UNASSIGNED: Chondrocyte viability, apoptosis, and migration are closely related to cartilage injury in osteoarthritis (OA) joints. Exosomes are identified as potential therapeutic agents for OA.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

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.

Staphylococcus aureus is a major causative pathogen of osteomyelitis. Intracellular infections of resident bone cells including osteocytes can persist despite gold-standard clinical intervention. The mechanisms by which intracellular S. aureus evades antibiotic therapy are unknown. In this study, we utilised an in vitro S. aureus infection model of human osteocytes to investigate whether antibiotic-mediated dysregulation of autophagy contributes to this phenomenon. Infected or non-infected osteocyte-like cells were exposed to combinations of rifampicin, vancomycin, and modulators of autophagy. Intracellular bacterial growth characteristics were assessed using colony-forming unit (CFU) analysis, viable bacterial DNA abundance, and the rate of escape into antibiotic-free medium, together with measures of autophagic flux. Rifampicin, alone or in combination with vancomycin, caused a rapid decrease in the culturability of intracellular bacteria, concomitant with stable or increased absolute bacterial DNA levels. Both antibiotics significantly inhibited autophagic flux. However, modulation of autophagic flux did not affect viable bacterial DNA levels. In summary, autophagy was shown to be a factor in the host-pathogen relationship in this model, as its modulation affected the growth state of intracellular S. aureus with respect to both their culturability and propensity to escape the intracellular niche. While rifampicin and vancomycin treatments moderately suppressed autophagic flux acutely, this did not explain the paradoxical response of antibiotic treatment in decreasing S. aureus culturability whilst failing to clear bacterial DNA and hence intracellular bacterial load. Thus, off-target effects of rifampicin and vancomycin on autophagic flux in osteocyte-like cells could not explain the persistent S. aureus infection in these cells.

Examination of bacteria/host cell interactions is important for understanding the aetiology of many infectious diseases. The colony forming unit (CFU) has been the standard for quantifying bacterial burden for the past century, however, this suffers from low sensitivity and is dependent on bacterial culturability in vitro. Our data demonstrate the discrepancy between the CFU and bacterial genome copy number in an osteomyelitis-relevant co-culture system and we confirm diagnosis and quantify bacterial load in clinical bone specimens. This study provides an improved workflow for the quantification of bacterial burden in such cases.

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.

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.

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.

UNASSIGNED: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models.Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year).
UNASSIGNED: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail.
UNASSIGNED: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture.
UNASSIGNED: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.

Adult bones are continuously remodeled by the balance between bone resorption by osteoclasts and subsequent bone formation by osteoblasts. Many studies have provided molecular evidence that bone remodeling is under the control of circadian rhythms. Circadian fluctuations have been reported in the serum and urine levels of bone turnover markers, such as digested collagen fragments and bone alkaline phosphatase. Additionally, the expressions of over a quarter of all transcripts in bones show circadian rhythmicity, including the genes encoding master transcription factors for osteoblastogenesis and osteoclastogenesis, osteogenic cytokines, and signaling pathway proteins. Serum levels of calcium, phosphate, parathyroid hormone, and calcitonin also display circadian rhythmicity. Finally, osteoblast- and osteoclast-specific knockout mice targeting the core circadian regulator gene Bmal1 show disrupted bone remodeling, although the results have not always been consistent. Despite these studies, however, establishing a direct link between circadian rhythms and bone remodeling in vivo remains a major challenge. It is nearly impossible to repeatedly collect bone materials from human subjects while following circadian changes. In addition, the differences in circadian gene regulation between diurnal humans and nocturnal mice, the main model organism, remain unclear. Filling the knowledge gap in the circadian regulation of bone remodeling could reveal novel regulatory mechanisms underlying many bone disorders including osteoporosis, genetic diseases, and fracture healing. This is also an important question for the basic understanding of how cell differentiation progresses under the influence of cyclically fluctuating environments.