osteocyte-derived exosomes may hold promise as a treatment for OA.

Iron overload is a risk factor for osteoporosis due to its oxidative toxicity. Previous studies have demonstrated that an excessive amount of iron increases osteocyte apoptosis and receptor activator of nuclear factor κ-B ligand (RANKL) production, which stimulates osteoclast differentiation in vitro. However, the effects of exogenous iron supplementation-induced iron overload on osteocytes in vivo and its role in iron overload-induced bone loss are unknown. This work aimed to develop an iron overloaded murine model of C57BL/6 mice by intraperitoneal administration of iron dextran for two months. The iron levels in various organs, bone, and serum, as well as the microstructure and strength of bone, apoptosis of osteocytes, oxidative stress in bone tissue, and bone formation and resorption, were assessed. The results showed that 2 months of exogenous iron supplementation significantly increased iron levels in the liver, spleen, kidney, bone tissue, and serum. Iron overload negatively affected bone microstructure and strength. Osteocyte apoptosis and empty lacunae rate were elevated by exogenous iron. Iron overload upregulated RANKL expression but had no significant impact on osteoprotegerin (OPG) and sclerostin levels. Static and dynamic histologic analyses and serum biochemical assay showed that iron overload increased bone resorption without significantly affecting bone formation. Exogenous iron promoted oxidative stress in osteocytes in vivo and in vitro. Additional supplementation of iron chelator (deferoxamine) or N-acetyl-l-cysteine (NAC) partially alleviated bone loss, osteocyte apoptosis, osteoclast formation, and oxidative stress due to iron overload. These findings, in line with prior in vitro studies, suggest that exogenous iron supplementation induces osteoclastogenesis and osteoporosis by promoting osteocyte apoptosis and RANKL production via 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.)

Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. While one formation is increased by removing the PI3K inhibitor (phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)), the effect of direct PI3K activation in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12 wk of age. Both sexes had smaller marrow areas from 6 wk of age. Female mice also exhibited greater cross-sectional area, which continued to increase until 24 wk of age, resulting in a further increase in bone strength. Although both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was more lamellar bone formation, including highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cell-to-cell mitochondrial transfer has recently been shown to play a role in maintaining physiological functions of cell. We previously illustrated that mitochondrial transfer within osteocyte dendritic network regulates bone tissue homeostasis. However, the mechanism of triggering this process has not been explored. Here, we showed that stressed osteocytes in mice release adenosine diphosphate (ADP), resulting in triggering mitochondrial transfer from healthy osteocytes to restore the oxygen consumption rate (OCR) and to alleviate reactive oxygen species accumulation. Furthermore, we identified that P2Y2 and P2Y6 transduced the ADP signal to regulate osteocyte mitochondrial transfer. We showed that mitochondrial metabolism is impaired in aged osteocytes, and there were more extracellular nucleotides release into the matrix in aged cortical bone due to compromised membrane integrity. Conditioned medium from aged osteocytes triggered mitochondrial transfer between osteocytes to enhance the energy metabolism. Together, using osteocyte as an example, this study showed new insights into how extracellular ADP triggers healthy cells to rescue energy metabolism crisis in stressed cells via mitochondrial transfer in tissue homeostasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li 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.)

Osteoporosis is the most common bone disorder, which is a highly dangerous condition that can promote bone metastases. As the current treatment for osteoporosis involves long-term medication therapy and a cure for bone metastasis is not known, ongoing efforts are required for drug development for osteoporosis. Animal experiments, traditionally used for drug development, raise ethical concerns and are expensive and time-consuming. Organ-on-a-chip technology is being developed as a tool to supplement such animal models. In this study, we developed a bone-on-a-chip by co-culturing osteoblasts, osteocytes, and osteoclasts in an extracellular matrix environment that can represent normal bone, osteopenia, and osteoporotic conditions. We then simulated bone metastases using breast cancer cells in three different bone conditions and observed that bone metastases were most active in osteoporotic conditions. Furthermore, it was revealed that the promotion of bone metastasis in osteoporotic conditions is due to increased vascular permeability. The bone-on-a-chip developed in this study can serve as a platform to complement animal models for drug development for osteoporosis and bone metastasis., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

Bone-forming osteoblasts, osteocytes, and bone-resorbing osteoclasts are responsible for life-long skeletal remodeling [...].

The treatment of critical-sized bone defects caused by tumor removal, skeletal injuries, or infections continues to pose a major clinical challenge. A popular potential alternative solution to autologous bone grafts is a tissue-engineered approach that utilizes the combination of mesenchymal stromal/stem cells (MSCs) with synthetic biomaterial scaffolds. This approach aims to support new bone formation by mimicking many of the biochemical and biophysical cues present within native bone. Regrettably, osteocyte cells, crucial for bone maturation and homeostasis, are rarely produced within MSC-seeded scaffolds, thereby restricting the development of fully mature cortical bone from these synthetic implants. In this work, we have constructed a multimodal scaffold by combining electrospun poly(lactic- co -glycolic acid) (PLGA) fibrous scaffolds with poly(ethylene glycol) (PEG)-based hydrogels that mimic the functional unit of cortical bone, osteon (osteon-mimetic) scaffolds. These scaffolds were decorated with a novel bone morphogenic protein-6 (BMP6) peptide (BMP6p) after our findings revealed that the BMP6p drives higher levels of Smad signaling than the full-length protein counterpart, soluble or when bound to the PEG hydrogel backbone. We show that our osteon-mimetic scaffolds, in presenting concentric layers of BMP6p-PEG hydrogel overlaid on MSC-seeded PLGA nanofibers, promoted the rapid formation of osteocyte-like cells with a phenotypic dendritic morphology, producing early osteocyte markers, including E11/gp38 (E11). Maturation of these osteocyte-like cells was further confirmed by the observation of significant dentin matrix protein 1 (DMP1) throughout our bilayered scaffolds after 3 weeks, even when cultured in a medium without dexamethasone (DEX) or any other osteogenic supplements. These results demonstrate that these osteon-mimetic scaffolds, in presenting biochemical and topographical cues reminiscent of the forming osteon, can drive the formation of osteocyte-like cells in vitro from hBMSCs without the need for any osteogenic factor media supplementation.

Introduction: Changes to bone physiology play a central role in the development of osteoarthritis with the mechanosensing osteocyte releasing factors that drive disease progression. This study developed a humanised in vitro model to detect osteocyte responses to either interleukin-6, a driver of degeneration and bone remodelling in animal and human joint injury, or mechanical loading, to mimic osteoarthritis stimuli in joints., Methods: Human MSC cells (Y201) were differentiated in 3-dimensional type I collagen gels in osteogenic media and osteocyte phenotype assessed by RTqPCR and immunostaining. Gels were subjected to a single pathophysiological load or stimulated with interleukin-6 with unloaded or unstimulated cells as controls. RNA was extracted 1-hour post-load and assessed by RNAseq. Markers of pain, bone remodelling, and inflammation were quantified by RT-qPCR and ELISA., Results: Y201 cells embedded within 3D collagen gels assumed dendritic morphology and expressed mature osteocytes markers. Mechanical loading of the osteocyte model regulated 7564 genes (Padj p<0.05, 3026 down, 4538 up). 93% of the osteocyte transcriptome signature was expressed in the model with 38% of these genes mechanically regulated. Mechanically loaded osteocytes regulated 26% of gene ontology pathways linked to OA pain, 40% reflecting bone remodelling and 27% representing inflammation. Load regulated genes associated with osteopetrosis, osteoporosis and osteoarthritis. 42% of effector genes in a genome-wide association study meta-analysis were mechanically regulated by osteocytes with 10 genes representing potential druggable targets. Interleukin-6 stimulation of osteocytes at concentrations reported in human synovial fluids from patients with OA or following knee injury, regulated similar readouts to mechanical loading including markers of pain, bone remodelling, and inflammation., Discussion: We have developed a reproducible model of human osteocyte like cells that express >90% of the genes in the osteocyte transcriptome signature. Mechanical loading and inflammatory stimulation regulated genes and proteins implicated in osteoarthritis symptoms of pain as well as inflammation and degeneration underlying disease progression. Nearly half of the genes classified as 'effectors' in GWAS were mechanically regulated in this model. This model will be useful in identifying new mechanisms underlying bone and joint pathologies and testing drugs targeting those mechanisms., Competing Interests: DM and CB hold patents for the use of glutamate receptor antagonists to prevent osteoarthritis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission., (Copyright © 2024 Gilbert, Jones, Egan, Bonnet, Evans and Mason.)

The traditional understanding of bone mechanosensation implicates osteocytes, canaliculi, and the lacunocanalicular network in biomechanical adaptation. However, recent findings challenge this notion, as shown in advanced teleost fish where anosteocytic bone lacking osteocytes are nevertheless responsive to mechanical load. To investigate specific molecular mechanisms involved in bone mechanoadaptation in osteocytic and anosteocytic fish bone, we conducted a 5-min single swim-training experiment with zebrafish and ricefish, respectively. Through RNASeq analysis of fish spines, analyzed at various time points following swim training, we uncovered distinct gene expression patterns in osteocytic and anosteocytic fish bones. Notably, osteocytic fish bone exhibited an early response to mechanical load, contrasting to a delayed response observed in anosteocytic fish bones, both within 8 h following stimulation. We identified an increase in osteoblast differentiation in anosteocytic bone following training, while chordoblast activity was delayed. This temporal response suggests a time-dependent adaptation in anosteocytic bone, indicating the presence of intricate feedback networks within bone that lacks osteocytes., Competing Interests: Declaration of competing interest Josephine T. Tauer: None. Tobias Thiele: None. Catherine Julien: None. Lior Ofer: None. Paul Zaslansky: None. Ron Shahar: None. Bettina M. Willie receives support for research studies unrelated to the presented study from Mesentech, Novartis, and Mereo BioPharma., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The notion that obesity can be a protective factor for bone health is a topic of ongoing debate. Increased body weight may have a positive impact on bone health due to its mechanical effects and the production of estrogen by adipose tissue. However, recent studies have found a higher risk of bone fracture and delayed bone healing in elderly obese patients, which may be attributed to the heightened risk of bone immune regulation disruption associated with obesity. The balanced functions of bone cells such as osteoclasts, osteoblasts, and osteocytes, would be subverted by aberrant and prolonged immune responses under obese conditions. This review aims to explore the intricate relationship between obesity and bone health from the perspective of osteoimmunology, elucidate the impact of disturbances in bone immune regulation on the functioning of bone cells, including osteoclasts, osteoblasts, and osteocytes, highlighting the deleterious effects of obesity on various diseases development such as rheumatoid arthritis (RA), osteoarthritis (AS), bone fracture, periodontitis. On the one hand, weight loss may achieve significant therapeutic effects on the aforementioned diseases. On the other hand, for patients who have difficulty in losing weight, the osteoimmunological therapies could potentially serve as a viable approach in halting the progression of these disease. Additional research in the field of osteoimmunology is necessary to ascertain the optimal equilibrium between body weight and bone health., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Renal cell carcinoma (RCC) bone metastatis progression is driven by crosstalk between tumor cells and the bone microenvironment, which includes osteoblasts, osteoclasts, and osteocytes. RCC bone metastases (RCCBM) are predominantly osteolytic and resistant to antiresorptive therapy. The molecular mechanisms underlying pathologic osteolysis and disruption of bone homeostasis remain incompletely understood. We previously reported that BIGH3/TGFBI (transforming growth factor-beta-induced protein ig-h3, shortened to BIGH3 henceforth) secreted by colonizing RCC cells drives osteolysis by inhibiting osteoblast differentiation, impairing healing of osteolytic lesions, which is reversible with osteoanabolic agents. Here, we report that BIGH3 induces osteocyte apoptosis in both human RCCBM tissue specimens and in a preclinical mouse model. We also demonstrate that BIGH3 reduces Cx43 expression, blocking gap junction (GJ) function and osteocyte network communication. BIGH3-mediated GJ inhibition is blocked by the lysosomal inhibitor hydroxychloroquine (HCQ), but not osteoanabolic agents. Our results broaden the understanding of pathologic osteolysis in RCCBM and indicate that targeting the BIGH3 mechanism could be a combinational strategy for the treatment of RCCBM-induced bone disease that overcomes the limited efficacy of antiresorptives that target osteoclasts., 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.)

Osteocytes perceive and process mechanical stimuli in the lacuno-canalicular network in bone. As a result, they secrete signaling molecules that mediate bone formation and resorption. To date, few three-dimensional (3D) models exist to study the response of mature osteocytes to biophysical stimuli that mimic fluid shear stress and substrate strain in a mineralized, biomimetic bone-like environment. Here we established a biomimetic 3D bone model by utilizing a state-of-art perfusion bioreactor platform where immortomouse/Dmp1-GFP-derived osteoblastic IDG-SW3 cells were differentiated into mature osteocytes. We evaluated proliferation and differentiation properties of the cells on 3D microporous scaffolds of decellularized bone (dBone), poly(L-lactide-co-trimethylene carbonate) lactide (LTMC), and beta-tricalcium phosphate (β-TCP) under physiological fluid flow conditions over 21 days. Osteocyte viability and proliferation were similar on the scaffolds with equal distribution of IDG-SW3 cells on dBone and LTMC scaffolds. After seven days, the differentiation marker alkaline phosphatase (Alpl), dentin matrix acidic phosphoprotein 1 (Dmp1), and sclerostin (Sost) were significantly upregulated in IDG-SW3 cells (p = 0.05) on LTMC scaffolds under fluid flow conditions at 1.7 ml/min, indicating rapid and efficient maturation into osteocytes. Osteocytes responded by inducing the mechanoresponsive genes FBJ osteosarcoma oncogene (Fos) and prostaglandin-endoperoxide synthase 2 (Ptgs2) under perfusion and dynamic compressive loading at 1 Hz with 5 % strain. Together, we successfully created a 3D biomimetic platform as a robust tool to evaluate osteocyte differentiation and mechanobiology in vitro while recapitulating in vivo mechanical cues such as fluid flow within the lacuno-canalicular network. STATEMENT OF SIGNIFICANCE: This study highlights the importance of creating a three-dimensional (3D) in vitro model to study osteocyte differentiation and mechanobiology, as cellular functions are limited in two-dimensional (2D) models lacking in vivo tissue organization. By using a perfusion bioreactor platform, physiological conditions of fluid flow and compressive loading were mimicked to which osteocytes are exposed in vivo. Microporous poly(L-lactide-co-trimethylene carbonate) lactide (LTMC) scaffolds in 3D are identified as a valuable tool to create a favorable environment for osteocyte differentiation and to enable mechanical stimulation of osteocytes by perfusion and compressive loading. The LTMC platform imitates the mechanical bone environment of osteocytes, allowing the analysis of the interaction with other cell types in bone under in vivo biophysical stimuli., 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 Author(s). Published by Elsevier Ltd.. All rights reserved.)

The maternal skeleton experiences significant bone loss during lactation, followed by rapid restoration post weaning. Parathyroid-related protein (PTHrP)-induced acidification of the perilacunar matrix by osteocytes is crucial in this process, yet its mechanism remains unclear. Here, we identify Cx43 hemichannels (HCs) as key mediators of osteocyte acidification and perilacunar-canalicular remodeling (PLR). Utilizing transgenic mouse models expressing dominant-negative Cx43 mutants, we show that mice with impaired Cx43 HCs exhibit attenuated lactation-induced responses compared to wild-type and only gap junction-impaired groups, including lacunar enlargement, upregulation of PLR genes, and bone loss with compromised mechanical properties. Furthermore, inhibition of HCs by a Cx43 antibody blunts PTHrP-induced calcium influx and protein kinase A activation, followed by impaired osteocyte acidification. Additionally, impeded HCs suppress bone recovery during the post-lactation period. Our findings highlight the pivotal role of Cx43 HCs in orchestrating dynamic bone changes during lactation and recovery by regulating acidification and remodeling enzyme expression., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bone tissue represents the most frequent site of cancer metastasis. We developed a hemichannel-activating antibody, Cx43-M2. Cx43-M2, directly targeting osteocytes in situ, activates osteocytic hemichannels and elevates extracellular ATP, thereby inhibiting the growth and migration of cultured breast and osteosarcoma cancer cells. Cx43-M2 significantly decreases breast cancer metastasis, osteosarcoma growth, and osteolytic activity, while improving survival rates in mice. The antibody's inhibition of breast cancer and osteosarcoma is dose dependent in both mouse and human cancer metastatic models. Furthermore, Cx43-M2 enhances anti-tumor immunity by increasing the population and activation of tumor-infiltrating immune-promoting effector T lymphocytes, while reducing immune-suppressive regulatory T cells. Our results suggest that the Cx43-M2 antibody, by activating Cx43 hemichannels and facilitating ATP release and purinergic signaling, transforms the cancer microenvironment from a supportive to a suppressive state. Collectively, our study underscores the potential of Cx43-M2 as a therapeutic for treating breast cancer bone metastasis and osteosarcoma., Competing Interests: Declaration of interests J.X.J. is a co-founder, shareholder, and a member of the Scientific Advisory Board of AlaMab Therapeutics. J.X.J., M.A.R., S.G., W.X., N.Z., and Z.A. are listed as inventors on relevant patent applications that were exclusively licensed to AlaMab Therapeutics by the Board of Regents of the University of Texas system. The University of Texas has a financial interest in AlaMab in the form of licensing., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

With exercise, muscle and bone produce factors with beneficial effects on brain, fat, and other organs. Exercise in mice increased fibroblast growth factor 23 (FGF23), urine phosphate, and the muscle metabolite L-β-aminoisobutyric acid (L-BAIBA), suggesting that L-BAIBA may play a role in phosphate metabolism. Here, we show that L-BAIBA increases in serum with exercise and elevates Fgf23 in osteocytes. The D enantiomer, described to be elevated with exercise in humans, can also induce Fgf23 but through a delayed, indirect process via sclerostin. The two enantiomers both signal through the same receptor, Mas-related G-protein-coupled receptor type D, but activate distinct signaling pathways; L-BAIBA increases Fgf23 through Gαs/cAMP/PKA/CBP/β-catenin and Gαq/PKC/CREB, whereas D-BAIBA increases Fgf23 indirectly through sclerostin via Gαi/NF-κB. In vivo, both enantiomers increased Fgf23 in bone in parallel with elevated urinary phosphate excretion. Thus, exercise-induced increases in BAIBA and FGF23 work together to maintain phosphate homeostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Glucocorticoids (GC) and parathyroid hormone (PTH) are widely used therapeutic endocrine hormones where their effects on bone and joint arise from actions on multiple skeletal cell types. In osteocytes, GC and PTH exert opposing effects on perilacunar canalicular remodeling (PLR). Suppressed PLR can impair bone quality and joint homeostasis, including in GC-induced osteonecrosis. However, combined effects of GC and PTH on PLR are unknown. Given the untapped potential to target osteocytes to improve skeletal health, this study sought to test the feasibility of therapeutically mitigating PLR suppression. Focusing on subchondral bone and joint homeostasis, we hypothesize that PTH(1-34), a PLR agonist, could rescue GC-suppressed PLR. The skeletal effects of GC and PTH(1-34), alone or combined, were examined in male and female mice by micro-computed tomography, mechanical testing, histology, and gene expression analysis. For each outcome, females were more responsive to GC and PTH(1-34) than males. GC and PTH(1-34) exerted regional differences, with GC increasing trabecular bone volume but reducing cortical bone thickness, stiffness, and ultimate force. Despite PTH(1-34)'s anabolic effects on trabecular bone, it did not rescue GC's catabolic effects on cortical bone. Likewise, cartilage integrity and subchondral bone apoptosis, tartrate-resistant acid phosphatase (TRAP) activity, and osteocyte lacunocanalicular networks showed no evidence that PTH(1-34) could offset GC-dependent effects. Rather, GC and PTH(1-34) each increased cortical bone gene expression implicated in bone resorption by osteoclasts and osteocytes, including Acp5, Mmp13, Atp6v0d2, Ctsk , differences maintained when GC and PTH(1-34) were combined. Since PTH(1-34) is insufficient to rescue GC's effects on young female mouse bone, future studies are needed to determine if osteocyte PLR suppression, due to GC, aging, or other factors, can be offset by a PLR agonist., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yee, Meliadis, Kaya, Chang and Alliston.)

Obesity can increase the risk of bone fragility, even when bone mass is intact. This fragility stems from poor bone quality, potentially caused by deficiencies in bone matrix material properties. However, cellular and molecular mechanisms leading to obesity-related bone fragility are not fully understood. Using male mouse models of obesity, we discovered TGF-β signaling plays a critical role in mediating the effects of obesity on bone. High-carbohydrate and high-fat diets increase TGF-β signaling in osteocytes, which impairs their mitochondrial function, increases cellular senescence, and compromises perilacunar/canalicular remodeling and bone quality. By specifically inhibiting TGF-β signaling in mouse osteocytes, some of the negative effects of high-fat and high-carbohydrate diets on bones, including the lacunocanalicular network, perilacunar/canalicular remodeling, senescence, and mechanical properties such as yield stress, were mitigated. DMP1-Cre-mediated deletion of TGF-β receptor II also blunted adverse effects of high-fat and high-carbohydrate diets on energy balance and metabolism. These findings suggest osteocytes are key in controlling bone quality in response to high-fat and high-carbohydrate diets. Calibrating osteocyte function could mitigate bone fragility associated with metabolic diseases while reestablishing energy balance.

Osteogenesis imperfecta (OI) increases fracture risk due to changes in bone quantity and quality caused by mutations in collagen and its processing proteins. Current therapeutics improve bone quantity, but do not treat the underlying quality deficiencies. Male and female G610C+/- mice, a murine model of OI, were treated with a combination of raloxifene and in vivo axial tibial compressive loading starting at 10 weeks of age and continuing for 6 weeks to improve bone quantity and quality. Bone geometry and mechanical properties were measured to determine whole bone and tissue-level material properties. A colocalized Raman/nanoindentation system was used to measure chemical composition and nanomechanical properties in newly formed bone compared to old bone to determine if bone formed during the treatment regimen differed in quality compared to bone formed prior to treatment. Lastly, lacunar geometry and osteocyte apoptosis were assessed. OI mice were able to build bone in response to the loading, but this response was less robust than in control mice. Raloxifene improved some bone material properties in female but not male OI mice. Raloxifene did not alter nanomechanical properties, but loading did. Lacunar geometry was largely unchanged with raloxifene and loading. However, osteocyte apoptosis was increased with loading in raloxifene treated female mice. Overall, combination treatment with raloxifene and loading resulted in positive but subtle changes to bone quality., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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

Osteocytes are embedded in lacunae and connected by canaliculi (lacuno-canalicular network, LCN). Bones from mice with X-linked hypophosphatemia (Hyp), which have impaired production of 1,25 dihydroxyvitamin D (1,25D) and hypophosphatemia, have abnormal LCN structure that is improved by treatment with 1,25D or an anti-FGF23 targeting antibody, supporting roles for 1,25D and phosphate in regulating LCN remodeling. Bones from mice lacking the vitamin D receptor (VDR) in osteocytes (Vdrf/f;Dmp1Cre+) and mice lacking the sodium phosphate transporter 2a (Npt2aKO), which have low serum phosphate with high serum 1,25D, have impaired LCN organization, demonstrating that osteocyte-specific actions of 1,25D and hypophosphatemia regulate LCN remodeling. In osteoclasts, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) is critical for stimulating bone resorption. Since osteocytes also resorb matrix, we hypothesize that NFATc1 plays a role in 1,25D and phosphate-mediated LCN remodeling. Consistent with this, 1,25D and phosphate suppress Nfatc1 mRNA expression in IDG-SW3 osteocytes, and knockdown of Nfatc1 expression in IDG-SW3 cells blocks 1,25D- and phosphate-mediated suppression of matrix resorption gene expression and 1,25D- and phosphate-mediated suppression of RANKL-induced acidification of the osteocyte microenvironment. To determine the role of NFATc1 in 1,25D- and phosphate-mediated LCN remodeling in vivo, histomorphometric analyses of tibiae from mice lacking osteocyte-specific Nfatc1 in Vdrf/f;Dmp1Cre+ and Npt2aKO mice were performed, demonstrating that bones from these mice have decreased lacunar size and expression of matrix resorption genes, and improved canalicular structure compared to Vdrf/f;Dmp1Cre+ and Npt2aKO control. This study demonstrates that NFATc1 is necessary for 1,25D- and phosphate-mediated regulation of LCN remodeling., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Osteocytes locally remodel their surrounding tissue through perilacunar canalicular remodeling (PLR). During lactation, osteocytes remove minerals to satisfy the metabolic demand, resulting in increased lacunar volume, quantifiable with synchrotron X-ray radiation micro-tomography (SRµCT). Although the effects of lactation on PLR are well-studied, it remains unclear whether PLR occurs uniformly throughout the bone and what mechanisms prevent PLR from undermining bone quality. We used SRµCT imaging to conduct an in-depth spatial analysis of the impact of lactation and osteocyte-intrinsic MMP13 deletion on PLR in murine bone. We found larger lacunae undergoing PLR are located near canals in the mid-cortex or endosteum. We show lactation-induced hypomineralization occurs 14 µm away from lacunar edges, past a hypermineralized barrier. Our findings reveal that osteocyte-intrinsic MMP13 is crucial for lactation-induced PLR near lacunae in the mid-cortex but not for whole-bone resorption. This research highlights the spatial control of PLR on mineral distribution during lactation., (© 2024. The Author(s).)

Osteoporotic fractures are a major complication of long-term glucocorticoid therapy. Glucocorticoids transiently increase bone resorption, but they predominantly inhibit bone formation and induce osteocyte apoptosis, leading to bone loss. Current treatments of glucocorticoid-induced osteoporosis aim mainly at reducing bone resorption and are, therefore, inadequate. We previously showed that signaling via the NO/cGMP/protein kinase G pathway plays a key role in skeletal homeostasis. Here, we show that pharmacological PKG activation with the guanylyl cyclase-1 activator cinaciguat or expression of a constitutively active, mutant PKG2R242Q restored proliferation, differentiation, and survival of primary mouse osteoblasts exposed to dexamethasone. Cinaciguat treatment of WT mice or osteoblast-specific expression of PKG2R242Q in transgenic mice prevented dexamethasone-induced loss of cortical bone mass and strength. These effects of cinaciguat and PKG2R242Q expression were due to preserved bone formation parameters and osteocyte survival. The basis for PKG2's effects appeared to be through recovery of Wnt/β-catenin signaling, which was suppressed by glucocorticoids but critical for proliferation, differentiation, and survival of osteoblast-lineage cells. Cinaciguat reduced dexamethasone activation of osteoclasts, but this did not occur in the PKG2R242Q transgenic mice, suggesting a minor role in osteoprotection. We propose that existing PKG-targeting drugs could represent a novel therapeutic approach to prevent glucocorticoid-induced osteoporosis.

Although mechanical loading is essential for maintaining bone health and combating osteoporosis, its practical application is limited to a large extent by the high variability in bone mechanoresponsiveness. Here, we found that gut microbial depletion promoted a significant reduction in skeletal adaptation to mechanical loading. Among experimental mice, we observed differences between those with high and low responses to exercise with respect to the gut microbial composition, in which the differential abundance of Lachnospiraceae contributed to the differences in bone mechanoresponsiveness. Microbial production of L-citrulline and its conversion into L-arginine were identified as key regulators of bone mechanoadaptation, and administration of these metabolites enhanced bone mechanoresponsiveness in normal, aged, and ovariectomized mice. Mechanistically, L-arginine-mediated enhancement of bone mechanoadaptation was primarily attributable to the activation of a nitric-oxide-calcium positive feedback loop in osteocytes. This study identifies a promising anti-osteoporotic strategy for maximizing mechanical loading-induced skeletal benefits via the microbiota-metabolite axis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose of Review: This review summarizes evidence on osteocyte support of extramedullary and bone marrow adipocyte development and discusses the role of endogenous osteocyte activities of nuclear receptors peroxisome proliferator-activated receptor gamma (PPARG) and alpha (PPARA) in this support., Recent Findings: PPARG and PPARA proteins, key regulators of glucose and fatty acid metabolism, are highly expressed in osteocytes. They play significant roles in the regulation of osteocyte secretome and osteocyte bioenergetics; both activities contributing to the levels of systemic energy metabolism in part through an effect on metabolic function of extramedullary and bone marrow adipocytes. The PPARs-controlled osteocyte endocrine/paracrine activities, including sclerostin expression, directly regulate adipocyte function, while the PPARs-controlled osteocyte fuel utilization and oxidative phosphorylation contribute to the skeletal demands for glucose and fatty acids, whose availability is under the control of adipocytes. Bone is an inherent element of systemic energy metabolism with PPAR nuclear receptors regulating osteocyte-adipocyte metabolic axes., (© 2024. The Author(s).)

Hypercholesterolaemia is a systemic metabolic disease, but the role of organs other than liver in cholesterol metabolism is unappreciated. The phenotypic characterization of the Tsc1 Dmp1 mice reveal that genetic depletion of tuberous sclerosis complex 1 (TSC1) in osteocytes/osteoblasts (Dmp1-Cre) triggers progressive increase in serum cholesterol level. The resulting cholesterol metabolic dysregulation is shown to be associated with upregulation and elevation of serum amyloid A3 (SAA3), a lipid metabolism related factor, in the bone and serum respectively. SAA3, elicited from the bone, bound to toll-like receptor 4 (TLR4) on hepatocytes to phosphorylate c-Jun, and caused impeded conversion of cholesterol to bile acids via suppression on cholesterol 7 α-hydroxylase (Cyp7a1) expression. Ablation of Saa3 in Tsc1 Dmp1 mice prevented the CYP7A1 reduction in liver and cholesterol elevation in serum. These results expand the understanding of bone function and hepatic regulation of cholesterol metabolism and uncover a potential therapeutic use of pharmacological modulation of SAA3 in hypercholesterolaemia., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background: Oxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway., Methods: MLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H 2 O 2 ) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining., Results: The experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H 2 O 2 -induced stress conditions., Conclusions: Nrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H 2 O 2 -induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes., 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 GmbH.. All rights reserved.)

This study aims to investigate the role and mechanism of Gusong Qianggu Decoction(GSQG) in attenuating bone loss in ovariectomized mice by targeting the endoplasmic reticulum stress(ERS)-induced apoptosis of osteocytes. After the modeling of osteoporosis in mice with bilateral ovary removal(OVX), 60 mice were randomized by the random number method into six groups: sham,model, low-, medium-, and high-dose GSQG(GSQG-L, GSQG-M, and GSQG-H, respectively), and estradiol(E&#95;2), with 10 mice in each group. The mice in each group were administrated with corresponding drugs by gavage one month after surgery and the administration lasted for 3 months. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the serum levels of osteocalcin(OCN), procollagen type Ⅰ N-terminal propeptide(PINP), carboxy-terminal cross-linked telopeptide of type Ⅰ collagen(CTX),and anti-tartarte acid phosphatase 5b(TRAcP-5b). Micro-CT was employed to observe the changes in bone microstructure of the distal femur. Hematoxylin-eosin(HE) staining was employed to observe the morphology of the bone tissue. RT-qPCR was conducted to determine the m RNA levels of tibial stem osteogenesis-associated genes [type Ⅰ collagen(Col-Ⅰ), alkaline phosphatase(ALP), Runtrelated transcription factor-2(Runx2), bone sialoprotein(BSP), and OCN] and bone-breaking related genes [tartrate-resistant acid phosphatase(TRAP), nuclear factor-activated T cell 1(NFATc1), and cathepsin K(CATK)]. TUNEL staining and immunohistochemistry were employed to detect the apoptosis of osteoblasts. Western blot was employed to measure the expression of ERS-related proteins glucose-regulated protein 78( Grp78), protein kinase RNA-like endoplasmic reticulum kinase( PERK), phosphorylated PERK(p-PERK),eukaryotic translation initiation factor 2 alpha(eIF2α), phosphorylated e IF2α(p-eIF2α), inositol-requiring enzyme 1 alpha(IRE1α), phosphorylated IRE1α(p-IRE1α), and activating transcription factor 6(ATF6) in the proximal tibial bone tissue. The results showed that GSQG significantly recovered the levels of OCN, PINP, TRAc P-5b, and CTX in the serum of ovariectomized mice, and Micro-CT showed that GSQG improved the bone microstructure of distal femur in a dose-dependent manner. Compared with the model group, GSQG widened and increased the bone trabeculae, restored the reticular structure with neat arrangement and enlarged interstitial gaps, and reduced the number of TUNEL-positive cells(P&lt;0. 05, P&lt;0. 01). Furthermore, GSQG down-regulated the expression levels of cysteine aspartate protease-3( caspase-3) and factor Bcl-2-associated X protein( Bax)(P&lt; 0. 05,P&lt;0. 01) and up-regulated the expression level of Bcl-2(P&lt;0. 05, P&lt;0. 01). The GSQG groups showed up-regulated m RNA levels of Col-Ⅰ, ALP, Runx2, BSP, and OCN(P&lt; 0. 01) and down-regulated m RNA levels of TRAP, NFATc1, and CATK(P&lt; 0. 05,P&lt;0. 01). In addition, GSQG, especially GSQG-H, down-regulated the protein levels of Grp78, p-PERK, p-eIF2, p-IRE1α, and ATF6(P&lt; 0. 05, P&lt; 0. 01). In conclusion, GSQG can inhibit the apoptosis of osteocytes by inhibiting the Grp78/PERK/e IF2α/IRE1α/ATF6 signaling pathway in the proximal tibia tissue, thus reducing bone loss in ovariectomized mice.