Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network. hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting. Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics. We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases

Abstract. Objectives. Unicompartmental and total knee arthroplasty (UKA and TKA) are successful treatments for osteoarthritis, but monolithic implants disrupt the natural homeostasis of bone which leads to bone loss over time. This can cause problems if the implant needs to be revised. This study aimed to demonstrate that tibial implants made from titanium lattice could replace the tibial condyle surface while minimising disruption of the bone's natural mechanical loading environment. A secondary aim was to determine whether implants perform better if they replicate more closely bone's mechanical modulus, anisotropy and spatial heterogeneity. This study was conducted in a human cadaveric model. Methods. In a cadaveric model, UKA and TKA procedures were performed on 8 fresh-frozen knee specimens by a board-certified consultant orthopaedic surgeon, using tibial implants made from conventional monolithic material and titanium lattice structures. Stress at the bone-implant interfaces was measured with pressure film and compared to the native knee. Results. Titanium lattice implants were able to restore the mechanical environment seen in the native tibia for both UKA and TKA designs. Maximum stress at the bone-implant interface ranged from 1.2–3.3MPa compared to 1.3–2.7MPa for the native tibia. The conventional UKA and TKA implants reduced the maximum stress in the bone by a factor of 10 and 9.7 respectively. The conventional UKA and TKA implants caused 71% and 77% of bone surface area to be underloaded compared to the native tibia. Conclusions. Titanium lattice implants can maintain the natural mechanical loading in the proximal tibia after UKA and TKA. This may help maintain normal bone homeostasis throughout the life of the implant. These encouraging data indicate normal bone homeostasis can be maintained after arthroplasty using manufacturing methods already in widespread use. This would maintain bone quality throughout the life of the implant and alleviate complications at revision surgery

Anatomically, bone consists of building blocks called osteons, which in turn comprise a central canal that contains nerves and blood vessels. This indicates that bone is a highly innervated and vascularized tissue. The function of vascularization in bone (development) is well-established: providing oxygen and nutrients that are necessary for the formation, maintenance, and healing. As a result, in the field of bone tissue engineering many research efforts take vascularization into account, focusing on engineering vascularized bone. In contrast, while bone anatomy indicates that the role of innervation in bone is equally important, the role of innervation in bone tissue engineering has often been disregarded. For many years, the role of innervation in bone was mostly clear in physiology, where innervation of a skeleton is responsible for sensing pain and other sensory stimuli. Unraveling its role on a cellular level is far more complex, yet more recent research efforts have unveiled that innervation has an influence on osteoblast and osteoclast activity. Such innervation activities have an important role in the regulation of bone homeostasis, stimulating bone formation and inhibiting resorption. Furthermore, due to their anatomical proximity, skeletal nerves and blood vessels interact and influence each other, which is also demonstrated by pathways cross-over and joint responses to stimuli. Besides those closely connected sytems, the immune system plays also a pivotal role in bone regeneration. Certain cytokines are important to attract osteogenic cells and (partially) inhibit bone resorption. Several leukocytes also play a role in the bone regeneration process. Overall, bone interacts with several systems. Aberrations in those systems affect the bone and are important to understand in the context of bone regeneration. This crosstalk has become more evident and is taken more into consideration. This leads to more complex tissue regeneration, but may recapitulate better physiological situations

In 2021 the bone grafting market was worth €2.72 billion globally. As allograft bone has a limited supply and risk of disease transmission, the demand for synthetic grafting substitutes (BGS) continues to grow while allograft bone grafts steadily decrease. Synthetic BGS are low in mechanical strength and bioactivity, inspiring the development of novel grafting materials, a traditionally laborious and expensive process. Here a novel BGS derived from sustainably grown coral was evaluated. Coral-derived scaffolds are a natural calcium carbonate bio-ceramic, which induces osteogenesis in bone marrow mesenchymal stem cells (MSCs), the cells responsible for maintaining bone homeostasis and orchestrating fracture repair. By 3D printing MSCs in coral-laden bioinks we utilise high throughput (HT) fabrication and evaluation of osteogenesis, overcoming the limitations of traditional screening methods. MSC and coral-laden GelXA (CELLINK) bioinks were 3D printed in square bottom 96 well plates using a CELLINK BIO X printer with pneumatic adapter Samples were non-destructively monitored during the culture period, evaluating both the sample and the culture media for metabolism (PrestoBlue), cytotoxicity (lactose dehydrogenase (LDH)) and osteogenic differentiation (alkaline phosphatase (ALP)). Endpoint, destructive assays used included qRT-PCR and SEM imaging. The inclusion of coral in the printed bioink was biocompatable with the MSCs, as reflected by maintained metabolism and low LDH release. The inclusion of coral induced osteogenic differentiation in the MSCs as seen by ALP secretion and increased RUNX2, collagen I and osteocalcin transcription. Sustainably grown coral was successfully incorporated into bioinks, reproducibly 3D printed, non-destructively monitored throughout culture and induced osteogenic differentiation in MSCs. This HT fabrication and monitoring workflow offers a faster, less labour-intensive system for the translation of bone substitute materials to clinic. Acknowledgements: This work was co-funded by Enterprise Ireland and Zoan Biomed through Innovation Partnership IP20221024

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

Osteoclasts (OCs) are multinucleated cells that play a pivotal role in skeletal development and bone remodeling. Abnormal activation of OCs contributes to the development of bone-related diseases, such as osteoporosis, bone metastasis and osteoarthritis. Restoring the normal function of OCs is crucial for bone homeostasis. Recently, RNA therapeutics emerged as a new field of research for osteoarticular diseases. The aim of this study is to use non-coding RNAs (ncRNAs) to molecularly engineer OCs and modulate their function. Specifically, we investigated the role of the microRNAs (namely miR-16) and long ncRNAs (namely DLEU1) in OCs differentiation and fusion. DLEU1/DLEU2 region, located at chromosome 13q14, also encodes miR-15 and miR-16. Our results show that levels of these ncRNA transcripts are differently expressed at distinct stages of the OCs differentiation. Specifically, silencing of DLEU1 by small interfering RNAs (siDLEU1) and overexpression of miR-16 by synthetic miRNA mimics (miR-16-mimics) led to a significant reduction in the number of OCs formed per field (OC/field), both at day 5 and 9 of the differentiation stage. Importantly, time-lapse analysis, used to track OCs behavior, revealed a significant decrease in fusion events after transfection with siDLEU1 or miR-16-mimics and an alteration in the fusion mode and partners. Next, we investigated the migration profile of these OCs, and the results show that only miR-16-mimics-OCs, but not siDLEU-OCs, have a lower percentage of immobile cells and an increase in cells with mobile regime, compared with controls. No differences in cell shape were found. Moreover, mass-spectrometry quantitative proteomic analysis revealed independent effects of siDLEU1 and miR-16-mimics at the protein levels. Importantly, DLEU1 and miR-16 act by distinct processes and pathways. Collectively, our findings support the ncRNAs DLEU1 and miR-16 as therapeutic targets to modulate early stages of OCs differentiation and, consequently, to impair OC fusion, advancing ncRNA-therapeutics for bone-related diseases. Acknowledgements: Authors would like to thank to AO CMF / AO Foundation (AOCMFS-21-23A). SRM and MIA are supported by FCT (SFRH/BD/147229/2019 and BiotechHealth Program; CEECINST/00091/2018/CP1500/CT0011, respectively)

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

Introduction and Objective. Bone remodelling is a continuous process whereby osteocytes regulate the activity of osteoblasts and osteoclasts to repair loading-induced microdamage. While many in vitro studies have established the role of paracrine factors (e.g., RANKL/OPG) and cellular pathways involved in bone homeostasis, these techniques are generally limited to two-dimensional cell culture, which neglects the role of the native extracellular matrix in maintaining the phenotype of osteocyte. Recently, ex vivo models have been used to understand cell physiology and mechanobiology in the presence of the native matrix. Such approaches could be applicable to study the mechanisms of bone repair, whilst also enabling exploration of biomechanical cues. However, to date an ex vivo model of bone remodelling in cortical bone has not been developed. In this study, the objective was to develop an ex vivo model where cortical bone was subjected to cyclic strains to study the remodelling of bone. Materials and Methods. Ex vivo model of bone remodelling induced by cyclic loading: At the day of culling, beam-shape bovine bone samples were cut and preserved in PBS + 5% Pen/Strep + 2 mM L-Glut overnight at 37°C. Cyclic strains were applied with a three-point bend system to induce damage with a regime at 16.66 mm/min for 5,000 cycles in sterile PBS in Evolve® bags (maximum strain 6%). A control group was cultured under static conditions. Metabolic activity: Alamar Blue assays were performed after 1 and 7 days of ex vivo culture for each group (Static, Loaded) and normalized to weight. Bone remodelling: ALP activity was assessed in the media at day 1 and 7. After 24 hours cell culture conditioned media (CM) was collected from each group and stored at −80°C. RAW264.7 cells were cultured with CM for 6 days, after which the samples were stained for TRAP, to determine osteoclastogenesis, and imaged. Histomorphometry: Samples were cultured with calcein for 3 days to label bone formation between day 4 and 7. Fluorescent images were captured at day 7. μCT scanning was performed at 3 μm resolution after labelling samples with BaSO. 4. precipitate to quantify bone damage. Results. Bone was sectioned and cultured to maintain live osteoblasts and osteocytes. CM that was obtained 24 hours after cyclic loading and added to RAW264.7 cells cultures, resulted in significantly increased osteoclastogenic potential compared to that from static samples (4.245±1.65% vs 0.88±0.48%, p<0.001). Calcein and HE staining indicated the presence of structures similar to bone remodelling cones in both groups after 7 days of culture. Also, 7 days post-loading, matrix microdamage in the stimulated area, detected with the BaSO. 4. precipitate, were not significantly increased under the load point in loaded samples (0.11±0.05% of bone volume), while at the support areas it was significantly higher (0.2387±0.06%, p<0.001) compared to the static (0.062±0.02%). Conclusions. This study demonstrates that (1) cyclic strains applied on ex vivo bovine cortical bone successfully induced remodelling as characterized by the formation of bone resorption cones, along with an increase of osteoclast formation, and (2) there was an induction of microdamage post loading as shown by the significant increases in microdamage labelled. This supports previous in vivo studies with an increase in osteoclastogenesis up to 7 days post loading. This is the first evidence of the development of an ex vivo model to study osteon remodelling that could be applied to study bone physiology and repair

Osteoporosis is a mineral bone disease arising from the predominance of osteoclastic bone resorption. Bisphosphonates which inhibit osteoclasts are commonly used in osteoporosis treatment, but are not without severe adverse effects like osteonecrosis of the jaw. The mechanisms behind the development of such phenomena is not well understood. Bone homeostasis is achieved through an intimate cross-talk between osteoclasts and osteoblasts. Thus, it is important to visualise activities of these cells simultaneously in situ. Currently, there are means to visualise osteoclast shape and numbers with tartrate-resistant alkaline phosphatase (TRAP) staining but no practical and accurate methods to quantify osteoclast activity in situ. This investigation aims to establish the use of ELF97, a substrate of TRAP, to visualise and quantify osteoclast activity. This provides vital clues to mechanisms of various bone disorders. TRAP dephosphorylation of ELF97 results in a detectable fluorescent product at areas of osteoclast activity. Osteoclastic activity was initiated in zebrafish by inducing crush injuries in tail fin rays. Colocalisation of ELF97 fluorescence with osteoclast-specific DsRed in transgenic zebrafish, visualised under confocal microscopy, is used to further establish the specificity of ELF97 to sites of osteoclastic activity. Quantification is established by comparing fluorescence between wild type, osteoclast-deficient mutants and bisphosphonate-treated zebrafish. The utility of ELF97 will also be investigated in terms of the stability of the florescent product. The investigation revealed that ELF97 and DsRed fluorescence were found commonly at crush sites with osteoclastic activity. Wild type zebrafish had greater fluorescence compared to osteoclast-deficient (p<0.0001) and bisphosphonate-treated zebrafish (p<0.0001) after 7 and 14 days post-crush, revealing that fluorescence from ELF97 corresponds to expected osteoclastic activity. Fluorescence of tail fins treated with ELF97 did not diminish over a period of 21 days of storage, demonstrating its stability. ELF97 is thus a useful means to visualise osteoclast activity, potentially crucial in more advanced investigations to understand bone disorders. It could be used in combination with other cellular markers in whole biological samples to study and experimentally manipulate bone remodelling

The current procedures being applied in the clinical setting to address osteoporosis-related delayed union and nonunion bone fractures have been found to present mostly suboptimal outcomes. As a result, bone tissue engineering (BTE) solutions involving the development of implantable biomimetic scaffolds to replace damaged bone and support its regeneration are gaining interest. The piezoelectric properties of the bone tissue, which stem primarily from the significant presence of piezoelectric type I collagen fibrils in the tissue's extracellular matrix (ECM), play a key role in preserving the bone's homeostasis and provide integral assistance to the regeneration process. However, despite their significant potential, these properties of bone tend to be overlooked in most BTE-related studies. In order to bridge this gap in the literature, novel hydroxyapatite (HAp)-filled osteoinductive and piezoelectric poly(vinylidene fluoride-co-tetrafluoroethylene) (PVDF-TrFE) electrospun nanofibers were developed to replicate the bone's fibrous ECM composition and electrical features. Different HAp nanoparticle concentrations (1–10%, wt%) were tested to assess their effect on the physicochemical and biological properties of the resulting fibers. The fabricated scaffolds displayed biomimetic collagen fibril-like diameters, while also presenting mechanical features akin to type I collagen. The increase in HAp presence was found to enhance both surface and piezoelectric properties of the fibers, with an improvement in scaffold wettability and increase in β-phase nucleation (translating to increased piezoelectricity) being observed. The HAp-containing scaffolds also exhibited an augmented bioactivity, with a more comprehensive surface mineralization of the fibers being obtained for the scaffolds with the highest HAp concentrations. Improved osteogenic differentiation of seeded human mesenchymal stem/stromal cells was achieved with the addition of HAp, as confirmed by an increased ALP activity, calcium deposition and upregulated expression of key osteogenic markers. Overall, our findings highlight, for the first time, the potential of combining PVDF-TrFE and HAp to develop electroactive and osteoinductive nanofibers for BTE. Acknowledgements: The authors thank FCT for funding through the projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), OptiBioScaffold (PTDC/EME-SIS/4446/2020) and BioMaterARISES (EXPL/CTM-CTM/0995/2021), the PhD scholarship (2022.10572.BD) and to the research institutions iBB (UIDB/04565/2020 and UIDP/04565/2020) and Associate Laboratory i4HB (LA/P/0140/2020)

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

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

Fatty marrow and bone loss are prominent pathologic features of osteoporosis. DNA hypermethylation shifts mesenchymal stem cells towards adipocytes impairing bone formation. Brown adipocytes produce growth factors advantageous to osteogenesis, whereas white adipocytes secrete pro-inflammatory cytokines deleterious to bone homeostasis. We assess DNA methylation inhibitor action to brown and white adipocyte formation in marrow fat of osteoporotic skeletons. Osteoporotic skeletons in mice were induced by glucocorticoid, ovariectomy or ageing. Marrow adipose volume and bone structure were quantified using OsO4 contrast-μCT imaging. Brown and white adipocytes were probed using immunostaining, RT-PCR and primary bone-marrow mesenchymal stem cell cultures. Abundant marrow fat and spare trabecular bone existed in osteoporotic skeletons. Osteoporosis increased expressions of general adipogenic markers PPARγ2 and FABP4 and white adipocyte markers TCF21 and HOXc9, whereas expressions of brown adipocyte markers PGC-1α and UCP-1 and osteogenic markers Runx2 and osteocalcin were significantly decreased. Number of UCP-1 immunostaining-positive brown adipocytes also reduced in osteoporotic bone. In vitro, DNA methylation inhibitor 5'-aza-deoxycystidine significantly increased brown adipocyte formation and osteogenic differentiation and mitigated dexamethasone-induced white adipocyte formation in mesenchymal stem cells. 5'-aza-deoxycystidine control of brown adipogenesis and white fat formation appeared to be regulated by increasing Wnt3a/β-catenin and reducing Dkk1. Disintegrated brown adipocyte and white fat cell differentiation contribute to osteoporosis pathogenesis. Maintaining DNA hypomethylation promotes Wnt signalling and brown adipocyte differentiation facilitating osteogenic differentiation. This study shed a new light to the contribution of brown adipocytic cells to bone metabolism during osteoporosis

Introduction and Objective. Senescent bone cell overburden accelerates osteoporosis. Epigenetic alteration, including microRNA signalling and DND methylation, is one of prominent features of cellular senescence. This study aimed to investigate what role microRNA-29a signalling may play in the development of senile osteoporosis. Materials and Methods. Bone biopsy and serum were harvested from 13 young patients and 15 senior patients who required spine surgery. Bone mass, microstructure, and biomechanics of miR-29a knockout mice (miR-29aKO) and miR-29a transgenic mice (miR-29aTg) were probed using mCT imaging and three-point bending material test. Senescent cells were probed using senescence-associated b-galactosidase (SA-b-gal) staining. Transcriptomic landscapes of osteoblasts were characterized using whole genome microarray and KEGG bioinformatics. miR-29a and senescence markers p16. INK4a. , p21. Waf/cipl. and inflammatory cytokines were quantified using RT-PCR. DNA methylome was probed using methylation-specific PCR and 5-methylcytosine immunoblotting. Results. Senescent osteoblast overburden, DNA hypermethylation and oxidative damage together with significant decreases in serum miR-29a levels were present in bone specimens of aged patients. miR-29aKO mice showed a phenotype of skeletal underdevelopment, low bone mineral density and weak biomechanics. miR-29a knockout worsened age-induced bone mass and microstructure deterioration. Of note, aged miR-29aTg mice showed less bone loss and fatty marrow than aged wild-type mice. Transgenic overexpression of miR-29s compromised age-dysregulated osteogenic differentiation capacity of bone-marrow mesenchymal cells. In vitro, miR-29a promoted transcriptomic landscapes of antioxidant proteins in osteoblasts. The microRNA interrupted DNA methyltransferase (Dnmt3b)-mediated DNA methylation, inhibiting reactive oxygen radicals burst, IL-6 and RANKL production, and a plethora of senescent activity, including increased p16. INK4a. , p21. Waf/cipl. signalling and SA-b-gal activity. Conclusions. miR-29a loss is correlated with human age-mediated osteoporosis. miR-29a signalling is indispensable in bone mase homeostasis and microstructure integrity. Gain of miR-29a function is advantageous to delay age-induced bone loss through promoting antioxidant proteins to inhibit DNA hypermethylation-mediated osteoblast senescence. Collective investigations shine light onto the anabolic effects miR-29a signalling to bone integrity and highlight a new epigenetic protection strategy through controlling microRNA signalling to delay osteoblast senescence and senile osteoporosis development

Background/Aims. Bisphosphonates play an important role in the treatment of catabolic bone diseases such as osteoporosis. In addition to their anti-resorptive activity exerted by their proapoptotic effect on osteoclasts, recent data suggest that nitrogen-containing bisphosphonates (N-BP) may also promote osteogenic differentiation by an unknown mechanism. Similar bone-anabolic effects have been attributed to cholesterol-lowering statins, which represent another class of mevalonate pathway inhibitors besides N-BP, suggesting a common mode of action. In vascular endothelial cells statins were recently shown to activate the Mek5/Erk5 mitogen-activated protein kinase cascade, which plays an important role in cellular differentiation, apoptosis or inflammatory processes. Here we evaluated whether N-BPs may also target the Mek5/Erk5 pathway and analysed the consequence of Erk5 activation on bone-relevant gene expression, calcification and osteoblast differentiation. Methods and Results. We show that N-BP dose-dependently activate Erk5 in primary human endothelial cells and osteoblasts. The mechanism likely involves farnesyldiphosphate synthase (FDPS) inhibition and subsequent inactivation of the small GTPase Cdc42 since siRNA-mediated knockdown of both genes could reproduce N-BP-induced ERK5 activation. ERK5 activation resulted in regulation of several bone-relevant genes and was required for calcification and osteoblastic differentiation of mesenchymal stems cells as evident by the lack of alkaline phosphatase induction and alizarin-red staining observed upon Erk5 knockdown or upon differentiation initiation in presence of a pharmacological Erk5 inhibitor. Conclusion. Our data provide first evidence that N-BP activate the Mek5/Erk5 cascade and reveal an essential role of Erk5 in the regulation of bone homeostasis by influencing bone mineralization and osteoblast differentiation

Osteoporosis is an international health and financial burden of ever increasing proportions. Current treatments limit the rate of bone resorption and reduce fracture risk, however they are often associated with significant and debilitating side effects. The most commonly used therapies also do not stimulate osteoblast activity. Much current research focus is aimed at the metabolic and epigenetic pathways involved in osteoporosis. MicroRNAs have been shown to play an important role in bone homeostasis and pathophysiological conditions of the musculoskeletal system. Upregulation of specific microRNAs has been identified in-vivo in osteoporotic patients. It is hypothesized that modulation of specific mircoRNA expression may have a key role in future targeted therapies of musculoskeletal diseases. The assessment and analysis of their potential therapeutic use in Osteoporosis is of great importance, due to the burden of the disease. We have developed a 3D osteoporotic model from human bone marrow, without the use of scaffold. Magnetic nanoparticles are utilised to form spheroids, which provides a closer representation of the in-vivo environment than monolayer culture. This model will provide the basis for analysing future microRNA experiments to assess the potential upregulation of osteoblastogenesis without cessation of osteoclast activity. The results of initial monolayer and spheroid experiments will be presented. Optimisation of the osteoporotic bone marrow culture conditions, involving response to differentiation medias, analysis of adipose and bone markers and cell migration in spheroid culture will be displayed. Quantitative and qualitative results, including fluorescence microscopy and in cell western, assessing the monolayer and spheroid cultures will be presented. The development of a pseudo osteoporosis model from healthy bone marrow will also be discussed. This model will form a basis of future work on miRNA targeting

Osteoporosis is an international health and financial burden of ever increasing proportions. Current treatments limit the rate of bone resorption and reduce fracture risk, however they are often associated with significant and debilitating side effects. The most commonly used therapies also do not stimulate osteoblast activity . 1,2,3. Much current research focus is aimed at the metabolic and epigenetic pathways involved in osteoporosis. MicroRNAs have been shown to play an important role in bone homeostasis and pathophysiological conditions of the musculoskeletal system. Up-regulation of specific microRNAs has been identified in-vivo in osteoporotic patients . 4,5. It is hypothesized that modulation of specific microRNA expression may have a key role in future targeted therapies of musculoskeletal diseases. The assessment and analysis of their potential therapeutic use in Osteoporosis is of great importance, due to the burden of the disease. We have developed a 3D osteoporotic model from human bone marrow, without the use of scaffold. Magnetic nanoparticles are utilised to form spheroids, which provides a closer representation of the in-vivo environment than monolayer culture. This model will provide the basis for analysing future microRNA experiments to assess the potential up-regulation of osteoblastogenesis without cessation of osteoclast activity. The results of initial monolayer and spheroid experiments will be presented. Optimisation of the osteoporotic bone marrow culture conditions, involving response to differentiation medias, analysis of adipose and bone markers and cell migration in spheroid culture will be displayed. Quantitative and qualitative results, including fluorescence microscopy and in cell western, assessing the monolayer and spheroid cultures will be presented. The development of a pseudo osteoporosis model from healthy bone marrow will also be discussed. This model will form a basis of future work on microRNA targeting. The development of improved therapies for osteoporosis is of great significance due to the predicted rise in incidence of the disease and associated fragility fractures. Targeted therapies, such as the manipulation of microRNA expression, offer the opportunity to increase osteoblastogenesis and decrease osteoclastogenesis, potentially without the associated side effects of older, systemic therapies. We believe our 3D human bone marrow derived osteoporotic model offers the closest relation to the in-vivo environment for assessment and manipulation of microRNA expression

The pathogenesis of falling bone mineral density (BMD) as a universal feature of advancing age is poorly understood. 1. Frequently culminating in the development of osteoporosis, the process is attributable to more than 500,000 fragility fractures occurring every year in the UK Such injuries are associated with great levels of morbidity, mortality and a £3.5 billion cost to the healthcare economy. 2. . With age, humans are known to accumulate somatic mitochondrial DNA (mtDNA) mutations in mitotic and post mitotic tissue, and stem cell precursors. 3. Compelling evidence in recent years, particularly that provided by animal models suggests that these mutations are intrinsic to the ageing process. 4–6. We provide evidence for the first time that mitochondrial dysfunction contributes significantly to the failure of bone homeostasis and falling BMD. We have utilised a mouse model that accumulates mtDNA mutations at 3–5 times the rate of normal mice, consequently ageing and developing osteoporosis prematurely. 7. , to clearly demonstrate that osteoblasts are vulnerable to mtDNA mutations. We have developed a new quadruple immunofluorescent assay to show that mitochondrial respiratory chain dysfunction occurs in osteoblasts as a consequence (p < 0.0001). We show that this mitochondrial dysfunction is associated with reduced BMD in female and male mice by 7 (p = 0.003) and 11 (p = 0.0003) months of age respectively. Using osteoblasts derived from mesenchymal stem cells extracted from male and female mice with mitochondrial dysfunction aged 4, 7 and 11 months, we demonstrate a vastly reduced capacity to produce new mineralised bone in vitro when compared to wild type cell lines (p < 0.0001). Exercise was found to have no beneficial effect on osteoblast and whole bone phenotype in this mouse model. It is likely that mtDNA mutations accumulating over a longer time period in human ageing have significantly detrimental effects on bone biology and diminishing BMD

Aims

This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation.

Mechanical loading is a potent stimulator of bone formation. A screen for genes associated with mechanically-induced osteogenesis implicated the glutamate transporter GLAST-1 (1), in the mechanoresponse. We are investigating whether modulation of glutamate transporters represents a potential anabolic therapy in bone. Bone cells express functional components from each stage of the glutamate signalling pathway and activation of ionotropic glutamate receptors on osteoblasts can increase bone forming activity (2). Five high affinity Na+-dependant excitatory amino acid transporters (EAATs 1-5) regulate glutamatergic signalling. EAAT1 (GLAST-1) is expressed by osteocytes and bone-forming osteoblasts in vivo. We quantified transcripts for EAATs 1-3 and two splice variants (EAAT1a and EAAT1ex9skip) in human osteoblasts (MG63, SaOS-2 and primary) using real time-PCR. EAAT1a expression was very low whilst levels of the dominant negative EAAT1ex9skip were much higher in all cell types. EAAT1 and EAAT3 proteins were detected by immunofluorescence. We also demonstrated that glutamate transporters function in human osteoblasts. Sodium-dependent 14C-labelled glutamate uptake, sensitive to pharmacological EAAT inhibitors (t-PDC, TBOA) and extracellular glutamate concentration (10-500μM) was detected in MG63 and SaOS-2 cells. To determine whether modulation of EAATs can influence bone formation, we used pharmacological inhibitors of EAATs 1-5 (t-PDC and TBOA) and also over-expressed EAAT1exon9skip using antisense oligonucleotides (AONs) targeted to splice donor sequence of exon 9. Experiments were performed in 0-500μM glutamate. Pharmacological inhibition of EAATs over 5-21 days increased alkaline phosphatase activity and mineralisation of SaOS-2 cells and human primary osteoblasts. Over-expression of EAAT1ex9skip significantly increased cell number and decreased cell death as well as significantly increasing PCNA, Osteonectin and Type I collagen mRNAs in MG63 cells. Furthermore, over-expression of EAAT1ex9skip increased mean alkaline phosphatase activity over 48hrs in SaOS-2 cells. These data show that EAATs are expressed and functional in osteoblasts and that pharmaceutical and genetic inhibition of their activity increases bone formation. These mechanically regulated glutamate transporters are important in regulating bone homeostasis and their manipulation may represent a new anabolic therapy for the treatment of disorders such as osteoporosis or non-union fractures