Superficial Zone Chondrocytes Research Articles

Response of Articular Cartilage to Hyperosmolar Stress: Report of an Ex Vivo Injury Model

Background: Physiological 0.9% saline is commonly used as an irrigation fluid in modern arthroscopy. There is a growing body of evidence that a hyperosmolar saline solution has chondroprotective effects, especially if iatrogenic injury occurs. Purpose: To (1) corroborate the superiority of a hyperosmolar saline solution regarding chondrocyte survival after mechanical injury and (2) observe the modulatory response of articular cartilage to osmotic stress and injury. Study Design: Controlled laboratory study. Methods: Osteochondral explants were isolated from bovine stifle joints and exposed to either 0.9% saline (308 mOsm) or hyperosmolar saline (600 mOsm) and then damaged with a sharp dermatome blade to attain a confined full-thickness cartilage injury site, incubated in the same fluids for another 3 hours, and transferred to chondropermissive medium for further culture for 1 week. Chondrocyte survival was assessed by confocal imaging, while the cellular response was evaluated over 1 week by relative gene expression for apoptotic and inflammatory markers and mediator release into the medium. Results: The full-thickness cartilage cut resulted in a confined zone of cell death that mainly affected superficial zone chondrocytes. Injured samples that were exposed to hyperosmolar saline showed less expansion of cell death in both the axial (P < .007) and the coronal (P < .004) plane. There was no progression of cell death during the following week of culture. Histological assessment revealed an intact cartilage matrix and normal chondrocyte morphology. Inflammatory and proapoptotic genes were upregulated on the first days postexposure with a notable downregulation toward day 7. Mediator release into the medium was concentrated on day 3. Conclusion: This in vitro cartilage injury model provides further evidence for the chondroprotective effect of a hyperosmolar saline irrigation fluid, as well as novel data on the capability of articular cartilage to quickly regain joint homeostasis after osmotic stress and injury. Clinical Relevance: Raising the osmolarity of an irrigating solution may be a simple and safe strategy to protect articular cartilage during arthroscopic surgery.

Passaged Articular Chondrocytes From the Superficial Zone and Deep Zone Can Regain Zone-Specific Properties After Redifferentiation

Background: Bioengineered cartilage is a developing therapeutic to repair cartilage defects. The matrix must be rich in collagen type II and aggrecan and mechanically competent, withstanding compressive and shearing loads. Biomechanical properties in native articular cartilage depend on the zonal architecture consisting of 3 zones: superficial, middle, and deep. The superficial zone chondrocytes produce lubricating proteoglycan-4, whereas the deep zone chondrocytes produce collagen type X, which allows for integration into the subchondral bone. Zonal and chondrogenic expression is lost after cell number expansion. Current cell-based therapies have limited capacity to regenerate the zonal structure of native cartilage. Hypothesis: Both passaged superficial and deep zone chondrocytes at high density can form bioengineered cartilage that is rich in collagen type II and aggrecan; however, only passaged superficial zone–derived chondrocytes will express superficial zone–specific proteoglycan-4, and only passaged deep zone–derived chondrocytes will express deep zone–specific collagen type X. Study Design: Controlled laboratory study. Methods: Superficial and deep zone chondrocytes were isolated from bovine joints, and zonal subpopulations were separately expanded in 2-dimensional culture. At passage 2, superficial and deep zone chondrocytes were seeded, separately, in scaffold-free 3-dimensional culture within agarose wells and cultured in redifferentiation media. Results: Monolayer expansion resulted in loss of expression for proteoglycan-4 and collagen type X in passaged superficial and deep zone chondrocytes, respectively. By passage 2, superficial and deep zone chondrocytes had similar expression for dedifferentiated molecules collagen type I and tenascin C. Redifferentiation of both superficial and deep zone chondrocytes led to the expression of collagen type II and aggrecan in both passaged chondrocyte populations. However, only redifferentiated deep zone chondrocytes expressed collagen type X, and only redifferentiated superficial zone chondrocytes expressed and secreted proteoglycan-4. Additionally, redifferentiated deep zone chondrocytes produced a thicker and more robust tissue compared with superficial zone chondrocytes. Conclusion: The recapitulation of the primary phenotype from passaged zonal chondrocytes introduces a novel method of functional bioengineering of cartilage that resembles the zone-specific biological properties of native cartilage. Clinical Relevance: The recapitulation of the primary phenotype in zonal chondrocytes could be a possible method to tailor bioengineered cartilage to have zone-specific expression.

The Actin Cytoskeleton as a Regulator of Proteoglycan 4.

The superficial zone (SZ) of articular cartilage is responsible for distributing shear forces for optimal cartilage loading and contributes to joint lubrication through the production of proteoglycan 4 (PRG4). PRG4 plays a critical role in joint homeostasis and is chondroprotective. Normal PRG4 production is affected by inflammation and irregular mechanical loading in post-traumatic osteoarthritis (PTOA). THe SZ chondrocyte (SZC) phenotype, including PRG4 expression, is regulated by the actin cytoskeleton in vitro. There remains a limited understanding of the regulation of PRG4 by the actin cytoskeleton in native articular chondrocytes. The filamentous (F)-actin cytoskeleton is a potential node in crosstalk between mechanical stimulation and cytokine activation and the regulation of PRG4 in SZCs, therefore developing insights in the regulation of PRG4 by actin may identify molecular targets for novel PTOA therapies. A comprehensive literature search on PRG4 and the regulation of the SZC phenotype by actin organization was performed. PRG4 is strongly regulated by the actin cytoskeleton in isolated SZCs in vitro. Biochemical and mechanical stimuli have been characterized to regulate PRG4 and may converge upon actin cytoskeleton signaling. Actin-based regulation of PRG4 in native SZCs is not fully understood and requires further elucidation. Understanding the regulation of PRG4 by actin in SZCs requires an in vivo context to further potential of leveraging actin arrangement to arthritic therapeutics.

Directed differentiation of human pluripotent stem cells into articular cartilage reveals effects caused by absence of WISP3, the gene responsible for progressive pseudorheumatoid arthropathy of childhood

ObjectivesProgressive pseudorheumatoid arthropathy of childhood (PPAC), caused by deficiency of WNT1 inducible signalling pathway protein 3 (WISP3), has been challenging to study because no animal model of the disease exists...

Depleting transforming growth factor beta receptor 2 signalling in the cartilage of itga1-null mice attenuates spontaneous knee osteoarthritis

ObjectivesIntegrin α1β1 protects against osteoarthritis (OA) when it is upregulated in the superficial zone of cartilage in the early stages of disease. However, the mechanism behind this protection is unknown. Integrin α1β1 moderates transforming growth factor β receptor II (TGFBR2) signalling, a critical regulator of chondrocyte anabolic activity. To this end, mice lacking integrin α1β1 have increased baseline activation of TGFBR2 signalling and overall fibrosis. The purpose of this study was to evaluate the interplay between integrin α1β1 and TGFBR2 in the development of spontaneous OA. We hypothesized that dampening TGFBR2 signalling in the cartilage of itga1-null mice would attenuate OA. MethodsBehavioural and histological manifestations of spontaneous knee OA were measured at 4, 8, 12 and 16 months in mice with and without a ubiquitous itga1 deletion and with and without a tamoxifen-induced cartilage specific TGFBR2 depletion. ResultsKnee cartilage degeneration, collateral ligament ossification and pain responses increased with age. Itga1-null mice with intact TGFBR2 signalling developed earlier and more severe OA compared to controls. In agreement with our hypothesis, depleting TGFBR2 signalling in the cartilage of itga1-null mice attenuated OA progression. ConclusionIntact TGFBR2 signalling drives early and worse knee OA in itga1-null mice. This result supports the hypothesis that the increased expression of integrin α1β1 by superficial zone chondrocytes early in OA development dampens TGFBR2 signalling and thus protects against degeneration.

Proteoglycan 4 (PRG4)/Lubricin and the Extracellular Matrix in Gout

Proteoglycan 4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes, released into synovial fluid, and adsorbed on cartilage and synovial surfaces. PRG4′s roles include cartilage boundary lubrication, synovial homeostasis, immunomodulation, and suppression of inflammation. Gouty arthritis is mediated by monosodium urate (MSU) crystal phagocytosis by synovial macrophages, with NLRP3 inflammasome activation and IL-1β release. The phagocytic receptor CD44 mediates MSU crystal uptake by macrophages. By binding CD44, PRG4 limits MSU crystal uptake and downstream inflammation. PRG4/CD44 signaling is transduced by protein phosphatase 2A, which inhibits NF-κB, decreases xanthine oxidoreductase (XOR), urate production, and ROS-mediated IL-1β secretion. PRG4 also suppresses MSU crystal deposition in vitro. In contrast to PRG4, collagen type II (CII) alters MSU crystal morphology and promotes the macrophage uptake of MSU crystals. PRG4 deficiency, mediated by imbalance in PRG4-degrading phagocyte proteases and their inhibitors, was recently implicated in erosive gout, independent of hyperuricemia. Thus, dysregulated extracellular matrix homeostasis, including deficient PRG4 and increased CII release, may promote incident gout and progression to erosive tophaceous joint disease. PRG4 supplementation may offer a new therapeutic option for gout.

Superficial zone chondrocytes can get compacted under physiological loading: A multiscale finite element analysis.

Recent in vivo and in vitro studies have demonstrated that superficial zone (SZ) chondrocytes within articular layers of diarthrodial joints die under normal physiologic loading conditions. In order to further explore the implications of this observation in future investigations, we first needed to understand the mechanical environment of SZ chondrocytes that might cause them to die under physiological sliding contact conditions. In this study we performed a multiscale finite element analysis of articular contact to track the temporal evolution of a SZ chondrocyte's interstitial fluid pressure, hydraulic permeability, and volume under physiologic loading conditions. The effect of the pericellular matrix modulus and permeability was parametrically investigated. Results showed that SZ chondrocytes can lose ninety percent of their intracellular fluid after several hours of intermittent or continuous contact loading, resulting in a reduction of intracellular hydraulic permeability by more than three orders of magnitude. These findings are consistent with loss of cell viability due to the impediment of cellular metabolic pathways induced by the loss of fluid. They suggest that there is a simple mechanical explanation for the vulnerability of SZ chondrocytes to sustained physiological loading conditions. Future studies will focus on validating these specific findings experimentally. STATEMENT OF SIGNIFICANCE: As with any mechanical system, normal 'wear and tear' of cartilage tissue lining joints is expected. Yet incidences of osteoarthritis are uncommon in individuals younger than 45. This counter-intuitive observation suggests there must be an intrinsic repair mechanism compensating for this wear and tear over many decades of life. Recent experimental studies have shown superficial zone chondrocytes die under physiologic loading conditions, suggesting that this repair mechanism may involve cell replenishment. To better understand the mechanical environment of these cells, we performed a multiscale computational analysis of articular contact under loading. Results indicated that normal activities like walking or standing can induce significant loss of intracellular fluid volume, potentially hindering metabolic activity and fluid transport properties, and causing cell death.

Induction of iPSC-derived Prg4-positive cells with characteristics of superficial zone chondrocytes and fibroblast-like synovial cells

BackgroundLubricin, a proteoglycan encoded by the PRG4 gene, is synthesised by superficial zone (SFZ) chondrocytes and synovial cells. It reduces friction between joints and allows smooth sliding of tendons. Although lubricin has been shown to be effective against osteoarthritis and synovitis in animals, its clinical application remains untested. In this study, we aimed to induce lubricin-expressing cells from pluripotent stem cells (iPSCs) and applied them locally via cell transplantation.MethodsTo generate iPSCs, OCT3/4, SOX2, KLF4, and L-MYC were transduced into fibroblasts derived from Prg4-mRFP1 transgenic mice. We established a protocol for the differentiation of iPSC-derived Prg4-mRFP1-positive cells and characterised their mRNA expression profile. Finally, we injected Prg4-mRFP1-positive cells into the paratenon, surrounding the Achilles tendons and knee joints of severe combined immunodeficient mice and assessed lubricin expression.ResultWnt3a, activin A, TGF-β1, and bFGF were applied to induce the differentiation of iPSC-derived Prg4-mRFP1-positive cells. Markers related to SFZ chondrocytes and fibroblast-like synovial cells (FLSs) were expressed during differentiation. RNA-sequencing indicated that iPSC-derived Prg4-mRFP1-positive cells manifested expression profiles typical of SFZ chondrocytes and FLSs. Transplanted iPSC-derived Prg4-mRFP1-positive cells survived around the Achilles tendons and in knee joints.ConclusionsThe present study describes a protocol for the differentiation of iPSC-derived Prg4-positive cells with characteristics of SFZ chondrocytes and FLSs. Transplantation of lubricin-expressing cells offers promise as a therapy against arthritis and synovitis.

Increased migratory activity and cartilage regeneration by superficial-zone chondrocytes in enzymatically treated cartilage explants

BackgroundLimited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage.MethodsFull-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, −AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-β were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared.ResultsSuperficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-β was associated with higher migratory activity of the superficial-zone chondrocytes.ConclusionsIn enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-β combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.

Eldecalcitol regulates the gene expressions of articular cartilage markers and differentiation markers in chondrocytes

Vitamin D has been shown to reduce symptoms in patients with osteoarthritis (OA). In a previous study, local administration of eldecalcitol, an active vitamin D3 analog, reduced degenerative changes in articular cartilage in the early phase of experimental OA. However, the target of vitamin D in OA remains unknown. Here, we investigated the effect of eldecalcitol treatment on chondrocytes, which were divided into superficial zone chondrocytes (SZC), deep zone chondrocytes (DZC), and differentiated chondrocytes. SZC and DZC were cultured in monolayer and 3D pellet cultures treated with eldecalcitol. The gene expressions of articular cartilage and chondrocyte differentiation markers were evaluated. Histological analysis of SZC and DZC 3D pellet cultures was performed. The results showed that the articular cartilage markers ETS-related gene (Erg) and lubricin/proteoglycan 4 (PRG4) were significantly increased in SZC, but not in DZC, in the monolayer culture treated with eldecalcitol. The chondrocyte differentiation markers type X collagen and alkaline phosphatase (ALP) were significantly decreased in the DZC pellet culture treated with eldecalcitol. Immunochemical analysis also showed that Erg and lubricin/PRG4 expressions were elevated in the SZC pellet culture treated with eldecalcitol, while type X collagen and ALP expressions were decreased in the DZC pellet culture treated with eldecalcitol. In conclusion, this study showed that eldecalcitol upregulated articular cartilage markers in SZC and suppressed differentiation markers in DZC. Such regulation of chondrocytes by eldecalcitol could be potentially effective against OA progression.

Hyaluronic acid synthesis, degradation, and crosslinking in equine osteoarthritis: TNF-\u03b1-TSG-6-mediated HC-HA formation

BackgroundTNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties.MethodsHA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3), TSG-6, and hyaluronan-degrading enzyme (HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively.ResultsTNF-α concentrations were greater in OA synovial fluid, and TSG6 expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity.ConclusionsSynovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6 gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA.

HMGB2 promotes chondrocyte proliferation under negative pressure through the phosphorylation of AKT

Cells in articular cartilage are zonal arranged. Cells in superficial zone cartilage are generally small and proliferative. Appropriate negative pressure stimulation is beneficial to cell survival and tissue repair. Whether negative pressure has promotive impact on the proliferation activity of the superficial zone chondrocytes is of interest. In this study, we isolated superficial chondrocytes from the mandibular condylar cartilage of rats. After negative pressure treatment, the cells were collected for RNA-sequencing, quantitative real-time PCR and western blotting assays, aiming to detect the proliferative responses of chondrocytes to negative pressure and explore the potential molecular mechanisms. Data from RNA-sequencing analysis indicated that the superficial chondrocytes responded to the 4 h −10 kPa treatment by a significant increase in proliferation. In addition, the expression of high-mobility group box 2 (HMGB2) and the phosphorylation of AKT were obviously promoted. Knockdown of HMGB2 decreased AKT phosphorylation and diminished the negative pressure-induced proliferation of chondrocytes, as shown by decreased expression of Ki67 and cyclin-dependent kinase 6 (CDK6). In contrast, overexpression of HMGB2 enhanced AKT phosphorylation and further promoted proliferative activity. Moreover, LY294002, an AKT inhibitor, suppressed the proliferative activity of chondrocytes under negative pressure, while SC79, an activator of AKT phosphorylation, enhanced the proliferation of chondrocytes. Our data demonstrated that HMGB2 exhibits a promotion impact on chondrocyte proliferation under negative pressure via the phosphorylation of AKT. These results provide a new perspective for superficial zone chondrocytes proliferation under negative pressure, which should be benefit for cartilage regeneration.

Using a big data approach to explore in situ chondrocyte response to impact loading in articular cartilage

Using a big data approach to explore in situ chondrocyte response to impact loading in articular cartilage

Proof-of-concept for the detection of early osteoarthritis pathology by clinically applicable endomicroscopy and quantitative AI-supported optical biopsy

Clinical trials for osteoarthritis (OA), the leading cause of global disability, are unable to pinpoint the early, potentially reversible disease with clinical technology. Hence, disease-modifying drug candidates cannot be tested early in the disease. To overcome this obstacle, we asked whether early OA-pathology detection is possible with current clinical technology. We determined the relationship between two sensitive early OA markers, atomic force microscopy (AFM)-measured human articular cartilage (AC) surface stiffness, and location-matched superficial zone chondrocyte spatial organizations (SCSOs), asking whether a significant loss of surface stiffness can be detected in early OA SCSO stages. We then tested whether current clinical technology can visualize and accurately diagnose the SCSOs using an approved probe-based confocal laser-endomicroscope and a random forest (RF) model. We demonstrated a correlation between AC surface stiffness and the SCSO (rrm=-0.91; 95%CI:-0.97,-0.73), and an extensive loss of surface stiffness specifically in those ACs with early OA-typical SCSO (95%CIs: string SCSO: 269-173kPa, double string SCSO: 77-46kPa). This established the SCSO as a visualizable, functionally relevant surrogate marker of early OA AC surface pathology. Moreover, SCSO-based stiffness discrimination worked well in each patient's AC. We then demonstrated feasibility of visualizing the SCSO by clinical laser-endomicroscopy and, importantly, accurate SCSO diagnosis using RF. We present the proof-of-concept of early OA-pathology detection with available clinical technology, introducing a future-oriented, AI-supported, non-destructive quantitative optical biopsy for early disease detection. Operationalizing SCSO recognition, this approach allows testing for correlations between local tissue architectures with other experimental and clinical read-outs, but needs clinical validation and a larger sample size for defining diagnostic thresholds.

Optimization and Validation of a Human Ex Vivo Femoral Head Model for Preclinical Cartilage Research and Regenerative Therapies.

ObjectiveArticular cartilage is incapable of effective repair following injury or during osteoarthritis. While there have been developments in cartilage repair technologies, there is a need to advance biologically relevant models for preclinical testing of biomaterial and regenerative therapies. This study describes conditions for the effective ex vivo culture of the whole human femoral head.DesignFresh, viable femoral heads were obtained from femoral neck fractures and cultured for up to 10 weeks in (a) Dulbecco’s modified Eagle’s medium (DMEM); (b) DMEM + mixing; (c) DMEM + 10% human serum (HS); (d) DMEM + 10% HS + mixing. The viability, morphology, volume, and density of fluorescently labelled in situ chondrocytes and cartilage surface roughness were assessed by confocal microscopy. Cartilage histology was studied for glycosaminoglycan content using Alcian blue and collagen content using picrosirius red.ResultsChondrocyte viability remained at >95% in DMEM + 10% HS. In DMEM alone, viability remained high for ~4 weeks and then declined. For the other conditions, superficial zone chondrocyte viability fell to <35% at 10 weeks with deeper zones being relatively unaffected. In DMEM + 10% HS at 10 weeks, the number of chondrocytes possessing cytoplasmic processes increased compared with DMEM (P = 0.017). Alcian blue labeling decreased (P = 0.02) and cartilage thinned (P ≤ 0.05); however, there was no change to surface roughness, chondrocyte density, chondrocyte volume, or picrosirius red labeling (P > 0.05).ConclusionsIn this ex vivo model, chondrocyte viability was maintained in human femoral heads for up to 10 weeks in culture, a novel finding not previously reported. This human model could prove invaluable for the exploration, development, and assessment of preclinical cartilage repair and regenerative therapies.

Superficial and deep zone articular chondrocytes exhibit differences in actin polymerization status and actin-associated molecules in vitro

The actin cytoskeleton regulates cell shape and plays a role in regulating chondrocyte phenotype. Most studies investigating regulation of the chondrocyte phenotype by the actin cytoskeleton use chondrocytes isolated from full-thickness (FT) cartilage, which has a heterogeneous cell population. Superficial zone chondrocytes (SZC) have an elongated morphology and account for 10-20% of chondrocytes, while the remaining chondrocytes in the deeper zones appear more rounded. This study characterizes the actin cytoskeleton and expression of actin-associated molecules in SZC and deep zone (DZ) chondrocytes (DZC) in vitro in order to identify molecules differentially expressed by SZC and DZC that may contribute to the observed differences in zonal chondrocyte shapes. SZ, DZ, and FT chondrocytes isolated from bovine metacarpal-phalangeal joints were cultured in monolayer for 48h. Macroscopic morphology, actin polymerization status, and expression of select actin-associated molecules (adseverin, cofilin, transgelin, vinculin, MRTF-A, and YAP/TAZ) were determined. SZC appeared more elongated and have more filamentous actin compared to DZC, as determined by quantifying cell circularity and G-/F-actin ratio. MRTF-A gene and protein levels were significantly higher in SZC compared to DZC while DZC more highly expressed transgelin and TAZ. Although there was differential gene expression, no significant differences in adseverin, cofilin, vinculin, or YAP protein levels were observed between the two cell populations. This study identifies differences in actin polymerization status and expression of actin-associated molecules in primary SZC and DZC in vitro. These findings further our understanding of candidate actin-related pathways that may be regulating zonal chondrocyte phenotype.

Proteoglycan 4 expression by superficial zone chondrocytes is regulated in part by JNK and P38 in vitro

Purpose: Osteoarthritis (OA) is a degenerative disease that affects joint homeostasis leading to cartilage degeneration. Inflammatory mediators, including interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), are key factors in this catabolic process that are known to activate the mitogen-activated protein kinases (MAPKs) and have been shown to play a central role in mediating this degradative response by OA chondrocytes. However, these studies typically use chondrocytes from full-thickness cartilage. A study by Rosenzweig et al. showed that the temporal and spatial expression of phosphorylated MAPKs in response to cartilage injury was zone-specific, and observed a chondroprotective effect of activation of the MAPK jun amino-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) in the superficial zone (SZ). The purpose of this study was to investigate the role of JNK and p38 in regulating key aspects of the SZ phenotype including cell morphology, actin polymerization status, expression of the chondroprotective SZ molecule proteoglycan 4 (PRG4), and localization of yes-associated protein and transcriptional co-activator with PDZ-binding motif, which we have previously shown to regulate PRG4 expression in SZ chondrocytes (SZC). Methods: Bovine chondrocytes were isolated from the SZ of the metacarpo-phalangeal joint of calves aged 6-9 months by sequential digestion of the tissue in 0.5% protease (45 minutes) followed by 0.1% collagenase (14-17 hours). SZC were seeded in monolayer in high glucose DMEM supplemented with ITS, 100nM dexamethasone, 40μg/mL proline, 100mM pyruvate, 100μg/mL ascorbic acid. After 24 hours of culture, SZC were treated with either SP600125 (JNK inhibitor (JKNi); 10μM) or SB203580 (p38 inhibitor (p38i); 5μM) and the effects on the SZ phenotype (cell shape, actin polymerization status, and PRG4 expression, YAP/TAZ localization) were assessed after 24 hours of treatment. Cell shape was evaluated by measuring cell circularity and area using Image J software analysis. Actin polymerization status was assessed by visualizing globular and filamentous actin by confocal microscopy and determining the ratio of globular to filamentous actin, which was extracted by differential triton extraction and separated by SDS-PAGE. PRG4 mRNA levels was determined by RT-PCR. YAP/TAZ localization was assessed by fractionating the cellular cytoplasmic and nuclear components (NE-PER Nuclear and Cytoplasmic Extraction Kit; ThermoFisher Scientific) and evaluating them by SDS-PAGE followed by western blot. Experiments were repeated with SZC from 3 independent cell isolations. Statistical analysis was performed using Student’s T-test with significance assigned as p<0.05. Results: SZC at 24 hours maintain their phenotype as demonstrated by their spindle cell shape and secretion of PRG4. To determine whether JNKi and p38i affect chondrocyte morphology and actin organization, SZC were treated with JNKi and p38i and cell shape and actin polymerization status was assessed. Treatment with JNKi did not significantly affect cell area (N=3; p=0.43) but resulted in an increase in cell circularity (N=3; p=0.047). No changes in actin organization were observed with confocal microscopy, and no significant changes in the G-/F-actin ratio were observed (N=4; p=0.5) with JNKi treatment. Treatment with p38i did not affect cell area (N=3; p=0.13) or circularity (N=3; p=0.91). Similarly, no changes in actin organization were observed with confocal microscopy, and no significant changes in the G-/F-actin ratio (N=4; p=0.49) were observed with p38i treatment. Despite no change in actin polymerization status with treatment, a decrease in nuclear TAZ was observed with JNKi (N=5; p=0.03) and p38i (N=4; p=0.009) treatment. No change in levels of cytoplasmic YAP was observed with JNKi treatment; however, treatment with the p38i resulted in an increase in cytoplasmic YAP (N=4; p=0.02). PRG4 mRNA levels decreased with JNKi (N=3; p<0.0001) and p38i (N=3; p<0.001) treatment. Conclusions: The data suggests that JNK and p38 regulates TAZ nuclear localization and PRG4 expression without affecting actin cytoskeleton status. Regulation of the SZ phenotype is complex and likely results from the convergence of several major regulatory pathways. While actin polymerization and nuclear localization of YAP/TAZ have been shown to regulate PRG4 expression, JNK and p38 also appear to regulate PRG4 expression and YAP/TAZ localization; however it has yet to be determined whether JNK and p38 act downstream of actin polymerization or independent of actin polymerization. Understanding the regulatory mechanisms governing the SZ phenotype and the expression of the chondroprotective molecule PRG4 may provide insight into therapeutic treatments to delay the onset or progression of OA.

YAP/TAZ regulates the expression of proteoglycan 4 and tenascin C in superficial-zone chondrocytes.

The roles of cell division control protein 42 homologue (CDC42) and actin polymerisation in regulating the phenotype of superficial-zone chondrocytes (SZCs) have been demonstrated in vitro; however, the signalling pathway(s) downstream have yet to be fully elucidated. The study hypothesis was that Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) act downstream to regulate proteoglycan 4 (PRG4) and tenascin C (TNC). Bovine SZCs grown in monolayer were treated with ML141 (CDC42 inhibitor) or the actin depolymerising agents, latrunculin B and cytochalasin D, to determine the effect on YAP/TAZ. Verteporfin (YAP/TAZ inhibitor) and YAP/TAZ siRNA-mediated knockdown were used to determine their role in regulating PRG4 and TNC. ML141 treatment reduced total YAP/TAZ protein, nuclear TAZ levels and the YAP/TAZ target gene, connective tissue growth factor (CTGF) mRNA levels. Latrunculin B decreased nuclear TAZ, while cytochalasin D treatment trended towards increased nuclear TAZ (p = 0.06), correlating with decreased and increased CTGF mRNA levels, respectively. Verteporfin treatment decreased PRG4 and TNC expression, with no effect on actin polymerisation. siRNA-mediated knockdown of YAP/TAZ revealed that PRG4 was regulated by YAP/TAZ while TNC was regulated by TAZ only. As cytochalasin D can activate myocardin-related transcription factor-A (MRTF-A), siRNA-mediated knockdown was performed to determine the role of MRTF-A in regulating YAP/TAZ. Although nuclear TAZ decreased, no significant changes in total protein levels were observed. Findings suggested that CDC42 and actin polymerisation regulated SZCs through multiple actin-regulated pathways. Understanding the regulation of these chondroprotective molecules may have important implications for prevention/treatment of osteoarthritis.

IL-1β regulates cell shape and expression of prg4 independent of actin polymerization in superficial zone chondrocytes

Purpose: The superficial zone (SZ) of articular cartilage plays a multifaceted role in joint homeostasis contributing the biomechanical integrity and metabolic homeostasis of the tissue. In the early stages of osteoarthritis (OA), one of the first observable changes is loss of the SZ. Although the pathogenesis of OA is multifactorial, elevated levels of inflammatory cytokines are one contributing factor to disease progression. Interleukin-1 is an inflammatory cytokine that contributes to joint remodeling by regulating the expression of collagenases, aggrecanases, and nitric oxide (NO). Additionally, decreased expression of the chondroprotective molecule PRG4 is observed in SZ chondrocytes (SZC) in vitro and in the SZ of cartilage explant culture with IL-1 treatment. Studies using chondrocytes isolated from full-thickness cartilage observed changes in cellular mechanics with IL-1β treatment including increased filamentous actin and cellular stiffness. Unlike full-thickness chondrocytes, where actin polymerization leads to loss of the chondrogenic phenotype, the polymerized form of actin is important for the maintenance of the SZ phenotype and expression of PRG4. Although a decrease in PRG4 expression has previously been observed with IL-1β treatment in SZC the mechanism has not yet been fully elucidated. The purpose of this study is to determine whether IL-1β regulates PRG4 expression through an actin-dependent mechanism. Methods: The SZ of articular cartilage was dissected from the metacarpal phalangeal joint of calves aged 6-9 months. SZC were isolated by sequential digestion in 0.5% protease for 45 minutes, followed by 0.1% collagenase for 14-17 hours. SZC were seeded in monolayer culture (52,000/cm2) in high glucose DMEM supplemented with ITS, 100nM dexamethasone, 40μg/mL proline, 100mM pyruvate, 100μg/mL ascorbic acid. After 24 hours in culture, SZC were treated with IL-1β (10ng/mL) and maintained for 24 hours prior to harvesting for analysis. The effect of IL-1β on cell morphology and the cytoskeleton was determined. Cellular morphology was assessed using Image J analysis software to calculate cell area and circularity. Gene and protein expression was determined by RT-PCR and Western blotting, respectively. The cytoskeleton polymerization status was determined by performing differential triton extraction containing the soluble portion (G-actin and M-tubulin) and insoluble portion (F-actin and P-tubulin). The fractions were separated by SDS-PAGE and the resultant bands were semi-quantified using Image J analysis software to determine the G-/F-actin ratio and M-/P-tubulin ratio. Confocal imaging of the actin cytoskeleton visualizing the G- and F-actin was performed by confocal microscopy. Experiments were performed with SZC from independent cell isolations. Each experiment was repeated 2-4 times; experiments were performed in triplicate, where appropriate. Student’s T-test was used to evaluate the results and significance assigned at p <0.05. Results: To confirm observations previously reported SZC treated with IL-1β were assessed by RT-PCR to determined PRG4 expression levels. PRG4 mRNA levels significantly decreased with treatment at 24 hours (p<0.05; N=4). The effect of IL-1β treatment on cellular morphology and actin organization was determined. Treatment with IL-1β resulted in no significant changes in cell area, but did result in a significant increase in cell circularity (p<0.001; N=2). Visualization of the G- and F-actin by confocal microscopy yielded no observable differences in the actin structure (N=2). To confirm this, the polymerization status of the actin cytoskeleton was determined by calculating the G-/F-actin ratio. Despite changes in cell circularity, no significant changes in the G-/F-actin ratio were observed (p=0.8; N=4). Changes in the microtubule structure can also induce changes in cellular morphology, thus we investigated whether the microtubule polymerization status was altered with IL-1β treatment, and observed no significant difference in the M-/P-tubulin ratio (p=0.3; N=3), suggesting another regulator of cell shape may be modulating PRG4 expression in SZC. Conclusions: The data suggests that the elongated morphology is an important regulator of PRG4 expression. Despite changes in cell circularity and reduced expression of PRG4, no changes in actin polymerization or tubulin polymerization were observed. Although actin polymerization has previously been shown to regulate PRG4 expression, additional regulators of cellular morphology may also be important for the maintenance of the SZ phenotype.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Increased Substance P Immunoreactivity in Ipsilateral Knee Cartilage of Rats Exposed to Lumbar Spine Injury

The present study aimed to investigate whether experimentally induced lumbar facet-joint OA lead to degenerative changes and enhanced SP expression within the ipsilateral neurosegmentally linked tibiofemoral cartilage. Adult male Sprague-Dawley rats were assigned to left side L5-L6 facet mechanical compression injury (surgery) (n = 6), L5-L6 facet exposure with no compression (sham) (n = 5), or naïve (no surgery) (n = 4) groups. The morphology of the tibiofemoral articular cartilage was assessed using a modified Mankin scoring system. Immunohistochemistry was used to examine the density of chondrocytes stained positive for SP (cells/cm2) in the ipsilateral tibiofemoral cartilage at 28 days postintervention. Tibiofemoral cartilage in the surgery group showed consistent loss of superficial zone chondrocytes, mild roughening of the articular surface and occasional chondrocyte clusters as well as a greater density of SP mainly in the superficial cartilage zone compared with sham and naïve groups, although they also had a basic SP-expression. Our results support the hypothesis that neurogenic mechanisms may mediate the spread of SP to neurosegmentally linked heterologous joints affecting the distal cartilage homeostasis. These findings contribute additional insight into the potential role of neurogenic inflammation with implications in the pathophysiology of chronic inflammatory joint disease and OA.