Aims. Osteoarthritis (OA) is a common degenerative joint disease. The osteocyte transcriptome is highly relevant to osteocyte biology. This study aimed to explore the osteocyte transcriptome in subchondral bone affected by OA. Methods. Gene expression profiles of OA subchondral bone were used to identify disease-relevant genes and signalling pathways. RNA-sequencing data of a bone loading model were used to identify the loading-responsive gene set. Weighted gene co-expression network analysis (WGCNA) was employed to develop the osteocyte mechanics-responsive gene signature. Results. A group of 77 persistent genes that are highly relevant to extracellular matrix (ECM) biology and bone remodelling signalling were identified in OA subchondral lesions. A loading responsive gene set, including 446 principal genes, was highly enriched in OA medial tibial plateaus compared to lateral tibial plateaus. Of this gene set, a total of 223 genes were identified as the main contributors that were strongly associated with osteocyte functions and signalling pathways, such as ECM modelling, axon guidance, Hippo, Wnt, and transforming growth factor beta (TGF-β) signalling pathways. We limited the loading-responsive genes obtained via the osteocyte transcriptome signature to identify a subgroup of genes that are highly relevant to osteocytes, as the mechanics-responsive osteocyte signature in OA. Based on WGCNA, we found that this signature was highly co-expressed and identified three clusters, including early, late, and persistently responsive genes. Conclusion. In this study, we identified the mechanics-responsive osteocyte signature in OA-lesioned subchondral bone. Cite this article: Bone Joint Res 2022;11(6):362–370

Aims. Gap junction intercellular communication (GJIC) in osteocytes is impaired by oxidative stress, which is associated with age-related bone loss. Ageing is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, it is still unknown whether AOPP accumulation is involved in the impairment of osteocytes’ GJIC. This study aims to investigate the effect of AOPP accumulation on osteocytes’ GJIC in aged male mice and its mechanism. Methods. Changes in AOPP levels, expression of connexin43 (Cx43), osteocyte network, and bone mass were detected in 18-month-old and three-month-old male mice. Cx43 expression, GJIC function, mitochondria membrane potential, reactive oxygen species (ROS) levels, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were detected in murine osteocyte-like cells (MLOY4 cells) treated with AOPPs. The Cx43 expression, osteocyte network, bone mass, and mechanical properties were detected in three-month-old mice treated with AOPPs for 12 weeks. Results. The AOPP levels were increased in aged mice and correlated with degeneration of osteocyte network, loss of bone mass, and decreased Cx43 expression. AOPP intervention induced NADPH oxidase activation and mitochondrial dysfunction, triggered ROS generation, reduced Cx43 expression, and ultimately impaired osteocytes’ GJIC, which were ameliorated by NADPH oxidase inhibitor apocynin, mitochondria-targeted superoxide dismutase mimetic (mito-TEMPO), and ROS scavenger N-acetyl cysteine. Chronic AOPP loading accelerated the degradation of osteocyte networks and decreased Cx43 expression, resulting in deterioration of bone mass and mechanical properties in vivo. Conclusion. Our study suggests that AOPP accumulation contributes to age-related impairment of GJIC in osteocytes of male mice, which may be part of the pathogenic mechanism responsible for bone loss during ageing. Cite this article: Bone Joint Res 2022;11(7):413–425

Abstract. OBJECTIVE. Changes in subchondral bone are one of few disease characteristics to correlate with pain in OA. 1. Profound neuroplasticity and nociceptor sprouting is displayed within osteoarthritic (OA) subchondral bone and is associated with pain and pathology. 2. The cause of these neural changes remains unestablished. Correct innervation patterns are indispensable for bone growth, homeostasis, and repair. Axon guidance signalling factor, Sema3A is essential for the correct innervation patterning of bony tissues. 3. , expressed in osteocytes. 4. and known to be downregulated in bone OA mechanical loading. 5. Bioinformatic analysis has also shown Sema3a as a differentially expressed pathway by bone in human OA patients. 6. HYPOTHESIS: Pathological mechanical load and inflammation of bone causes dysregulation of Sema3A signalling leading to perturbed sensory nerve plasticity and pain. METHODS. Human KOLF2-C1 iPSC derived nociceptors were generated by TALEN-mediated insertion of transcription factors NGN2+Brn3A and modified chambers differentiation protocol to produce nociceptor-like cells. Nociceptor phenotype was confirmed by immunocytochemistry. Human Y201-MSC cells were embedded in 3D type-I collagen gels (0.05 × 106 cell/gel), in 48-well plates and silicone plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) and soluble IL-6 receptor (sIL-6r (40ng/ml), IL6/sIL6r and mechanical load mimetic Yoda1 (5μM) or unstimulated (n=5/group) (48-well plates) or were mechanically loaded in silicone plates (5000μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). Conditioned media transfer was performed from osteocyte to nociceptor cultures assessed by continuous 24-hour phase contrast confocal microscopy. 24-hours after stimulation RNA was quantified by RT-qPCR (IL6) or RNAseq whole transcriptome analysis/DEseq2 analysis (Load). Protein release was quantified by ELISA. Normally distributed data with homogenous variances was analysed by two-tailed t test. RESULTS. IPSC-derived nociceptor-like cells display elongated (>5mm) dendritic projections and nociceptive molecular markers such as TUJ1, PrPH and Neun and TrkA. Sema3A signalling ligands were expressed in 100% of osteocyte cultures. Mechanical loading regulated the Sema3 pathway; Sema3A (0.4-fold, p<0.001), Sema3B (13-fold, p<0.001), Sema3C (0.4-fold, p<0.001). Under inflammatory stimulation by IL6/IL6sR, SEMA3A (7-fold, p=0.01) and receptor Plexin1 (3-fold, p=0.03) show significant regulation. Sema3A protein release showed a significant downregulation of Sema3A release by IL6/sIL6r+Yoda1 (2-fold, p=0.02). Continuous 24-hour phase contrast confocal microscopy measuring the number of extending/retreating dendritic projections revealed that sensory nerve cultures exposed to media from osteocytes stimulated with IL-6/sIL-6R+Yoda1 displayed significantly more invading dendritic projections (p=0.0175, 12-fold±SEM 3.5) across 3 random fields of view within a single stimulated neural culture and significantly fewer retracting dendritic projections (p=0.0075, 2-fold±SEM 0.33) compared to controls. CONCLUSIONS. Here we show osteocytic regulation of Sema3A under pathological mechanical loading and the ability of media pathologically loaded osteocyte cultures to induce the branching and invasion of cultured nociceptor-like cells as displayed in OA subchondral bone. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Periprosthetic joint infection (PJI) is a potentially devastating complication of joint replacement surgery. Osteocytes comprise 90–95% of all cells in hard bone tissue, are long-lived and are becoming increasingly recognised as a critical cell type in the regulation of bone and systemic physiology. The purpose of this study was to examine role of these cells in PJI pathophysiology and aetiology, with the rationale that their involvement could contribute to the difficulty in detecting and clearing PJI. This study examined the ability of human osteocytes to become infected by Staphylococcus aureus and the responses of both the host cell and pathogen in this scenario. Several S. aureus (MRSA) strains were tested for their ability to infect human primary osteocyte-like cells in vitro and human bone samples ex vivo. Bone biopsies were retrieved from patients undergoing revision total hip arthroplasty for either aseptic loosening associated with osteolysis, or for PJI. Retrieved bacterial colony number from cell lysates and colony morphology were determined. Gene expression was measured by microarray/bioinformatics analysis and/or real-time RT-PCR. Exposure to planktonic S. aureus (approx. 100 CFU/cell) resulted in intracellular infection of human osteocyte-like cells. We found no evidence of increased rates of osteocyte cell death in bacteria exposed cultures. Microarray analysis of osteocyte gene expression 24h following exposure revealed more than 1,500 differentially expressed genes (fold-change more than 2, false discovery rate p < 0.01). The gene expression patterns were consistent with a strong innate immune response and altered functionality of the osteocytes. Consistent patterns of host gene expression were observed between experimentally infected osteocyte-like cultures and human bone, and in PJI patient bone samples. Internalised bacteria switched to the quasi-dormant small colony variant (SCV) form over a period of 5d, and the ensuing infection appeared to reach a stable state. S. aureus infection of viable osteocytes was also identified in bone taken from PJI patients. We have demonstrated [1] that human osteocytes can become infected by S. aureus and respond robustly by producing immune mediators. The bony location of the infected osteocyte may render them refractory to clearance by immune cells, and osteocytes may therefore be an immune-privileged cell type. The phenotypic switch of S. aureus to SCV, a form less sensitive to most antibiotics and one associated with intracellular survival, suggests that infection of osteocytes may contribute to a chronic disease state. The osteocyte may therefore serve as a reservoir of bacteria for reinfection, perhaps explaining the high prevalence of infections that only become apparent after long periods of time or recur following surgical/medical treatment. Our findings also provide a biological rationale for the recognised need for aggressive bone debridement in the surgical management of PJI

Osteocytes are terminally differentiated long-lived cells and account for greater than 95% of the bone cell population. It has been established that osteocytes are connected through their highly developed dendritic network, which is necessary for the maintenance of optimal bone homeostasis. However, little is known on how osteocytes use the network to coordinate their cellular function and communication that requires energy and protein turnover. Here using super-resolution confocal imaging on both live and fixed osteocytes, we demonstrated conclusively that mitochondria are widely distributed and dynamically shared between osteocytes. Using confocal live cell imaging analysis we showed that inhibiting the contact between mitochondria and endoplasmic reticulum (ER) by the knockdown of MFN2 in osteocytes impedes the transfer of mitochondria suggesting the involvement of ER contact with mitochondria in the transfer process. Moreover, we showed that transport of mitochondria between osteocytes within the network enables rescue of osteocytes with dysfunction of mitochondria. Using the 3D tetraculture system with confocal imaging, we identify the transfer of mitochondria from healthy osteocytes enables recovery of mitochondria activities in osteocytes that devoid of mitochondrial DNA by ethidium bromide. The results indicated that when osteocytes are depleted of functional mitochondria, normal parental osteocytes can transfer mitochondria to these stressed osteocytes to provide them with energy. Collectively we show for the first time that the utilisation of mitochondrial transfer enables osteocytes to function with a network and coordinate their cellular activities in response to different energy demands

The expression of the mechanosensor, integrin αvβ3, is reduced in osteoporotic bone cells compared to controls. MLO-Y4 osteocytes experience altered mechanotransduction under estrogen deficiency and it is unknown whether this is associated with defective αvβ3 expression or signalling. The objectives of this study are to (1) investigate αvβ3 expression and spatial organisation in osteocytes during estrogen deficiency, and (2) establish whether altered responses of osteocytes under estrogen deficiency correlate to defective αvβ3 expression and functionality. MLO-Y4 cells were cultured as follows: Ctrl (no added estradiol), E+ (10nM 17β-estradiol for 5 days), and Ew (10nM 17β-estradiol for 3 days and withdrawal for 2 days). Cells were cultured with/without 0.5µM IntegriSense750 (αvβ3 antagonist). Laminar oscillatory fluid flow of 1Pa at 0.5Hz was applied for 1hr. αvβ3 content was quantified using an ELISA. The location and quantity of αvβ3 and focal-adhesions was determined by immunocytochemistry. Estrogen withdrawal under static conditions led to lower cell and focal-adhesion area (p<0.05), compared to E+ cells. Fluid flow led to higher αvβ3 content (p<0.05) in all groups, compared to static counterparts, with αvβ3 blocking altering this response. Fluid flow on Ew cells had the highest αvβ3 levels (p<0.05), but αvβ3 did not localise at focal-adhesions sites. Cell morphologies were similar after treatment with the αvβ3 antagonist to the Ew group. These results suggest there are fewer functional focal-adhesion sites at which αvβ3 integrins localise to facilitate mechanotransduction. To further understand these results, we are analysing osteocyte mechanotransduction by quantifying PGE2 and gene expression (COX-2, RANKL, OPG, SOST)

Aims. There is an increasing concern of osteoporotic fractures in the ageing population. Low-magnitude high-frequency vibration (LMHFV) was shown to significantly enhance osteoporotic fracture healing through alteration of osteocyte lacuno-canalicular network (LCN). Dentin matrix protein 1 (DMP1) in osteocytes is known to be responsible for maintaining the LCN and mineralization. This study aimed to investigate the role of osteocyte-specific DMP1 during osteoporotic fracture healing augmented by LMHFV. Methods. A metaphyseal fracture was created in the distal femur of ovariectomy-induced osteoporotic Sprague Dawley rats. Rats were randomized to five different groups: 1) DMP1 knockdown (KD), 2) DMP1 KD + vibration (VT), 3) Scramble + VT, 4) VT, and 5) control (CT), where KD was performed by injection of short hairpin RNA (shRNA) into marrow cavity; vibration treatment was conducted at 35 Hz, 0.3 g; 20 minutes/day, five days/week). Assessments included radiography, micro-CT, dynamic histomorphometry and immunohistochemistry on DMP1, sclerostin, E11, and fibroblast growth factor 23 (FGF23). In vitro, murine long bone osteocyte-Y4 (MLO-Y4) osteocyte-like cells were randomized as in vivo groupings. DMP1 KD was performed by transfecting cells with shRNA plasmid. Assessments included immunocytochemistry on osteocyte-specific markers as above, and mineralized nodule staining. Results. Healing capacities in DMP1 KD groups were impaired. Results showed that DMP1 KD significantly abolished vibration-enhanced fracture healing at week 6. DMP1 KD significantly altered the expression of osteocyte-specific markers. The lower mineralization rate in DMP1 KD groups indicated that DMP1 knockdown was associated with poor fracture healing process. Conclusion. The blockage of DMP1 would impair healing outcomes and negate LMHFV-induced enhancement on fracture healing. These findings reveal the importance of DMP1 in response to the mechanical signal during osteoporotic fracture healing. Cite this article: Bone Joint Res 2022;11(7):465–476

Objectives. The exact aetiology and pathogenesis of microdamage-induced long bone fractures remain unknown. These fractures are likely to be the result of inadequate bone remodelling in response to damage. This study aims to identify an association of osteocyte apoptosis, the presence of osteocytic osteolysis, and any alterations in sclerostin expression with a fracture of the third metacarpal (Mc-III) bone of Thoroughbred racehorses. Methods. A total of 30 Mc-III bones were obtained; ten bones were fractured during racing, ten were from the contralateral limb, and ten were from control horses. Each Mc-III bone was divided into a fracture site, condyle, condylar groove, and sagittal ridge. Microcracks and diffuse microdamage were quantified. Apoptotic osteocytes were measured using TUNEL staining. Cathepsin K, matrix metalloproteinase-13 (MMP-13), HtrA1, and sclerostin expression were analyzed. Results. In the fracture group, microdamage was elevated 38.9% (. sd 2.6. ) compared with controls. There was no difference in the osteocyte number and the percentage of apoptotic cells between contralateral limb and unraced control; however, there were significantly fewer apoptotic cells in fractured samples (p < 0.02). Immunohistochemistry showed that in deep zones of the fractured samples, sclerostin expression was significantly higher (p < 0.03) than the total number of osteocytes. No increase in cathepsin K, MMP-13, or HtrA1 was present. Conclusion. There is increased microdamage in Mc-III bones that have fractured during racing. In this study, this is not associated with osteocyte apoptosis or osteocytic osteolysis. The finding of increased sclerostin in the region of the fracture suggests that this protein may be playing a key role in the regulation of bone microdamage during stress adaptation. Cite this article: N. Hopper, E. Singer, F. Henson. Increased sclerostin associated with stress fracture of the third metacarpal bone in the Thoroughbred racehorse. Bone Joint Res 2018;7:94–102. DOI: 10.1302/2046-3758.71.BJR-2016-0202.R4

Aims. To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism. Methods. In a series of in vitro experiments, we used tert-Butyl hydroperoxide (TBHP) to induce senescence of MLO-Y4 cells successfully, and collected conditioned medium (CM) and senescent MLO-Y4 cell-derived exosomes, which were then applied to MC3T3-E1 cells, separately, to evaluate their effects on osteogenic differentiation. Furthermore, we identified differentially expressed microRNAs (miRNAs) between exosomes from senescent and normal MLO-Y4 cells by high-throughput RNA sequencing. Based on the key miRNAs that were discovered, the underlying mechanism by which senescent osteocytes regulate osteogenic differentiation was explored. Lastly, in the in vivo experiments, the effects of senescent MLO-Y4 cell-derived exosomes on age-related bone loss were evaluated in male SAMP6 mice, which excluded the effects of oestrogen, and the underlying mechanism was confirmed. Results. The CM and exosomes collected from senescent MLO-Y4 cells inhibited osteogenic differentiation of MC3T3-E1 cells. RNA sequencing detected significantly lower expression of miR-494-3p in senescent MLO-Y4 cell-derived exosomes compared with normal exosomes. The upregulation of exosomal miR-494-3p by miRNA mimics attenuated the effects of senescent MLO-Y4 cell-derived exosomes on osteogenic differentiation. Luciferase reporter assay demonstrated that miR-494-3p targeted phosphatase and tensin homolog (PTEN), which is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overexpression of PTEN or inhibition of the PI3K/AKT pathway blocked the functions of exosomal miR-494-3p. In SAMP6 mice, senescent MLO-Y4 cell-derived exosomes accelerated bone loss, which was rescued by upregulation of exosomal miR-494-3p. Conclusion. Reduced expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenic differentiation and accelerates age-related bone loss via PTEN/PI3K/AKT pathway. Cite this article: Bone Joint Res 2024;13(2):52–65

Nuclear factor erythroid 2–related factor 2 (Nrf2) is a crucial transcription factor to maintain cellular redox homeostasis, but is also affecting bone metabolism. As the association between Nrf2 and osteoporosis in elderly females is not fully elucidated, our aim was to shed light on the potential contribution of Nrf2 to the development of age-dependent osteoporosis using a mouse model. Female wild-type (WT, n=18) and Nrf2-knockout (KO, n=12) mice were sacrificed at different ages (12 weeks=young mature adult, and 90 weeks=old), morphological cortical and trabecular properties of femoral bone analyzed by micro-computed tomography (µCT), and compared to histochemistry. Mechanical properties were derived from quasi-static compression tests and digital image correlation (DIC) used to analyze full-field strain distribution. Bone resorbing cells and aromatase expression by osteocytes were evaluated immunohistochemically and empty osteocyte lacunae counted in cortical bone. Wilcoxon rank sum test was used for data comparison and differences considered statistically significant at p<0.05. When compared to old WT mice, old Nrf2-KO mice revealed a significantly reduced trabecular bone mineral density (BMD), cortical thickness (Ct.Th), cortical area (Ct.Ar), and cortical bone fraction (Ct.Ar/Tt.Ar). Surprisingly, these parameters were not different in skeletally mature young adult mice. Metaphyseal trabeculae were thin but present in all old WT mice, while no trabecular bone was detectable in 60% of old KO mice. Occurrence of empty osteocyte lacunae did not differ between both groups, but a significantly higher number of osteoclast-like cells and fewer aromatase-positive osteocytes were found in old KO mice. Furthermore, female Nrf2-KO mice showed an age-dependently reduced fracture resilience when compared to age-matched WT mice. Our results confirmed lower bone quantity and quality as well as an increased number of bone resorbing cells in old female Nrf2-KO mice. Additionally, aromatase expression in osteocytes of old Nrf2-KO mice was compromised, which may indicate a chronic lack of estrogen in bones of old Nrf2-deficient mice. Thus, chronic Nrf2 loss seems to contribute to age-dependent progression of female osteoporosis

Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and SNPs in the Piezo1 locus are associated with changes in fracture risk. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. The current study used a human, cell-based physiological, 3D in vitro model of bone to determine whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Human Y201 MSCs, embedded in type I collagen gels and differentiated to osteocytes for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and assessed by RNAseq analysis. To mimic mechanical load and activate Piezo1, cells were differentiated to osteocytes for 13 days and treated ± Yoda1 (5µM, 2- and 24-hs, n=4); vehicle treated cells served as controls (n=4). RNA was subjected to RT-qPCR and data normalised to the housekeeping gene, YWHAZ. Media was analysed for IL6 release by ELISA. Mechanical load upregulated Piezo1 gene expression (16.5-fold, p<0.001) and expression of the transcription factor NFATc1, and matricellular protein CYR61, known regulators of Piezo1 mechanotransduction (3-fold; p= 5.0E-5 and 6.8-fold; p= 6.0E-5, respectively). After 2-hrs, Yoda1 increased the expression of the early mechanical response gene, cFOS (11-fold; p=0.021), mean Piezo1 expression (2.3-fold) and IL-6 expression (103-fold, p<0.001). Yoda1 increased the release of IL6 protein after 24 hours (7.5-fold, p=0.001). This study confirms Piezo1 as an important mechanosensor in osteocytes. Piezo1 activation mediated an increase in IL6, a cytokine that drives inflammation and bone resorption providing a direct link between mechanical activation of Piezo1, bone remodeling and inflammation, which may contribute to mechanically induced joint degeneration in diseases such as osteoarthritis. Mechanistically, we hypothesize this may occur through promoting Ca2+ influx and activation of the NFATc1 signaling pathway

Abstract. Objectives. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and deletion of Piezo1 in osteoblasts and osteocytes decreases bone mass and bone strength in mice. This study determined whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Methods. Human MSC cells (Y201), embedded in type I collagen gels and differentiated to osteocytes in osteogenic media for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and Piezo1 activation assessed by RNAseq analysis (NovaSeq S1 flow cell 2 × 100bp PE reads). To mimic mechanical load and activate Piezo1, Y201s were differentiated to osteocytes in 3D gels for 13 days and treated, with Yoda1 (5µM, 2 hours, n=4); vehicle treated cells served as controls (n=4). Extracted RNA was subjected to RT-qPCR and data analysed by Minitab. Results. Low mRNA expression of PIEZO1 in unloaded cells was upregulated 5-fold following 1-hr of mechanical load (p=0.003). In addition, the transcription factor NFATc1, a known regulator of Piezo1 mechanotransduction, was also upregulated by load (2.4-fold; p=0.03). Y201 cells differentiated in gels expressed the osteocyte marker, SOST. Yoda1 upregulated PIEZO1 (1.7-fold; p=0.057), the early mechanical response gene, cFOS (4-fold; p=0.006), COL1A1 (3.9-fold; p=0.052), and IL-6 expression (7.7-fold; p=0.001). Discussion. This study reveals PIEZO1 as an important mechanosenser in osteocytes. Piezo 1 mediated increases in the bone matrix protein, type I collagen, and IL-6, a cytokine that drives inflammation and bone resorption. This provides a direct link between mechanical activation of Piezo 1, bone remodelling and inflammation, which may contribute to mechanically-induced joint degeneration in osteoarthritis. Mechanistically, we hypothesise this may occur through promoting Ca2+ influx and activation of the NFAT1 signalling pathway

Osteoarthritis (OA) is a common cause of chronic pain. Subchondral bone is highly innervated, and bone structural changes directly correlate with pain in OA. Mechanisms underlying skeletal–neural interactions are under-investigated. Bone derived axon guidance molecules are known to regulate bone remodelling. Such signals in the nervous system regulate neural plasticity, branching and neural inflammation. Perturbation of these signals during OA disease progression may disrupt sensory afferents activity, affecting tissue integrity, nociception, and proprioception. Osteocyte mechanical loading and IL-6 stimulation alters axon guidance signalling influencing innervation, proprioception, and nociception. Human Y201 MSC cells, embedded in 3D type I collagen gels (0.05 × 106 cell/gel) in 48 well plastic or silicone (load) plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) with soluble IL-6 receptor (sIL-6r (40ng/ml) or unstimulated (n=5/group), or mechanically loaded (5000 μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). RNA extracted 1hr and 24hrs post load was quantified by RNAseq whole transcriptome analysis (NovaSeq S1 flow cell 2 × 100bp PE reads and differentially expressed neurotransmitters identified (>2-fold change in DEseq2 analysis on normalised count data with FDR p<0.05). After 24 hours, extracted IL-6 stimulated RNA was quantified by RT-qPCR for neurotrophic factors using 2–∆∆Ct method (efficiency=94-106%) normalised to reference gene GAPDH (stability = 1.12 REfinder). Normally distributed data with homogenous variances was analysed by two-tailed t test. All detected axonal guidance genes were regulated by mechanical load. Axonal guidance genes were both down-regulated (Netrin1 0.16-fold, p=0.001; Sema3A 0.4-fold, p<0.001; SEMA3C (0.4-fold, p<0.001), and up-regulated (SLIT2 2.3-fold, p<0.001; CXCL12 5-fold, p<0.001; SEMA3B 13-fold, p<0.001; SEMA4F 2-fold, p<0.001) by mechanical load. IL6 and IL6sR stimulation upregulated SEMA3A (7-fold, p=0.01), its receptor Plexin1 (3-fold, p=0.03). Neutrophins analysed in IL6 stimulated RNA did not show regulation. Here we show osteocytes regulate multiple factors which may influence innervation, nociception, and proprioception upon inflammatory or mechanical insult. Future studies will establish how these factors may combine and affect nerve activity during OA disease progression

Abstract. INTRODUCTION. The mechanisms underlying abnormal joint mechanics are poorly understood despite it being a major risk factor for developing osteoarthritis. Glutamate signalling has been implicated in osteoarthritic bone changes and AMPA/kainate glutamate receptor (GluR) antagonists alleviate degeneration in rodent models of osteoarthritis. We investigated whether glutamate signalling molecules are mechanically regulated in a human, cell-based 3D model of bone. METHODS. Human Y201 MSC cells embedded in 3D type I collagen gels (0.05 × 106 cell/gel) differentiated to osteocytes were mechanically loaded in silicone plates (5000 µstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). RNA extracted 1-hr post load was quantified by RTqPCR and RNAseq whole transcriptome analysis (NovaSeq S1 flow cell 2 × 100bp PE reads). Differentially expressed GluRs and glutamate transporters (GluTs) were identified using DEseq2 analysis on normalised count data. Genes were considered differentially expressed if >2 fold change and FDR p<0.05. RESULTS. Cells expressed mature osteocyte markers (E11, sclerostin, DMP-1). DEseq2 analysis, revealed 981 mechanically regulated genes. Mechanical loading upregulated kainate GluRs, GRIK2 (1.6 fold, p=0.024) and GRIK5 (4.2 fold, p=0.045); the NMDA GluR GRIN3B (3.25 fold, p=0.047) and the GluT SLC1A1 (3 fold, p=0.037). Conversely, AMPA GRIA3, NMDA GluRs GRIN2A&C, and the GluT SLC1A2 were down regulated by 50–60%, although not significant. Kainate GRIK3&4; AMPA GRIA2, NMDA GRIN1, and GluTs SLC1A6&A7 were not expressed in control or loaded osteocytes, whereas GluRs (GRIK1, GRIA1&4, GRIN2B&2D&3A) and GluT SLC1A3 were expressed but not regulated by mechanical loading. DISCUSSION. Mechanical loading of human osteocytes in 3D revealed that they regulated expression of glutamate receptors and transporters. This is consistent with our observation that mechanical perturbation after joint injury in rodent models of OA regulates glutamatergic signalling in the bone thus linking mechanical stimuli to inflammatory and nociceptive pathways mediated by glutamate receptors. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Introduction and Objective. The osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances that occur during bone remodeling, caused by hormone perturbations or alterations in mechanical loading, can induce bone disease as osteoporosis. Due to limited understanding of the underlying mechanisms, current therapies for osteoporosis cannot adequately address this imbalance because current studies of osteocytes rely on conventional cell culture that cannot recapitulate local in vivo microenvironments for the lack of control of the spatial/temporal distribution of cells and biomolecules. Microfluidics is the science and technology of microscale fluid manipulating and sensing and can help fill this gap. Materials and Methods. We used a microfluidic device to enable the culture of osteocyte-like cells (MLO-Y4 and MLO-A5) in a 3D fashion. Osteocytes were cultured in a perfused and 160 μm high channel and embedded in a bone-like extracellular matrix: osteocytes were embedded in a matrigel- and collagen-based hydrogel enriched with nanostructured hydroxypatite crystals (HA-NP) to mimic bone. To set up the best combination of matrigel enriched with Type I collagen we used fluorescent microspheres and confocal analysis. To evaluate the viability and the expression of osteocytic markers, we used live-dead assay amd immunofluorescent staining and confocal analysis combined with automated quantification. For mineralization, we performed alizarin red staining. Results. Osteocytes in the organ-on-a-chip model showed high viability and, in respect to 2D conventional cell cultures an increased differentiation, as assessed by a live-dead assay and the staining of the osteocytic markers connexin-43 and alkaline phosphatase and the increased mineralization activity. Furthermore, the addition of HA-NP significantly increased the formation of dendrite-like structures spreading through the xyz-axes, as assessed after G-actin immunofluorescence. Conclusions. Using a microfluidic device for MLO-Y4 and MLO-A5 cell cultures, compared to the 2D surfaces, we demonstrated a significant difference in cell differentiation and morphology. In particular, 3D cultures allowed the formation of 3D cell networks and the osteogenic phenotype. As a platform technology, this microfluidic device can function as a novel cell culture model that enables further studies of osteocytes and 3D co-culturing with other bone cells for the screening of anti-osteoporotic drugs

Osteoporosis is a progressive, chronic disease of bone metabolism, characterized by decreased bone mass and mineral density, predisposing individuals to an increased risk of fractures. The use of animal models, which is the gold standard for the screening of anti-osteoporosis drugs, raises numerous ethical concerns and is highly debated because the composition and structure of animal bones is very different from human bones. In addition, there is currently a poor translation of pre-clinical efficacy in animal models to human trials, meaning that there is a need for an alternative method of screening and evaluating new therapeutics for metabolic bone disorders, in vitro. The aim of this project is to develop a 3D Bone-On-A-Chip that summarizes the spatial orientation and mutual influences of the key cellular components of bone tissue, in a citrate and hydroxyapatite-enriched 3D matrix, acting as a 3D model of osteoporosis. To this purpose, a polydimethylsiloxane microfluidic device was developed by CAD modelling, stereolithography and replica molding. The device is composed by two layers: (i) a bottom layer for a 3D culture of osteocytes embedded in an osteomimetic collagen-enriched matrigel matrix with citrate-doped hydroxyapatite nanocrystals, and (ii) a upper layer for a 2D perfused co-culture of osteoblasts and osteoclasts seeded on a microporous PET membrane. Cell vitality was evaluated via live/dead assay. Bone deposition and bone resorption was analysed respectively with ALP, Alizarin RED and TRACP staining. Osteocytes dendrite expression was evaluated via immunofluorescence. Subsequently, the model was validated as drug screening platform inducing osteocytes apoptosis and administrating standard anti-osteoporotic drugs. This device has the potential to substitute or minimize animal models in pre-clinical studies of osteoporosis, contributing to pave the way for a more precise and punctual personalized treatment

Osteoporotic fracture has become a major problem in ageing population and often requires prolonged healing time. Low Intensity Pulsed Ultrasound (LIPUS) can significantly enhance fracture healing through alteration of osteocyte lacuno-canalicular network (LCN). DMP1 in osteocytes is responsible for maintaining LCN and mineralisation. This study aims to investigate osteocyte-specific DMP1's role in enhanced osteoporotic fracture healing in response to mechanical stimulation. Bilateral ovariectomy was performed in 6-month-old female SD rats to induce osteoporosis. Metaphyseal fracture was created at left distal femur using oscillating micro-saw. Rats were randomised to groups: (1) DMP1 KD, (2) DMP1 KD + LIPUS, (3) Control, or (4) Control + LIPUS, where KD stands for knockdown by injection of shRNA into marrow cavity 2 weeks before surgery. Assessments included weekly radiography, microCT and immunohistochemistry on DMP1, E11, FGF23 and sclerostin. DMP1 KD significantly impaired LIPUS-accelerated fracture healing when comparing KD + LIPUS group to Control + LIPUS group. The X-ray relative opacity showed less tissue growth at all timepoints (Week 1, 3 & 6; p=0.000, 0.001 and 0.003 respectively) and the bone volume fraction was decreased after DMP1 KD at Week 3 (p=0.006). DMP1 KD also significantly altered the expression levels of osteocyte-specific DMP1, E11, FGF23 and sclerostin during healing process. The lower relative opacity and bone volume fraction in DMP1 KD groups indicated that knockdown of DMP1 was associated with poorer fracture healing process compared to non-knockdown groups. The similar results between knockdown group with and without LIPUS showed that blockage of DMP1 would negate LIPUS-induced enhancement on fracture healing. Acknowledgment: General Research Fund (Ref: 14113018)

Age-related fragility fractures are highly correlated with the loss of bone integrity and deteriorated morphology of the osteocytes. Previous studies have reported low-magnitude high-frequency vibration(LMHFV) promotes osteoporotic diaphyseal fracture healing to a greater extent than in age-matched normal fracture healing, yet how osteoporotic fractured bone responds to the mechanical signal has not been explored. As osteocytes are prominent for mechanosensing and initiating bone repair, we hypothesized that LMHFV could enhance fracture healing in ovariectomized metaphyseal fracture through morphological changes and mineralisation in the osteocyte Lacuno-canalicular Network(LCN). As most osteoporotic fractures occur primarily at the metaphysis, an osteoporotic metaphyseal fracture model was established. A total of 72 six-month old female Sprague-Dawley rats (n=72) were obtained(animal ethical approval ref: 16–037-MIS). Half of the rats underwent bilateral ovariectomy(OVX) and kept for 3 months for osteoporosis induction. Metaphyseal fracture on left distal femur was created by osteotomy and fixed by a plate. Rats were then randomized to (1) OVX+LMHFV(20 mins/day and 5 days/week, 35Hz, 0.3g), (2) OVX control, (3) SHAM+LMHFV, (4) SHAM control. Assessments of morphological structural changes, functional markers of the LCN(Scanning Electron Microscopy, FITC-Imaris, immunohistochemistry), mineralization status(EDX, dynamic histomorphometry) and healing outcomes(X-ray, microCT, mechanical testing) were performed at week 1, 2 and 6 post-fracture. One‐way ANOVA with post-hoc test was performed. Statistical significance was set at p < 0.05. Our results showed LMHFV could significantly enhance the morphology of the LCN. There was a 65.3% increase in dendritic branch points(p=0.03) and 93% increase in canalicular length(p=0.019) in the OVX-LMHFV group at week 2 post-fracture. Besides, a similar trend was also observed in the SHAM+LMHFV group, with a 43.4% increase in branch points and 53% increase in canaliculi length at week 2. A significant increase of E11 and DMP1 was observed in the LMHFV groups, indicating the reconstruction of the LCN. The decreasing sclerostin and increasing FGF23 at week 1 represented the active bone formation phase while the gradual increase at week 6 signified the remodelling phase. Furthermore, Ca/P ratio, mineral apposition rate and bone formation rate were all significantly enhanced in the OVX+LMHFV group. The overall bone mineral density in BV was significantly raised in the OVX+LMHFV group at week 2(p=0.043) and SHAM+LMHFV at week 6(p=0.04). Quantitative analysis of microCT showed BV/TV was significantly increased at week 2 in OVX+LMHFV group(p=0.008) and week 6(p=0.001) in both vibration groups. In addition, biomechanical testing revealed that the OVX+LMHFV group had a significantly higher ultimate load(p=0.03) and stiffness(p=0.02) at week 2. To our best knowledge, this is the first report to illustrate LMHFV could enhance osteocytes' morphology, mineralisation status and healing outcome in a new osteoporotic metaphyseal fracture animal model. Our cumulative data supports that the mechanosensitivity of bone would not impair due to osteoporosis. The revitalized osteocyte LCN and upregulated osteocytic protein markers implied a better connectivity and transduction of signals between osteocytes, which may foster the osteoporotic fracture healing process through an enhanced mineralisation process. This could stimulate further mechanistic investigations with potential translation of LMHFV to our fragility fracture patients

Abstract. Objectives. The mechanisms underlying abnormal joint mechanics are poorly understood despite it being a major risk factor for developing osteoarthritis. This study investigated the response of a 3D in vitro bone cell model to mechanical load. Methods. Human MSC cells (Y201) embedded in 3D type I collagen gels were differentiated in osteogenic media for 7-days in deformable, silicone plates. Gels were loaded once (5000 µstrain, 10Hz, 3000 cycles), RNA extracted 1-hr post load and assessed by RT-qPCR and RNAseq analysis (n=5/treatment). Cell shape and phenotype were assessed by immunocytochemistry and phalloidin staining. Data was analysed by Minitab. Results. RTqPCR revealed cells expressed markers of mature osteocytes (E11, sclerostin, DMP-1) and osteoprotegerin (OPG), alkaline phosphatase and type I collagen (COL1A1). Immunolocalisation of sclerostin and DMP-1 protein along with phalloidin staining confirmed a dendritic osteocyte phenotype. Load almost abolished sclerostin gene expression (p=0.05) and reduced E11 (2-fold p=0.03); COL1A1 was unchanged (p=0.349). Using DEseq2 analysis, of the 981 genes differentially regulated more than 2-fold at FDR p<0.05, 159 were downregulated and 821 upregulated by load. These were involved in processes important in bone biology including the inflammatory response (56 genes), ECM organisation (27), ageing (30), response to mechanical load (23), ER stress (34), regulation of ossification (26), bone morphogenesis (14), cartilage development (14), programmed cell death (161), and positive regulation of bone mineralisation (6). Discussion. Y201 cells were successfully differentiated to osteocytes. The osteocytes’ mechanical response revealed regulation of factors that contribute to bone remodelling and inflammation. Since the biological mechanisms underlying mechanically induced joint degeneration are unclear, there is a need for humanised, cell models to delineate molecular pathways activated by mechanical load. Such pathways may reveal the molecular basis for genetic predispositions to osteoarthritis and identify new therapeutic targets. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Osteocytes direct bone adaptation to mechanical loading (e.g., exercise), but the ways in which osteocytes detect loading remain unclear. We recently showed that osteocytes develop repairable plasma membrane disruptions (PMD) in response to treadmill-running exercise, and that these PMD initiate mechanotransduction. As treadmill running is a non-voluntary activity for rodents, our current goal was to determine whether osteocytes develop PMD with voluntary wheel running as a better model of physiological exercise. Male and female Hsd:ICR mice from lines selectively bred (>75 generations) to demonstrate high voluntary wheel running (“High Runners”) or non-selected control lines (“Control”) were studied (n=9 to 12 mice per sex per line, 4 lines each). At 12 weeks of age, half of the animals within each group were provided access to running wheels for 6 days while remaining mice had no wheel access. Tibias were collected at sacrifice and bone mineral density was analyzed by DXA. Osteocyte PMD were quantified by immunochemistry for intracellular albumin. Groups were compared with 3-factor ANOVA. Voluntary exercise (wheel access) significantly increased osteocyte PMD (+16.4%, p=0.013). PMD-labelled osteocytes did not differ between sexes (p=0.415). Male mice had significantly greater BMD (p=0.0007) and BMC (<0.0001) than females. Interestingly, mice with wheel access had significantly lower BMD and BMC compared to mice without wheel access (p<0.004), and high runner lines had significantly lower BMD (p=0.001) and BMC (p<0.0001) than control lines. This may reflect new bone formation in the exercising mice, as newly formed bone is less mineralized than older bone. Data from this experiment support the idea that loading-induced disruptions develop in the osteocyte plasma membrane during both voluntary (wheel running) and forced (treadmill, shown previously) physical activity. These studies support the role of plasma membrane disruptions as a mechanosensation mechanism in osteocytes