Objective The purpose of this study was to screen FDA-approved compounds for potential disease-modifying osteoarthritis drugs. Design An FDA-approved library of 2679 compounds was used for a high throughput screen (HTS) of primary human chondrocytes activated by a fibronectin fragment (FN7-10) used to model the chondrocyte OA phenotype. Inhibition of matrix metalloproteinase-13 (MMP-13) production measured using a fluorescent probe was the HTS readout. Secondary testing included dose response studies, an anabolic assay measuring chondrocyte glycosaminoglycan (GAG) production, and effects of compounds on expression and production of OA mediators by chondrocytes and synovial fibroblasts. Results 230 compounds in the HTS blocked MMP-13 production by >70% with low cytotoxicity. Mitogen-activated protein kinase kinase (MEK1/2) inhibitors were among the most enriched and potent inhibitors of MMP-13 production and the top enhancers of GAG production. The MEK1/2 inhibitor trametinib enhanced COL2A1 and ACAN expression and decreased levels of MMP-13 and IL-6 in chondrocytes isolated from OA patients. Trametinib also blocked production of pro-inflammatory cytokines and chemokines in cultures of chondrocytes and synovial fibroblasts, including CCL20, CCL7, LIF, CCL4, CXCL5, CCL5, and TNF-α. Signaling pathway analysis revealed trametinib blocked mitogen-activated protein kinase (MAPK) and c-Fos activation caused by FN7-10. Inhibition of the MAPK-activated transcription factor AP-1 was found to reduce MMP-13 and IL-6 in chondrocytes and synovial fibroblasts. A PROteolysis Targeting Chimera (PROTAC) that mediates degradation of MEK1/2 also reduced production of OA catabolic and inflammatory markers. Conclusions These results suggest MEK1/2 should be explored as a potential therapeutic target for OA disease modification.

OBJECTIVE:To investigate the effects of a matrikine, fibronectin fragment (FN7-10), on human osteoarthritic (OA) synovial fibroblasts and implications for inflammation and cartilage degradation. DESIGN:Joint tissue was obtained from 50 OA patients undergoing knee arthroplasty. Isolated synovial fibroblasts were treated with 1 µM of FN7-10 or PBS as control. Cytokine protein arrays were used to identify differentially secreted inflammatory mediators in conditioned media (CM). Bulk RNA-seq was used to evaluate transcriptional changes. Monocytic THP-1 cells in transwell assays were used to evaluate chemotactic activity of CM. Macrophage differentiation of THP-1 cells was assessed via qPCR and flow cytometry. Chondrocyte gene expression was evaluated by qPCR and glycosaminoglycan (GAG) production by Alcian blue assays. RESULTS:FN7-10 stimulation of synovial fibroblasts induced an inflammatory secretome, including production of chemokines. RNA-seq analysis confirmed FN7-10 induced upregulation of chemokines, including CCL5, CCL7, CCL8, CCL20, CXCL5 and CXCL10, which displayed mean transcriptional log-2-fold changes of 3.89 (CI, 2.81 - 4.97). CM from FN7-10-stimulated synovial fibroblasts increased THP-1 chemotaxis by 7.8-fold (CI, 3.26 - 12.35) which was reduced by CCR2 or CXCR2 inhibitors and promoted monocyte to macrophage differentiation characterized by an increase in pro-inflammatory gene expression and CD14. Chondrocytes treated with FN7-10 synovial fibroblast CM showed elevated IL6 and MMP1 and decreased ACAN and COL2A1 expression, along with reduced GAG levels. CONCLUSIONS:The matrikine FN7-10 promotes an inflammatory synovial fibroblast phenotype. Proinflammatory mediators, including chemokines, released from activated synovial fibroblasts recruit monocytes that may exacerbate joint inflammation and can also promote increased catabolic activity in chondrocytes.

DNA damage lesions can result in mutations and genome rearrangements that are associated with cellular aging and diseases. The landscape of somatic mutations in individual tissue and cell types are dictated by their unique physiological states, cellular functions, mutagenic exposures, and efficiency of DNA repair. Articular chondrocytes and skin fibroblasts are two cell types of mesodermal origin with distinct exposure to internal and external sources of DNA damage. While somatic genome instability features of skin fibroblasts have been well detailed, knowledge about mechanisms underlying genome changes in chondrocytes is scarce. Here, we took a whole-genome sequencing approach to evaluate the load, sources, and patterns of genome changes in 18 primary human chondrocyte clones from donors with and without osteoarthritis (OA). Findings in chondrocyte clones largely agreed with a recent study of 100 single-cell sequenced chondrocytes. We compared genome changes in chondrocytes with clonally-expanded human skin fibroblasts sequenced in our previous studies. We demonstrated that skin fibroblasts show a higher burden of somatic mutations, with an increased rate of mutation accumulation per cell division. Motif-centered analyses of mutation catalogues identified only endogenous sources of mutations in chondrocytes, as opposed to skin fibroblasts which also showed a heavy burden of UV-induced mutations. Spontaneous deamination of meCpG and mutagenesis by exposure to small epoxides and SN2 electrophiles showed higher mutagenic activities in chondrocytes compared to skin fibroblasts. Chondrocytes showed ubiquitous prevalence of indels in homonucleotide runs of ≥5 bases, while skin fibroblasts showed high contributions of UV-associated deletions of ≥5 bp not in repeats. Structural variants in rearrangement hotspots colocalized with human common fragile sites in skin fibroblasts, but not in chondrocytes. Together, our study comprehensively recorded genome instability features in chondrocytes and highlighted the unique mutagenesis landscapes of two mesenchymal cell types.

OBJECTIVE:We examined whether 18 months of strength training in individuals with knee varus alignment and medial tibiofemoral osteoarthritis (OA) reduced knee joint loads during walking compared to an attention control group. METHODS:This study was a secondary analysis of a randomized clinical trial that compared the effects of strength training to a control group in adults with knee OA. For this analysis, control participants had knee varus malalignment (≥2° varus; N = 49); participants in the strength training group met the varus malalignment criterion and increased their hip abductor strength by ≥20% from baseline to 18-month follow-up (N = 39). Linear regressions were used to compare means between groups at 18 months. RESULTS:The strength training group had greater increases in strength in the quadriceps (45%), hamstrings (68%), and hip abductors (42%) than the control group (16%, 11%, 4%, respectively; P < 0.05). There were no significant differences in the mean peak internal knee abduction moment or mean peak knee compressive force between groups at 18-month follow-up. The adjusted means at 18-month follow-up for the internal knee extension moment were significantly less (27%) in the strength training group (Padjusted = 0.03). CONCLUSION:Among older adults with knee OA and varus alignment, long-term lower extremity strength training results in significant increases in strength but does not significantly alter most measures of knee joint loading during walking relative to an attention control group. The results cast doubt on whether clinically meaningful improvement in lower extremity muscle strength translates to clinically important attenuation in knee joint loading.

PurposeTo investigate the longitudinal relationships between serum biomarkers of joint metabolism, knee injury, and Knee Injury and Osteoarthritis Outcome Score (KOOS) using novel methodologies.MethodsData were collected from military officers who enrolled as cadets between 2004-2009, with follow-up conducted between 2015-2017. Analyses included 234 officers who had no history of knee ligament/meniscal injury at the time of military academy matriculation, had serum biomarker measurements at matriculation and graduation, demographic data, and KOOS assessment at follow-up. Biomarkers included Collagen Type II (C2C) and Type I and II (C1,2C) collagenase-generated cleavage epitopes, C-terminal propeptide of Type II collagen (CPII), and C- and N-terminal telopeptides of type I collagen (CTX and NTX). Angle-based Joint and Individual Variation Explained (AJIVE) was used to determine demographic determinants of biomarker levels and individual modes of variation specific to biomarker levels at matriculation and graduation, stratified by sex.ResultsWe confirmed known associations of joint metabolism biomarkers with age in both sexes and with smoking in males. Matriculation biomarker data in males suggested a protective biomarker profile characterized by high cartilage synthesis and low cleavage of type I and II collagen in association with healthy KOOS scores at follow-up. CPII measured at matriculation was negatively associated with incident injuries after adjustment for smoking status (p = 0.03, logistic regression), confirming results from AJIVE.ConclusionThese exploratory analyses suggest that CPII alone, or in combination with other joint metabolism biomarkers, may help identify individual risk of knee injury.

Context:A history of anterior cruciate ligament (ACL) injury and high body mass index (BMI) are strong risk factors for incident knee osteoarthritis (KOA). Limited research has evaluated the interaction between ACL injury and high BMI on early deleterious changes in cartilage health. Objective:To determine differences in T1ρ relaxation time and serum cartilage oligomeric matrix protein (sCOMP) concentration between individuals with high and normal BMI following an ACL injury. Design:Cross-sectional study. Setting:A controlled laboratory setting. Patients or Other Participants:Forty-two participants with primary ACL injuries were assigned to either the high-BMI (>25 kg/m2, n = 20, age: 22.9 ± 5.1 years, time between injury and visit: 3.6 ± 2.2 weeks) or normal-BMI (≤25 kg/m2, n = 22, age: 21.1 ± 4.2 years, time between injury and visit: 3.3 ± 1.7 weeks) group based on their BMI. Main Outcome Measures:T1ρ relaxation time for the medial and lateral tibia (MTC and LTC) and femur for each participant, sCOMP concentrations, and the Knee Injury and Osteoarthritis Outcome Score (KOOS). Results:The high-BMI group, regardless of limbs, demonstrated greater T1ρ relaxation times in the LTC (mean difference: 2.1 ± 0.1 milliseconds, P = .004, d = 0.77) and MTC (mean difference: 1.7 ± 0.4 milliseconds, P = .04, d = 0.44) knees compared with the normal-BMI group. The high-BMI group showed greater concentrations in sCOMP compared with the normal-BMI group (mean difference: 26.4 ± 15.8 ng/mL, P = .02, d = .72). There were no differences in KOOS scores between groups. Conclusions:Overweight individuals experiencing primary ACL injuries exhibit higher T1ρ relaxation times in both their injured and uninjured limbs and sCOMP concentrations compared with normal-weight individuals with primary ACL injuries. Our findings indicate that BMI significantly impacts cartilage composition and biochemical changes associated with KOA development in individuals with ACL injuries within 3.5 weeks of injury.

Obesity amplifies osteoarthritis (OA) pain disproportionately to joint damage, creating a major unmet clinical need for non-opioid interventions that act beyond the joint. Using OA as a translational model, we integrated serum multi-omics in obese mice with surgically induced OA and genetic and adipose-reconstitution models of complement factor D (FD). In humans, we analyzed longitudinal metabolomics data from the IDEA weight-loss trial and conducted functional studies in dorsal root ganglion (DRG) neurons. Adipose-derived FD emerged as a regulator of systemic immunometabolic state: FD deficiency in obese mice worsened pain sensitivity whereas restoring circulating FD normalized pain and inflammatory markers without altering joint structure. Cross-species lipid profiling identified conserved shifts in linoleic acid versus arachidonic acid-derived lipids that were associated with pain phenotypes in mice and with pain improvement in humans. Defined lipid cocktails modulated excitability and TRPV1 sensitivity in human DRG neurons, and transcriptomics of knee-innervating DRGs revealed diet and FD-dependent activation of complement and neuronal excitability pathways. Together, these findings define an adipose-complement-lipid axis that regulates nociceptive vulnerability independent of joint damage and identify extra-articular targets for translational, non-opioid OA pain therapies. One Sentence Summary:We identify an adipose-complement-lipid axis that systemically regulates sensory neuron sensitization, providing a mechanistic basis for pain-structure discordance in obesity-associated osteoarthritis.

The combination of hydrogels and polymeric nanoparticles (NPs) offers a versatile strategy to engineer multifunctional nanocomposite systems for advanced drug delivery applications. In this work, three amphiphilic block copolymers were synthesized through controlled/living polymerizations, affording macromolecules with distinct end-chain functionalities. These copolymers self-assembled into core-shell NPs, which were subsequently embedded within a cross-linked agarose-carbomer-hyaluronic acid hydrogel via physical, chemical, or ionic interactions. The incorporation of NPs within the hydrogel matrix enabled the co-delivery of both hydrophobic and hydrophilic therapeutic cargos, confining dexamethasone (DEX) in the hydrophobic NP core and a model protein within the water-rich hydrogel network. The resulting hybrid systems exhibited tunable rheological and NP release properties, depending on the NP surface moieties and the encapsulation method. Sustained DEX release was displayed over several days, and controllable protein release was achieved according to the NP surface properties. The nanocomposite showed excellent cytocompatibility, demonstrating a relevant reduction of pro-inflammatory cytokines expression in vitro. Overall, the proposed strategy highlights the potential of polymer chemistry-driven design to tailor hydrogel-NP interactions, providing a promising platform for targeted, sustained co-delivery of therapeutics suitable for several applications.

Fetuin-A is a glycoprotein with high affinity for calcium-phosphates, with a role in cartilage and bone metabolism, and an anti-inflammatory role in injury. Studies have shown decreasing serum fetuin-A levels in patients with severe osteoarthritis (OA), and lower amounts of fetuin-A in OA sclerotic osteoblasts. Therefore, decreasing fetuin-A during OA might be responsible for increased inflammation, cartilage mineralization and subchondral bone thickness. To assess the therapeutic potential of fetuin-A in post-traumatic OA (PTOA), we used micrometric hyaluronic-acid particles (µHA) to achieve a sustained intra-articular release of fetuin-A into diseased joint knees and followed PTOA progression over time. Because OA progression may lead to muscle degeneration, we also assessed muscle strength. Shape-defined hyaluronic-acid microparticles were fabricated and associated with fetuin-A, generating a fetuin-A µHA complex (Fet-µHA). After physicochemical characterization and biocompatibility studies on chondrocytes, the release profile of fetuin-A from Fet-µHA was established. The therapeutic efficacy of Fet-µHA on PTOA was assessed using the destabilization of the medial meniscus (DMM) model. We intra-articularly injected Fet-µHA (20 mg/kg, every 3wks), empty-µHA, or saline into DMM knees of C57BL/6 J mice, following OA outcomes at early and severe PTOA (4 weeks and 12 weeks post-DMM). Outcomes included cartilage structure (ACS score, H E), matrix loss (Safranin-O score), articular cartilage (AC) thinning, osteophyte development, bone histomorphometry, synovial hyperplasia and maximal tetanic force. All group analyses were performed with ordinary two-way ANOVA (cell viability) or one-way ANOVA (in vivo studies), followed by Tukey’s post-hoc test for multiple comparisons (statistical significance at P < 0.05). The in vitro studies confirmed the biocompatibility of Fet-µHA and established a release profile up to 45 days. In vivo intra-articular administration of the Fet-µHA into DMM knees was beneficial for OA cartilage,bone damage and synovial hyperplasia. Furthermore, Fet-µHA treatment led to a significative improvement of tetanic max contraction force at the severe stage. This pre-clinical study not only opens new perspectives for the potential use of fetuin-A in OA treatment but confirms µHA as a promising drug carrier in OA.

Objective: To investigate potentially novel and modifiable mechanisms of the effects of gut microbiome composition on obesity-related osteoarthritis (OA), focusing on cross-sectional relationships between microbiota, cytokines, and lipopolysaccharide (LPS). Design: Johnston County OA Project participants (n ​= ​64) with (cases) and without (controls) OA in hands and knees, with age ≥55 years and obesity (BMI ≥30 ​kg/m2), provided samples for multiplex cytokine, LPS, and fecal microbiota analysis. Latent Dirichlet Allocation (LDA), a machine learning method to detect latent groups within data, was used to identify microbial enterotypes. LDA regression models were used to evaluate associations of enterotypes with demographics, cytokines associated with OA, and LPS. Results: We identified 5 enterotypes. Enterotypes 3, 4 (most prevalent in our sample), and 5, dominated respectively by genera Akkermansia, Bacteroides, Ruminococcus/Phascolarctobacterium, were positively associated with control status, and inversely associated with levels of at least two cytokines associated with OA in our sample. We observed no associations of enterotypes with LPS levels. Enterotype 3 was inversely associated with thrombopoietin and IL-4 levels (b [95 ​% CIs] −0.19 [−0.43, 0.05] and −0.17 [−0.42, 0.08]), enterotype 5 with osteopontin and thrombopoietin (−0.23 [−0.49, 0.03] and −0.24 [−0.51, 0.04]), and enterotype 4 was inversely associated with all 3 of these cytokines (b −0.20 to −0.35). Conclusion: Three of five identified enterotypes were inversely associated with OA status and levels of OA associated cytokines. These exploratory analyses revealed associations between the gut microbiome, cytokines, and OA outcomes, suggesting potentially cytokine-mediated mechanisms of the effects of gut composition on OA in obese individuals, and providing a basis for further investigation of the underlying causal mechanisms.

IntroductionRheumatoid arthritis (RA) primarily affects the joints but can also affect multiple organs and profoundly impacts patients’ ability to carry out daily activities, mental health, and life expectancy. Current treatments for RA are limited in terms of duration, efficacy, and adverse effects. PD-L1 is a checkpoint protein that plays important roles in immune regulation and has been implicated in the initiation and progression of multiple autoimmune diseases.MethodIn a previous study, we demonstrated that intra-articular injection with adeno-associated virus (AAV) vectors encoding wild type PD-L1 improved local inflammation in the joint in the collagen-induced arthritis (CIA) mouse model of RA. To further improve efficacy, we explored AAV-mediated delivery of the soluble PD-L1 (sPD-L1) to CIA mice.ResultAfter intra-articular injection of AAV6 vectors expressing the optimal isoform of sPD-L1 (shPD-L1), more potency was observed when compared to wild type PD-L1, with a lower dose of AAV6/shPD-L1 needed for arthritis improvement. To study the therapeutic effect of systemic expression of sPD-L1, we administered AAV8/shPD-L1 gene therapy in CIA mice via retro-orbital injection and found significant improvements in joint inflammation and paw swelling, exhibiting similar phenotypes to that in naïve mice. The levels of total immunoglobulin and anti-collagen specific antibodies were lower in AAV8/shPD-L1 treated CIA mice than those in controls. The levels of pro-inflammatory cytokines in blood were also significantly decreased in shPD-L1 treated mice. Additionally, T cell apoptosis rates in the spleen showed a 2-fold increase in treated mice. Finally, we investigated the therapeutic effect of AAV/shPD-L1 via intramuscular injection. After injection of AAV6/shPD-L1, decreased paw swelling, reduced joint inflammation, and lower levels of pro-inflammatory cytokines in blood were achieved. The therapeutic effect of shPD-L1 was dose dependent via intramuscular treatment with AAV vectors.ConclusionIn conclusion, the findings in this study suggest that intra-articular injection of AAV vectors encoding sPD-L1 results in greater therapeutic benefit on arthritis, and systemic AAV/sPD-L1 is able to block the development of inflammatory arthritis with inhibition of the systemic immune response, underlining the potential of gene therapy with systemic delivery of shPD-L1 via AAV vectors in RA.

Osteoarthritis (OA) is a highly prevalent and painful joint disease in desperate need of disease-modifying therapeutics. Decline in the activity of the Forkhead box O (FOXO) family of transcriptional regulators in articular chondrocytes may contribute to the development of OA. In a study in this issue of the JCI, Kurakazu et al. screened compounds for FOXO activators and discovered that the antihistamine cyproheptadine activated FOXO3 through inhibition of the histamine H1 receptor. Cyproheptadine modulated the activity of OA-relevant pathways and reduced the severity of joint damage and pain behavior in a mouse model of OA, thus showing potential for development as a disease-modifying OA drug.