YAP activation inhibits inflammatory signalling and cartilage breakdown associated with reduced primary cilia expression.

OBJECTIVE: To clarify the role of YAP in modulating cartilage inflammation and degradation and the involvement of primary cilia and associated intraflagellar transport (IFT). METHODS: Isolated primary chondrocytes were cultured on substrates of different stiffness (6-1000 kPa) or treated with YAP agonist lysophosphatidic acid (LPA) or YAP antagonist verteporfin (VP), or genetically modified by YAP siRNA, all ± IL1ß. Nitric oxide (NO) and prostaglandin E2 (PGE2) release were measured to monitor IL1ß response. YAP activity was quantified by YAP nuclear/cytoplasmic ratio and percentage of YAP-positive cells. Mechanical properties of cartilage explants were tested to confirm cartilage degradation. The involvement of primary cilia and IFT was analysed using IFT88 siRNA and ORPK cells with hypomorphic mutation of IFT88. RESULTS: Treatment with LPA, or increasing polydimethylsiloxane (PDMS) substrate stiffness, activated YAP nuclear expression and inhibited IL1ß-induced release of NO and PGE2, in isolated chondrocytes. Treatment with LPA also inhibited IL1ß-mediated inflammatory signalling in cartilage explants and prevented matrix degradation and the loss of cartilage biomechanics. YAP activation reduced expression of primary cilia, knockdown of YAP in the absence of functional cilia/IFT failed to induce an inflammatory response. CONCLUSIONS: We demonstrate that both pharmaceutical and mechanical activation of YAP blocks pro-inflammatory signalling induced by IL1ß and prevents cartilage breakdown and the loss of biomechanical functionality. This is associated with reduced expression of primary cilia revealing a potential anti-inflammatory mechanism with novel therapeutic targets for treatment of osteoarthritis (OA).

Activation of TRPV4 by mechanical, osmotic or pharmaceutical stimulation is anti-inflammatory blocking IL-1ß mediated articular cartilage matrix destruction.

OBJECTIVE: Cartilage health is maintained in response to a range of mechanical stimuli including compressive, shear and tensile strains and associated alterations in osmolality. The osmotic-sensitive ion channel Transient Receptor Potential Vanilloid 4 (TRPV4) is required for mechanotransduction. Mechanical stimuli inhibit interleukin-1ß (IL-1ß) mediated inflammatory signalling, however the mechanism is unclear. This study aims to clarify the role of TRPV4 in this response. DESIGN: TRPV4 activity was modulated glycogen synthase kinase (GSK205 antagonist or GSK1016790 A (GSK101) agonist) in articular chondrocytes and cartilage explants in the presence or absence of IL-1ß, mechanical (10% cyclic tensile strain (CTS), 0.33 Hz, 24hrs) or osmotic loading (200mOsm, 24hrs). Nitric oxide (NO), prostaglandin E2 (PGE2) and sulphated glycosaminoglycan (sGAG) release and cartilage biomechanics were analysed. Alterations in post-translational tubulin modifications and primary cilia length regulation were examined. RESULTS: In isolated chondrocytes, mechanical loading inhibited IL-1ß mediated NO and PGE2 release. This response was inhibited by GSK205. Similarly, osmotic loading was anti-inflammatory in cells and explants, this response was abrogated by TRPV4 inhibition. In explants, GSK101 inhibited IL-1ß mediated NO release and prevented cartilage degradation and loss of mechanical properties. Upon activation, TRPV4 cilia localisation was increased resulting in histone deacetylase 6 (HDAC6)-dependent modulation of soluble tubulin and altered cilia length regulation. CONCLUSION: Mechanical, osmotic or pharmaceutical activation of TRPV4 regulates HDAC6-dependent modulation of ciliary tubulin and is anti-inflammatory. This study reveals for the first time, the potential of TRPV4 manipulation as a novel therapeutic mechanism to supress pro-inflammatory signalling and cartilage degradation.

Mechanical loading inhibits cartilage inflammatory signalling via an HDAC6 and IFT-dependent mechanism regulating primary cilia elongation.

OBJECTIVE: Physiological mechanical loading reduces inflammatory signalling in numerous cell types including articular chondrocytes however the mechanism responsible remains unclear. This study investigates the role of chondrocyte primary cilia and associated intraflagellar transport (IFT) in the mechanical regulation of interleukin-1ß (IL-1ß) signalling. DESIGN: Isolated chondrocytes and cartilage explants were subjected to cyclic mechanical loading in the presence and absence of the cytokine IL-1ß. Nitric oxide (NO) and prostaglandin E2 (PGE2) release were used to monitor IL-1ß signalling whilst Sulphated glycosaminoglycan (sGAG) release provided measurement of cartilage degradation. Measurements were made of HDAC6 activity and tubulin polymerisation and acetylation. Effects on primary cilia were monitored by confocal and super resolution microscopy. Involvement of IFT was analysed using ORPK cells with hypomorphic mutation of IFT88. RESULTS: Mechanical loading suppressed NO and PGE2 release and prevented cartilage degradation. Loading activated HDAC6 and disrupted tubulin acetylation and cilia elongation induced by IL-1ß. HDAC6 inhibition with tubacin blocked the anti-inflammatory effects of loading and restored tubulin acetylation and cilia elongation. Hypomorphic mutation of IFT88 reduced IL-1ß signalling and abolished the anti-inflammatory effects of loading indicating the mechanism is IFT-dependent. Loading reduced the pool of non-polymerised tubulin which was replicated by taxol which also mimicked the anti-inflammatory effects of mechanical loading and prevented cilia elongation. CONCLUSIONS: This study reveals that mechanical loading suppresses inflammatory signalling, partially dependent on IFT, by activation of HDAC6 and post transcriptional modulation of tubulin.

Cobalt ions stimulate a fibrotic response through matrix remodelling, fibroblast contraction and release of pro-fibrotic signals from macrophages.

Many studies report the adverse responses to metal-on-metal (MoM) hip prostheses, with tissues surrounding failed MoM hip prostheses revealing abundant tissue necrosis and fibrosis. These local effects appear to be initiated by metal ions released from the prosthesis causing the secretion of inflammatory mediators. However, little is known about the effect of the metal ions on tissue remodelling and pseudotumor formation, which are also associated with the failure of MoM hip prostheses. The peri-prosthetic soft tissue masses can lead to pain, swelling, limited range of joint movement and extensive tissue lesion. To elucidate this cellular response, a multidisciplinary approach using both two- and three-dimensional (2D and 3D) in vitro culture systems was employed to study the effects of Co2+ and Cr3+ on human fibroblast activation and mechanobiology. Co2+ induced a fibrotic response, characterised by cytoskeletal remodelling and enhanced collagen matrix contraction. This was associated with increased cell stiffness and contractile forces as measured by atomic force microscopy and traction force microscopy, respectively. These effects were triggered by the generation of reactive oxygen species (ROS). Moreover, this fibrotic response was enhanced in the presence of macrophages, which increased the prevalence of a-smooth muscle actin (a-SMA)-positive fibroblasts and collagen synthesis. Cr3+ did not show any significant effect on fibroblast activation. Co2+ promoted matrix remodelling by fibroblasts that was further enhanced by macrophage signalling. Use of alternative implant materials or manipulation of this fibrotic response could provide an opportunity for enhancing the success of prostheses utilising CoCr alloys.

Chondrocyte expansion is associated with loss of primary cilia and disrupted hedgehog signalling.

Tissue engineering-based therapies targeting cartilage diseases, such as osteoarthritis, require in vitro expansion of articular chondrocytes. A major obstacle for these therapies is the dedifferentiation and loss of phenotype accompanying chondrocyte expansion. Recent studies suggest that manipulation of hedgehog signalling may be used to promote chondrocyte re-differentiation. Hedgehog signalling requires the primary cilium, a microtubule-based signalling compartment, the integrity of which is linked to the cytoskeleton. We tested the hypothesis that alterations in cilia expression occurred as consequence of chondrocyte dedifferentiation and influenced hedgehog responsiveness. In vitro chondrocyte expansion to passage 5 (P5) was associated with increased actin stress fibre formation, dedifferentiation and progressive loss of primary cilia, compared to primary (P0) cells. P5 chondrocytes exhibited ~50 % fewer cilia with a reduced mean length. Cilia loss was associated with disruption of ligand-induced hedgehog signalling, such that P5 chondrocytes did not significantly regulate the expression of hedgehog target genes (GLI1 and PTCH1). This phenomenon could be recapitulated by applying 24 h cyclic tensile strain, which reduced cilia prevalence and length in P0 cells. LiCl treatment rescued cilia loss in P5 cells, partially restoring hedgehog signalling, so that GLI1 expression was significantly increased by Indian hedgehog. This study demonstrated that monolayer expansion disrupted primary cilia structure and hedgehog signalling associated with chondrocyte dedifferentiation. This excluded the possibility to use hedgehog ligands to stimulate re-differentiation without first restoring cilia expression. Furthermore, primary cilia loss during chondrocyte expansion would likely impact other cilia pathways important for cartilage health and tissue engineering, including transforming growth factor (TGF), Wnt and mechanosignalling.

Experimental validation of a flat punch indentation methodology calibrated against unconfined compression tests for determination of soft tissue biomechanics.

Mechanical characterisation of soft biological tissues using standard compression or tensile testing presents a significant challenge due to specimen geometrical irregularities, difficulties in cutting intact and appropriately sized test samples, and issues with slippage or damage at the grips. Indentation can overcome these problems but requires fitting a model to the resulting load-displacement data in order to calculate moduli. Despite the widespread use of this technique, few studies experimentally validate their chosen model or compensate for boundary effects. In this study, viscoelastic hydrogels of different concentrations and dimensions were used to calibrate an indentation technique performed at large specimen-strain deformation (20%) and analysed with a range of routinely used mathematical models. A rigid, flat-ended cylindrical indenter was applied to each specimen from which 'indentation moduli' and relaxation properties were calculated and compared against values obtained from unconfined compression. Only one indentation model showed good agreement (<10% difference) with all moduli values obtained from compression. A sample thickness to indenter diameter ratio ≥1:1 and sample diameter to indenter diameter ratio ≥4:1 was necessary to achieve the greatest accuracy. However, it is not always possible to use biological samples within these limits, therefore we developed a series of correction factors. The approach was validated using human diseased omentum and bovine articular cartilage resulting in mechanical properties closely matching compression values. We therefore present a widely useable indentation analysis method to allow more accurate calculation of material mechanics which is important in the study of soft tissue development, ageing, health and disease.

IFT88 influences chondrocyte actin organization and biomechanics.

OBJECTIVES: Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. METHODS: The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. RESULTS: IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. CONCLUSIONS: This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology.

Chondrocyte dedifferentiation increases cell stiffness by strengthening membrane-actin adhesion.

OBJECTIVE: Chondrocyte dedifferentiation is known to influence cell mechanics leading to alterations in cell function. This study examined the influence of chondrocyte dedifferentiation in monolayer on cell viscoelastic properties and associated changes in actin organisation, bleb formation and membrane-actin cortex interaction. METHOD: Micropipette aspiration was used to estimate the viscoelastic properties of freshly isolated articular chondrocytes and the same cells after passage in monolayer. Studies quantified the cell membrane-actin cortex adhesion by measuring the critical pressure required for membrane detachment and bleb formation. We then examined the expression of ezrin, radixin and moesin (ERM) proteins which are involved in linking the membrane and actin cortex and combined this with theoretical modelling of bleb dynamics. RESULTS: Dedifferentiated chondrocytes at passage 1 (P1) were found to be stiffer compared to freshly isolated chondrocytes (P0), with equilibrium modulus values of 0.40 and 0.16 kPa respectively. The critical pressure increased from 0.59 kPa at P0 to 0.74 kPa at P1. Dedifferentiated cells at P1 exhibited increased cortical F-actin organisation and increased expression of total and phosphorylated ERM proteins compared to cells at P0. Theoretical modelling confirmed the importance of membrane-actin cortex adhesion in regulating bleb formation and effective cellular elastic modulus. CONCLUSION: This study demonstrates that chondrocyte dedifferentiation in monolayer strengthens membrane-actin cortex adhesion associated with increased F-actin organisation and up-regulation of ERM protein expression. Thus dedifferentiated cells have reduced susceptibility to bleb formation which increases cell modulus and may also regulate other fundamental aspects of cell function such as mechanotransduction and migration.

The rate of hypo-osmotic challenge influences regulatory volume decrease (RVD) and mechanical properties of articular chondrocytes.

OBJECTIVES: Osteoarthritis (OA) is associated with a gradual reduction in the interstitial osmotic pressure within articular cartilage. The aim of this study was to compare the effects of sudden and gradual hypo-osmotic challenge on chondrocyte morphology and biomechanics. METHODS: Bovine articular chondrocytes were exposed to a reduction in extracellular osmolality from 327 to 153 mOsmol/kg applied either suddenly (<5 s) or gradually (over 180 min). Temporal changes in cell diameter and the existence of regulatory volume decrease (RVD) were quantified along with changes in cortical actin and chromatin condensation. The cellular viscoelastic mechanical properties were determined by micropipette aspiration. RESULTS: In response to a sudden hypo-osmotic stress, 66% of chondrocytes exhibited an increase in diameter followed by RVD, whilst 25% showed no RVD. By contrast, cells exposed to gradual hypo-osmotic stress exhibited reduced cell swelling without subsequent RVD. There was an increase in the equilibrium modulus for cells exposed to sudden hypo-osmotic stress. However, gradual hypo-osmotic challenge had no effect on cell mechanical properties. This cell stiffening response to sudden hypo-osmotic challenge was abolished when actin organization was disrupted with cytochalasin D or RVD inhibited with REV5901. Both sudden and gradual hypo-osmotic challenge reduced cortical F-actin distribution and caused chromatin decondensation. CONCLUSIONS: Sudden hypo-osmotic challenge increases chondrocyte mechanics by activation of RVD and interaction with the actin cytoskeleton. Moreover, the rate of hypo-osmotic challenge is shown to have a profound effect on chondrocyte morphology and biomechanics. This important phenomenon needs to be considered when studying the response of chondrocytes to pathological hypo-osmotic stress.

The primary cilium influences interleukin-1ß-induced NFκB signalling by regulating IKK activity.

The primary cilium is an organelle acting as a master regulator of cellular signalling. We have previously shown that disruption of primary cilia assembly, through targeting intraflagellar transport, is associated with muted nitric oxide and prostaglandin responses to the inflammatory cytokine interleukin-1ß (IL-1ß). Here, we show that loss of the primary cilium disrupts specific molecular signalling events in cytosolic NFκB signalling. The induction of cyclooxygenase 2 (COX2) and inducible nitrous oxide synthase (iNOS) protein is abolished. Cells unable to assemble cilia exhibit unaffected activation of IκB kinase (IKK), but delayed and reduced degradation of IκB, due to diminished phosphorylation of inhibitor of kappa B (IκB) by IKK. This results in both delayed and reduced NFκB p65 nuclear translocation and nuclear transcript binding. We also demonstrate that heat shock protein 27 (hsp27), an established regulator of IKK, is localized to the ciliary axoneme and cellular levels are dramatically disrupted with loss of the primary cilium. These results suggest that the primary cilia compartment exerts influence over NFκB signalling. We propose that the cilium is a locality for regulation of the molecular events defining NFκB signalling events, tuning signalling as appropriate.

Primary cilia disassembly down-regulates mechanosensitive hedgehog signalling: a feedback mechanism controlling ADAMTS-5 expression in chondrocytes.

OBJECTIVE: Hedgehog signalling is mediated by the primary cilium and promotes cartilage degeneration in osteoarthritis. Primary cilia are influenced by pathological stimuli and cilia length and prevalence are increased in osteoarthritic cartilage. This study aims to investigate the relationship between mechanical loading, hedgehog signalling and cilia disassembly in articular chondrocytes. METHODS: Primary bovine articular chondrocytes were subjected to cyclic tensile strain (CTS; 0.33 Hz, 10% or 20% strain). Hedgehog pathway activation (Ptch1, Gli1) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS-5) expression were assessed by real-time PCR. A chondrocyte cell line generated from the Tg737(ORPK) mouse was used to investigate the role of the cilium in this response. Cilia length and prevalence were quantified by immunocytochemistry and confocal microscopy. RESULTS: Mechanical strain upregulates Indian hedgehog expression and activates hedgehog signalling. Ptch1, Gli1 and ADAMTS-5 expression were increased following 10% CTS, but not 20% CTS. Pathway activation requires a functioning primary cilium and is not observed in Tg737(ORPK) cells lacking cilia. Mechanical loading significantly reduced cilium length such that cilia became progressively shorter with increasing strain magnitude. Inhibition of histone deacetylase 6 (HDAC6), a tubulin deacetylase, prevented cilia disassembly and restored mechanosensitive hedgehog signalling and ADAMTS-5 expression at 20% CTS. CONCLUSIONS: This study demonstrates for the first time that mechanical loading activates primary cilia-mediated hedgehog signalling and ADAMTS-5 expression in adult articular chondrocytes, but that this response is lost at high strains due to HDAC6-mediated cilia disassembly. The study provides new mechanistic insight into the role of primary cilia and mechanical loading in articular cartilage.

Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells.

The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering ß-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear ß-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active ß-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation.

Stent encrustation in feline and human artificial urine: does the low molecular weight composition account for the difference?

Anecdotal evidence suggests that the rate of encrustation on JJ stents placed in domesticated cats appears to be decreased as compared to humans. Our study tests the hypothesis that this may be due to specific differences in the chemical composition of human and feline urine. Artificial human and feline urine solutions were used in an in vitro encrustation model where an 80 % stent encrustation could be expected after 7 weeks of incubation. Scanning electron microscopy (SEM) was used to analyse crystal morphology. Energy dispersive X-ray spectrometry (EDS) was used to assess composition weight. The percentage of surface coverage of encrustation on the respective stents was quantified using image J Java plug-in software. No significant difference was observed between both solutions with regard to quality and quantity of stent encrustation. Crystals were formed in both solutions as a mixture of Ca-dihydrate and Ca-monohydrate. The study shows that there is no significant difference in the rate of encrustations on JJ stents incubated in artificial feline or human urine. This suggests that a possible difference in stent encrustation between cats and humans is due to factors other than the inorganic biochemical composition of the urines alone. Keeping in mind a true species difference, analysis of urinary macromolecules and proteins will be the logical next step.

Cell mechanics, structure, and function are regulated by the stiffness of the three-dimensional microenvironment.

This study adopts a combined computational and experimental approach to determine the mechanical, structural, and metabolic properties of isolated chondrocytes cultured within three-dimensional hydrogels. A series of linear elastic and hyperelastic finite-element models demonstrated that chondrocytes cultured for 24 h in gels for which the relaxation modulus is <5 kPa exhibit a cellular Young's modulus of ∼5 kPa. This is notably greater than that reported for isolated chondrocytes in suspension. The increase in cell modulus occurs over a 24-h period and is associated with an increase in the organization of the cortical actin cytoskeleton, which is known to regulate cell mechanics. However, there was a reduction in chromatin condensation, suggesting that changes in the nucleus mechanics may not be involved. Comparison of cells in 1% and 3% agarose showed that cells in the stiffer gels rapidly develop a higher Young's modulus of ∼20 kPa, sixfold greater than that observed in the softer gels. This was associated with higher levels of actin organization and chromatin condensation, but only after 24 h in culture. Further studies revealed that cells in stiffer gels synthesize less extracellular matrix over a 28-day culture period. Hence, this study demonstrates that the properties of the three-dimensional microenvironment regulate the mechanical, structural, and metabolic properties of living cells.

Dynamic compressive strain influences chondrogenic gene expression in human periosteal cells: a case study.

Physical stimuli play a crucial role in skeletogenesis and osteochondral repair and regeneration. Although the periosteum and periosteum-derived cells offer considerable therapeutic potential, the molecular mechanisms that control their differentiation are still not fully understood. As an initial case study, this work explores the hypothesis that dynamic compression might selectively enhance chondrogenic and/or osteogenic differentiation in human periosteal cells from two donors. Donor derived human periosteal cells were expanded in monolayer culture before seeding in 3% (w/v) agarose constructs. The ability of this in vitro culture model to support cell viability, chondrogenesis, and mechanotransduction was optimised. The time course of early chondrogenic differentiation was assessed by real time RT-PCR of mRNA expression levels for bone and cartilage specific gene markers. Intermittent dynamic compression (1 Hz, 15% strain) was applied to constructs, in the presence or absence of 10 ng/ml TGF-ß3, for up to 4 days. The combined effect of TGF-ß3 and compressive loading on the expression levels of the Sox-9, Runx-2, ALP, Collagen X, and collagen type I genes was donor dependent. A synergistic effect was noted only in donor two, with peak mRNA expression levels at 24 h, particularly Sox-9 which increased 59.0-fold. These findings suggest that the interactions between mechanical stimuli and TGF-ß signalling may be an important mechanotransduction pathway for human periosteal cells and that, importantly, this cellular mechanosensitivity varies between donors.

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response.

Primary cilia are singular, cytoskeletal organelles present in the majority of mammalian cell types where they function as coordinating centres for mechanotransduction, Wnt and hedgehog signalling. The length of the primary cilium is proposed to modulate cilia function, governed in part by the activity of intraflagellar transport (IFT). In articular cartilage, primary cilia length is increased and hedgehog signaling activated in osteoarthritis (OA). Here, we examine primary cilia length with exposure to the quintessential inflammatory cytokine interleukin-1 (IL-1), which is up-regulated in OA. We then test the hypothesis that the cilium is involved in mediating the downstream inflammatory response. Primary chondrocytes treated with IL-1 exhibited a 50% increase in cilia length after 3 h exposure. IL-1-induced cilia elongation was also observed in human fibroblasts. In chondrocytes, this elongation occurred via a protein kinase A (PKA)-dependent mechanism. G-protein coupled adenylate cyclase also regulated the length of chondrocyte primary cilia but not downstream of IL-1. Chondrocytes treated with IL-1 exhibit a characteristic increase in the release of the inflammatory chemokines, nitric oxide and prostaglandin E2. However, in cells with a mutation in IFT88 whereby the cilia structure is lost, this response to IL-1 was significantly attenuated and, in the case of nitric oxide, completely abolished. Inhibition of IL-1-induced cilia elongation by PKA inhibition also attenuated the chemokine response. These results suggest that cilia assembly regulates the response to inflammatory cytokines. Therefore, the cilia proteome may provide a novel therapeutic target for the treatment of inflammatory pathologies, including OA.

The effect of mechanical strain or hypoxia on cell death in subpopulations of rat dorsal root ganglion neurons in vitro.

Spinal nerves and their associated dorsal root ganglion (DRG) cells can be subject to mechanical deformation and hypoxia associated with pathology such as disc herniation, spinal stenosis and spine trauma. There is very limited information on the response of adult DRG neurons to such stressors. In this study we used an in vitro approach to examine the response of adult DRG cells to (a) mechanical, hypoxic, and combined injuries; and (b) to compare the effects on injury on nociceptive and non-nociceptive neurons, as well as on non-neuronal cells. Mechanical injury (20% tensile strain) led to significant neuronal cell death (assessed by ethidium homodimer-1 labelling), which was proportional to strain duration (5 min, 1 h, 6 h or 18 h). Hypoxia (2% O(2) for 24 h) also promoted death of DRG neurons, and was further enhanced when mechanical strain and hypoxia were combined. Both mechanical strain and hypoxia significantly decreased the maximum neurite length. Conversely, death of non-neuronal cells was only increased by hypoxia and not by mechanical strain. Total cell death in response to mechanical injury or hypoxia was similar in both non-nociceptive (neurofilament, NF-200 immunoreactive) and nociceptive (calcitonin gene-related peptide, CGRP immunoreactive) neurons, but apoptosis (assessed by activated caspase-3 immunostaining) was significantly higher in CGRP than NF-200 neurons. Surprisingly, cell death of non-peptidergic nociceptors (identified by Griffonia simplicifolia IB4 lectin binding) was already high in control cultures, and was not increased further by either mechanical stretch or hypoxia. These results provide detailed information on the response of adult DRG subpopulations to hypoxia and mechanical strain, and describe in vitro models that could be useful for screening potential neuroprotective agents.

Articular chondrocytes express connexin 43 hemichannels and P2 receptors - a putative mechanoreceptor complex involving the primary cilium?

Mechanical loading is essential for the health and homeostasis of articular cartilage, although the fundamental mechanotransduction pathways are unclear. Previous studies have demonstrated that cyclic compression up-regulates proteoglycan synthesis via an intracellular Ca(2+) signalling pathway, mediated by the release of ATP. However, the mechanism(s) of ATP release has not been elucidated. The present study examines expression of the putative mechanosensitive ATP-release channel, connexin 43 and whether it is expressed on the chondrocyte primary cilium, which acts as a mechanosensor in a variety of other cell types. In addition the study characterized the expression of a range of purine receptors through which ATP may activate downstream signalling events controlling cell function. Bovine articular chondrocytes were isolated by sequential enzyme digestion and seeded in agarose constructs. To verify the presence of functional hemichannels, Lucifer yellow (LY) uptake into viable cells was quantified following treatment with a hemichannel agonist (EGTA) and antagonist (flufenamic acid). LY uptake was observed in 45% of chondrocytes, increasing to 83% following EGTA treatment (P < 0.001). Treatment with the hemichannel blocker, flufenamic acid, significantly decreased LY uptake to less than 5% with and without EGTA. Immunofluorescence and confocal microscopy confirmed the presence of primary cilia and the expression of connexin 43. Approximately 50% of bovine chondrocyte primary cilia were decorated with connexin 43. Human chondrocytes in situ within cartilage explants also expressed connexin 43 hemichannels. However, expression was confined to the upper 200 microm of the tissue closest to the articular surface. Immunofluorescence revealed the expression of a range of P2X and P2Y receptor subtypes within human articular cartilage. In conclusion, the expression of functional hemichannels by articular chondrocytes may represent the mechanism through which mechanical loading activates ATP release as part of a purinergic mechanotransduction pathway. Furthermore, the expression of connexin 43 on the chondrocyte primary cilium suggests the possible involvement of the cilium in this pathway.

Biomechanical analysis of structural deformation in living cells.

Most tissues are subject to some form of physiological mechanical loading which results in deformation of the cells triggering intracellular mechanotransduction pathways. This response to loading is generally essential for the health of the tissue, although more pronounced deformation may result in cell and tissue damage. In order to determine the biological response of cells to loading it is necessary to understand how cells and intracellular structures deform. This paper reviews the various loading systems that have been adopted for studying cell deformation both in situ within tissue explants and in isolated cell culture systems. In particular it describes loading systems which facilitate visualisation and subsequent quantification of cell deformation. The review also describes the associated microscopy and image analysis techniques. The review focuses on deformation of chondrocytes with additional information on a variety of other cell types including neurons, red blood cells, epithelial cells and skin and muscle cells.

An improved confocal FRAP technique for the measurement of long-term actin dynamics in individual stress fibers.

The present study describes an improved fluorescent recovery after photobleaching (FRAP) technique, which has been successfully used to quantify actin dynamics within individual fibers. Chondrocytes were transfected with an eGFP-actin plasmid and cultured on glass coverslips. In cells expressing eGFP-actin, confocal microscopy was used to bleach 3 x 1 microm regions accurately positioned along individual stress fibers. The subsequent fluorescent recovery over a 10-min imaging period was assessed from a series of intensity profiles, positioned along the length of the stress fibers and spanning the bleach region. From these profiles, the normalized fluorescent intensity values were plotted against time. In this way, the technique provided sufficient spatial precision to describe the long-term behavior within individual stress fibers while accounting for the inherent movement. An identical procedure was used to examine FRAP for eGFP-actin within the interfiber region. The FRAP curves for stress fibers were accurately modeled by two phase exponentials which indicated only partial recovery with a mobile fraction of 46%. This suggests that some of the F-actin molecules were in a tightly bound configuration with negligible turnover. The interfiber region exhibited similar two phase exponential FRAP with a mobile fraction of 68%. This partial recovery may be due to the presence, within the interfiber region, of both G-actin and fine F-actin fibers beneath the resolution of the confocal microscope. In conclusion, the present FRAP methodology overcomes many of the limitations of previous studies in order to provide new data describing long-term actin dynamics within individual stress fibers.