Osteoarthritis and Cartilage

ObjectiveEarly knee osteoarthritis (KOA) presents as minor structural abnormalities in joint tissues, such as cartilage and subchondral bone, that cannot be assessed radiographically. Identification of a sensitive and convenient marker for early disease has the potential to enhance patient outcomes. This study determined 1) whether fat infiltration in muscle (i.e., muscle quality), as measured by ultrasound, is associated with structural abnormalities seen in early KOA and 2) which quadriceps muscles are appropriate as a novel marker for early KOA. MethodsParticipants with early symptomatic KOA (Kellgren Lawrence grade 1-2) underwent ultrasound assessment to measure the echo intensity of the vastus medialis and rectus femoris. The echo intensity corrected for ultrasound wave attenuation caused by subcutaneous fat was then calculated (i.e., corrected echo intensity). Structural abnormalities were assessed using the whole-organ magnetic resonance score (WORMS). A generalized linear mixed model was used to assess the relationship between the corrected echo intensity and WORMS score. ResultsForty-nine participants (ages: 44-78 years, 65.3% women) with 52 knees were included. After adjustment for covariates, increased corrected echo intensity (i.e., poor muscle quality) in the vastus medialis muscle was significantly associated with greater structural abnormalities, including disrupted cartilage integrity in the medial tibiofemoral joint. The association was not significant in the rectus femoris muscle. ConclusionIndividuals with poor muscle quality in the vastus medialis displayed compromised joint integrity. This study suggests that fat infiltration in vastus medialis assessed by ultrasound is an indicator of early symptomatic KOA.

BACKGROUNDNerve growth factor (NGF), a key mediator of pain and inflammation, is increased in joints with osteoarthritis (OA). Neutralizing NGF with monoclonal antibodies has shown analgesic effects in painful knee OA, but clinical development was stopped due to side effects in the joints. Knowledge about the biological effects of long-term exposure of joint tissues to NGF is limited. Therefore, we aimed to explore the effects of repeated intra-articular (IA) injections of NGF into the knee joints of healthy mice on pain and sensitization, as well as joint innervation and structure. METHODSWe conducted five experiments in male C57BL/6 mice. In Experiment 1, NGF (50ng or 500ng) or vehicle was injected IA into the knee of naive wildtype (WT) mice, twice a week for 4 weeks. We assessed knee swelling, knee hyperalgesia and histopathology. In Experiment 2, mice were injected with 500ng NGF or vehicle, twice a week for 4 weeks and microCT of the knee was performed. In Experiment 3, NaV1.8-tdTomato reporter mice were injected with 500ng NGF or vehicle, twice a week for 4 weeks, and joint innervation was assessed. In Experiment 4, WT mice received 500ng NGF or vehicle twice a week for 4 weeks and were used for single cell RNA sequencing (scRNAseq) of the synovium. In Experiment 5, L3-L5 DRGs of mice that received 3 IA injections of 500ng NGF or vehicle twice a week were used for bulk RNA sequencing. RESULTSRepeated bi-weekly IA injections of NGF caused knee hyperalgesia in naive mice. NGF caused dose-dependent knee swelling, synovial pathology, increased bone mineral density and trabecular bone thickness in the medial subchondral bone, growth of pre-osteophytes in the medial compartment, but no cartilage degeneration. NGF injection caused sprouting of NaV1.8+ neurons in the medial but not the lateral synovium. ScRNAseq of the synovium revealed upregulated genes related to neuronal sprouting, synovial fibrosis and ossification, confirming histopathological findings. Bulk RNA seq of DRG showed upregulated pathways related to axonal growth. CONCLUSIONSIn healthy mouse knees, NGF induced mechanical sensitization, synovitis, neoinnervation in the medial synovium, subchondral bone changes and pre-osteophyte growth in the medial compartment, thus capturing many pathological changes observed in OA, except cartilage damage.

ObjectiveKnee joints are densely innervated by nociceptors. Sprouting of nociceptors has been reported in late-stage osteoarthritis (OA), both in human knees and in rodent models. Here, we sought to describe progressive nociceptor remodeling in four mouse models of knee OA, capturing early and late-stage disease. MethodsSham surgery, destabilization of the medial meniscus (DMM), partial meniscectomy (PMX), or non-invasive anterior cruciate ligament rupture (ACLR) was performed in the right knee of 10-12-week old male C57BL/6 NaV1.8-tdTomato mice. Mice were euthanized (1) 4, 8 or 16 weeks after DMM or sham surgery; (2) 4 or 12 weeks after PMX or sham; (3) 1 or 4 weeks after ACLR injury or sham. Additionally, a cohort of naive male wildtype mice was evaluated at 6 and 24 months. Twenty-m thick mid-joint cryosections were assessed qualitatively and quantitatively for NaV1.8+ and PGP9.5+ innervation. Cartilage damage (using a modified OARSI score), synovitis, and osteophytes were assessed blindly. ResultsProgressive OA developed in the medial compartment after DMM, PMX, and ACLR. Synovitis and associated neo-innervation by nociceptors peaked in early-stage OA. In the subchondral bone, channels containing sprouting nociceptors appeared early, and progressed with worsening joint damage. Two-year old mice developed primary OA in both the medial and the lateral compartment, accompanied with neuroplasticity in the synovium and the subchondral bone. All 4 models had an increased nerve signal in osteophytes. ConclusionAnatomical neuroplasticity of nociceptors was observed in association with joint damage in 4 distinct mouse models, suggesting that it is intrinsic to OA pathology.

ObjectivesOsteoarthritis (OA) is a debilitating joint disease that affects millions of people worldwide, with the temporomandibular joint (TMJ) and knee joint being prominently affected. Despite its prevalence, TMJ-OA remains understudied. This study aimed to investigate the transcriptional signature of the TMJ compared to that of the knee joint and to explore transcriptional differences in the medial and superficial layers of the TMJ-OA. DesignSix-month-old C57BL/6J mice TMJ and knee samples were collected. Goat TMJ superficial and medial layer cartilage was separated and treated with IL-1{beta}. All samples were subjected to bulk RNA sequencing followed by differential expression and gene set enrichment analysis. ResultsWe identified 4,031 protein-coding genes differentially expressed in the TMJ compared to the knee, with significant enrichment of neuronal system genes and lower enrichment of innate immune system genes. Key osteoarthritis biomarkers such as Mmp13, Postn, and Col1a1 were more highly expressed in the TMJ, indicating a higher vulnerability to OA development. IL-1{beta} treatment in goat TMJ chondrocytes mimicked the natural TMJ-OA-like transcriptional changes and immune responses, which are also observed in the rabbit TMJ-OA model. This validated the in vitro goat TMJ-OA model. The IL-1{beta}-treated goat TMJ medial cartilage layer was enriched in OA-associated transcription factors (TFs), senescence genes, and epigenetic regulators. ConclusionOur study demonstrated the unique transcriptomic signature of the TMJ compared with the knee joint, highlighting its vulnerability to OA and pain. These findings provide valuable insights into the molecular mechanisms of TMJ and offer a resource for potential therapeutic target selection for TMJ-OA treatment. HighlightsO_LISignificant enrichment of neuronal system genes and lower enrichment of innate immune system genes in temporomandibular joint. C_LIO_LIKey osteoarthritis biomarkers such as Mmp13, Postn, and Col1a1 have higher expression in temporomandibular joint, indicating a higher vulnerability to osteoarthritis development. C_LIO_LIInterleukin-1beta treatment in goat temporomandibular joint medial layer cartilage mimics natural temporomandibular joint osteoarthritis-like transcriptional changes and immune responses observed in rabbit temporomandibular joint osteoarthritis model. C_LI

ObjectiveTo characterize the relationship between OA frequency and a host of demographic characteristics, comorbidities, military service history, and physical health variables in a veteran population. MethodsWe investigated the Million Veteran Program (MVP) cohort to outline frequency of OA across six joint sites (knee, spine, hip, hand, finger, thumb) in veterans with respect to demographics (age, sex, race/ethnicity, etc.), military service data, and detailed electronic health records profiling OA and other comorbidities. The large veteran contingent provided the unique opportunity to investigate the association of OA with prior service across military branches and war eras. ResultsWe validated previous reports of sex- and age-dependent differences in OA frequency, and we identified that generalized OA was associated with a higher frequency of sixteen Deyo-Charlson comorbidities. These associations generally persisted within each isolated joint site-specific OA. Depending on military branch, prior military engagement was differentially associated with frequency of OA. Prior Army and Navy service were associated with higher and lower risk, respectively of OA across all joint sites. However, multivariable-adjusted models adjusting for a range of covariates (including age, sex, and ancestry) reversed the apparent protective effect of prior Navy service ConclusionThese findings highlight the breadth of factors associated with OA in the MVP veteran population and suggest that physical status may be a modifiable risk factor for OA. This work may contribute to designing strategies to optimize appropriate detection, intervention, treatment, and even rehabilitation strategies for OA in veterans and the general population.

ObjectivesAlthough rodent models of traumatically or chemically induced intervertebral facet joint osteoarthritis (FJOA) were previously described, the characteristics of spontaneous FJOA animal models have not been documented. This study aimed to identify the characteristics of a murine model of spontaneous FJOA and its underlying mechanisms. MethodsThe lumbar facet joints of mutant mice carrying a disrupted NFAT1 (nuclear factor of activated T cells 1) allele and of wild-type control mice were examined by histochemistry, quantitative gene expression analysis, immunohistochemistry, and histomorphometry using a novel FJOA scoring system at 2, 6, 12, and 18 months of age. The reproducibility of the FJOA scoring system was analyzed by inter-observer and intra-observer variability tests. Tissue-specific histomorphometric and gene expression changes were statistically analyzed. ResultsNFAT1-mutant facet joints displayed dysfunction of articular chondrocytes and synovial cells with aberrant gene and protein expression in cartilage and synovium as early as 2 months, followed by osteoarthritic structural changes such as articular surface fissuring and chondro-osteophyte formation at 6 months. Deeper cartilage lesions, synovitis, separation of cartilage from thickened subchondral bone, and tissue-specific molecular and cellular alterations in NFAT1-mutant facet joints became evident at 12 and 18 months. Osteoarthritic structural changes were not detected in wild-type facet joints at any ages, though age-related cartilage degeneration was observed at 18 months. ConclusionsUsing NFAT1-mutant mice, this study has identified for the first time an animal model of spontaneous FJOA with age-dependent osteoarthritic characteristics, developed the first FJOA scoring system, and elucidated the molecular mechanisms of NFAT1 mutation-mediated FJOA.

ObjectiveTo validate SERPINB2 and SERPINA9 as chondrogenic biomarker candidates across independent transcriptomic platforms and cell sources, to characterise the complete SERPIN expression landscape during kartogenin (KGN)-induced chondrogenic differentiation of human mesenchymal stem cells (hMSCs), and to identify novel SERPIN biomarker candidates and their signalling context during cartilage lineage commitment. DesignMulti-platform transcriptomic analysis across three independent datasets: (i) Affymetrix HGU133+2 microarray of KGN-induced chondrocytes versus undifferentiated hMSCs (ATCC source); (ii) Affymetrix Clariom D whole-transcriptome array of KGN-treated versus control hMSCs from an independent Mexican source (SINREG Laboratories); and (iii) previously published qPCR validation. Differential expression was computed using limma with Benjamini,Hochberg correction. SERPIN-focused cross-platform correlation and targeted pathway analysis were performed. ResultsThe Clariom D dataset yielded 1,869 differentially expressed genes (925 upregulated, 944 downregulated; FDR < 0.05) from 29,124 transcripts tested. SERPINB2 was concordantly upregulated across all three platforms (Clariom D: fold-change [FC] +3.54, FDR = 0.006; HGU133+2: log2FC = +3.29, nominal P = 0.027; qPCR confirmed), establishing it as one of the most reproducible transcriptomic signals in chondrogenic differentiation. In the direct Bone versus Cart comparison, SERPINB2 showed [~]45-fold chondrogenic enrichment (log2FC = -5.45, adjusted P < 0.0001). Cross-platform SERPIN correlation was significant (Pearson r = 0.54, P = 0.0025; n = 29 shared genes). Four additional SERPINs reached genome-wide significance on Clariom D: SERPINE2 (FC +2.57), SERPING1, SERPIND1, and SERPINE1. SERPINA9 was not replicated in the independent SINREG source, identifying it as a context-dependent marker. ConclusionsSERPINB2 is a robust, cross-platform chondrogenic biomarker with translational potential for osteoarthritis (OA) monitoring. The coordinated SERPIN programme activates a multi-layered proteolytic and signalling network during cartilage lineage commitment, positioning SERPINB2 as a functional regulator of the chondro-osteogenic lineage decision.

ObjectivesThe etiology of osteoarthritis revolves around the interplay between genetic predisposition and perturbing environmental cues, such as mechanical stress. The pericellular matrix, with its hallmark proteins collagen type VI and fibronectin, surrounds chondrocytes and is critical in transducing the biomechanical cues. The objective is to study the functional effects of an OA disease-risk mutation in COL6A3 in interaction with hyper-physiological mechanical cues in a tailored human induced pluripotent stem cells (hiPSCs) derived cartilage organoid model. MethodTo identify pathogenic OA mutations exome sequencing in symptomatic OA patients was performed. To study functional effects, CRISPR-Cas9 genome engineering was used to introduce the mutation in our established human induced pluripotent stem cell-derived in-vitro neo-cartilage organoid model in interaction with hyper-physiological mechanical loading conditions. ResultsA high-impact mutation in COL6A3 was identified that resulted in significantly lower binding between the PCM proteins COLVI and fibronectin (FN) and provoked an osteoarthritic chondrocyte state. Moreover, aberrant function of the PCM, secondary to the COL6A3 mutation, abolished the initial stress responses marked particularly by upregulation of PTGS2 encoding cyclooxygenase-2 (COX-2), after hyper-physiological mechanical loading conditions. ConclusionThese findings demonstrate that ablating the characteristic transient COX-2 response after injurious mechanical cues may have a direct negative impact on chondrocyte health. What is already knownO_LIThe etiology of osteoarthritis revolves around the interplay between genetic predisposition and perturbing environmental cues, such as mechanical stress. C_LIO_LIThe pericellular matrix, with its hallmark proteins collagen type VI and fibronectin, surrounds the chondrocytes and is critical in transducing biomechanical cues from the extracellular matrix to chondrocytes henceforth it determines the chondrocyte mechanical environment. C_LIO_LIThe mechanical environment of the chondrocytes is a critical factor that influences chondrocyte health as it determines the balance between synthesis and degradation of the articular cartilage extracellular matrix. C_LI What this study addsO_LIA sustainable human induced pluripotent stem cell-derived in-vitro neo-cartilage organoid model that is tailored to study detailed biologic effects of mechanical cues to chondrocytes. C_LIO_LIAn OA disease-risk mutation in COL6A3 reduces the binding between collagen type VI to fibronectin and provoked an osteoarthritic chondrocyte state. C_LIO_LIUpon hyper-physiological mechanical loading, aberrant function of the pericellular matrix, secondary to the COL6A3 mutation, ablates the initial transient inflammatory response, characterized particularly by PTGS2 encoding cyclooxygenase-2 (COX-2). C_LI How this study might affect research practice or policyO_LIInhibiting COX-2, as an important transient inflammatory response after hyper-physiological mechanical cues, could worsen the loss of structural integrity of the cartilage in osteoarthritis patients. Henceforth, prescription of COX-2 inhibitors as pain treatment for OA patients should be reconsidered. C_LI

ObjectivesShift work-induced circadian rhythm disruption has been identified as a risk factor for specific diseases. Additionally, physically demanding work has been linked to osteoarthritis. This study investigated the independent associations of shift work and physical work with risk of large joint osteoarthritis. DesignUK Biobank participants completed questionnaires detailing their employment status, including shift work, night shifts, heavy manual work and prolonged non-sedentary work. Responses were categorised into binary and categorical variables. Knee and hip osteoarthritis diagnoses were extracted from hospital records and osteoarthritis (any site) was self-reported. Logistic regression models, adjusted for age, sex, BMI, Townsend Deprivation Index and other work factors, were used to investigate the relationships between work characteristics and osteoarthritis outcomes. ResultsThis study included 285,947 participants (mean age 52.7 years; males 48.0%). Shift work and night shifts were associated with knee osteoarthritis (fully adjusted OR: 1.12 [95% CI:1.07-1.17] and 1.12 [1.04-1.20], respectively), and self-reported osteoarthritis but there was little evidence of an association with hip osteoarthritis (1.01 [0.95-1.08] and 1.03 [0.93-1.14]). Heavy manual work and prolonged non-sedentary work were associated with increased risk of all osteoarthritis outcomes. ConclusionsShift work showed independent associations with knee osteoarthritis and self-reported osteoarthritis but not hip osteoarthritis, suggesting circadian rhythm dysfunction may play a role in knee osteoarthritis pathogenesis. Heavy manual work and prolonged non-sedentary work were associated with all outcomes, with stronger associations in knee osteoarthritis, possibly reflecting the knees higher susceptibility to biomechanical stress. Further research is needed to explore workplace interventions for reducing these risks.

ObjectiveAs the progression of knee osteoarthritis (OA) is associated with large biomechanical loads, an optimally designed intervention is needed to prevent disease progression and symptoms. We aimed to investigate the effect of exercise therapy--the gold standard intervention--on biomechanical loads in patients with knee OA and identify its modification factors. DesignSystematic review and meta-analysis Data sourcesPubMed, PEDro, and CINAHL; from study inception to May 2021 Eligibility criteriaStudies evaluating the first peak knee adduction moment (KAM), peak knee flexion moment (KFM), maximal knee joint compression force (KCF), or co-contraction during walking before and after exercise therapy in patients with knee OA Risk of BiasPEDro scale and NIH scale. ResultsAmong 11 RCTs and nine non-RCTs, 1100 patients with knee OA were included. First peak KAM (SMD 0.11; 95% CI: -0.03-0.24), peak KFM (SMD 0.13; 95% CI: -0.03-0.29), and maximal KCF (SMD 0.09; 95% CI -0.05-0.22) tended to increase. An increased first peak KAM was significantly associated with a larger improvement in knee muscle strength and WOMAC pain. The quality of evidence regarding the biomechanical loads was low-to-moderate according to the GRADE approach. ConclusionsExercise therapy tends to increase biomechanical loads. The improvement in pain and knee muscle strength may mediate the increase in first peak KAM, suggesting difficulty in balancing symptom relief and biomechanical load reduction. Therefore, exercise therapy may satisfy both aspects simultaneously when combined with biomechanical interventions, such as a valgus knee brace or insoles. FundingGrant-in-Aid for JSPS Research Fellows, 19J23020. RegistrationPROSPERO (CRD42021230966)

Knee osteoarthritis (OA) is the most common form of OA which affects knee joints and there is currently no disease-modifying treatment available for OA. Therefore, an ideal strategy to prevent the development of OA is to identify and intervene at the modifiable risk factors for the development and progression of OA. Early-life factors such as obesity and malalignment may affect the mechanical aspect of the knee (i.e. alterations in normal knee kinematics) and could be the risk factor for the development of knee OA in later life. Identifying early-life (gestational factors, congenital defects, childhood, adolescence, early adulthood) factors which affect the development of knee OA in later stages of the life may help to develop targeted prevention programs in early-life itself to prevent the development of knee OA. Hence, this systematic review protocol provides the method to be used to comprehensively summarise the existing evidence on early life modifiable risk factors associated with the development and progression of knee OA.

Osteoarthritis (OA) is a progressive degenerative articular disease, and knee orthoses are widely used to reduce the external knee adduction moment (EKAM). However, quantitative evidence regarding the corrective moments exerted by orthoses during gait and their relationship with EKAM is scarce. This study directly measured the corrective moments generated by a knee orthosis using a six-axis force sensor and assessed the influence of orthosis structure on these moments and EKAM. Sixteen individuals with medial knee OA (Kellgren-Lawrence grade II or III) affecting the left knee performed walking assessments across three test states: without an orthosis, with an orthosis incorporating a 152-mm medial lower-leg strut, and with an orthosis incorporating a 192-mm strut. Kinematic data and ground reaction forces were used to calculate EKAM, while corrective moments were collected concurrently. The 192-mm orthosis generated greater corrective moments than the 152-mm orthosis throughout all gait phases. EKAM during the loading response declined markedly, being lowest with the 192-mm orthosis, intermediate with the 152-mm orthosis, and highest without an orthosis. An inverse association was observed between corrective moments and EKAM, indicating that higher corrective moments corresponded to lower EKAM. Although walking speed slightly decreased with greater corrective moments, stride length was unchanged. These results demonstrate that the length of the medial lower-leg strut determines the magnitude of corrective moments and is pivotal for lowering EKAM. Collectively, the results highlight the importance of optimizing orthosis design to meet the specific biomechanical requirements of patients with medial knee OA.

ObjectiveAltered mechanical loading is a known risk factor for osteoarthritis. Destabilization of the medial meniscus (DMM) is a preclinical gold standard model for post-traumatic osteoarthritis and is thought to induce instability and locally increased loading. However, the joint- and tissue-level mechanical environment underlying cartilage degeneration remains poorly documented. DesignUsing a custom multiscale modeling approach, we assessed joint and tissue biomechanics in rats undergoing sham surgery and DMM. High-fidelity experimental gait data were collected in a setup combining biplanar fluoroscopy and a ground reaction force plate. Knee poses and joint-level loading were estimated through musculoskeletal modeling, using bony landmarks, semi-automatically tracked via deep learning on fluoroscopic images, and ground reaction forces. A musculoskeletal model of the rat hindlimb was adapted to represent knee flexion-extension, valgus-varus, and internal-external rotation. The tissue-level cartilage mechanical environment was then spatially estimated, using the musculoskeletal modeling parameters as inputs into a dedicated finite element (FE) model of the rat knee, comprising cartilage and meniscal tissues. Experimental gait data and modeling workflows, including musculoskeletal models and FE meshes, are openly shared through a data repository. ResultsIn rats with DMM, the frontal plane knee pose was altered, yet there was no indication of joint-level overloading. Tissue-level mechanical cues typically linked with cartilage degeneration were not increased in the medial tibial cartilage, despite evidence of tissue structural changes. ConclusionDMM did not increase joint and tissue mechanical responses in the knee medial compartment, suggesting that mechanical loading alone does not explain the observed osteoarthritis-like structural changes.

Objectivesi) develop and use a new cryogenically-enhanced phase contrast method to visualise hyaline articular cartilage (HAC); ii) to measure HAC, articular calcified cartilage (ACC) and total articular cartilage thicknesses in male STR/Ort (osteoarthritis, OA) and CBA (healthy) mouse tibial epiphyses, reflecting divergent OA predisposition, at three age timepoints chosen to reflect pre-OA, OA onset and late-progression; iii) to compare HAC, trans-zonal and ACC 3D chondrocyte anatomy in tibial epiphyses. MethodsSTR/Ort and CBA mouse knees (n=4 per age and strain group) were synchrotron-CT scanned at high-resolution while fresh frozen, without staining, fixation, dissection or dehydration of the joint capsule. Both cartilage thickness and cellular characteristics (chondrocyte n=420) were manually measured and statistically compared (SPSS). ResultsCryo-enhanced phase contrast allowed cartilage to be seen in full thickness with cellular detail. HAC was thicker in STR/Ort than age-matched CBA mice in 16/24 knee joint compartments and timepoints (all p<0.04). In contrast, HAC was thicker only in the posterior lateral femur of CBA mice at 10weeks (p<0.001, Table 1). ACC and total cartilage were also thicker in STR/Orts. Trans-zonal chondrocytes were smaller than ACC and HAC chondrocytes (p-values<0.001, volumes 878, 1,567m3 and 1,348m3 respectively). O_TBL View this table: org.highwire.dtl.DTLVardef@191d5a9org.highwire.dtl.DTLVardef@162523borg.highwire.dtl.DTLVardef@4b7caorg.highwire.dtl.DTLVardef@1be6005org.highwire.dtl.DTLVardef@192143d_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTABLE 1:C_FLOATNO O_TABLECAPTIONCondylar compartments in which HAC thickness was measured, and the presence or absence of a significant difference between STR/Ort and CBA mouse strains. * indicates a significant difference between strains (p<0.05). Note that the posterior lateral femur at 10 weeks of age is the only region in which STR/Ort mice have significantly thinner HAC than CBA mice. C_TABLECAPTION C_TBL ConclusionsCryogenically-enhanced phase-contrast imaging allowed cellular detail to be seen in 3D as never before in HAC in this (or any other) model. Our findings challenge current understanding by associating STR/Ort OA vulnerability with regions of thick, rather than thinning-with-age, cartilage. Our data affirm an association between excessively hypertrophic chondrocytes and OA is present in STR/Ort mice.

While advanced age has long been recognized as the greatest risk factor for osteoarthritis (OA), the biological mechanisms behind this connection remain unclear. Previous work has demonstrated that chondrocytes from older cadaveric donors have elevated levels of DNA damage as compared to chondrocytes from younger donors. The purpose of this study was to determine whether a decline in DNA repair efficiency is one explanation for the accumulation of DNA damage with age, and to quantify the improvement in repair with activation of Sirtuin 6 (SIRT6). Using an acute irradiation model to bring the baseline level of all donors to the same starting point, this study demonstrates a decline in repair efficiency during aging when comparing chondrocytes from young ([&le;]45 years old), middle-aged (50-65 years old), or older (>70 years old) cadaveric donors with no known history of OA or macroscopic cartilage degradation at isolation. Activation of SIRT6 in middle-aged chondrocytes with MDL-800 (20 M) improved the repair efficiency, while inhibition with EX-527 (10 M) inhibited the rate of repair and the increased the percentage of cells that retained high levels of damage. Treating chondrocytes from older donors with MDL-800 for 48 hours significantly reduced the amount of DNA damage, despite this damage having accumulated over decades. Lastly, chondrocytes isolated from the proximal femurs of mice between 4 months and 22 months of age revealed both an increase in DNA damage with aging, and a decrease in DNA damage following MDL-800 treatment.

ObjectivesOsteoarthritis (OA) is a prevalent age related joint disease-causing chronic pain. This study investigated how nociceptive and sympathetic nerve innervation in the mouse knee joint changes with age and OA progression, and how these changes relate to pain and disease severity. MethodsThirty-eight mice were assigned to four groups: young male (My), aged male (Ma), young female (Fy), and aged female (Fa). Pain sensitivity was evaluated via Pressure Application Measurement (PAM), and joint damage was graded using OARSI scoring. CGRP and PIEZO2 expressions in dorsal root ganglia (DRG) were also assessed. We employed iDISCO tissue clearing and 3D light sheet fluorescence microscopy to visualize total (PGP9.5), nociceptive (CGRP), and sympathetic (TH) nerve fibers in anterior regions of mouse knee joints. A MATLAB-based tool quantified nerve architecture. ResultsThe Ma group displayed the highest OA severity and markedly lower mechanical withdrawal thresholds compared with My (H = 11.59, {varepsilon}{superscript 2} = 0.64), suggesting an age effect on pain-related behavior. This phenotype was accompanied by a higher total PGP9.5 nerve fiber density in the knee joint (mean difference -0.3127, 95% CI -0.4551 to -0.1704) and increased CGRP nociceptive innervation. In contrast, female mice showed no age-dependent change in PAM withdrawal thresholds, consistent with preserved cartilage integrity and stable OARSI scores, and no detectable age-related differences in PGP9.5 or CGRP innervation. The TH sympathetic fiber distribution was comparable across sexes and ages. Consistent with joint-level findings, DRG analyses demonstrated increased CGRP and PIEZO2 expression in the Ma group, whereas females exhibited no significant change. ConclusionEnhanced nociceptive but not sympathetic nerve remodeling in knee cavities is associated with increased OA severity and knee pain in the Ma group. These findings emphasize the role of peripheral sensory plasticity in OA pain and demonstrate the value of 3D imaging for visualizing neuroanatomical changes in joint disorders.

Proteomic studies have implicated clusterin as a potential biomarker of osteoarthritis (OA). However, there are two isoforms of clusterin with opposing functions, and their roles in OA have not previously been clarified. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone which prevents protein aggregation and enhances cell proliferation and viability, whereas nuclear clusterin (nCLU) acts as a pro-death signal. In this study, we focused on the role of sCLU and used established, pathophysiologically relevant, in vitro culture models to validate this potential biomarker of cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and chondrocytes was analysed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with or without pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}) and tumour necrosis factor- (TNF-). The amount of sulphated glycosaminoglycans (sGAG) released into the medium was determined by dimethylmethylene blue (DMMB) analysis. Clusterin mRNA expression was quantified by real-time PCR. MMP-3, MMP-13, COMP and sGAG released from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. Release of sCLU was attenuated with cytokine treatment in all three in vitro models. Expression of clusterin mRNA in cartilage explants and chondrocytes was down-regulated 7-days post cytokine stimulation. Cytokine stimulation attenuated expression and secretion of sCLU, therefore potentially limiting the cytoprotection which sCLU provides. These observations further implicate sCLU as having a role in OA, and diagnostic value as a potential biomarker for cartilage degradation.

Osteoarthritis (OA) is a leading cause of chronic pain and disability, for which there is no cure. Mesenchymal stromal cells (MSCs) have been used in clinical trials for treating OA due to their unique functions to send paracrine anti-inflammatory and trophic signals. Interestingly, these studies have shown mainly short-term effects of MSCs in improving pain and joint function, rather than sustained and consistent benefits. This may reflect a change or loss in the therapeutic effects of MSCs after intra-articular injection. This study aimed to unravel the reasons behind the variable efficacy of MSC injections for OA using an in vitro co-culture model. Osteoarthritic human synovial fibroblasts (OA-HSFs) exposed to MSCs showed short-term downregulation of pro-inflammatory and pro-catabolic genes, but the MSCs showed upregulation of pro-inflammatory genes and impaired ability to undergo osteogenesis and chondrogenesis in the presence of OA-HSFs. Moreover, short-term exposure of OA-HSFs to MSCs was insufficient for inducing sustained changes to their diseased behaviour. These findings suggest MSCs may not provide long-term effects in correcting the OA joint environment due to adopting the diseased phenotype of the surrounding tissues, which have important implications in the future development of effective stem cell-based OA treatments with long-term therapeutic efficacy.

ObjectiveNovel targets for osteoarthritis therapy are urgently needed, and sensory nerve fibres and their neuropeptides are increasingly recognised for their contribution to structural aspects of joint pathology. The nociceptive sensory neuropeptide alpha calcitonin gene-related peptide (CGRP) was previously detected in synovial fluid and serum of osteoarthritis patients and was also described as trophic factor for chondrocytes, affecting ECM organisation and biomechanical properties. Here, we investigated the potential of CGRP to alter the chondrocyte mechanoresponse, and thus to affect the resilience of cartilage towards mechanical loading. MethodsTissue-engineered neocartilage based on human articular chondrocytes was treated with 1{micro}M CGRP for 24 hours and subjected to an anabolic loading protocol (intermittent dynamic compression, 1Hz, 25%) for the last 3 hours before analysing its molecular mechano-response. ResultsMechanotransduction was largely unaltered by CGRP as demonstrated by ERK activation and stimulation of mechano-regulated gene expression, yet load-stimulated glycosaminoglycan synthesis was disturbed by CGRP. Presence of CGRP did not affect stimulation of WNT5A expression by loading, but decreased DKK3 expression under loading. Importantly, WNT inhibition prevented the negative effect of CGRP on load-stimulated glycosaminoglycan synthesis. ConclusionIdentifying WNT5A as a novel mechano-response gene, we reveal that CGRP can block the load-stimulated proteoglycan production of human chondrocytes in the presence of WNT pathway activity. Thus, our data propose a novel, negative role for the pain-mediator CGRP in the cartilage loading response, compromising its resilience to loading. Overall, our study implicates CGRP as a potential target for osteoarthritis treatment, but also in patient stratification.

Temporomandibular joint osteoarthritis (TMJ-OA) affects a significant proportion of the population worldwide. However, there has been no substantial progress in the development of FDA-approved drugs for treatment due to a lack of understanding of the specific factors regulating key TMJ-OA molecular mechanisms. Lysyl Oxidase Like-2 (LOXL2) promotes knee joint cartilage protection, and it is downregulated in TMJ-OA animal model. We evaluated the role of LOXL2 in TMJ cartilage, its molecular mechanism and gene networks using in vivo Loxl2 knockout mice (Acan-Cre; Loxl2flox/flox) and ex vivo goat TMJ cartilage. Our results show that Loxl2 knockout in mice cartilage upregulates Il1b, Mmp9, Mmp13, Adamts4, and Adamts5, whereas it reduces the levels of aggrecan and proteoglycan. Loxl2 deleted TMJ cartilage show a higher enrichment of inflammatory response, TNFA signaling via NF-kB, extracellular matrix (ECM), and collagen degradation pathway network. Conversely, LOXL2 treatment reduces interleukin-1 beta (IL-1{beta})-induced expression of Mmp13, protects mitochondrial function and ECM from degeneration. Importantly, LOXL2 attenuates IL-1{beta}-induced chondrocyte apoptosis via phosphorylation of NF-{kappa}B and expression of pain-related gene PTGS2 (encodes COX2). Taken together, Loxl2 knockout mice exacerbate TMJ-OA through cartilage/ECM degradation, mitochondrial dysfunction, chondrocyte apoptosis, and inflammatory gene expression, whereas LOXL2 treatment mitigates these effects. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/653519v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@cb7540org.highwire.dtl.DTLVardef@17ebe2eorg.highwire.dtl.DTLVardef@1f7fd59org.highwire.dtl.DTLVardef@19fdd7_HPS_FORMAT_FIGEXP M_FIG C_FIG