The effects of physiological and injurious hydrostatic pressure on murine ex vivo articular and growth plate cartilage explants: an RNAseq study.

Introduction: Chondrocytes are continuously exposed to loads placed upon them. Physiological loads are pivotal to the maintenance of articular cartilage health, while abnormal loads contribute to pathological joint degradation. Similarly, the growth plate cartilage is subject to various loads during growth and development. Due to the high-water content of cartilage, hydrostatic pressure is considered one of the main biomechanical influencers on chondrocytes and has been shown to play an important role in the mechano-regulation of cartilage. Methods: Herein, we conducted RNAseq analysis of ex vivo hip cap (articular), and metatarsal (growth plate) cartilage cultures subjected to physiological (5 MPa) and injurious (50 MPa) hydrostatic pressure, using the Illumina platform (n = 4 replicates). Results: Several hundreds of genes were shown to be differentially modulated by hydrostatic pressure, with the majority of these changes evidenced in hip cap cartilage cultures (375 significantly upregulated and 322 downregulated in 5 MPa versus control; 1022 upregulated and 724 downregulated in 50 MPa versus control). Conversely, fewer genes were differentially affected by hydrostatic pressure in the metatarsal cultures (5 significantly upregulated and 23 downregulated in 5 MPa versus control; 7 significantly upregulated and 19 downregulated in 50 MPa versus control). Using Gene Ontology annotations for Biological Processes, in the hip cap data we identified a number of pathways that were modulated by both physiological and injurious hydrostatic pressure. Pathways upregulated in response to 50 MPa versus control, included those involved in the generation of precursor metabolites and cellular respiration. Biological processes that were downregulated in this tissue included ossification, connective tissue development, and chondrocyte differentiation. Discussion: Collectively our data highlights the divergent chondrocyte phenotypes in articular and growth plate cartilage. Further, we show that the magnitude of hydrostatic pressure application has distinct effects on gene expression and biological processes in hip cap cartilage explants. Finally, we identified differential expression of a number of genes that have previously been identified as osteoarthritis risk genes, including Ctsk, and Chadl. Together these data may provide potential genetic targets for future investigations in osteoarthritis research and novel therapeutics.

Increased PHOSPHO1 and alkaline phosphatase expression during the anabolic bone response to intermittent parathyroid hormone delivery.

The administration of intermittent parathyroid hormone (iPTH) is anabolic to the skeleton. Recent studies with cultured osteoblasts have revealed that the expression of PHOSPHO1, a bone-specific phosphatase essential for the initiation of mineralisation, is regulated by PTH. Therefore, this study sought to determine whether the bone anabolic response to iPTH involves modulation of expression of Phospho1 and of other enzymes critical for bone matrix mineralisation. To mimic iPTH treatment, primary murine osteoblasts were challenged with 50 nM PTH for 6 h in every 48 h period for 8 days (4 cycles), 14 days (7 cycles) and 20 days (10 cycles) in total. The expression of both Phospho1 and Smpd3 was almost completely inhibited after 4 cycles, whereas 10 cycles were required to stimulate a similar response in Alpl expression. To explore the in vivo role of PHOSPHO1 in PTH-mediated osteogenesis, the effects of 14- and 28-day iPTH (80 µg/kg/day) administration was assessed in male wild-type (WT) and Phospho1-/- mice. The expression of Phospho1, Alpl, Smpd3, Enpp1, Runx2 and Trps1 expression was enhanced in the femora of WT mice following iPTH administration but remained unchanged in the femora of Phospho1-/- mice. After 28 days of iPTH administration, the anabolic response in the femora of WT was greater than that noted in Phospho1-/- mice. Specifically, cortical and trabecular bone volume/total volume, as well as cortical thickness, were increased in femora of iPTH-treated WT but not in iPTH-treated Phospho1-/- mice. Trabecular bone osteoblast number was also increased in iPTH-treated WT mice but not in iPTH-treated Phospho1-/-  mice. The increased levels of Phospho1, Alpl, Enpp1 and Smpd3 in WT mice in response to iPTH administration is consistent with their contribution to the potent anabolic properties of iPTH in bone. Furthermore, as the anabolic response to iPTH was attenuated in mice deficient in PHOSPHO1, this suggests that the osteoanabolic effects of iPTH are at least partly mediated via bone mineralisation processes.

UK Nutrition Research Partnership 'Hot Topic' workshop: Vitamin D-A multi-disciplinary approach to (1) elucidate its role in human health and (2) develop strategies to improve vitamin D status in the UK population.

Vitamin D is truly unique-not a 'vital' amine in the true sense of the word, but rather a prohormone, which is produced in the skin during exposure to sunlight (UVB radiation at 290-315 nm) and which can also be obtained from food and from supplements. A high prevalence of low vitamin D status has been reported across the world in a wide range of population groups, and this includes communities living in low latitude areas despite the abundance of sunlight. It is accepted that vitamin D status is reflected by the level of the circulating metabolite 25-hydroxyvitamin D (25[OH]D), which is produced by hepatic hydroxylation of vitamin D, derived either from the skin from UV exposure or the gut from oral intake. Vitamin D has been associated with a wide range of health outcomes, but controversies remain as to their exact nature and extent and whether associations are in the causal pathway. In order to enable wider discussions on this nutrient, a 'Hot Topic' Vitamin D Workshop achieved funding from the UK Nutrition Research Partnership Medical Research Council call. The objectives of the workshop were (1) to elucidate the role of vitamin D in human health and (2) develop strategies to improve vitamin D status in the UK population. This paper provides a detailed resume of the discussions of the workshop; of the presentations and concomitant Q&As; and of identified areas for future research.

Increased PHOSPHO1 expression mediates cortical bone mineral density in renal osteodystrophy.

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities, a condition known as renal osteodystrophy (ROD). While tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 are critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both WT and Phospho1 knockout (P1KO) mice through dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (P < 0.05) but decreased in trabecular bone (P < 0.05). These changes were accompanied by decreased TNAP (P < 0.01) and increased PHOSPHO1 (P < 0.001) expression in WT CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expressions to be downregulated in response to phosphate and PTH. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism. Increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

PALMD regulates aortic valve calcification via altered glycolysis and NF-κB-mediated inflammation.

Recent genome-wide association and transcriptome-wide association studies have identified an association between the PALMD locus, encoding palmdelphin, a protein involved in myoblast differentiation, and calcific aortic valve disease (CAVD). Nevertheless, the function and underlying mechanisms of PALMD in CAVD remain unclear. We herein investigated whether and how PALMD affects the pathogenesis of CAVD using clinical samples from CAVD patients and a human valve interstitial cell (hVIC) in vitro calcification model. We showed that PALMD was upregulated in calcified regions of human aortic valves and calcified hVICs. Furthermore, silencing of PALMD reduced hVIC in vitro calcification, osteogenic differentiation, and apoptosis, whereas overexpression of PALMD had the opposite effect. RNA-Seq of PALMD-depleted hVICs revealed that silencing of PALMD reduced glycolysis and nuclear factor-κB (NF-κB)-mediated inflammation in hVICs and attenuated tumor necrosis factor α-induced monocyte adhesion to hVICs. Having established the role of PALMD in hVIC glycolysis, we examined whether glycolysis itself could regulate hVIC osteogenic differentiation and inflammation. Intriguingly, the inhibition of PFKFB3-mediated glycolysis significantly attenuated osteogenic differentiation and inflammation of hVICs. However, silencing of PFKFB3 inhibited PALMD-induced hVIC inflammation, but not osteogenic differentiation. Finally, we showed that the overexpression of PALMD enhanced hVIC osteogenic differentiation and inflammation, as opposed to glycolysis, through the activation of NF-κB. The present study demonstrates that the genome-wide association- and transcriptome-wide association-identified CAVD risk gene PALMD may promote CAVD development through regulation of glycolysis and NF-κB-mediated inflammation. We propose that targeting PALMD-mediated glycolysis may represent a novel therapeutic strategy for treating CAVD.

Ciliary IFT88 Protects Coordinated Adolescent Growth Plate Ossification From Disruptive Physiological Mechanical Forces.

Compared with our understanding of endochondral ossification, much less is known about the coordinated arrest of growth defined by the narrowing and fusion of the cartilaginous growth plate. Throughout the musculoskeletal system, appropriate cell and tissue responses to mechanical force delineate morphogenesis and ensure lifelong health. It remains unclear how mechanical cues are integrated into many biological programs, including those coordinating the ossification of the adolescent growth plate at the cessation of growth. Primary cilia are microtubule-based organelles tuning a range of cell activities, including signaling cascades activated or modulated by extracellular biophysical cues. Cilia have been proposed to directly facilitate cell mechanotransduction. To explore the influence of primary cilia in the mouse adolescent limb, we conditionally targeted the ciliary gene Intraflagellar transport protein 88 (Ift88fl/fl ) in the juvenile and adolescent skeleton using a cartilage-specific, inducible Cre (AggrecanCreERT2 Ift88fl/fl ). Deletion of IFT88 in cartilage, which reduced ciliation in the growth plate, disrupted chondrocyte differentiation, cartilage resorption, and mineralization. These effects were largely restricted to peripheral tibial regions beneath the load-bearing compartments of the knee. These regions were typified by an enlarged population of hypertrophic chondrocytes. Although normal patterns of hedgehog signaling were maintained, targeting IFT88 inhibited hypertrophic chondrocyte VEGF expression and downstream vascular recruitment, osteoclastic activity, and the replacement of cartilage with bone. In control mice, increases to physiological loading also impair ossification in the peripheral growth plate, mimicking the effects of IFT88 deletion. Limb immobilization inhibited changes to VEGF expression and epiphyseal morphology in Ift88cKO mice, indicating the effects of depletion of IFT88 in the adolescent growth plate are mechano-dependent. We propose that during this pivotal phase in adolescent skeletal maturation, ciliary IFT88 protects uniform, coordinated ossification of the growth plate from an otherwise disruptive heterogeneity of physiological mechanical forces. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The Effects of Exercise on Bone Mineral Density in Men: A Systematic Review and Meta-Analysis of Randomised Controlled Trials.

The aim of this systematic review and meta-analysis was to provide an updated analysis, including the use of more robust methods, on the effects of exercise on bone mineral density in men. Randomised Control Trials of > 24 weeks and published in English up to 01/05/20 were retrieved from 3 electronic databases, cross-referencing, and expert review. The primary outcome measures were changes in FN, LS, and lower limb BMD Standardised effect sizes were calculated from each study and pooled using the inverse heterogeneity model. A statistically significant benefit of exercise was observed on FN BMD [g = 0.21 (0.03, 0.40), Z = 2.23 p = 0.03], with no observed statistically significant benefit of exercise on LS BMD [g = 0.10 (- 0.07, 0.26), Z = 1.15 p = 0.25]. This analysis provided additional evidence to recommend ground- and/or joint-reaction force exercises for improving or maintaining FN, but not LS BMD. Additional well-designed RCTs are unlikely to alter this evidence, although interventions that include activities that directly load the lumbar spine are needed to ensure this is not a potential method of improving LS BMD.

Characterisation of Growth Plate Dynamics in Murine Models of Osteoarthritis.

The purpose of this study was to investigate growth plate dynamics in surgical and loading murine models of osteoarthritis, to understand whether abnormalities in these dynamics are associated with osteoarthritis development. 8-week-old C57BL/6 male mice underwent destabilisation of medial meniscus (DMM) (n = 8) surgery in right knee joints. Contralateral left knee joints had no intervention (controls). In 16-week-old C57BL/6 male mice (n = 6), osteoarthritis was induced using non-invasive mechanical loading of right knee joints with peak force of 11N. Non-loaded left knee joints were internal controls. Chondrocyte transiency in tibial articular cartilage and growth plate was confirmed by histology and immunohistochemistry. Tibial subchondral bone parameters were measured using microCT and correlated to 3-dimensional (3D) growth plate bridging analysis. Higher expression of chondrocyte hypertrophy markers; Col10a1 and MMP13 were observed in tibial articular cartilage chondrocytes of DMM and loaded mice. In tibial growth plate, Col10a1 and MMP13 expressions were widely expressed in a significantly enlarged zone of proliferative and hypertrophic chondrocytes in DMM (p=0.002 and p<0.0001, respectively) and loaded (both p<0.0001) tibiae of mice compared to their controls. 3D quantification revealed enriched growth plate bridging and higher bridge densities in medial compared to lateral tibiae of DMM and loaded knee joints of the mice. Growth plate dynamics were associated with increased subchondral bone volume fraction (BV/TV; %) in medial tibiae of DMM and loaded knee joints and epiphyseal trabecular bone volume fraction in medial tibiae of loaded knee joints. The results confirm articular cartilage chondrocyte transiency in a surgical and loaded murine models of osteoarthritis. Herein, we reveal spatial variation of growth plate bridging in surgical and loaded osteoarthritis models and how these may contribute to anatomical variation in vulnerability of osteoarthritis development.

Proton Pump Inhibitors Inhibit PHOSPHO1 Activity and Matrix Mineralisation In Vitro.

Proton pump inhibitors (PPIs) have been associated with an increased risk of fragility fractures in pharmaco-epidemiological studies. The mechanism is unclear, but it has been speculated that by neutralising gastric acid, they may reduce intestinal calcium absorption, causing secondary hyperparathyroidism and bone loss. Here we investigated that hypothesis that the skeletal effects of PPI might be mediated by inhibitory effects on the bone-specific phosphatase PHOSPHO1. We found that the all PPIs tested inhibited the activity of PHOSPHO1 with IC50 ranging between 0.73 µM for esomeprazole to 19.27 µM for pantoprazole. In contrast, these PPIs did not inhibit TNAP activity. We also found that mineralisation of bone matrix in primary osteoblast cultures was inhibited by several PPIs in a concentration dependent manner. In contrast, the histamine-2 receptor antagonists (H2RA) nizatidine, famotidine, cimetidine and ranitidine had no inhibitory effects on PHOSPHO1 activity. Our experiments show for the first time that PPIs inhibit PHOSPHO1 activity and matrix mineralisation in vitro revealing a potential mechanism by which these widely used drugs are associated with the risk of fractures.

Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.

Zinc ameliorates human aortic valve calcification through GPR39 mediated ERK1/2 signalling pathway.

AIMS: Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. It has been reported that zinc is accumulated in calcified human aortic valves. However, whether zinc directly regulates CAVD is yet to be elucidated. The present study sought to determine the potential role of zinc in the pathogenesis of CAVD. METHODS AND RESULTS: Using a combination of a human valve interstitial cell (hVIC) calcification model, human aortic valve tissues, and blood samples, we report that 20 µM zinc supplementation attenuates hVIC in vitro calcification, and that this is mediated through inhibition of apoptosis and osteogenic differentiation via the zinc-sensing receptor GPR39-dependent ERK1/2 signalling pathway. Furthermore, we report that GPR39 protein expression is dramatically reduced in calcified human aortic valves, and there is a significant reduction in zinc serum levels in patients with CAVD. Moreover, we reveal that 20 µM zinc treatment prevents the reduction of GPR39 observed in calcified hVICs. We also show that the zinc transporter ZIP13 and ZIP14 are significantly increased in hVICs in response to zinc treatment. Knockdown of ZIP13 or ZIP14 significantly inhibited hVIC in vitro calcification and osteogenic differentiation. CONCLUSIONS: Together, these findings suggest that zinc is a novel inhibitor of CAVD, and report that zinc transporter ZIP13 and ZIP14 are important regulators of hVIC in vitro calcification and osteogenic differentiation. Zinc supplementation may offer a potential therapeutic strategy for CAVD.

Conditional deletion of E11/Podoplanin in bone protects against ovariectomy-induced increases in osteoclast formation and activity.

E11/Podoplanin (Pdpn) is implicated in early osteocytogenesis and the formation of osteocyte dendrites. This dendritic network is critical for bone modelling/remodelling, through the production of receptor activator of nuclear factor κ B (RANK)-ligand (RANKL). Despite this, the role of Pdpn in the control of bone remodelling is yet to be established in vivo. Here we utilised bone-specific Pdpn conditional knockout mice (cKO) to examine the role of Pdpn in the bone loss associated with ovariectomy (OVX). MicroCT revealed that Pdpn deletion had no significant effect on OVX-induced changes in trabecular microarchitecture. Significant differences between genotypes were observed in the trabecular pattern factor (P<0.01) and structure model index (P<0.01). Phalloidin staining of F-actin revealed OVX to induce alterations in osteocyte morphology in both wild-type (WT) and cKO mice. Histological analysis revealed an expected significant increase in osteoclast number in WT mice (P<0.01, compared with sham). However, cKO mice were protected against such increases in osteoclast number. Consistent with this, serum levels of the bone resorption marker Ctx were significantly increased in WT mice following OVX (P<0.05), but were unmodified by OVX in cKO mice. Gene expression of the bone remodelling markers Rank, Rankl, Opg and Sost were unaffected by Pdpn deletion. Together, our data suggest that an intact osteocyte dendritic network is required for sustaining osteoclast formation and activity in the oestrogen-depleted state, through mechanisms potentially independent of RANKL expression. This work will enable a greater understanding of the role of osteocytes in bone loss induced by oestrogen deprivation.

Raman spectroscopy links differentiating osteoblast matrix signatures to pro-angiogenic potential.

Mineralization of bone is achieved by the sequential maturation of the immature amorphous calcium phase to mature hydroxyapatite (HA) and is central in the process of bone development and repair. To study normal and dysregulated mineralization in vitro, substrates are often coated with poly-l-lysine (PLL) which facilitates cell attachment. This study has used Raman spectroscopy to investigate the effect of PLL coating on osteoblast (OB) matrix composition during differentiation, with a focus on collagen specific proline and hydroxyproline and precursors of HA. Deconvolution analysis of murine derived long bone OB Raman spectra revealed collagen species were 4.01-fold higher in OBs grown on PLL. Further, an increase of 1.91-fold in immature mineral species (amorphous calcium phosphate) was coupled with a 9.32-fold reduction in mature mineral species (carbonated apatite) on PLL versus controls. These unique low mineral signatures identified in OBs were linked with reduced alkaline phosphatase enzymatic activity, reduced Alizarin Red staining and altered osteogenic gene expression. The promotion of immature mineral species and restriction of mature mineral species of OB grown on PLL were linked to increased cell viability and pro-angiogenic vascular endothelial growth factor (VEGF) production. These results demonstrate the utility of Raman spectroscopy to link distinct matrix signatures with OB maturation and VEGF release. Importantly, Raman spectroscopy could provide a label-free approach to clinically assess the angiogenic potential of bone during fracture repair or degenerative bone loss.

In situ characterization of nanoscale strains in loaded whole joints via synchrotron X-ray tomography.

Imaging techniques for quantifying changes in the hierarchical structure of deforming joints are constrained by destructive sample treatments, sample-size restrictions and lengthy scan times. Here, we report the use of fast low-dose pink-beam synchrotron X-ray tomography in combination with mechanical loading at nanometric precision for in situ imaging, at resolutions below 100 nm, of the mechanical strain in intact untreated joints under physiologically realistic conditions. We show that in young, older and osteoarthritic mice, hierarchical changes in tissue structure and mechanical behaviour can be simultaneously visualized, and that the tissue structure at the cellular level correlates with the mechanical performance of the whole joint. We also use the tomographic approach to study the colocalization of tissue strains to specific chondrocyte lacunar organizations within intact loaded joints and to explore the role of calcified-cartilage stiffness on the biomechanics of healthy and pathological joints.

Azathioprine Has a Deleterious Effect on the Bone Health of Mice with DSS-Induced Inflammatory Bowel Disease.

Patients with inflammatory bowel disease (IBD) often present poor bone health and are 40% more at risk of bone fracture. Studies have implicated autophagy in IBD pathology and drugs used to treat IBD stimulate autophagy in varying degrees, however, their effect on the skeleton is currently unknown. Here, we have utilised the dextran sulphate sodium (DSS) model of colitis in mice to examine the effects of the thiopurine drug azathioprine on the skeleton. Ten-week-old male mice (n = 6/group) received 3.0% DSS in their drinking water for four days, followed by a 14-day recovery period. Mice were treated with 10 mg/kg/day azathioprine or vehicle control. Histopathological analysis of the colon from DSS mice revealed significant increases in scores for inflammation severity, extent, and crypt damage (p < 0.05). Azathioprine provided partial protection to the colon, as reflected by a lack of significant difference in crypt damage and tissue regeneration with DSS treatment. MicroCT of vehicle-treated DSS mice revealed azathioprine treatment had a significant detrimental effect on the trabecular bone microarchitecture, independent of DSS treatment. Specifically, significant decreases were observed in bone volume/tissue volume (p < 0.01), and trabecular number (p < 0.05), with a concurrent significant increase in trabecular pattern factor (p < 0.01). Immunohistochemical labelling for LC3 revealed azathioprine to induce autophagy in the bone marrow. Together these data suggest that azathioprine treatment may have a deleterious effect on IBD patients who may already be at increased risk of osteoporotic bone fractures and thus will inform on future treatment strategies for patient stratification.

How To Build a Bone: PHOSPHO1, Biomineralization, and Beyond.

Since its characterization two decades ago, the phosphatase PHOSPHO1 has been the subject of an increasing focus of research. This work has elucidated PHOSPHO1's central role in the biomineralization of bone and other hard tissues, but has also implicated the enzyme in other biological processes in health and disease. During mineralization PHOSPHO1 liberates inorganic phosphate (Pi) to be incorporated into the mineral phase through hydrolysis of its substrates phosphocholine (PCho) and phosphoethanolamine (PEA). Localization of PHOSPHO1 within matrix vesicles allows accumulation of Pi within a protected environment where mineral crystals may nucleate and subsequently invade the organic collagenous scaffold. Here, we examine the evidence for this process, first discussing the discovery and characterization of PHOSPHO1, before considering experimental evidence for its canonical role in matrix vesicle-mediated biomineralization. We also contemplate roles for PHOSPHO1 in disorders of dysregulated mineralization such as vascular calcification, along with emerging evidence of its activity in other systems including choline synthesis and homeostasis, and energy metabolism. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Conditional deletion of E11/podoplanin in bone protects against load-induced osteoarthritis.

BACKGROUND: Subchondral bone (SCB) thickening is one of the earliest detectable changes in osteoarthritic joints and is considered a potential trigger for subsequent articular cartilage degeneration. In this manuscript, we examine whether disruption to the SCB osteocyte network contributes to the initiation and pathogenesis of osteoarthritis. METHODS: We examined expression patterns of the glycoprotein E11/podoplanin by immunohistochemical labelling in murine, human and canine osteoarthritis models. We also examined the effects of twice-weekly administration of Bortezomib, a proteasome inhibitor which stabilises osteocyte E11 levels, to C57/BL6 wild-type male mice (1 mg/kg/day) for 8 weeks after surgical destabilisation of the medial meniscus. By inducing osteoarthritis-like changes in the right knee joint of 12-week-old male E11 hypomorphic mice (and corresponding controls) using a post-traumatic joint loading model, we also investigated whether a bone-specific E11 deletion in mice increases joint vulnerability to osteoarthritis. Articular cartilage degradation and osteophyte formation were assessed by histology and in line with the OARSI grading system. RESULTS: Our studies reveal increased E11 expression in osteocytes of human and canine osteoarthritic SCB. We found that Bortezomib administration had no effect on surgically-induced osteoarthritis, potentially due to a lack of the expected stabilisation of E11 in the SCB. We also found, in concordance with our previous work, wild-type mice exhibited significant load-induced articular cartilage lesions on the lateral femoral condyle (p < 0.01) and osteophyte formation. In contrast, E11 hypomorphic mice did not develop osteophytes or any corresponding articular lesions. CONCLUSIONS: Overall, these data suggest that an intact osteocyte network in the SCB contributes to the development of mechanically-driven osteoarthritis. Further, the data presented here indicate that the molecular pathways that preserve the osteocyte network, such as those driven by E11, may be targeted to limit osteoarthritis pathogenesis.

The Inflammatory Bowel Disease Drug Azathioprine Induces Autophagy via mTORC1 and the Unfolded Protein Response Sensor PERK.

BACKGROUND: Genetic studies have strongly linked autophagy to Crohn's disease (CD), and stimulating autophagy in CD patients may be therapeutically beneficial. The aim of this study was to evaluate the effect of current inflammatory bowel disease (IBD) drugs on autophagy and investigate molecular mechanisms of action and functional outcomes in relation to this cellular process. METHODS: Autophagy marker LC3 was evaluated by confocal fluorescence microscopy and flow cytometry. Drug mechanism of action was investigated by polymerase chain reaction (PCR) array with changes in signaling pathways examined by immunoblot and quantitative reverse transcription PCR (RT-qPCR). Clearance of adherent-invasive Escherichia coli (AIEC) and levels of pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) were evaluated by gentamicin protection assays and RT-qPCR, respectively. The marker LC3 was analyzed in peripheral blood mononuclear cells (PBMCs) from pediatric patients by flow cytometry. RESULTS: Azathioprine induces autophagy via mechanisms involving modulation of mechanistic target of rapamycin (mTORC1) signaling and stimulation of the unfolded protein response (UPR) sensor PERK. Induction of autophagy with azathioprine correlated with the enhanced clearance of AIEC and dampened AIEC-induced increases in TNFα. Azathioprine induced significant increase in autophagosome bound LC3-II in PBMC populations ex vivo, supporting in vitro findings. In patients, the CD-associated ATG16L1 T300A single-nucleotide polymorphism did not attenuate azathioprine induction of autophagy. CONCLUSIONS: Modulation of autophagy via mTORC1 and the UPR may contribute to the therapeutic efficacy of azathioprine in IBD.

The Ex Vivo Organ Culture of Bone.

The ex vivo organ culture of bone provides many of the advantages of both the whole organism and isolated cell strategies and can deliver valuable insight into the network of processes and activities that are fundamental to bone and cartilage biology. Through maintaining the bone and/or cartilage cells in their native environment, this model system provides the investigator with a powerful experimental protocol to address specific facets of skeletal growth and development. In this chapter, we outline the basic protocols and possible readouts of organ culture models to replicate; (a) linear bone growth (murine metatarsal culture model), (b) bone and cartilage metabolism (murine femoral head culture model), (c) bone response to mechanical stimulation (bovine trabecular core culture model), and (d) bone resorption and formation (murine calvaria culture model).

A Computed Microtomography Method for Understanding Epiphyseal Growth Plate Fusion.

The epiphyseal growth plate is a developmental region responsible for linear bone growth, in which chondrocytes undertake a tightly regulated series of biological processes. Concomitant with the cessation of growth and sexual maturation, the human growth plate undergoes progressive narrowing, and ultimately disappears. Despite the crucial role of this growth plate fusion "bridging" event, the precise mechanisms by which it is governed are complex and yet to be established. Progress is hindered by the current methods for growth plate visualization; these are invasive and largely rely on histological procedures. Here, we describe our non-invasive method utilizing synchrotron X-ray computed microtomography for the examination of growth plate bridging, which ultimately leads to its closure coincident with termination of further longitudinal bone growth. We then apply this method to a dataset obtained from a benchtop micro computed tomography scanner to highlight its potential for wide usage. Furthermore, we conduct finite element modeling at the micron-scale to reveal the effects of growth plate bridging on local tissue mechanics. Employment of these 3D analyses of growth plate bone bridging is likely to advance our understanding of the physiological mechanisms that control growth plate fusion.