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

During in vitro sub-culturing, tenocytes lose their phenotype which ultimately affects their functioning. As spindle-shaped fibroblasts, tenocytes have a unique thin elongated phenotype and they possess more spread-out shape through phenomena named dedifferentiation1. Given the link between cell shape and cell function, in this study, we first aimed to dedifferentiate tenocytes through in vitro sub-culturing in order to have a model system for dedifferentiation. For this, we isolated human flexor tendon cells from healthy female flexor digitorum longus and seeded at 5000 cells/cm² cell density, passaged every two days for six passages. In order to assess cell phenotype, we fixed with 4% paraformaldehyde and stained with phalloidin and DAPI to visualize the actin cytoskeleton and DNA respectively. We noted that in each passage, cells lost their spindle-shaped phenotype and became more pancake-shaped. At passage 1 and 2, the main cell phenotype is spindle-shaped. However, as the cells are further passaged, the phenotype of the cell population becomes more heterogeneous and at passage 5 and 6, they already display a more spread-out shape. Based on these results, we further hypothesized that they can be re-differentiated through matrix-mediated mechano-transduction and regain their morphology and function. For this aim, we generated decellularized tendon from porcine Achilles tendon and setup a mechanical loading system where we can provide mechanical loadings at physiological levels. This system will provide a new approach on in vitro tenocyte culturing.

Distal arthrogryposis (DA) is a collection of rare developmental disorders characterized by congenital joint contractures. Most arthrogryposis mutations are in muscle- and joint-related genes, and the anatomical defects originate cell-autonomously within the musculoskeletal tissues. However, gain-of-function (GOF) mutations in PIEZO2, a principal mechanosensor in somatosensation, cause DA subtype 5 via unknown mechanisms. We show that expression of a GOF PIEZO2 mutation in proprioceptive sensory neurons mainly innervating muscle spindles and tendons is sufficient to induce DA5-like phenotypes in mice. Overactive PIEZO2 causes anatomical defects via increased activity within the peripheral nervous system during postnatal development. Surprisingly, overactive PIEZO2 is likely to cause joint abnormalities via increased exocytosis from sensory neuron endings without involving motor circuitry. This reveals a role for somatosensory neurons: excessive mechanosensation within these neurons disrupts musculoskeletal development. We also present proof-of-concept that Botox injection or dietary treatment can counteract the effect of overactive PIEZO2 function to evade DA-like phenotypes in mice when applied during a developmental critical period. These approaches might have clinical applications. Beyond this, our findings call attention to the importance of considering sensory mechanotransduction when diagnosing and treating other musculoskeletal disorders. Acknowledgements: Our work is supported by National Institutes of Health grant (R35 NS105067, R01 DE022358, R25 SC3GM127195, R25 GM07138, R01GM133845, intramural) and Howard Hughes Medical Institute

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

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

Introduction. Hyaluronan (HA) is assumed to have a regulatory role in the bone remodelling process by influencing the behaviour of mesenchymal stem cells (MSCs), osteoblasts and osteoclasts. The hyaluronan synthases (HAS1, HAS2 and HAS3) which are responsible for the formation of HA are expressed in human MSCs (hMSCs). Although HAS are only active when they are located in the plasma membrane and an intracellular storage pool of the HAS is assumed, the mechanisms controlling the intracellular traffic of HAS are hardly investigated. Since chitin synthases and cellulose synthases, members of the same enzyme family like the HAS, are regulated by interaction with the cytoskeleton, we hypothesize that HAS interrelate somehow with the cytoskeleton and that their expression, their transport and/or their activity are regulated via mechanotransduction. Methods and Results. We generated immortalized hMSCs (SCP-1) constitutively expressing eGFP-tagged HAS by lentiviral gene transfer (SCP1-HAS1-eGFP, SCP1-HAS2-eGFP and SCP1-HAS3-eGFP). The expression of the transgene HAS was verified by RT-PCR, western blot, FACS analysis and direct fluorescence microscopy or immunofluorence. The enzymatic activity of the transgene HAS was determined by HA-ELISA and by staining of HA. hMSCs expressing lifeact-RFPruby and HAS-eGFP were investigated in a video timelapse analysis in order to study the putative interaction of HAS-eGFP with the actin cytoskeleton. The HAS-eGFP proteins are globular structured and aligned along the actin filaments. The timelapse pictures show that the HAS-eGFP moves without loss of their alignment to actin. In addition we investigated the impact of shear stress on hMSCs under defined flow conditions. The upregulation of the expression levels of the three HAS isoforms was shown by quantitative real time RT-PCR after exposure to the stimulus. Discussion. Here, we were able to show the regulation of HAS expression via mechanotransduction. At the moment we investigate if HAS activity and their transport towards the plasma membrane are changed by shear stress. Furthermore we generate hMSCs expressing eGFP tagged HAS in their active form. We have first hints for an interaction of the transgene HAS with the actin cytoskeleton. Our cells can be used for further investigation of the functional and regulatory role of HAS in the bone microenvironment. In some bone diseases such as osteogenesis imperfecta, multiple myeloma and osteoporosis, the HA content in the bone or HAS expression in the hMSCs are changed. Understanding the role of HA in bone regeneration and the regulatory mechanisms of HAS in the hMSCs will provide therapeutic starting points for an improved fracture healing in patients suffering from one of these bone diseases

Tendon injuries are a worldwide problem affecting several age groups and stem cell based therapies hold potential for tendon strategies guiding tendon regeneration. Tendons rely on mechano-sensing mechanisms that regulate homeostasis and influence regeneration. The mechanosensitive receptors available in cell membranes sense the external stimuli and initiate mechanotransduction processes. Activins are members of the TGF-β superfamily which participate in several tendon biological processes. It is envisioned that the activation of the activin receptor, trigger downstream Smad2/3 pathway thus regulating the transcription of tenogenic genes driving stem cell differentiation. In this work, we propose to target the Activin receptor type IIA (ActRIIA) in human adipose stem cells (hASCs), inducing hASCs commitment towards the tenogenic lineage. Since mechanotransduction can be remotely triggered through magnetic actuation combined with magnetic nanoparticles (MNPs), we stimulated hASCs tagged complexes using a vertical oscillating magnetic bioreactor (MICA Biosystems Ltd). Carboxyl functionalised MNPs (Micromod) were coated with anti-ActRIIA antibody (Abcam) by carbodiimide activation. hASCs were then cultured with MNPs-anti-ActRIIA for 14days with or without magnetic exposure (1Hz, 1h/every other day). hASCs cultured alone in αMEM (negative control) or in αMEM supplemented with ActivinA (R&D systems) (positive control of ActRIIA activation) were used as experimental controls. The tenogenic commitment of hASCs was assessed by real time RT-PCR, immunocytochemistry and quantification of collagen and non-collagenous proteins. Moreover, the phosphorylation of Smad2/3 was also evaluated on hASCs incubated for 2, 10, or 30min under magnetic stimulated (1Hz) and non-stimulated conditions. The increased gene expression of tendon related markers and higher ECM proteins deposition suggests that remote magnetic activation of ActRIIA promotes effectively hASCs tenogenic commitment. Furthermore, the detection of phospho-Smad2/3 proteins by ELISA (Cell Signaling Technology) was significantly more intense after 10min in hASCs under magnetic stimulation and in comparison to the control groups. These outcomes suggest that ActRIIA is a mechanosensitive receptor that can be remotely activated upon magnetic stimulation. In conclusion, remotely activation of MNPs tagged hASCs has potential for modulating tenogenic differentiation of stem cells envisioning successful cell therapies for tendon regeneration. Acknowledgements. FCT/MCTES PD/59/2013 (fellowship PD/BD/113802/2015), FCT post-doctoral grant SFRH/BPD/111729/2015, FCT grant IF/00685/2012, and EU-ITN MagneticFun

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of time in culture. Biomaterial-based tendon implants are designed to withstand high physiological loads but often lack the appropriate biochemical, biophysical and biological structure to drive tendon regeneration by populating cells. The objective of this study is to use tendon main component, collagen type I, to create scaffolds that reproduce tendon natural anisotropy and rigidity, in an effort to engineer functional tendon tissue with native organization and strength, able to maintain tenocyte phenotype and to differentiate stem cells towards the tenogenic lineage. Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 um) or planar PDMS moulds and air-dried to obtain 5 mg/ml collagen films. Surface topography and elastic modulus were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were cultured on the micro-grooved/planar scaffolds for up to 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry. Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP (0, 0.5, 1 and 1.5mM) at low pH and temperature. The elastic modulus of the scaffolds cross-linked with 4SP (0.5mM) matched the values previously reported to induce tenogenic differentiation in stem cells (50–90 kPa). Approximately eighty percent of the human tendon cells cultured on the micro-grooved collagen films aligned in the direction of the anisotropy for 10 days in culture, mimicking the alignment of tenocytes in the native tissue. Cell nuclei morphology, known to play a central role in the process of mechanotransduction, was significantly more elongated for the tenocytes cultured on the micro-grooved scaffolds after 4 days in culture for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively on the micro-grooved and rigid scaffolds after 10 days in culture, respectively. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment. Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities

Introduction. The hierarchical structure of tendon results in a complex mechanical strain environment, with tenocytes experiencing both tension and shear during loading. The mechanotransduction mechanisms involved in sensing these environments is currently unclear. To better understand the effects of shear and tension on cell behaviour, a fibre composite system able to recapitulate the physiological shear-tension ratio found in tendons, was used. Cell attachment within the composite was achieved by using either a collagen type I mimetic peptide, DGEA, or a fibronectin associated peptide, YRGDS, and the gene expression response analysed after loading. Materials and Methods. Fibre composites with 4 different shear-tension (S-T) ratios were made using both PEG-DGEA and PEG-YRGDS fibres. 4 composites were made for each S-T ratio, of which 2 were loaded and 2 used as non-strained controls. Bovine digital extensor tendon tenocytes were seeded within composites, with 3 biological repeats from different donors. Loaded samples were exposed to 5% cyclic strain (1Hz) for 24 hours maintained in an incubator. The gene expression of 14 matrix related genes were analysed after loading via RT-qPCR. Results. Tenocytes seeded on PEG-DGEA fibres were more mechano-sensitive than those seeded on PEG-YRGDS fibres; tenocytes in PEG-DGEA composites exhibited upregulation of COL-3, MMP-3 and IL-6, and downregulation of SCX with shear, while tenocytes in PEG-YRGDS composites downregulated TIMP-3 with shear. Discussion. The main integrin involved in DGEA binding is α2β1 while the integrins associated with YRGDS attachment include α5β1, αVβ3 and αIIbβ3. Consequently, the findings of this study emphasise the importance of integrins in the role of mechanotransduction, and suggest integrins involved in collagen type I binding induce functionally different responses in tenocytes to those not involved in collagen type I binding when sensing mechanical stimuli comprised of shear and tension. This information is critical in future studies investigating tenocyte behaviour and tissue engineering approaches, as physiological integrin binding may be key in maintaining normal tenocyte pathways

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

Introduction. Low back pain is a major public health problem in our society. Degeneration of intervertebral disc (IVD) appears to be the leading cause of chronic low-back pain [1]. Mechanical stimulations including compressive and tensional forces are directly implicated in IVD degeneration. Several studies have implicated the cytoskeleton in mechanotransduction [2, 3], which is important for communication and transport between the cells and extracellular matrix (ECM). However, the potential roles of the cytoskeletal elements in the mechanotransduction pathways in IVD are largely unknown. Methods. Outer annulus fibrosus (OAF) and nucleus pulposus (NP) cells from skeletally mature bovine IVD were either seeded onto Flexcell¯ type I collagen coated plates or seeded in 3% agarose gels, respectively. OAF cells were subjected to cyclic tensile strain (10%, 1Hz) and NP cells to cyclic compressive strain (10%, 1Hz) for 60 minutes. Post-loading, cells were processed for immunofluorescence microscopy and RNA extracted for quantitative PCR analysis. Results. F-actin reorganisation was evident in OAF and NP cells subjected to tensile and compressive strain respectively and is likely due to load-induced differential mRNA expression of actin-binding proteins. The vimentin network was also more intricately organised in loaded NP cells. Compressive strain increased type II collagen and aggrecan transcription in NP cells, whereas levels decreased in OAF cells under tension. mRNA levels of ECM-degrading enzymes were significantly reduced in both cell populations after loading. Conclusion. Tensile and compressive strains induce different mechano-responses in the organisation/expression of cytoskeletal elements and on markers of IVD metabolism. Differential mechano-regulation of anabolic and catabolic ECM components in the OAF and NP populations reflects their respective mechanical environments in situ

While the phenomena of bone adaption to mechanical loading has been long observed, the mechanisms governing bone mechanotransduction during health and disease are not well understood. Our multidisciplinary experimental and computational research strives to enhance understanding of bone mechanobiology, and in particular how this process is affected at the onset of osteoporosis. We have provided an enhanced understanding of bone cell mechanosensation. We have characterised the local mechanical environment of MSCs, osteoblasts and osteocytes in vivo. Most importantly, we have discovered that the matrix composition, expression of mechanosensors and the mechanical environment of osteocytes is altered during osteoporosis. Interestingly, a mechanobiological response restores the homeostatic mechanical environment of the cells in the longer term. Our recent in vitro studies have revealed that estrogen withdrawal from bone cells alters calcium signalling, mineralisation, biochemical responses and osteogenic gene expression when these cells are exposed to an applied fluid shear stress. Our ongoing research is investigating mechanobiology-based therapeutic approaches for treatment of bone pathologies, by (1) targeting mechanoregulatory signalling pathways and (2) developing in vitro tissue regeneration strategies that seek to optimise the mechanical environment (through matrix stiffness, bioreactors) to stimulate osteogenesis

Osteoporosis affects millions globally and current anti-catabolic treatments are limited by significant side-effects. Osteoporosis arises when skeletal stem cells (SSC) no longer sufficiently replenish osteoblasts, leading to net bone loss. A key regulator of SSC behaviour is physical loading, yet the mechanisms by which SSCs sense and respond to changes in their mechanical environment are virtually unknown. Primary cilia are nearly ubiquitous ‘antennae-like’ cellular organelles that have very recently emerged as extracellular chemo/mechano-sensors and thus, are strong candidates to play an important role in regulating SSC responses in bone. This paper will demonstrate that the SSC primary cilium plays an important role in loading-induced bone formation via initial chemosensation and transduction of the potent chemokine TGFβ1 regulating SSC recruitment to the bone surface and secondly it will be shown that the primary cilium is a cAMP responsive mechanosensor directly regulating SSC mechanotransduction via localisation of adenylyl cyclase 6 to the ciliary microdomain. Finally, it will be shown that targeting the cilium therapeutically can be an effective approach to enhance both biochemical and biophysically induced SSC osteogenesis contributing to bone formation, demonstrating a novel anabolic therapy for bone loss diseases such as osteoporosis

Summary. This study helps to elucidate how ColVI and Dcn within the pericellular matrix (PCM) of differentiating hMSCs directly impacts dynamic cytoskeletal response to load, and demonstrates an important role for the PCM in mechanotransduction during chondrogenesis. Introduction. Mechanosignaling events in differentiating human mesenchymal stem cells (hMSCs) are dependent on their temporally changing micromechanical environment and their dynamic cytoskeleton. During chondrogenic differentiation, hMSCs develop a matrix composed of type VI collagen (ColVI) and proteoglycans such as decorin (Dcn). We have previously demonstrated that this developing PCM is important in cellular mechanotransduction. The aim of this study was to determine the functional roles of ColVI and Dcn in modulating load-induced changes in the organization of vimentin intermediate filaments (VIF), actin microfilaments (AM), and vinculin. Methods. hMSCs were transduced with shRNA targeting either col6a1 (shColVI) or dcn (shDcn) and then cultured in 2% alginate beads for 14 days in chondrogenic media. GFP-transduced hMSCs were cultured in parallel. Cells with their intact PCM were isolated with 100mM sodium citrate, 30mM EDTA, and re-embedded in alginate discs. These hMSC-alginate constructs underwent unconfined compression at 0.1Hz for 1 hour from 0–10% strain. Free Swelling (FS) and loaded discs were fixed either immediately following (0hr) or 4 hours post-load. Discs were cryosectioned and fluorescently labeled for VIF, AM, or vinculin with DAPI counterstain. Confocal image-stacks were collected and corrected total cell fluorescence (CTCF) per area was calculated using ImageJ (NIH) to quantify cytoskeletal organization. Results. VIF fluorescence showed a dynamic cytoskeletal response to load in non-infected and GFP-transduced controls with an increase in intensity. In both ColVI and Dcn knockdown groups, VIF exhibited higher fluorescence in FS, which then didn't significantly change following load, possibly due to its higher baseline concentration. AM showed a similar response to load in shColVI knockdown samples, with a significantly higher intensity in FS samples, which wasn't affected by loading and remained cortically localised in all samples. In shDcn samples, there was a significant increase in actin content immediately following load, similar to control. Vinculin staining showed significantly lower staining intensity in shColVI knockdown samples than non-infected and GFP-transduced samples at all timepoints, and exhibited a different trend in response to loading. Following loading, vinculin was diffusely stained across the cytoskeleton in all samples. Discussion. This study demonstrates through targeted shRNA knockdown that the involvement of ColVI and Dcn in mechanosignaling events of differentiating hMSCs could be due to their interactions with and control over the dynamic cytoskeleton. Vimentin and actin have previously been shown to contribute to the viscoelastic mechanical properties of hMSCs and are important in the ability for cells to resist load. Our experiments indicate that specific components in the PCM can affect cytoskeletal dynamics, with knockdown samples lacking the significantly dynamic response to load seen in control samples. Actin cytoskeletal intensity and vinculin intensity show an inverse relationship to load. Since vinculin mediates interactions between actin cytoskeleton and integrins at focal adhesions, these data suggest that ColVI and Dcn regulation of cytoskeletal response may be governed more by the changing external micromechanical environment, rather than altered cell-matrix interactions. Further studies are needed to elucidate the roles of ColVI and Dcn in downstream intracellular mechanosignaling events of differentiating hMSCs

Dynamic loading is necessary for the preservation of native cartilage, but mechanical disuse is one major risk factor for osteoarthritis (OA). As post-transcriptional regulators, microRNAs (miRs) represent promising molecules to quickly adjust the cellular transcriptome in a stimulus-dependent manner. Several miR clusters were related to skeletal development, joint homeostasis and OA pathophysiology but whether miRs are associated with mechanosensitivity and regulated by mechanotransduction is so far unknown. We aimed to investigate the influence of mechanical loading on miR expression and to identify mechanosensitive miR clusters characteristic for non-beneficial loading regimes which may serve as future tools for improved diagnosis or intervention during OA development. Loading regimes leading to an anabolic or catabolic chondrocyte response were established based on an increase or decrease of proteoglycan synthesis after loading of human engineered cartilage. miR microarray profiling at termination of loading revealed only small changes of miR expression (7 significantly upregulated miRs) by an anabolic loading protocol while catabolic stimulation produced a significant regulation of 80 miRs with a clear separation of control and compressed samples by hierarchical clustering. Overall regulation of 8/14 miR was confirmed by qRT-PCR with mean amplitudes of up to 2.5-fold for catabolic loading. Cross-testing revealed that 2 miRs were upregulated by both loading conditions and 6 were specifically elevated by the catabolic loading regime. Conclusively, this study defines the first mechanosensitive miR cluster associated with non-beneficial compressive cyclic loading of human engineered cartilage which can now be tested for its diagnostic potential in healthy versus OA-affected human cartilage

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

Among the very large number of polymeric materials that have been proposed in the field of orthopedics, polyethylene terephthalate (PET) is one of the most attractive thanks to its flexibility, thermal resistance, mechanical strength and durability. Several studies were proposed that interface nano- or micro-structured surfaces with mesenchymal stromal cells (MSCs), demonstrating the potential of this technology for promoting osteogenesis. All these studies were carried out on other biomaterials than PET, which remains almost un-investigated in terms of cell shaping, alignment and differentiation. In a previous study, we developed a hot-embossing method to transfer nano-textures (down to below 100 nm lateral size) onto PET substrate, and demonstrated that PET nanogratings (NGs) can optimally stimulate hMSC mechanotransduction mechanism. Specifically, we showed that cell and nuclear morphology, and cytoskeletal components are similarly affected by NGs, and that NG ridge sizes of 500 nm and 1 μm were both effective in stimulating cell polarization, without compromising cell viability. We study the effect of PET 350-depth nanogratings (NGs) having ridge and groove lateral size of 500 nm (T1) or 1 µm (T2), on bone-marrow human MSC (hMSC) differentiation towards the osteogenic fate. In particular, we cultured hMSCs on PET NGs having different periodicity and measured the expression of a complete set of genes characterizing osteo-differentiation, at different time-points from day 3 up to day 21. In order to evaluate how the contact interaction with PET NGs affects hMSC differentiation, the expression of a set of genes (RUNX2, COL1A1, ALPL, BMP2 and IBSP) characterizing osteogenesis was measured by RT-qPCR. BMP2 and IBSP were the most sensitive to the presence of the engineered surfaces The production of bone matrix was finally evaluated at the end of the differentiation period in terms of morphology, substrate coverage and alignment to the underlying topography. Overall, the data show that among the tested genes, BMP2 and IBSP are the most sensitive to the presence of the engineered surfaces. Although for RUNX2, COL1A1 and ALPL we measured only small modifications, upregulation of BMP2 and IBSP was relevant, especially in case of Osteogenic Medium (OM) and for the T2 geometry. This result suggests the T2 substrate as the most promising structure for stimulating hMSCs towards osteogenic maturation. We demonstrate that these substrates, especially the T2, can promote the osteogenic phenotype more efficiently than standard flat surfaces and that this effect is more marked if cells are cultured in osteogenic medium than in basal medium. Finally, we show that the shape and disposition of calcium hydroxyapatite granules on the different substrates was influenced by the substrate symmetry, being more elongated and spatially organized on NGs than on flat surfaces. This study demonstrates that PET nanogratings can promote osteogenic differentiation of hMSC in vitro. Since PET is an FDA approved material and we did not use any surface chemical treatment for cell adhesion and spreading, PET NGs can be considered promising for clinical translation in the field of orthopedic tissue engineering

Introduction. Sclerostin has been implicated in mechanotransduction in bone and recent data show a lack of response to loading in the sclerostin transgenic mouse. Sclerostin, the protein product of the SOST gene, is an attractive therapeutic target for low bone mass conditions, including osteoporosis. It is expressed exclusively by mature osteocytes in bone and we have shown that sclerostin targets pre-osteocytes/osteocytes to regulate bone mineralization and osteoclast activity, as well as inducing catabolic gene expression in osteocytes themselves and promoting osteocyte-mediated bone loss (osteocytic osteolysis). The aim of this study was to examine the direct effects of sclerostin on anabolic responses to loading in bone ex vivo. Methods. 10 × 5mm bovine sternum trabecular bone cores were perfused with osteogenic media at 37°C for up to 3 weeks in individual bone culture chambers. The cores were divided into 3 groups; a) mechanically loaded (300 cycles, 4000 μstrain, 1 Hz/day), b) identical loading regime with continuous perfusion of 50 ng/ml recombinant human sclerostin and c) unloaded controls. Loading was accomplished using a second-generation Zetos™ bone loading system. Daily measurements of bone stiffness (Young's modulus), media pH and ionic calcium concentrations were made. Histomorphometric assessment, including fluorochrome labelling analysis, was made of resin-embedded, non-decalcified samples at the end of the experiment. Gene expression in the bovine bone was examined by real-time RT-PCR. Results. Bovine bone cores showed a steady increase in Young's modulus with daily application of mechanical loading. This increase in stiffness was blocked by the co-addition of sclerostin. Sclerostin also induced bone acidification and a net release of bone calcium, indicated by the decrease in media pH and the relative increase in ionic calcium concentrations in the presence of sclerostin. Sclerostin also completely abrogated loading-induced calcium/calcein uptake. Sclerostin induced an increase in the expression of the bone resorption genes, tartrate resistant acid phosphatase (TRAP), carbonic anhydrase and cathepsin K and induced the release of β-CTX. Histological examination revealed a significant increase in the size of the osteocyte lacunae in sclerostin-treated bone cores, suggesting a role for osteocytic osteolysis in this effect. Discussion/Conclusion. The observation that sclerostin abrogated the loading-induced increase in bone stiffness constitutes direct evidence for a negative effect of sclerostin on the anabolic response to mechanical loading. Our findings may be explained in part by the observation that sclerostin negatively controls mineralization by late osteoblasts and pre-osteocytes (1). It is also possible that osteocytes themselves are capable of releasing bone mineral in response to sclerostin. This study demonstrates that sclerostin directly antagonises the anabolic effects of mechanical loading in the absence of external (circulating, neural, hormonal) influences. The mechanisms, by which sclerostin exerts these effects, warrant further study

Summary Statement. We have shown that integrin mRNA expression is regulated by the application of mechanical load. This indicates that mechanical loading may modify cell sensitivity to perceive further load through increased interaction with the ECM. Introduction. Tendinopathies are a range of diseases characterised by pain and insidious degeneration. Although poorly understood, onset is often associated with physical activity. We have previously investigated the regulation by mechanical strain of metalloproteinase gene expression in human tenocyte in a 3D collagen matrix. Integrins are important in cellular interaction with the ECM and are reported to mediate mechanotransduction in various non-tendon tissues. We have reported that TGFbeta activation is a key player in the regulation of metalloproteinases in response to mechanical load, which may be mediated by integrins. This project aims to investigate the effect of cyclic loading and TGFbeta stimulation on integrin expression by human tenocytes, in collagen and fibrin matrices. Methods. Human tenocytes were seeded at 1.5×106 cells/ml into collagen (rat tail type I, 1mg/ml) or fibrin (fibrinogen 6mg/ml, Thrombin 0.2u/ml) gels and stretched using a sinusoidal waveform of 0–5% at 1Hz using the Flexcell FX4000T(trade mark) system. Cultures were treated with or without 1ng/ml TGFbeta1 and load for 0–48 hours. Taqman Low density Array was used to asses a range of integrin, including ITGA1-6, ITGA10 and ITGA11 as well as ITGB1-5 (n=3). Results. In collagen cultures all integrins assayed were detectable (Ct < 35). ITGB1 was increased 2 fold with 48 hours of cyclic strain (p=0.006). ITGA6 and ITGA10 were decreased 1.4 and 2 fold with TGFbeta treatment after 24 hours (p=0.019, p=0.006). ITGA3 and ITGB3 were significantly decreased 7.6 and 8.3 fold with TGFbeta treatment after 48 hours (p=0.012, p=0.023). ITGA5 and ITGB1 showed similar responses with strain and TGFbeta, i.e. an increased trend. However, the other integrins showed a dissimilar response to strain and TGFbeta. Here we compare these responses to those in fibrin under the same conditions. Discussion. We have shown that integrin mRNA expression is regulated by the application of mechanical load. This indicates that mechanical loading may modify cell sensitivity to perceive further load through increased interaction with the ECM. Any differences in the cellular response to load in collagen and fibrin cultures, indicates that cellular interaction with the ECM is an important factor in the detection of load. Due to the differential regulation of some of the integrins with strain and TGFbeta, it appears that TGFbeta may not be responsible for the regulation of all integrins with strain. However this remains unconfirmed and may be explained by a temporal difference. Further analysis of how integrins are regulated in response to mechanical load and how this expression is translated to the protein level is required

Objectives

The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known.