Bone metastasis is a devastating complication for advanced-stage prostate cancer patients. Osteocytes, as the primary mechanosensors in bone, have been recently investigated for their role in prostate cancer bone metastasis. In vivo findings show potential benefits of exercise as a preventative intervention strategy for bone metastasis. In contrast, in vitro studies indicate direct prostate cancer-osteocyte interactions under mechanical loading promote prostate cancer growth and migration. These findings are not consistent with in vivo results and may be more reflective of late-stage metastatic colonization. Here, the role of flow-stimulated osteocytes during early-stage bone metastasis, particularly prostate cancer-endothelial interactions, is examined. Flow-stimulated osteocytes reduce PC-3 prostate cancer cell adhesion and trans-endothelial migration by 32.3% and 40% compared to static controls. Both MLO-Y4 and primary murine osteocytes under mechanical loading regulate the extravasation distance and frequency of PC-3 cells in a microfluidic tissue model. Application of vascular cellular adhesion molecule 1 (VCAM-1) neutralizing antibody abolishes the difference in cancer cell adhesion, extravasation frequency, and number of extravasated PC-3 cells between static and flow-stimulated groups. Taken together, the role of osteocytes in early-stage bone metastasis using PC-3 cells as a model is demonstrated here, bridging the gap between in vitro and in vivo findings.

Aging-related bone loss significantly impacts the growing elderly population globally, leading to debilitating conditions such as osteoporosis. Senescent osteocytes play a crucial role in the aging process of bone. This longitudinal study examines the impact of continuous local and paracrine exposure to senescence-associated secretory phenotype (SASP) factors on biophysical and biomolecular markers in osteocytes. Significant cytoskeletal stiffening in irradiated (IR) osteocytes are found, accompanied by expansion of F-actin areas and a decline in dendritic integrity. These changes, correlating with alterations in pro-inflammatory cytokine levels and osteocyte-specific gene expression, support the reliability of biophysical markers for identifying senescent osteocytes. Notably, local accumulation of SASP factors have a more pronounced impact on osteocyte biophysical properties than paracrine effects, suggesting that the interplay between local and paracrine exposure can substantially influence cellular aging. This study underscores the importance of osteocyte mechanical and morphological properties as biophysical markers of senescence, highlighting their time dependence and differential effects of local and paracrine SASP exposure. Collectively, the investigation into biophysical senescence markers offers unique and reliable functional hallmarks for the non-invasive identification of senescent osteocytes, providing insights that can inform therapeutic strategies to mitigate aging-related bone loss.

To explore the effects of Icaritin (ICA) on the regulation of osteocyte exosomal miRNAs and to promote the understanding of the potential molecular mechanisms involved in bone repair by ICA.

MLO-Y4 cells were treat with PBS or 10 µM ICA for 24 h and the supernatant was collected. Exosomes were isolated and purified according to standard methods, and identified by transmission electron microscopy, nanoparticle tracking analysis and protein blotting. Exosomal miRNAs were analysed by RNA sequencing.

Osteocyte exosomes were successfully isolated and characterised. MiRNA sequencing showed that two known exosomal miRNAs (miR-128-3p, miR-30a-5p) were significantly up-regulated and two were significantly down-regulated (miR-5112, miR-1285) after ICA intervention.

Based on the findings, ICA regulates several miRNAs of osteocytes, which deepen our understanding of the therapeutic effects and mechanisms of ICA on skeletal diseases.

Osteocytes are the most abundant cell type in bone tissue, of which the impact on bone homeostasis is still not clear. This study explored the impact of icaritin on osteocytes and their derived exosomes. By doing so, we hope to contribute to the understanding the therapeutic potential of ICA and osteocytes in maintaining bone health and treating conditions such as osteoporosis.

Background/Objective: Osteocytes are the most abundant cell type in the skeleton, with key endocrine functions, particularly in regulating osteoblast and osteoclast activity to maintain bone quality. Angiotensin II (Ang II), a critical component of the renin-angiotensin-aldosterone system, is well-known for its role in vasoconstriction during hypertension. Beyond its cardiovascular functions, Ang II participates in various biological processes, including bone metabolism. While its influence on osteoblast proliferation, differentiation, and osteoclastogenesis has been documented, its effects on osteocytes remain unexplored. This study hypothesized that Ang II enhances the osteoclastogenic activity of osteocytes. Methods: Mouse calvariae were cultured ex vivo in an Ang II-containing medium, analyzed via immunohistochemistry, and evaluated for osteoclastogenic gene expression through real-time PCR. Western blotting was employed to assess protein levels and signaling pathway activation in the MLO-Y4 osteocytic cell line in vitro. Results: Ang II significantly increased the expression of receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). These effects were abrogated by azilsartan, a blocker targeting Ang II type 1 receptors (AT1R). p38 and ERK1/2 in the MAPK pathway were also activated by Ang II. Conclusions: Ang II enhances osteocyte-mediated osteoclastogenesis via AT1R activation, highlighting its potential as a therapeutic target for bone diseases.

Aging-related bone loss significantly impacts the growing elderly population globally, leading to debilitating conditions such as osteoporosis. Senescent osteocytes play a crucial role in the aging process of bone. This longitudinal study examines the impact of continuous local and paracrine exposure to senescence-associated secretory phenotype (SASP) factors on senescence-associated biophysical and biomolecular markers in osteocytes. We found significant cytoskeletal stiffening in irradiated osteocytes, accompanied by expansion of F-actin areas and a decline in dendritic integrity. These changes, correlating with alterations in pro-inflammatory cytokine levels and osteocyte-specific gene expression, support the reliability of biophysical markers for identifying senescent osteocytes. Notably, local accumulation of SASP factors had a more pronounced impact on osteocyte properties than paracrine effects, suggesting that the interplay between local and paracrine exposure could substantially influence cellular aging. This study underscores the importance of osteocyte mechanical and morphological properties as biophysical markers of senescence, highlighting their time-dependence and differential effects of local and paracrine SASP exposure. Collectively, our investigation into biophysical senescence markers offer unique and reliable functional hallmarks for non-invasive identification of senescent osteocytes, providing insights that could inform therapeutic strategies to mitigate aging-related bone loss.

In this study, we developed a hydrostatic pressurizing chamber capable of applying hydrostatic pressure to osteocytic spheroids derived from mouse osteoblastic MC3T3-E1 cells. Our results demonstrate that a 4-h exposure to 200 kPa of hydrostatic pressure did not alter the apparent morphology of the spheroids. However, gene expression analysis revealed a significant up-regulation of Sost, a marker of late-stage osteocyte differentiation.

Osteocytes are the main cells in mineralized bone tissue. Elevated osteocyte apoptosis has been observed in lytic bone lesions of patients with multiple myeloma. However, their precise contribution to bone metastasis remains unclear. Here, we investigated the pathogenic mechanisms driving melanoma-induced osteocyte death. Both in vivo models and in vitro assays were combined with untargeted RNA sequencing approaches to explore the pathways governing melanoma-induced osteocyte death. We could show that ferroptosis is the primary mechanism behind osteocyte death in the context of melanoma bone metastasis. HMOX1 was identified as a crucial regulatory factor in this process, directly involved in inducing ferroptosis and affecting osteocyte viability. We uncover a non-canonical pathway that involves excessive autophagy-mediated ferritin degradation, highlighting the complex relationship between autophagy and ferroptosis in melanoma-induced osteocyte death. In addition, HIF1α pathway was shown as an upstream regulator, providing a potential target for modulating HMOX1 expression and influencing autophagy-dependent ferroptosis. In conclusion, our study provides insight into the pathogenic mechanisms of osteocyte death induced by melanoma bone metastasis, with a specific focus on ferroptosis and its regulation. This would enhance our comprehension of melanoma-induced osteocyte death.

Bioactive cerium-doped (Ce-BGs) glasses with proven antioxidant properties, which may reduce post-implant oxidative stress, were studied in vitro and in vivo to evaluate their application in bone regeneration. Based on the Kokubo (K) composition, they contain 3.6 and 5.3 mol% cerium (referred to as K3.6 and K5.3, respectively).

Ce-BGs were synthesized by melting and sieved to produce granules (size range = 200-500 µm). In vitro studies were conducted against MLO-Y4 cells using direct Neutral Red (NR) and indirect Bromo-2-deoxyUridine (BrdU) assays to assess cell viability and proliferation respectively. In vivo studies were carried out using a New Zealand white rabbit model to evaluate bone healing potential.

NR results showed a significant increase in cell viability for Ce-BGs: 77% for K and 79 and 85% for K3.6 and K5.3, after 24 h. After 72 h, cell viability decreased for K to 58% and increased for K3.6 and K5.3 (76% and 116% respectively). Cerium inhibits cell proliferation in BrdU assay as explainable by the increased durability of Ce-BGs. In vivo studies, after 30 and 60 days, revealed a delayed degradation for Ce-BGs that can stimulate the osteo-regeneration without inflammatory or degenerative effects. Moreover, the new bone area (NBA) was higher for Ce-BGs compared to control; after 60 days 32% for K5.3 versus 21% for K.

Ce-BGs granules show improved direct cytocompatibility in vitro and enhance the long-term bone remodeling process in vivo, contributing to a more controlled and effective bone healing compared to the K granules. This improved behavior can be linked to the antioxidant and anti-inflammatory properties of cerium, that can assist bone regeneration and reduce implant-associated inflammation, and to their slower dissolution rate that supports the controlled release of ions. These results suggest Ce-BGs as a promising device for therapeutic applications on hard tissues.

Renal osteodystrophy (ROD) leads to increased fractures, potentially due to underlying low bone turnover in chronic kidney disease (CKD). We hypothesized that indoxyl sulfate (IS), a circulating toxin elevated in CKD and a ligand for the aryl hydrocarbon receptor (AhR), may target the osteocytes leading to bone cell uncoupling in ROD. The IDG-SW3 osteocytes were cultured for 14 days (early) and 35 days (mature osteocytes) and incubated with 500 μM of IS after dose finding studies to confirm AhR activation. Long-term incubation of IS for 14 days led to decreased expression of Tnfsf11/Tnfrsf11b ratio (RANKL/OPG), which would increase osteoclast activity, and increased expression of Wnt inhibitors Sost and Dkk1, which would decrease bone formation in addition to decreased mineralization and alkaline phosphatase (ALP) activity. When osteocytes were incubated with IS and the AhR translocation inhibitor CH223191, mineralization and ALP activity were restored. However, the Tnfsf11/Tnfrsf11b ratio and Sost, Dkk1 expression were not altered compared with IS alone, suggesting more complex signaling. In both early and mature osteocytes, co-culture with parathyroid hormone (PTH) and IS reversed the IS-induced upregulation of Sost and Dkk1, and IS enhanced the PTH-induced increase of the Tnfsf11/Tnfrsf11b ratio. Co-culture of IS with PTH additively enhanced the AhR activity assessed by Cyp1a1 and Cyp1b1 expression. In summary, IS in the absence of PTH increased osteocyte messenger RNA (mRNA) Wnt inhibitor expression in both early and mature osteocytes, decreased mRNA expression ofTnfsf11/Tnfrsf11b ratio and decreased mineralization in early osteocytes. These changes would lead to decreased resorption and formation resulting in low bone remodeling. These data suggest IS may be important in the underlying low turnover bone disease observed in CKD when PTH is not elevated. In addition, when PTH is elevated, IS interacts to further increase Tnfsf11/Tnfrsf11b ratio for osteoclast activity in both early and mature osteocytes, which would worsen bone resorption.

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.

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.

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/cm2), 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.

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.

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.

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 (H2O2) 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.

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 H2O2-induced stress conditions.

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 H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

Bone metastasis is a common complication of certain cancers such as melanoma. The spreading of cancer cells into the bone is supported by changes in the bone marrow environment. The specific role of osteocytes in this process is yet to be defined. By RNA-seq and chemokines screening we show that osteocytes release the chemokine CXCL5 when they are exposed to melanoma cells. Osteocytes-mediated CXCL5 secretion enhanced the migratory and invasive behaviour of melanoma cells. When the expression of the CXCL5 receptor, CXCR2, was down-regulated in melanoma cells in vitro, we observed a significant decrease in melanoma cell migration in response to osteocytes. Furthermore, melanoma cells with down-regulated CXCR2 expression showed less bone metastasis and less bone loss in the bone metastasis model in vivo. Furthermore, when simultaneously down-regulating CXCL5 in osteocytes and CXCR2 in melanoma cells, melanoma progression was abrogated in vivo. In summary, these data suggest a significant role of osteocytes in bone metastasis of melanoma, which is mediated through the CXCL5-CXCR2 pathway.

Yerba mate (YM, Ilex paraguariensis) consumption beneficially affects the bones. However, whether YM components exert their effect on bone cells directly remains elusive.

We evaluated how main YM components affect osteoblastic (MC3T3-E1) and osteocytic (MLO-Y4) cells in vitro when administered separately or in an aqueous extract. MC3T3-E1 and MLO-Y4 cells were exposed to three different experimental conditions: (1) Caffeine, chlorogenic acid, and their combinations; (2) Caffeine, rutin, and their combinations; (3) Aqueous YM extract.

All polyphenol and caffeine concentrations as well as that of their tested combinations significantly increased MC3T3-E1 cell viability from 16.6% to 34.8% compared to the control. In MLO-Y4 cells, the lowest rutin and the two highest caffeine concentrations significantly increased cell viability by 11.9, 14.9, and 13.7%, respectively. While rutin and caffeine combinations tended to increase MLO-Y4 cell viability, different chlorogenic acid and caffeine combinations did not affect it. Finally, the aqueous YM extract significantly increased MLO-Y4, MC3T3-E1, and differentiated MC3T3-E1 cell viability compared to the control without treatment.

YM components (rutin, chlorogenic acid, and caffeine) positively affected bone cells, mainly pre-osteoblast cells. Moreover, the aqueous YM extract significantly increased MLO-Y4, MC3T3-E1, and differentiated MC3T3-E1 cell viabilities indicating an additional relevant nutritional property of YM infusion. Further studies would be required to elucidate the underlying effector mechanism of YM on the bones and its relationship with previously described in vivo positive effects.

During bone formation, osteoblasts are embedded in a collagen-rich osteoid tissue and differentiate into an extensive 3D osteocyte network throughout the mineralizing matrix. However, how these cells dynamically remodel the matrix and undergo 3D morphogenesis remains poorly understood. Although previous reports investigated the impact of matrix stiffness in osteocyte morphogenesis, the role of matrix viscoelasticity is often overlooked. Here, we report a viscoelastic alginate-collagen interpenetrating network (IPN) hydrogel for 3D culture of murine osteocyte-like IDG-SW3 cells. The IPN hydrogels consist of an ionically crosslinked alginate network to tune stress relaxation as well as a permissive collagen network to promote cell adhesion and matrix remodeling. Two IPN hydrogels were developed with comparable stiffnesses (4.4-4.7 kPa) but varying stress relaxation times (t1/2, 1.5 s and 14.4 s). IDG-SW3 cells were pre-differentiated in 2D under osteogenic conditions for 14 days to drive osteoblast-to-osteocyte transition. Cellular mechanosensitivity to fluid shear stress (2 Pa) was confirmed by live-cell calcium imaging. After embedding in the IPN hydrogels, cells remained highly viable following 7 days of 3D culture. After 24 h, osteocytes in the fast-relaxing hydrogels showed the largest cell area and long dendritic processes. However, a significantly larger increase of some osteogenic markers (ALP, Dmp1, hydroxyapatite) as well as intercellular connections via gap junctions were observed in slow-relaxing hydrogels on day 14. Our results imply that fast-relaxing IPN hydrogels promote early cell spreading, whereas slow relaxation favors osteogenic differentiation. These findings may advance the development of 3D in vivo-like osteocyte models to better understand bone mechanobiology.

Bioassembly techniques for the application of scaffold-free tissue engineering approaches have evolved in recent years toward producing larger tissue equivalents that structurally and functionally mimic native tissues. This study aims to upscale a 3-dimensional bone in-vitro model through bioassembly of differentiated rat osteoblast (dROb) spheroids with the potential to develop and mature into a bone macrotissue.

dROb spheroids in control and mineralization media at different seeding densities (1 × 104, 5 × 104, and 1 × 105 cells) were assessed for cell proliferation and viability by trypan blue staining, for necrotic core by hematoxylin and eosin staining, and for extracellular calcium by Alizarin red and Von Kossa staining. Then, a novel approach was developed to bioassemble dROb spheroids in pillar array supports using a customized bioassembly system. Pillar array supports were custom-designed and printed using Formlabs Clear Resin® by Formlabs Form2 printer. These supports were used as temporary frameworks for spheroid bioassembly until fusion occurred. Supports were then removed to allow scaffold-free growth and maturation of fused spheroids. Morphological and molecular analyses were performed to understand their structural and functional aspects.

Spheroids of all seeding densities proliferated till day 14, and mineralization began with the cessation of proliferation. Necrotic core size increased over time with increased spheroid size. After the bioassembly of spheroids, the morphological assessment revealed the fusion of spheroids over time into a single macrotissue of more than 2.5 mm in size with mineral formation. Molecular assessment at different time points revealed osteogenic maturation based on the presence of osteocalcin, downregulation of Runx2 (p < 0.001), and upregulated alkaline phosphatase (p < 0.01).

With the novel bioassembly approach used here, 3D bone macrotissues were successfully fabricated which mimicked physiological osteogenesis both morphologically and molecularly. This biofabrication approach has potential applications in bone tissue engineering, contributing to research related to osteoporosis and other recurrent bone ailments.

Metformin is an anti-glycemic agent, which is widely prescribed to diabetes patients. Although its alleged role on bone strength has been reported for some time, this review focuses primarily on the recent mechanistical insights of metformin on osteocytes, osteoblasts, and osteoclasts.

Overall, metformin contributed to steering anabolic activity in osteocytes. It caused lower expression in osteocytes of the negative regulators of bone formation sclerostin and DKK1. Likewise, the osteoclastogenesis function of osteoblasts was also skewed towards lower RANKL and higher OPG expressions. Osteoblast lineage cells generally responded to metformin by activating bone formation parameters, such as alkaline phosphatase activity, higher expression of anabolic members of the Wnt pathway, transcription factor Runx2, bone matrix protein proteins, and subsequent mineralization. Metformin affected osteoclast formation and activity in a negative way, reducing the number of multinucleated cells in association with lower expression of typical osteoclast markers and with inhibited resorption. A common denominator studied in all three cell types is its beneficial effect on activating phosphorylated AMP kinase (AMPK) which is associated with the coordination of energy metabolism. Metformin differentially affects bone cells, shifting the balance to more bone formation. Although metformin is a drug prescribed for diabetic patients, the overall bone anabolic effects on osteocytes and osteoblasts and the anti-catabolic effect on osteoclast suggest that metformin could be seen as a promising drug in the bone field.

Radix Asteris, the root of Aster tataricus L. f., is historically significant in East Asian medicine for treating respiratory conditions. Yet, its implications on bone health remain uncharted. This research investigated the impact of an aqueous ethanol extract of Radix Asteris (EERA) on osteoclast differentiation and its prospective contribution to osteoporosis management. We discerned that EERA retards osteoclast differentiation by inhibiting receptor activator of nuclear factor kappa-B ligand (RANKL) expression and obstructing RANKL-induced osteoclastogenesis. EERA markedly suppressed RANKL-induced expression of NFATc1, a pivotal osteoclastogenic factor, via modulating early RANK signaling. EERA's therapeutic potential was underscored by its defense against trabecular bone degradation and its counteraction to increased body and perigonadal fat in ovariectomized mice, mirroring postmenopausal physiological changes. In the phytochemical analysis of EERA, we identified several constituents recognized for their roles in regulating bone and fat metabolism. Collectively, our findings emphasize the potential of EERA in osteoclast differentiation modulation and in the management of osteoporosis and associated metabolic changes following estrogen depletion, suggesting its suitability as an alternative therapeutic strategy for postmenopausal osteoporosis intertwined with metabolic imbalances.

Osteocytes play an important role as regulators of both osteoclasts and osteoblasts, and some proteins that are secreted from them play a role in bone remodeling and modeling. LPS affects bone structure because it is an inflammatory factor, despite verbascoside's potential for bone preservation and healing. Osteocytes may also be involved in the control of the bone's response to immunological changes in inflammatory situations. MLO-Y4 cells were cultured in either supplemented -MEM alone with a low serum to inhibit cell growth or media with LPS (10 ng/mL) and/or verbascoside (50 g/mL) to show the LPS effect. In our research, LPS treatment increased RANKL levels while decreasing OPG and RUNX2 expression. Treatment with verbascoside reduced RANKL expression. In our work, verbascoside increased the expression of OPG and RUNX2. In MLO-Y4 cells exposed to verbascoside, SOD, CAT, and GSH activities as well as the expression levels of bone mineralization proteins like PHEX, RUNX2, and OPG were all elevated.

Anethum graveolens L., known as European dill, is a versatile herb widely used in both traditional medicine and culinary practices. Despite its long-standing history, the potential impact of the water extract of A. graveolens seeds (WEAG) on bone health remains unexplored. In this study, we investigated the influence of WEAG on osteoclast differentiation and assessed its potential as an anti-osteoporotic agent. WEAG hindered osteoclast differentiation through the suppression of receptor activator of nuclear factor-κB ligand (RANKL) expression in osteoclast-supporting cells and by directly targeting osteoclast precursor cells. WEAG significantly reduced the expression of key osteoclastogenic transcription factors, namely c-Fos and NFATc1, typically induced by RANKL in osteoclast precursors. This reduction was attributed to the suppression of both MAPKs and NF-κB pathways in response to RANKL. In vivo experiments further revealed that WEAG administration effectively reduces trabecular bone loss and weight gain triggered by ovariectomy, mimicking postmenopausal osteoporosis. Furthermore, our comprehensive phytochemical analysis of WEAG identified a range of phytochemical constituents, associated with bone health and weight regulation. Notably, we discovered a specific compound, isorhamnetin-3-O-glucuronide, within WEAG that exhibits anti-osteoclastogenic potential. Overall, this research elucidated the beneficial effects and mechanistic basis of WEAG on osteoclast differentiation and bone loss, indicating its potential as a viable alternative to address bone loss in conditions like postmenopause.

The differentiation and function of osteocytes are controlled by surrounding cells and mechanical stress; however, the detailed mechanisms are unknown. Recent findings suggest that IL-33 is highly expressed in periodontal tissues in orthodontic tooth movement. The present study aimed to elucidate the effect of IL-33 on the expression of regulatory factors for bone remodeling and their molecular mechanisms in the osteocyte-like cell line MLO-Y4. MLO-Y4 cells were treated with IL-33, and the activation of intracellular signaling molecules and transcriptional factors was determined using Western blot analysis and chromatin immunoprecipitation assay. IL-33 treatment enhanced the expression of IL-6 in MLO-Y4 cells, which was suppressed by the knockdown of the IL-33 receptor ST2L. Additionally, IL-33 treatment induced activation of NF-κB, JNK/AP-1, and p38 MAPK signaling pathways in MLO-Y4 cells. Moreover, pretreatment with specific inhibitors of NF-κB, p38 MAPK, and JNK/AP-1 attenuated the IL-33-induced expression of IL-6. Furthermore, chromatin immunoprecipitation indicated that IL-33 increased c-Jun recruitment to the IL-6 promoter. Overall, these results suggest that IL-33 induces IL-6 expression and regulates osteocyte function via activation of the NF-κB, JNK/AP-1, and p38 MAPK pathways through interaction with ST2L receptors on the plasma membrane.

Mouse ligature-induced periodontitis (LIP) has been used to study bone loss in periodontitis. However, the role of osteocytes in LIP remains unclear. Furthermore, there is no consensus on the choice of alveolar bone parameters and time points to evaluate LIP. Here, we investigated the dynamics of changes in osteoclastogenesis and bone volume (BV) loss in LIP over 14 days. Time-course analysis revealed that osteoclast induction peaked on days 3 and 5, followed by the peak of BV loss on day 7. Notably, BV was restored by day 14. The bone formation phase after the bone resorption phase was suggested to be responsible for the recovery of bone loss. Electron microscopy identified bacteria in the osteocyte lacunar space beyond the periodontal ligament (PDL) tissue. We investigated how osteocytes affect bone resorption of LIP and found that mice lacking receptor activator of NF-κB ligand (RANKL), predominantly in osteocytes, protected against bone loss in LIP, whereas recombination activating 1 (RAG1)-deficient mice failed to resist it. These results indicate that T/B cells are dispensable for osteoclast induction in LIP and that RANKL from osteocytes and mature osteoblasts regulates bone resorption by LIP. Remarkably, mice lacking the myeloid differentiation primary response gene 88 (MYD88) did not show protection against LIP-induced bone loss. Instead, osteocytic cells expressed nucleotide-binding oligomerization domain containing 1 (NOD1), and primary osteocytes induced significantly higher Rankl than primary osteoblasts when stimulated with a NOD1 agonist. Taken together, LIP induced both bone resorption and bone formation in a stage-dependent manner, suggesting that the selection of time points is critical for quantifying bone loss in mouse LIP. Pathogenetically, the current study suggests that bacterial activation of osteocytes via NOD1 is involved in the mechanism of osteoclastogenesis in LIP. The NOD1-RANKL axis in osteocytes may be a therapeutic target for bone resorption in periodontitis. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The periosteum is a thin tissue surrounding each skeletal element that contains stem and progenitor cells involved in bone development, postnatal appositional bone growth, load-induced bone formation, and fracture repair. BMP and TGFβ signaling are important for periosteal activity and periosteal cell behavior, but thorough examination of the influence of these pathways on specific cell populations resident in the periosteum is lacking due to limitations associated with primary periosteal cell isolations and in vitro experiments. Here we describe the generation of a novel periosteum-derived clonal cell (PDC) line from postnatal day 14 mice and use it to examine periosteal cell behavior in vitro. PDCs exhibit key characteristics of periosteal cells observed during skeletal development, maintenance, and bone repair. Specifically, PDCs express established periosteal markers, can be expanded in culture, demonstrate the ability to differentiate into chondrocytes, osteoblasts, and adipocytes, and exhibit an osteogenic response to physical stimulation. PDCs also engage in BMP and/or TGFβ signaling when treated with the activating ligands BMP2 and TGFβ-1, and in response to mechanical stimulation via fluid shear. We believe that this PDC line will be useful for large-scale, long-term experiments that were not feasible when using primary periosteal cells. Anticipated future uses include advancing our understanding of the signaling interactions that occur during appositional bone growth and fracture repair and developing drug screening platforms to discover novel growth and fracture healing factors.

Introduction: This study aimed to identify and analyze in vitro studies investigating the biological effect of fluid-flow shear stress (FSS) on cells found in the periodontal ligament and bone tissue. Method: We followed the PRISMA guideline for systematic reviews. A PubMed search strategy was developed, studies were selected according to predefined eligibility criteria, and the risk of bias was assessed. Relevant data related to cell source, applied FSS, and locus-specific expression were extracted. Based on this evidence synthesis and, as an original part of this work, analysis of differential gene expression using over-representation and network-analysis was performed. Five relevant publicly available gene expression datasets were analyzed using gene set enrichment analysis (GSEA). Result: A total of 6,974 articles were identified. Titles and abstracts were screened, and 218 articles were selected for full-text assessment. Finally, 120 articles were included in this study. Sample size determination and statistical analysis related to methodological quality and the ethical statement item in reporting quality were most frequently identified as high risk of bias. The analyzed studies mostly used custom-made fluid-flow apparatuses (61.7%). FSS was most frequently applied for 0.5 h, 1 h, or 2 h, whereas FSS magnitudes ranged from 6 to 20 dyn/cm2 depending on cell type and flow profile. Fluid-flow frequencies of 1 Hz in human cells and 1 and 5 Hz in mouse cells were mostly applied. FSS upregulated genes/metabolites responsible for tissue formation (AKT1, alkaline phosphatase, BGLAP, BMP2, Ca2+, COL1A1, CTNNB1, GJA1, MAPK1/MAPK3, PDPN, RUNX2, SPP1, TNFRSF11B, VEGFA, WNT3A) and inflammation (nitric oxide, PGE-2, PGI-2, PTGS1, PTGS2). Protein-protein interaction networks were constructed and analyzed using over-representation analysis and GSEA to identify shared signaling pathways. Conclusion: To our knowledge, this is the first review giving a comprehensive overview and discussion of methodological technical details regarding fluid flow application in 2D cell culture in vitro experimental conditions. Therefore, it is not only providing valuable information about cellular molecular events and their quantitative and qualitative analysis, but also confirming the reproducibility of previously published results.

Skeletal muscle and bone interact at the level of mechanical loading through the application of force by muscles to the skeleton and more recently focus has been placed on molecular/biochemical coupling of these two tissues. We sought to determine if muscle and muscle-derived factors were essential to the osteocyte response to loading. Botox® induced muscle paralysis was used to investigate the role of muscle contraction during in vivo tibia compression loading. 5-6 month-old female TOPGAL mice had their right hindlimb muscles surrounding the tibia injected with either BOTOX® or saline. At four days post injections when muscle paralysis peaked, the right tibia was subjected to a single session of in vivo compression loading at ∼2600 με. At 24 h post-load we observed a 2.5-fold increase in β-catenin signaling in osteocytes in the tibias of the saline injected mice, whereas loading of tibias from Botox® injected mice failed to active β-catenin signaling in osteocytes. This suggests that active muscle contraction produces a factor(s) that is necessary for or conditions the osteocyte's ability to respond to load. To further investigate the role of muscle derived factors, MLO-Y4 osteocyte-like cells and a luciferase based β-catenin reporter (TOPflash-MLO-Y4) cell line we developed were treated with conditioned media (CM) from C2C12 myoblasts (MB) and myotubes (MT) and ex vivo contracted Extensor Digitorum Longus (EDL) and Soleus (Sol) muscles under static or loading conditions using fluid flow shear stress (FFSS). 10 % C2C12 myotube CM, but not myoblast or NIH3T3 fibroblast cells CM, induced a rapid activation of the Akt signaling pathway, peaking at 15 min and returning to baseline by 1-2 h under static conditions. FFSS applied to MLO-Y4 cells for 2 h in the presence of 10 % MT-CM resulted in a 6-8 fold increase in pAkt compared to a 3-4 fold increase under control or when exposed to 10 % MB-CM. A similar response was observed in the presence of 10 % EDL-CM, but not in the presence of 10 % Sol-CM. TOPflash-MLO-Y4 cells were treated with 10 ng/ml Wnt3a in the presence or absence of MT-CM. While MT-CM resulted in a 2-fold activation and Wnt3a produced a 10-fold activation, the combination of MT-CM + Wnt3a resulted in a 25-fold activation of β-catenin signaling, implying a synergistic effect of factors in MT-CM with Wnt3a. These data provide clear evidence that specific muscles and myotubes produce factors that alter important signaling pathways involved in the response of osteocytes to mechanical load. These data strongly suggest that beyond mechanical loading there is a molecular coupling of muscle and bone.

Age-related musculoskeletal disorders, including osteoporosis, are frequent and associated with long lasting morbidity, in turn significantly impacting on healthcare system sustainability. There is therefore a compelling need to develop reliable preclinical models of disease and drug screening to validate novel drugs possibly on a personalized basis, without the need ofin vivoassay. In the context of bone tissue, although the osteocyte (Oc) network is a well-recognized therapeutic target, currentin vitropreclinical models are unable to mimic its physiologically relevant and highly complex structure. To this purpose, several features are needed, including an osteomimetic extracellular matrix, dynamic perfusion, and mechanical cues (e.g. shear stress) combined with a three-dimensional (3D) culture of Oc. Here we describe, for the first time, a high throughput microfluidic platform based on 96-miniaturized chips for large-scale preclinical evaluation to predict drug efficacy. We bioengineered a commercial microfluidic device that allows real-time visualization and equipped with multi-chips by the development and injection of a highly stiff bone-like 3D matrix, made of a blend of collagen-enriched natural hydrogels loaded with hydroxyapatite nanocrystals. The microchannel, filled with the ostemimetic matrix and Oc, is subjected to passive perfusion and shear stress. We used scanning electron microscopy for preliminary material characterization. Confocal microscopy and fluorescent microbeads were used after material injection into the microchannels to detect volume changes and the distribution of cell-sized objects within the hydrogel. The formation of a 3D dendritic network of Oc was monitored by measuring cell viability, evaluating phenotyping markers (connexin43, integrin alpha V/CD51, sclerostin), quantification of dendrites, and responsiveness to an anabolic drug. The platform is expected to accelerate the development of new drug aimed at modulating the survival and function of osteocytes.

Introduction: Myosin IXB (MYO9B) is an unconventional myosin with RhoGAP activity and thus is a regulator of actin cytoskeletal organization. MYO9B was previously shown to be necessary for skeletal growth and health and to play a role in actin-based functions of both osteoblasts and osteoclasts. However, its role in responses to mechanical stimulation of bone cells has not yet been described. Therefore, experiments were undertaken to determine the role of MYO9B in bone cell responses to mechanical stress both in vitro and in vivo. Methods: MYO9B expression was knocked down in osteoblast and osteocyte cell lines using RNA interference and the resulting cells were subjected to mechanical stresses including cyclic tensile strain, fluid shear stress, and plating on different substrates (no substrate vs. monomeric or polymerized collagen type I). Osteocytic cells were also subjected to MYO9B regulation through Slit-Robo signaling. Further, wild-type or Myo9b -/- mice were subjected to a regimen of whole-body vibration (WBV) and changes in bone quality were assessed by micro-CT. Results: Unlike control cells, MYO9B-deficient osteoblastic cells subjected to uniaxial cyclic tensile strain were unable to orient their actin stress fibers perpendicular to the strain. Osteocytic cells in which MYO9B was knocked down exhibited elongated dendrites but were unable to respond normally to treatments that increase dendrite length such as fluid shear stress and Slit-Robo signaling. Osteocytic responses to mechanical stimuli were also found to be dependent on the polymerization state of collagen type I substrates. Wild-type mice responded to WBV with increased bone tissue mineral density values while Myo9b -/- mice responded with bone loss. Discussion: These results demonstrate that MYO9B plays a key role in mechanical stress-induced responses of bone cells in vitro and in vivo.

Due to the aging of population, materials able to repair damaged tissues are needed. Among others, bioactive glasses (BGs) have attracted a lot of interest due to their outstanding properties both for hard and soft tissues. Here, for the first time, two new BGs, which gave very promising results in preliminary in vitro-tests, were implanted in animals in order to evaluate their regenerative potential. The new BGs, named BGMS10 and Bio_MS and containing specific therapeutic ions, were produced in granules and implanted in rabbits' femurs for up to 60 days, to test their biocompatibility and osteoconduction. Additionally, granules of 45S5 Bioglass® were employed and used as a standard reference for comparison. The results showed that, after 30 days, the two novel BGs and 45S5 displayed a similar behavior, in terms of bone amount, thickness of new bone trabeculae and affinity index. On the contrary, after 60 days, 45S5 granules were mainly surrounded by wide and scattered bone trabeculae, separated by large amounts of soft tissue, while in BGMS10 and Bio_MS the trabeculae were thin and uniformly distributed around the BG granules. This latter scenario could be considered as more advantageous, since the features of the two novel BG granules allowed for the neo-formation of a uniformly distributed bony trabeculae, predictive of more favorable mechanical behavior, compared to the less uniform coarse trabeculae, separated by large areas of soft tissue in 45S5 granules. Thus, BGMS10 and Bio_MS could be considered suitable products for tissue regeneration in the orthopedic and dental fields.

The mechanistic regulation of bone mass in aged animals is poorly understood. In this study, we examined the role of SIRT6, a longevity-associated factor, in osteocytes, using mice lacking Sirt6 in Dmp-1-expressing cells (cKO mice) and the MLO-Y4 osteocyte-like cell line. cKO mice exhibited increased osteocytic expression of Sost, Fgf23 and senescence inducing gene Pai-1 and the senescence markers p16 and Il-6, decreased serum phosphate levels, and low-turnover osteopenia. The cKO phenotype was reversed in mice that were a cross of PAI-1-null mice with cKO mice. Furthermore, senescence induction in MLO-Y4 cells increased the Fgf23 and Sost mRNA expression. Sirt6 knockout and senescence induction increased HIF-1α binding to the Fgf23 enhancer sequence. Bone mass and serum phosphate levels were higher in PAI-1-null aged mice than in wild-type mice. Therefore, SIRT6 agonists or PAI-1 inhibitors may be promising therapeutic options for aging-related bone metabolism disruptions.

As we age, our bones undergo a process of loss, often accompanied by muscle weakness and reduced physical activity. This is exacerbated by decreased responsiveness to mechanical stimulation in aged skeleton, leading to the hypothesis that decreased mechanical stimulation plays an important role in age-related bone loss. Piezo1, a mechanosensitive ion channel, is critical for bone homeostasis and mechanotransduction. Here, we observed a decrease in Piezo1 expression with age in both murine and human cortical bone. Furthermore, loss of Piezo1 in osteoblasts and osteocytes resulted in an increase in age-associated cortical bone loss compared to control mice. The loss of cortical bone was due to an expansion of the endosteal perimeter resulting from increased endocortical resorption. In addition, expression of Tnfrsf11b, encoding anti-osteoclastogenic protein OPG, decreases with Piezo1 in vitro and in vivo in bone cells, suggesting that Piezo1 suppresses osteoclast formation by promoting Tnfrsf11b expression. Our results highlight the importance of Piezo1-mediated mechanical signaling in protecting against age-associated cortical bone loss by inhibiting bone resorption in mice.

The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost.

We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene.

We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells.

2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

Long cytoplasmic processes of osteocytes orchestrate bone activity by integration of biochemical and mechanical signals and regulate load-induced bone adaptation. Low-Intensity Pulsed Ultrasound (LIPUS) is a clinically used technique for fracture healing that delivers mechanical impulses to the damaged bone tissue in a non-invasive and non-ionizing manner. The mechanism of action of LIPUS is still controversially discussed in the scientific community. In this study, the effect of focused LIPUS (FLIPUS) on the survival of starved MLO-Y4 osteocytes was investigated in vitro. Osteocytes stimulated for 10 min with FLIPUS exhibited extended dendrites, which formed frequent connections to neighboring cells and spanned longer distances. The sonicated cells displayed thick actin bundles and experienced increase in expression of connexin 43 (Cx43) proteins, especially on their dendrites, and E11 glycoprotein, which is responsible for the elongation of cellular cytoplasmic processes. After stimulation, expression of cell growth and survival genes as well as genes related to cell-cell communication was augmented. In addition, cell viability was improved after the sonication, and a decrease in ATP release in the medium was observed. In summary, FLIPUS mitigated apoptosis of starved osteocytes, which is likely related to the formation of the extensive dendritic network that ensured cell survival.

Though magnetic iron oxide nanoparticles (IONPs) are approved for clinical use as contrast agents for MR imaging in United States and Europe, and are widely used to label cells in research, the relationship between IONPs and mesenchymal stem cells (MSCs) is not fully addressed. Here the effects of consistently appeared γ-Fe₂ O₃ on the lineage commitment of MSCs were studied to optimize applications of IONPs in MSCs upon verification of viability. 30 nm 10 μg/mL induced highest promotions on osteogenesis, while 30 and 50 nm of 100 μg/mL elicited most chondrogensis in 14 days, where the effects on ALP, GAG and SOX9 appeared after 7 days, while on RUNX2 came out after 10 days. γ-Fe₂ O₃ enhanced intracellular and extracellular Fe³⁺ and ROS, modulated F-actin and decreased Lamin A of MSCs at different time scale. The disturbances of F-actin, Lamin A or ROS altered the effects of γ-Fe₂ O₃ on MSC differentiation. Our results demonstrate that different size, concentration and modulation of γ-Fe₂ O₃ are needed in its MSC applications for bone and cartilage tissues. Furthermore, an undocumented phenomenon that the modulation of F-actin affected the Lamin A expression in MSCs was observed.

A large number of studies in recent years indicate that osteocytes are the orchestrators of bone remodeling by regulating both osteoblast and osteoclast activities. Oxidative stress-induced osteocyte apoptosis plays critical roles in the pathological processes of postmenopausal osteoporosis. Resveratrol is a natural polyphenolic compound that ameliorates postmenopausal osteoporosis. However, whether resveratrol regulates osteocyte apoptosis via autophagy remains largely unknown. The effects of resveratrol on regulating osteocyte apoptosis and autophagy were analyzed both in vivo and in vitro. In vitro, cultured MLO-Y4 cells were exposed to H₂O₂ with or without resveratrol. In vivo, an ovariectomy-induced osteoporosis model was constructed in rats with or without daily intraperitoneal injection of 10 mg/kg body weight resveratrol. It was found that resveratrol attenuated H₂O₂-induced apoptosis through activating autophagy in cultured MLO-Y4 cells, which was mediated by the dissociation of Beclin-1/Bcl-2 complex in AMPK/JNK1-dependent pathway, ultimately regulating osteocytes function. Furthermore, it was shown that resveratrol treatment reduced osteocytes oxidative stress, inhibited osteocytes apoptosis and promoted autophagy in ovariectomized rats. Our study suggests that resveratrol protects against oxidative stress by restoring osteocytes autophagy and alleviating apoptosis via AMPK/JNK1 activation, therefore dissociating Bcl-2 from Beclin-1.

Although the formation of bone-like nodules is regarded as the differentiation process from stem cells to osteogenic cells, including osteoblasts and osteocytes, the precise biological events during nodule formation are unknown. Here we performed the osteogenic induction of human induced pluripotent stem cells using a three-dimensional (3D) culture system using type I collagen gel and a rapid induction method with retinoic acid. Confocal and time-lapse imaging revealed the osteogenic differentiation was initiated with vigorous focal proliferation followed by aggregation, from which cells invaded the gel. Invading cells changed their morphology and expressed osteocyte marker genes, suggesting the transition from osteoblasts to osteocytes. Single-cell RNA sequencing analysis revealed that 3D culture-induced cells with features of periosteal skeletal stem cells, some of which expressed TGFβ-regulated osteoblast-related molecules. The role of TGFβ signal was further analyzed in the transition from osteoblasts to osteocytes, which revealed that modulation of the TGFβ signal changed the morphology and motility of cells isolated from the 3D culture, suggesting that the TGFβ signal maintains the osteoblastic phenotype and the transition into osteocytes requires down-regulation of the TGFβ signal.

Although two-dimensional (2D) cultures from bone lineage cells are often used, it is well-known that this culture system is completely different from the in vivo bone matrix environment. In this paper, we describe a 3D culture method using both the mouse osteocytic cell line, MLO-Y4, and an osteocyte-enriched population of the cells isolated from mice. These cells are embedded in collagen gel with recombinant cellular communication network (CCN) factor proteins; then, osteoblasts or osteoclasts are inoculated and cultured on the collagen gel. Because this method mimics the in vitro bone matrix environment, it is useful for understanding the detailed mechanism of actions of CCN proteins in the bone matrix.

Our understanding of how osteocytes, the principal mechanosensors within bone, sense and perceive force remains unclear. Previous work identified "tethering elements" (TEs) spanning the pericellular space of osteocytes and transmitting mechanical information into biochemical signals. While we identified the heparan sulfate proteoglycan perlecan (PLN) as a component of these TEs, PLN must attach to the cell surface to induce biochemical responses. As voltage-sensitive calcium channels (VSCCs) are critical for bone mechanotransduction, we hypothesized that PLN binds the extracellular α₂δ₁ subunit of VSCCs to couple the bone matrix to the osteocyte membrane. Here, we showed co-localization of PLN and α₂δ₁ along osteocyte dendritic processes. Additionally, we quantified the molecular interactions between α₂δ₁ and PLN domains and demonstrated for the first time that α₂δ₁ strongly associates with PLN via its domain III. Furthermore, α₂δ₁ is the binding site for the commonly used pain drug, gabapentin (GBP), which is associated with adverse skeletal effects when used chronically. We found that GBP disrupts PLN::α₂δ₁ binding in vitro, and GBP treatment in vivo results in impaired bone mechanosensation. Our work identified a novel mechanosensory complex within osteocytes composed of PLN and α₂δ₁, necessary for bone force transmission and sensitive to the drug GBP.