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

Degeneration of the intervertebral disc results in patent cracks [1] and a decrease in osmotic pressure associated with loss of fixed charges. The relationship between mechanical load and damage in the disc is very poor [2]. This finding is at odds with physical intuition. The subject of this study is relationship between the development of patent cracks and the decrease in osmotic pressure in the degenerating disc in the light of the physics of swelling [3–7]. We restrict the experimental part of this study to hydrogel, thus avoiding complications associated with biological variability. The finite element modelling [6,7] used in this study catches salient features of stress profiles measured by Mc Nally and al. Thin hydrogel samples with a crack of 5 mm are used. The crack opens as a result of decreasing osmotic pressure in the experiments and in the simulation. The initial uniform stress distribution turns into a distribution with a decreased average stress level and a high stress around the crack tip. A decrease in osmotic pressure opens an existing crack in swelling materials independently from external mechanical load. Hence, disc degeneration causes the overall stress to decrease, while local stress around a crack tip increases. This mechanism may explain why damage in the disc is so poorly correlated with mechanical load [3] and why the degenerated disc is characterized by patent cracks [1]. The process of crack opening in the degenerating disc is comparable to the crack development in an aging oaken beam, while loosing its turgor

Summary Statement. An organ culture experiment was simulated to explore the mechanisms that can link cell death to mechanical overload in the intervertebral disc. Coupling cell nutrition and tissue deformations led to altered metabolic transport that largely explained cell viability measurements. Introduction. Part of intervertebral disc (IVD) maintenance relies on limited nutrient availability to the cells and on mechanical loads, but effective implication of these two factors is difficult to quantify. Theoretical models have helped to understand the link between solute transport and cell nutrition in deforming IVD, but omitted the direct link between tissue mechanics and cell metabolism. Hence, we explored numerically the relation between disc mechanics and cell death in relation to an organ culture experiment. Methods. A finite element model of a caudal bovine IVD was created to reproduce an organ culture experiment. All subtissues were modelled, and coupled to cell metabolism in two ways: (i) mechanical strains and metabolic reactions were simply coupled to the diffusions of oxygen, lactate and glucose through a mechano-transport algorithm (IND model). (ii), a hypermetabolism model based on in vitro data involved a 30% increase in glucose consumption by the cells, activated either as a Step or as a Gaussian function over 15% strain (DIR model). Exponential decays of cell density occurred below 0.5 mM of glucose and/or below pH 6.78. Concentrations of 21 kPa oxygen and 4.5 mM glucose were imposed at the boundary, and a combination of 0.2 MPa compression and 10° bending was applied over 7 days. Results. The highest hypermetabolic response was given by the Step activation. For all models, cell death mostly occurred in the compressed area of the flexed IVD, and steady-state cell viability was reached in about two days of load. In the outer annulus fibrosus (AF), the DIR model with Step activation led to increased cell death, in line with the cell viability measured in vitro. In the inner AF, all cell viability results matched the reported measurements. Discussion/Conclusion. This study focused on elucidating the links between mechanical stimulation and cell survival in the IVD, and simulation of nutrition issues allowed reproducing the results of an organ culture experiment. Results suggest that mechano-regulated metabolism can play a significant role in the nutrition-related cell death. Truly, the IND model gave both low glucose and low pH, and altered metabolic transport represented the main cell death mechanism. Yet, the role of hypermetabolism was increased nearby the nutrient supply at the outer AF, meaning that cell death could occur, even in regions where nutrient supply seems ensured by short diffusion distances. Though further mechanistic developments must be considered, this novel mechano-regulated metabolism model permits mechano-transport models to be used to explore important interactions between tissue biophysics and multiphysics. In particular, the extracellular matrix degradation along degeneration and cell death can be coupled to the poromechanical parameters introduced, e.g. initial porosity and osmotic pressure values that largely depend on the proteoglycan concentration

The skeletal system exhibits functional adaptation. For bone the mechanotransduction mechanisms have been well elucidated; in contrast, the response of tendon to its mechanical environment is much more poorly understood despite tendon disorders being commonly encountered in clinical practice. This study presents a novel approach to developing an isolated tendon system in vivo. This model is used to test the hypothesis that stress-shielding, and subsequent restressing, causes significant biomechanical changes. We propose a control mechanism that governs this process.

A custom-built external fixator was used to functionally isolate the ovine patellar tendon(PT). In group 1 animals(n=5) the right PT was stress-shielded for 6 weeks. This was achieved by drawing the patella towards the tibial tubercle, thus slackening the PT. In group 2 (n=5) the PT was stress-shielded for 6 weeks. The external fixator was then removed and the PT physiologically loaded for a further 6 weeks. In each case, the PT subsequently underwent tensile testing and measurement of length(L) and cross-sectional area(CSA). The untreated left PTs acted as controls (n=10).

6 weeks of stress-shielding significantly decreased material and structural properties of tendon compared to controls (elastic modulus(E) 76.2%, ultimate tensile strength(UTS) 69.3%, stiffness(S) 79.2%, ultimate load(UL) 68.5%, strain energy(SE) 60.7%; p< 0.05). Ultimate strain(US), L and CSA were not significantly changed. 6 weeks of subsequent functional loading (Group 2) caused some improvement in material properties, but greater recovery in structural properties (E 79.8%, UTS 91.8%, S 96.7%, UL 92.7%, SE 96.5%). CSA was significantly greater than Group 1 tendons at 114% of control value.

Previous models of tendon remodelling have relied on either joint immobilization or direct surgical procedures. This model allows close control of the tendon’s mechanical environment whilst allowing normal joint movement and avoiding surgical insult to the tendon itself. The hypothesis that stress-shielding, and subsequent restressing, causes significant biomechanical changes has been upheld. We propose that the biomechanical changes observed are governed by a strain homeostasis feedback mechanism.

Introduction. Mechanical loading has been hypothesized to play an important role in the development, remodeling and in diseases of many skeletal tissues, including cartilage. In order to study the metabolic response of cartilage to physical forces, in vitro systems have often been used because of the precise control with which mechanical loads can be applied. We developed a new mechanical loading system, in which we were able to load the intact femoral condyle in order to preserve the native cartilage/subchondral bone structure. This system represents a more ‚in vivo‘ situation than cartilage explants or chondrocyte cell culture systems. Our approach focused on changes in mRNA expression of type II collagen, type VI collagen, and aggrecan in loaded versus adjacent unloaded cartilage in order to analyse the early response of chondrocytes to well-defined mechanical stresses. Methods. Femoral condyles were obtained from two-year-old cows. The integrity of the cartilage surface was controlled by staining with safranin O. The femoral condyles were compressed in an Instron 8501 material testing machine. Cyclic compression pressure was applied for 2000 cycles in a sinusoidal waveform of 0. 5 Hz-frequency with a peak stress of 0. 2 to12. 5 MPa. Following loading, full depth cartilage sections were cut out and one half immediately frozen in liquid nitrogen for RNA isolation and the other half soaked in 4% paraformaldehyde for paraffin embedding. As control, the adjacent unloaded cartilage was collected and treated in the same way. Total RNA was isolated and changes in mRNA expression were quantitated by competitive quantitative PCR, using an internal standard of a C-terminal truncated version of the corresponding genes. The PCR-reactions were separated by agarose gel electrophoresis and amplified fragments quantified using video-densitometry analysis. The results were expressed as the ratio of mRNA from loaded to unloaded cartilage. Results. Cyclic compression with peak stresses of 12. 5, 6. 3, 2. 5 and 0. 6 MPa lead to a two-fold decrease in the mRNA expression of type II collagen and aggrecan and a threefold decrease of type VI collagen, in consideration of the intra-assay variability of about 30%. Compression with peak stresses of 0. 3 and 0. 2 MPa lead to a three-fold increase of the mRNA expression of type II collagen, a four-fold increase of aggrecan and a slight decrease of type VI collagen. Low compression strength leads to an increase of the mRNA expression of the major components of cartilage, type II collagen and aggrecan, whereas high loading leads to a decrease of the mRNA expression. Conclusion. The results show that our system can be used to analyze early responses of chondrocytes to well-defined mechanical stresses in an intact cartilage/bone-system and therefore will enable us to investigate the role of physiological and non-physiological high loading on the induction of cartilage degradation and regeneration in joint trauma and osteoarthritis. Since the cartilage/bone samples are incubated in medium during the experiment, this system will also offer us the opportunity to investigate additives to the medium as potential pharmacological therapeutics in osteoarthritis

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

Mechanical loading is important to maintain the homeostasis of the intervertebral disc (IVD) under physiological conditions but can also accelerate cell death and tissue breakdown in a degenerative state. Bioreactor loaded whole organ cultures are instrumental for investigating the effects of the mechanical environment on the IVD integrity and for preclinical testing of new therapies under simulated physiological conditions. Thereby the loading parameters that determine the beneficial or detrimental reactions largely depend on the IVD model and its preparation. Within this symposium we are discussing the use of bovine caudal IVD culture models to reproduce tissue inflammation or matrix degradation with or without bioreactor controlled mechanical loading. Furthermore, the outcome parameters that define the degenerative state of the whole IVD model will be outlined. Besides the disc height, matrix integrity, cell viability and phenotype expression, the tissue secretome can provide indications about potential interactions of the IVD with other cell types such as neurons. Finally, a novel multiaxial bioreactor setup capable of mimicking the six degrees-of-freedom loading environment of IVDs will be introduced that further advances the relevance of preclinical ex-vivo testing

Abstract. Objectives. Prediction of bone adaptation in response to mechanical loading is useful in the clinical management of osteoporosis. However, few studies have investigated the effect of repeated mechanical loading in the mouse tibia. Therefore, this study uses a combined experimental and computational approach to evaluate the effect of mechanical loading on bone adaptation in a mouse model of osteoporosis. Methods. Six female C57BL/6 mice were ovariectomised (OVX) at week 14 and scanned using in vivo micro computed tomography (10.4µm/voxel) at week 14, 16, 18, 20 and 22. The right tibiae were mechanically loaded in vivo at week 19 and 21 with a 12N peak load, 40 cycles/day, 3 days/week. Linear isotropic homogeneous finite element (microFE) models were created from the tissue mineral density calibrated microCT images. Changes in bone adaptation, densitometric and spatial analyses were measured by comparing the longitudinal images after image registration. Results. Mechanical loading increased periosteal apposition between weeks 18–20, which reduced slightly between weeks 20–22. Periosteal resorption reduced between weeks 18–20. At weeks 20–22, it remained lower than before treatment, but was up to 70% higher than after the first week of loading. Average SED increased due to OVX before decreasing due to mechanical loading. The highest increase in SED was at the proximal tibia between weeks 14 to 16 (102%), whereas the highest reduction (40%) occurred after the second week of loading in the proximal tibia. Conclusions. The decrease/increase in bone apposition/resorption between weeks 20–22, despite the similar strain distributions between weeks 18–20 and 20–22, suggests that the first application of mechanical loading had a greater effect on reversing the adverse effects of the disease than the second. This imply that a systematic increase in peak load or loading rate may be required to achieve a similar bone adaptation rate with time. 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

Introduction. Within articular cartilage, chondrocytes reside within the pericellular matrix (PCM), collectively constituting the microanatomical entity known as a chondron. The PCM functions as a pivotal protective shield and mediator of biomechanical and biochemical cues. In the context of Osteoarthritis (OA), enzymatic degradation of the PCM is facilitated by matrix metalloproteinases (MMPs). This study delves into the functional implications of PCM structural integrity decline on the biomechanical properties of chondrons and impact on Ca. 2+. signaling dynamics. Method. Chondrons isolated from human cartilage explants were incubated with activated MMP-2, -3, or -7. Structural degradation of the pericellular matrix (PCM) was assessed by immunolabelling (collagen type VI and perlecan, n=5). Biomechanical properties of chondrons (i.e. elastic modulus (EM)) were analyzed using atomic force microscopy (AFM). A fluorescent calcium indicator (Fluo-4-AM) was used to record and quantify the intracellular Ca. 2+. influx of chondrons subjected to single cell mechanical loading (500nN) with AFM (n=7). Result. Each of the three MMPs disrupted the structural integrity of the PCM, leading to attenuated fluorescence intensity for both perlecan and collagen VI. A significant decrease of EM was observed for all MMP groups (p<0.005) with the most notable decrease observed for MMP-2 and MMP-7 (p<0.001). In alignment with the AFM results, there was a significant alteration in Ca. 2+. influx observed for all MMP groups (p<0.05), in particular for MMP-2 and MMP-7 (p<0.001). Conclusion. Proteolysis of the PCM by MMP-2, -3, and -7 not only significantly alters the biomechanical properties of articular chondrons but also affects their mechanotransduction profile and response to mechanical loading, indicating a close interconnection between these processes. These findings underscore the influence of an intact pericellular matrix (PCM) in protecting cells from high stress profiles and carry implications for the transmission of mechanical signaling during OA onset and progression

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. The aim of this study was to investigate whether mechanical loading induced by physical activity can reduce risk of sarcopenia in middle-aged adults. Methods. This was a longitudinal study based on a subset of UK Biobank data consisting of 1,918 participants (902 men and 1,016 women, mean age 56 years) who had no sarcopenia at baseline (assessed between 2006 and 2010). The participants were assessed again after 6 years at follow-up, and were categorized into no sarcopenia, probable sarcopenia, or sarcopenia according to the definition and algorithm developed in 2018 by European Working Group on Sarcopenia in Older People (EWGSOP). Physical activity was assessed at a time between baseline and follow-up using 7-day acceleration data obtained from wrist worn accelerometers. Raw acceleration data were then analysed to study the mechanical loading of physical activity at different intensities (i.e. very light, light, moderate-to-vigorous). Multinominal logistic regression was employed to examine the association between the incidence of sarcopenia and physical activity loading, between baseline and follow up, controlled for other factors at baseline including age, gender, BMI, smoking status, intake of alcohol, vitamin D and calcium, history of rheumatoid arthritis, osteoarthritis, secondary osteoporosis, and type 2 diabetes. Results. Among the 1918 participants with no sarcopenia at baseline, 230 (69 men and 161 women) developed probable sarcopenia and 37 (14 men and 23 women) developed sarcopenia at follow-up. Physical activity loading at moderate-to-vigorous intensity was higher in men (p<0.05), while women had higher physical activity loading at very light intensity (p<0.05). No significant difference was found in physical activity loading at light intensity between men and women (p>0.05). Logistic regression models showed that increase in physical activity loading at moderate-to-vigorous intensity significantly reduced the risk of sarcopenia (odds ratio = 0.368, p<0.05), but not probable sarcopenia (odds ratio = 0.974, p>0.05), while loading at light or very light activity intensity were not associated with the risk of sarcopenia or probable sarcopenia (p>0.05). Conclusion. Loading of physical activity at moderate-to-vigorous intensity could reduce risk of sarcopenia in middle-aged adults. 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

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

In a healthy joint, mechanical loading increases matrix synthesis and maintains cell phenotype, while reducing catabolic activities. It activates several pathways, most of them yet largely unknown, with integrins, TGF-β, canonical (Erk 1/2) and stress-activated (JNK) MAPK playing a key role. Degenerative joint diseases are characterized by Wnt upregulation and by the presence of proteolytic fibronectin fragments (FB-fs). Despite they are known to impair some of the aforementioned pathways, little is known on their modulatory effect on cartilage mechanoresponsiveness. This study aims at investigating the effect of mechanical loading in healthy and in vitro diseased cartilage models using pro-hypertrophic Wnt agonist CHIR99021 and the pro-catabolic FB-fs 30 kDa. Human primary chondrocytes from OA patients have been grown in alginate hydrogels for one week, prior to be incubated for 4 days with 3μM CHIR99021 or 1 μM FB-fs. Human cartilage explants isolated from OA patients have incubated 4 days with 3 μM CHIR99021 or 1 μM FB-fs. Both groups have then been mechanically stimulated (unconfined compression, 10% displacement, 1.5 hours, 1 Hz), using a BioDynamic bioreactor 5270 from TA Instruments. Expression of collagen type I, II and X, aggrecan, ALK-1, ALK-5, αV, α5 and β1 integrins, TGF-β1 have been assessed by Real Time-PCR and normalized with the expression of S29. Percentage of phosphorylated Smad2, Smad1 and JNK were determined through western blot. TGF-β1 content was quantified by sandwich ELISA; MMP-13 and GAG by western blot and DMMB assay, respectively. At least three biological replicates were used. ANOVA test was used for parametric analysis; Kruskal-Wallis and Mann-Whitney post hoc test for non-parametric. Preliminary data show that compression increased collagen II expression in control, but not in CHIR99021 and FB-fs pre-treated group (Fig. 1A-B). This was associated with downregulation of β1-integrin expression, which is the main collagen receptor and further regulates collagen II expression, suggesting inhibition of Erk1/2 pathway. A trend of increase expression of collagen type X after mechanical loading was observed in CHIR and FB-fs group. ALK-1 and ALK-5 showed a trend toward stronger upregulation in CHIR99021 group after compression, suggesting the activation of both Smad1/5/8 and Smad 2/3 pathways. To further investigate pathways leading to these different mechano-responses, the phosphorylation levels of Smad1 and Smad2, Erk1/2 and JNK proteins are currently being studied. Preliminary results show that Smad2, Smad1 and JNK protein levels increased in all groups after mechanical loading, independently of an increase in TGF-β1 expression or content. Compression further increased phosphorylation of Smad2, but not of Smad1, in all groups

It is well known that environmental cues such as mechanical loading and/or cell culture medium composition affect tissue-engineered constructs resembling natural bone. These studies are mostly based on an initial setting of the influential parameter that will not be further changed throughout the study. Through the growth of the cells and the deposition of the extracellular matrix (ECM) the initial environmental conditions of the cells will change, and with that also the loads on the cells will change. This study investigates how changes of mechanical load or media composition during culture influences the differentiation and ECM production of mesenchymal stromal cells seeded on porous 3D silk fibroin scaffolds. ECM formation, ECM mineralization and cell differentiation in 3D tissue-engineered bone were analyzed using microscopic tools. Our results suggest that mechanical stimuli are necessary to differentiate human mesenchymal stromal cells of both bone marrow and adipose tissue origin into ECM producing osteoblasts which ultimately become ECM-embedded osteocytes. However, the influence of this stimulus seems to fade quickly after the onset of the culture. Constructs which were initially cultured under mechanical loading continued to deposit minerals at a similar growth rate once the mechanical stimulation was stopped. On the other hand, cell culture medium supplementation with FBS was identified as an extremely potent biochemical cue that influences the mechanosensitivity of the cells with regards to cell differentiation, ECM secretion and mineral deposition. Only through a thorough understanding on these influences over time will we be able to predictably control tissue development in vitro

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

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

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 the most common joint disease, which is characterized by a progressive loss of proteoglycans and the destruction of extracellular matrix (ECM), leading to a loss of cartilage integrity and joint function. During OA development, chondrocytes alter ECM synthesis and change their gene expression profile including upregulation of hypertrophic markers known from the growth plate. Although physiological mechanical loading can support cartilage formation and maintenance, mechanical overload represents one major risk factor for OA development. To date, little is known on how an OA-like hypertrophic chondrocyte phenotype alters the response of cartilage tissue to mechanical loading. The aim of this study was to investigate whether a hypertrophic phenotype change of chondrocytes affects the response to physiological mechanical loading and to reveal differences compared to normal control cartilage. Cartilage replacement tissue was generated using human articular chondrocytes (normal control cartilage, n=3–5) or human mesenchymal stromal cells which develop a hypertrophic phenotype similar to the one observed in OA (OA cartilage model, n=3–6). Cells were seeded in a collagen type I/III carrier and attached to a beta-TCP bone replacement phase, building an osteochondral unit for simulation of natural conditions. After 21 and 35 days of chondrogenic (re)differentiation, a single physiological mechanical compression episode (1 Hz, 25 %, 3 h) was applied, imitating three hours of normal walking in ten-minute intervals. Proteoglycan and collagen synthesis, gene expression and activation of signaling pathways were assessed. Cartilage replacement tissue of both groups had similar proteoglycan and collagen type II content as well as hardness properties. During (re)differentiation, both cell types showed a comparable upregulation of the chondrogenic marker genes COL2A1 and ACAN. As expected, hypertrophic marker genes (COL10A1, ALPL, MEF2C, IBSP) were only upregulated in the OA cartilage model. Mechanotransduction in both tissues was confirmed by load-induced activation of pERK1/2 signaling. While the 3 h loading episode significantly increased proteoglycan synthesis in normal control cartilage at day 35, the same protocol resulted in a suppression of proteoglycan and collagen synthesis in the OA cartilage model, which was accompanied by a downregulation of COL2A1 gene expression. In addition, hypertrophic marker genes COL10A1, ALPL and IBSP were significantly reduced after loading. Along lower load-induced SOX9 mRNA and protein stimulation in the OA cartilage tissue, a weaker induction of mechanosensitive BMP2, BMP6, FOS and FOSB gene expression was observed. While stable cartilage showed anabolic effects after physiological loading, the hypertrophic chondrocytes reacted with a reduced extracellular matrix synthesis. This could be explained by a lower mechanoinduction of the BMP signaling cascade and insufficient SOX9 stimulation. Progressive OA development could thus be influenced by a reduced mechanocompetence of osteoarthritic chondrocytes

Tendinopathy is a disease associated with pain and tendon degeneration, leading to a decreased range of motion and an increased risk of tendon rupture. The etiology of this frequent disease is still unknown. In other musculoskeletal tissues like cartilage and intervertebral discs, transient receptor potential channels (TRP- channels) were shown to play a major role in the progression of degeneration. Due to their responsiveness to a wide range of stimuli like temperature, pH, osmolarity and mechanical load, they are potentially relevant factors in tendon degeneration as well. We therefore hypothesize that TRP- channels are expressed in tendon cells and respond to degeneration inducing stimuli. By immunohistochemistry, qRT-PCR and western blot analyses, we found three TRP channel members, belonging to the vanilloid (TRPV), and ankyrin (TRPA) subfamily, respectively, to be expressed in healthy human tendon tissue as well as in rodent tendon, with expression being located to cells within the dense tendon proper, as well as to endotenon resident cells. In vitro-inflammatory and ex vivo-mechanical stimulation led to a significant upregulation of TRPA1 expression in tendon cells, which correlates well with the fact that TRPA1 is considered as mechanosensitive channel being sensitized by inflammatory mediators. This is the first description of TRP- channels in human and rodent tendon. As these channels are pharmacologically targetable by both agonists and antagonists, they may represent a promising target for novel treatments of tendinopathy

Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are considered when performing in vivo studies, in vitro studies are still largely performed under static conditions. Therefore, an increasing complexity of in vitro culture models is required, with the ultimate aim to recreate the articulating joint as accurately as possible. We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. Using this bioreactor, we have been able to demonstrate the mechanical induction of human bone marrow stromal cell (BMSC) chondrogenesis in the absence of exogenous growth factors. Building on previous bioreactor studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs, we have been further increasing the complexity of in vitro models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition. The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable product development pathways