Joint-specific regulation of homeobox D10 expression in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a systemic immune-mediated disease characterized by joint inflammation and destruction. The disease typically affects small joints in the hands and feet, later progressing to involve larger joints such as the knees, shoulders, and hips. While the reasons for these joint-specific differences are unclear, distinct epigenetic patterns associated with joint location have been reported. In this study, we evaluated the unique epigenetic landscapes of fibroblast-like synoviocytes (FLS) from hip and knee synovium in RA patients, focusing on the expression and regulation of Homeobox (HOX) transcription factors. These highly conserved genes play a critical role in embryonic development and are known to maintain distinct expression patterns in various adult tissues. We found that several HOX genes, especially HOXD10, were differentially expressed in knee FLS compared with hip FLS. Epigenetic differences in chromatin accessibility and histone marks were observed in HOXD10 promoter between knee and hip FLS. Histone modification, particularly histone acetylation, was identified as an important regulator of HOXD10 expression. To understand the mechanism of differential HOXD10 expression, we inhibited histone deacetylases (HDACs) with small molecules and siRNA. We found that HDAC1 blockade or deficiency normalized the joint-specific HOXD10 expression patterns. These observations suggest that epigenetic differences, specifically histone acetylation related to increased HDAC1 expression, play a crucial role in joint-specific HOXD10 expression. Understanding these mechanisms could provide insights into the regional aspects of RA and potentially lead to therapeutic strategies targeting specific patterns of joint involvement during the course of disease.

Multi-microdialysis analytical system to monitor acetaminophen and its pharmacokinetic interaction with A. bidentata in rat blood, forelimb extensor muscle, brain striatum, and the knee joint cavity.

Acetaminophen (APAP), or paracetamol, is one of the most widespread and commonly used non-prescription pain medication in the world, and is effective at managing wide range of pain, including headache, muscle ache, and minor arthritic pain. While the pharmacokinetics of APAP is generally understood, there is a lack of data for its transfer ratio especially into the knee. A novel multi-microdialysis model was developed to simultaneously sample from blood, forelimb extensor muscle, brain striatum, and the knee joint cavity in the same experimental subject to investigate the potential interaction between APAP and Achyranthes bidentata Blume (A. bidentata), another widely used traditional Chinese medicininal herb especially for pain in the lower extremity. Rats were pre-treated with A. bidentata extract (ABex), APAP was then administered (60 mg/kg, i.v.), dialysates then subsequently analyzed using HPLC-PDA. Our analysis demonstrated that APAP concentrations, achieved after its administration either alone or in combination with ABex (1 and 3 g/kg, q.d. gavage), could be modelled effectively with a one-compartment model. The distribution ratio (AUCorgan/AUCblood) of blood-to-muscle, blood-to-brain and blood-to-knee was 0.372 ± 0.053, 0.277 ± 0.095 and 0.191 ± 0.042, respectively after administration of APAP (60 mg/kg, i.v.). No significant difference was observed between the pharmacokinetics of APAP administered alone and in combination with ABex; and APAP concentration exceed the half maximal effective concentration (EC50) in all sampled organs for close to 3 hours with one single dose of drug administration, providing evidence for its broad-range analgesic effect.

Role of hypoxia-mediated pyroptosis in the development of extending knee joint contracture in rats.

Joint contracture is one of the common diseases clinically, and joint capsule fibrosis is considered to be one of the most important pathological changes of joint contracture. However, the underlying mechanism of joint capsule fibrosis is still controversial. The present study aims to establish an animal model of knee extending joint contracture in rats, and to investigate the role of hypoxia-mediated pyroptosis in the progression of joint contracture using this animal model. 36 male SD rats were selected, 6 of which were not immobilized and were used as control group, while 30 rats were divided into I-1 group (immobilized for 1 week following 7 weeks of free movement), I-2 group (immobilized for 2 weeks following 6 weeks of free movement), I-4 group (immobilized for 4 weeks following 4 weeks of free movement), I-6 group (immobilized for 6 weeks following 2 weeks of free movement) and I-8 group (immobilized for 8 weeks) according to different immobilizing time. The progression of joint contracture was assessed by the measurement of knee joint range of motion, collagen deposition in joint capsule was examined with Masson staining, protein expression levels of HIF-1α, NLRP3, Caspase-1, GSDMD-N, TGF-ß1, α-SMA and p-Smad3 in joint capsule were assessed using western blotting, and the morphological changes of fibroblasts were observed by transmission electron microscopy. The degree of total and arthrogenic contracture progressed from the first week and lasted until the first eight weeks after immobilization. The degree of total and arthrogenic contracture progressed rapidly in the first four weeks after immobilization and then progressed slowly. Masson staining indicated that collagen deposition in joint capsule gradually increased in the first 8 weeks following immobilization. Western blotting analysis showed that the protein levels of HIF-1α continued to increase during the first 8 weeks of immobilization, and the protein levels of pyroptosis-related proteins NLRP3, Caspase-1, GSDMD-N continued to increase in the first 4 weeks after immobilization and then decreased. The protein levels of fibrosis-related proteins TGF-ß1, p-Smad3 and α-SMA continued to increase in the first 8 weeks after immobilization. Transmission electron microscopy showed that 4 weeks of immobilization induced cell membrane rupture and cell contents overflow, which further indicated the activation of pyroptosis. Knee extending joint contracture animal model can be established by external immobilization orthosis in rats, and the activation of hypoxia-mediated pyroptosis may play a stimulating role in the process of joint capsule fibrosis and joint contracture.

Human Infrapatellar Fat Pad Mesenchymal Stem Cell-derived Extracellular Vesicles Purified by Anion Exchange Chromatography Suppress Osteoarthritis Progression in a Mouse Model.

BACKGROUND: Extracellular vesicles derived from mesenchymal stem cells (MSCs) show great promise in treating osteoarthritis (OA). However, studies from the perspective of clinical feasibility that consider an accessible cell source and a scalable preparation method for MSC-extracellular vesicles are lacking. QUESTIONS/PURPOSES: (1) Does an infrapatellar fat pad obtained from patients undergoing TKA provide a suitable source to provide MSC-extracellular vesicles purified by anion exchange chromatography? Using an in vivo mouse model for OA in the knee, (2) how does injection of the infrapatellar fat pad-derived MSC-extracellular vesicles alter gait, cartilage structure and composition, protein expression (Type II collagen, MMP13, and ADAMTS5), subchondral bone remodeling and osteophytes, and synovial inflammation? METHODS: The infrapatellar fat pad was collected from three patients (all female; 62, 74, 77 years) during TKA for infrapatellar fat pad-derived MSC culturing. Patients with infection, rheumatic arthritis, and age > 80 years were excluded. MSC-extracellular vesicles were purified by anion exchange chromatography. For the animal study, we used 30 male C57BL/6 mice aged 10 weeks, divided into six groups. MSC-extracellular vesicles were injected weekly into the joint of an OA mouse model during ACL transection (ACLT). To answer our first research question, we characterized MSCs based on their proliferative potential, differentiation capacity, and surface antigen expression, and we characterized MSC-extracellular vesicles by size, morphology, protein marker expression, and miRNA profile. To answer our second research question, we evaluated the effects of MSC-extracellular vesicles in the OA mouse model with quantitative gait analysis (mean pressure, footprint area, stride length, and propulsion time), histology (Osteoarthritis Research Society International Score based on histologic analysis [0 = normal to 24 = very severe degeneration]), immunohistochemistry staining of joint sections (protein expression of Type II collagen, MMP13, and ADAMTS5), and micro-CT of subchondral bone (BV/TV and Tb.Pf) and osteophyte formation. We also examined the mechanism of action of MSC-extracellular vesicles by immunofluorescent staining of the synovium membrane (number of M1 and M2 macrophage cells) and by analyzing their influence on the expression of inflammatory factors (relative mRNA level and protein expression of IL-1ß, IL-6, and TNF-α) in lipopolysaccharide-induced macrophages. RESULTS: Infrapatellar fat pads obtained from patients undergoing TKA provide a suitable cell source for producing MSC-extracellular vesicles, and anion exchange chromatography is applicable for isolating MSC-extracellular vesicles. Cultured MSCs were spindle-shaped, proliferative at Passage 4 (doubling time of 42.75 ± 1.35 hours), had trilineage differentiation capacity, positively expressed stem cell surface markers (CD44, CD73, CD90, and CD105), and negatively expressed hematopoietic markers (CD34 and CD45). MSC-extracellular vesicles purified by anion exchange chromatography had diameters between 30 and 200 nm and a typical cup shape, positively expressed exosomal marker proteins (CD63, CD81, CD9, Alix, and TSG101), and carried plentiful miRNA. Compared with the ACLT group, the ACLT + extracellular vesicle group showed alleviation of pain 8 weeks after the injection, indicated by increased area (0.67 ± 0.15 cm 2 versus 0.20 ± 0.03 cm 2 , -0.05 [95% confidence interval -0.09 to -0.01]; p = 0.01) and stride length (5.08 ± 0.53 cm versus 6.20 ± 0.33 cm, -1.12 [95% CI -1.86 to -0.37]; p = 0.005) and decreased propulsion time (0.22 ± 0.06 s versus 0.11 ± 0.04 s, 0.11 [95% CI 0.03 to 0.19]; p = 0.007) in the affected hindlimb. Compared with the ACLT group, the ACLT + extracellular vesicles group had lower Osteoarthritis Research Society International scores after 4 weeks (8.80 ± 2.28 versus 4.80 ± 2.28, 4.00 [95% CI 0.68 to 7.32]; p = 0.02) and 8 weeks (16.00 ± 3.16 versus 9.60 ± 2.51, 6.40 [95% CI 2.14 to 10.66]; p = 0.005). In the ACLT + extracellular vesicles group, there was more-severe OA at 8 weeks than at 4 weeks (9.60 ± 2.51 versus 4.80 ± 2.28, 4.80 [95% CI 0.82 to 8.78]; p = 0.02), indicating MSC-extracellular vesicles could only delay but not fully suppress OA progression. Compared with the ACLT group, the injection of MSC-extracellular vesicles increased Type II collagen expression, decreased MMP13 expression, and decreased ADAMTS5 expression at 4 and 8 weeks. Compared with the ACLT group, MSC-extracellular vesicle injection alleviated osteophyte formation at 8 weeks and inhibited bone loss at 4 weeks. MSC-extracellular vesicle injection suppressed inflammation; the ACLT + extracellular vesicles group had fewer M1 type macrophages than the ACLT group. Compared with lipopolysaccharide-treated cells, MSC-extracellular vesicles reduced mRNA expression and inhibited IL-1ß, IL-6, and TNF-α in cells. CONCLUSION: Using an OA mouse model, we found that infrapatellar fat pad-derived MSC-extracellular vesicles could delay OA progression via alleviating pain and suppressing cartilage degeneration, osteophyte formation, and synovial inflammation. The autologous origin of extracellular vesicles and scalable purification method make our strategy potentially viable for clinical translation. CLINICAL RELEVANCE: Infrapatellar fat pad-derived MSC-extracellular vesicles isolated by anion exchange chromatography can suppress OA progression in a mouse model. Further studies with large-animal models, larger animal groups, and subsequent clinical trials are necessary to confirm the feasibility of this technique for clinical OA treatment.

The experimental study of mir-99a-5p negative regulation of TLR8 receptor mediated-mediated innate immune response in rabbit knee cartilage injury.

BACKGROUND: Traumatic cartilage injury is an important cause of osteoarthritis (OA) and limb disability, and toll-like receptors (TLRs) mediated innate immune response has been confirmed to play a crucial role in cartilage injury. In the previous study, we found that the activation of TLR8 molecules in injured articular cartilage was more obvious than other TLRs by establishing an animal model of knee impact injury in rabbits, and the changes of TLR8 molecules could significantly affect the process of articular cartilage injury and repair. OBJECTIVE: To verify how mir-99a-5p regulates TLR8 receptor mediated innate immune response to treat traumatic cartilage injury. METHODS: The impact of a heavy object on the medial condyle of the rabbit's knee joint caused damage to the medial condylar cartilage. Through pathological and imaging analysis, it was demonstrated whether the establishment of an animal model of traumatic cartilage injury was successful. Establishing a cell model by virus transfection of chondrocytes to demonstrate the role of TLR8 in the innate immune response to impact cartilage injury. Through transcriptome sequencing, potential targets of TLR8, mir-99a-5p, were predicted, and basic experiments were conducted to demonstrate how they interact with innate immune responses to impact cartilage damage. RESULTS: TLR8 is a receptor protein of the immune system, which is widely expressed in immune cells. In our study, we found that TLR8 expression is localized in lysosomes and endosomes. Mir-99a-5p can negatively regulate TLR8 to activate PI3K-AKT molecular pathway and aggravate cartilage damage. Inhibiting TLR8 expression can effectively reduce the incidence of articular cartilage damage. CONCLUSION: Based on the results from this study, mir-99a-5p may be an effective molecular marker for predicting traumatic cartilage injury and targeting TLR8 is a novel and promising approach for the prevention or early treatment of cartilage damage.

Whole-Transcriptome Sequencing of Knee Joint Cartilage from Kashin-Beck Disease and Osteoarthritis Patients.

The aim of this study was to provide a comprehensive understanding of similarities and differences in mRNAs, lncRNAs, and circRNAs within cartilage for Kashin-Beck disease (KBD) compared to osteoarthritis (OA). We conducted a comparison of the expression profiles of mRNAs, lncRNAs, and circRNAs via whole-transcriptome sequencing in eight KBD and ten OA individuals. To facilitate functional annotation-enriched analysis for differentially expressed (DE) genes, DE lncRNAs, and DE circRNAs, we employed bioinformatic analysis utilizing Gene Ontology (GO) and KEGG. Additionally, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we validated the expression levels of four cartilage-related genes in chondrocytes. We identified a total of 43 DE mRNAs, 1451 DE lncRNAs, and 305 DE circRNAs in KBD cartilage tissue compared to OA (q value < 0.05; |log2FC| > 1). We also performed competing endogenous RNA network analysis, which identified a total of 65 lncRNA-mRNA interactions and 4714 miRNA-circRNA interactions. In particular, we observed that circRNA12218 had binding sites for three miRNAs targeting ACAN, while circRNA12487 had binding sites for seven miRNAs targeting COL2A1. Our results add a novel set of genes and non-coding RNAs that could potentially serve as candidate diagnostic biomarkers or therapeutic targets for KBD patients.

Nanoparticle-based inhibition of vascular endothelial growth factor receptors alleviates osteoarthritis pain and cartilage damage.

Osteoarthritis (OA) is characterized by cartilage damage, inflammation, and pain. Vascular endothelial growth factor receptors (VEGFRs) have been associated with OA severity, suggesting that inhibitors targeting these receptors alleviate pain (via VEGFR1) or cartilage degeneration (via VEGFR2). We have developed a nanoparticle-based formulation of pazopanib (Votrient), an FDA-approved anticancer drug that targets both VEGFR1 and VEGFR2 (Nano-PAZII). We demonstrate that a single intraarticular injection of Nano-PAZII can effectively reduce joint pain for a prolonged time without substantial side effects in two different preclinical OA rodent models involving either surgical (upon partial medial meniscectomy) or nonsurgical induction (with monoiodoacetate). The injection of Nano-PAZII blocks VEGFR1 and relieves OA pain by suppressing sensory neuronal ingrowth into the knee synovium and neuronal plasticity in the dorsal root ganglia and spinal cord. Simultaneously, the inhibition of VEGFR2 reduces cartilage degeneration. These findings provide a mechanism-based disease-modifying drug strategy that addresses both pain symptoms and cartilage loss in OA.

Specific-cytokine associations with outcomes in knee osteoarthritis subgroups: breaking down disease heterogeneity with phenotyping.

BACKGROUND: Despite existing extensive literature, a comprehensive and clinically relevant classification system for osteoarthritis (OA) has yet to be established. In this study, we aimed to further characterize four knee OA (KOA) inflammatory phenotypes (KOIP) recently proposed by our group, by identifying the inflammatory factors associated with KOA severity and progression in a phenotype-specific manner. METHODS: We performed an analysis within each of the previously defined four KOIP groups, to assess the association between KOA severity and progression and a panel of 13 cytokines evaluated in the plasma and synovial fluid of our cohort's patients. The cohort included 168 symptomatic female KOA patients with persistent joint effusion. RESULTS: Overall, our analyses showed that associations with KOA outcomes were of higher magnitude within the KOIP groups than for the overall patient series (all p-values < 1.30e-16) and that several of the cytokines showed a KOIP-specific behaviour regarding their associations with KOA outcomes. CONCLUSION: Our study adds further evidence supporting KOA as a multifaceted syndrome composed of multiple phenotypes with differing pathophysiological pathways, providing an explanation for inconsistencies between previous studies focussed on the role of cytokines in OA and the lack of translational results to date. Our findings also highlight the potential clinical benefits of accurately phenotyping KOA patients, including improved patient stratification, tailored therapies, and the discovery of novel treatments.

Knee Osteotomy Decreases Joint Inflammation Based on Synovial Histology and Synovial Fluid Analysis.

PURPOSE: To examine the biological changes in the joints of patients with knee osteoarthritis (OA) before and after around-knee osteotomy (AKO), focusing on synovial fluid (SF) and synovial pathological changes. METHODS: Patients who underwent AKO for medial compartment knee OA between 2019 and 2021 were examined. SF and synovium were obtained at the time of AKO and plate removal after bone union (mean, 16.8 months [range: 11-38 months] postoperatively). SF volume and interleukin (IL)-6 concentrations in SF were assayed using enzyme-linked immunosorbent assay. Synovitis was assessed histologically using a semiquantitative scoring system. Macrophage infiltration was assessed by immunohistochemistry using a semiquantitative score for F4/80 expression. The M1/M2 ratio was calculated using percentage of cells positive for CD80 and CD163. The expression of proinflammatory cytokines was assessed by the percentage of IL-1ß- and IL-6-positive cells. The number of vascular endothelial growth factor-positive luminal structures was counted to assess angiogenesis. The change in each parameter was compared before and after AKO using the Wilcoxon matched-pairs signed-rank test. RESULTS: Twenty-four knees of 21 patients were included. SF volume and IL-6 concentration significantly decreased postoperatively (12.6 ± 2.1 mL vs 4.2 ± 0.6 mL; P < .0001 and 50.5 ± 8.6 pg/mL vs 20.7 ± 3.8 pg/mL; P = .0001, respectively). A significant reduction in synovitis score (P = .0001), macrophage infiltration (P < .0003), M1/M2 ratio (P < .0007), angiogenesis (P < .0001), and the percentage of IL-1ß- and IL-6-positive cells in the intima (P < .008 and P < .002, respectively) was found after AKO. CONCLUSIONS: SF volume and IL-6 concentrations in the SF decreased and inflammatory synovium pathology improved after AKO. In addition to biomechanical changes, the biological environment of the joint can be improved after AKO. LEVEL OF EVIDENCE: Level IV, retrospective therapeutic case series.

Comparative Analysis of Differentially Expressed Genes in Chondrocytes from Rats Exposed to Low Selenium and T-2 Toxin.

The aim of this study was to construct rat models of environmental risk factors for Kashin-Beck disease (KBD) with low selenium and T-2 toxin levels and to screen the differentially expressed genes (DEGs) between the rat models exposed to environmental risk factors. The Se-deficient (SD) group and T-2 toxin exposure (T-2) group were constructed. Knee joint samples were stained with hematoxylin-eosin, and cartilage tissue damage was observed. Illumina high-throughput sequencing technology was used to detect the gene expression profiles of the rat models in each group. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were performed and five differential gene expression results were verified by quantitative real-time polymerase chain reaction (qRT‒PCR). A total of 124 DEGs were identified from the SD group, including 56 upregulated genes and 68 downregulated genes. A total of 135 DEGs were identified in the T-2 group, including 68 upregulated genes and 67 downregulated genes. The DEGs were significantly enriched in 4 KEGG pathways in the SD group and 9 KEGG pathways in the T-2 group. The expression levels of Dbp, Pc, Selenow, Rpl30, and Mt2A were consistent with the results of transcriptome sequencing by qRT‒PCR. The results of this study confirmed that there were some differences in DEGs between the SD group and the T-2 group and provided new evidence for further exploration of the etiology and pathogenesis of KBD.

[ 11 C]-PBR28 positron emission tomography signal as an imaging marker of joint inflammation in knee osteoarthritis.

ABSTRACT: Although inflammation is known to play a role in knee osteoarthritis (KOA), inflammation-specific imaging is not routinely performed. In this article, we evaluate the role of joint inflammation, measured using [ 11 C]-PBR28, a radioligand for the inflammatory marker 18-kDa translocator protein (TSPO), in KOA. Twenty-one KOA patients and 11 healthy controls (HC) underwent positron emission tomography/magnetic resonance imaging (PET/MRI) knee imaging with the TSPO ligand [ 11 C]-PBR28. Standardized uptake values were extracted from regions-of-interest (ROIs) semiautomatically segmented from MRI data, and compared across groups (HC, KOA) and subgroups (unilateral/bilateral KOA symptoms), across knees (most vs least painful), and against clinical variables (eg, pain and Kellgren-Lawrence [KL] grades). Overall, KOA patients demonstrated elevated [ 11 C]-PBR28 binding across all knee ROIs, compared with HC (all P 's < 0.005). Specifically, PET signal was significantly elevated in both knees in patients with bilateral KOA symptoms (both P 's < 0.01), and in the symptomatic knee ( P < 0.05), but not the asymptomatic knee ( P = 0.95) of patients with unilateral KOA symptoms. Positron emission tomography signal was higher in the most vs least painful knee ( P < 0.001), and the difference in pain ratings across knees was proportional to the difference in PET signal ( r = 0.74, P < 0.001). Kellgren-Lawrence grades neither correlated with PET signal (left knee r = 0.32, P = 0.19; right knee r = 0.18, P = 0.45) nor pain ( r = 0.39, P = 0.07). The current results support further exploration of [ 11 C]-PBR28 PET signal as an imaging marker candidate for KOA and a link between joint inflammation and osteoarthritis-related pain severity.

Extracellular Vesicles in Infrapatellar Fat Pad from Osteoarthritis Patients Impair Cartilage Metabolism and Induce Senescence.

Infrapatellar fat pad (IPFP) is closely associated with the development and progression of knee osteoarthritis (OA), but the underlying mechanism remains unclear. Here, it is find that IPFP from OA patients can secret small extracellular vesicles (sEVs) and deliver them into articular chondrocytes. Inhibition the release of endogenous osteoarthritic IPFP-sEVs by GW4869 significantly alleviated IPFP-sEVs-induced cartilage destruction. Functional assays in vitro demonstrated that IPFP-sEVs significantly promoted chondrocyte extracellular matrix (ECM) catabolism and induced cellular senescence. It is further demonstrated that IPFP-sEVs induced ECM degradation in human and mice cartilage explants and aggravated the progression of experimental OA in mice. Mechanistically, highly enriched let-7b-5p and let-7c-5p in IPFP-sEVs are essential to mediate detrimental effects by directly decreasing senescence negative regulator, lamin B receptor (LBR). Notably, intra-articular injection of antagomirs inhibiting let-7b-5p and let-7c-5p in mice increased LBR expression, suppressed chondrocyte senescence and ameliorated the progression of experimental OA model. This study uncovers the function and mechanism of the IPFP-sEVs in the progression of OA. Targeting IPFP-sEVs cargoes of let-7b-5p and let-7c-5p can provide a potential strategy for OA therapy.

AdipoRon reduces TGFß1-mediated collagen deposition in vitro and alleviates knee stiffness in vivo.

Arthrofibrosis, which causes joint motion restrictions, is a common complication following total knee arthroplasty (TKA). Key features associated with arthrofibrosis include myofibroblast activation, knee stiffness, and excessive scar tissue formation. We previously demonstrated that adiponectin levels are suppressed within the knee tissues of patients affected by arthrofibrosis and showed that AdipoRon, an adiponectin receptor agonist, exhibited anti-fibrotic properties in human mesenchymal stem cells. In this study, the therapeutic potential of AdipoRon was evaluated on TGFß1-mediated myofibroblast differentiation of primary human knee fibroblasts and in a mouse model of knee stiffness. Picrosirius red staining revealed that AdipoRon reduced TGFß1-induced collagen deposition in primary knee fibroblasts derived from patients undergoing primary TKA and revision TKA for arthrofibrosis. AdipoRon also reduced mRNA and protein levels of ACTA2, a key myofibroblast marker. RNA-seq analysis corroborated the anti-myofibrogenic effects of AdipoRon. In our knee stiffness mouse model, 6 weeks of knee immobilization, to induce a knee contracture, in conjunction with daily vehicle (DMSO) or AdipoRon (1, 5, and 25 mg/kg) via intraperitoneal injections were well tolerated based on animal behavior and weight measurements. Biomechanical testing demonstrated that passive extension angles (PEAs) of experimental knees were similar between vehicle and AdipoRon treatment groups in mice evaluated immediately following immobilization. Interestingly, relative to vehicle-treated mice, 5 mg/kg AdipoRon therapy improved the PEA of the experimental knees in mice that underwent 4 weeks of knee remobilization following the immobilization and therapy. Together, these studies revealed that AdipoRon may be an effective therapeutic modality for arthrofibrosis.

PSD95 as a New Potential Therapeutic Target of Osteoarthritis: A Study of the Identification of Hub Genes through Self-Contrast Model.

Osteoarthritis (OA) is a worldwide joint disease. However, the precise mechanism causing OA remains unclear. Our primary aim was to identify vital biomarkers associated with the mechano-inflammatory aspect of OA, providing potential diagnostic and therapeutic targets for OA. Thirty OA patients who underwent total knee arthroplasty were recruited, and cartilage samples were obtained from both the lateral tibial plateau (LTP) and medial tibial plateau (MTP). GO and KEGG enrichment analyses were performed, and the protein-protein interaction (PPI) assessment was conducted for hub genes. The effect of PSD95 inhibition on cartilage degeneration was also conducted and analyzed. A total of 1247 upregulated and 244 downregulated DEGs were identified. Significant differences were observed between MTP and LTP in mechanical stress-related genes and activated sensory neurons based on a self-contrast model of human knee OA. Cluster analysis identified DLG4 as the hub gene. Cyclic loading stress increased PSD95 (encoded by DLG4) expression in LTP cartilage, and PSD95 inhibitors could alleviate OA progression. This study suggests that inhibiting PSD95 could be a potential therapeutic strategy for preventing articular cartilage degradation.

CD10-Bound Human Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles Possess Immunomodulatory Cargo and Maintain Cartilage Homeostasis under Inflammatory Conditions.

The onset and progression of human inflammatory joint diseases are strongly associated with the activation of resident synovium/infrapatellar fat pad (IFP) pro-inflammatory and pain-transmitting signaling. We recently reported that intra-articularly injected IFP-derived mesenchymal stem/stromal cells (IFP-MSC) acquire a potent immunomodulatory phenotype and actively degrade substance P (SP) via neutral endopeptidase CD10 (neprilysin). Our hypothesis is that IFP-MSC robust immunomodulatory therapeutic effects are largely exerted via their CD10-bound small extracellular vesicles (IFP-MSC sEVs) by attenuating synoviocyte pro-inflammatory activation and articular cartilage degradation. Herein, IFP-MSC sEVs were isolated from CD10High- and CD10Low-expressing IFP-MSC cultures and their sEV miRNA cargo was assessed using multiplex methods. Functionally, we interrogated the effect of CD10High and CD10Low sEVs on stimulated by inflammatory/fibrotic cues synoviocyte monocultures and cocultures with IFP-MSC-derived chondropellets. Finally, CD10High sEVs were tested in vivo for their therapeutic capacity in an animal model of acute synovitis/fat pad fibrosis. Our results showed that CD10High and CD10Low sEVs possess distinct miRNA profiles. Reactome analysis of miRNAs highly present in sEVs showed their involvement in the regulation of six gene groups, particularly those involving the immune system. Stimulated synoviocytes exposed to IFP-MSC sEVs demonstrated significantly reduced proliferation and altered inflammation-related molecular profiles compared to control stimulated synoviocytes. Importantly, CD10High sEV treatment of stimulated chondropellets/synoviocyte cocultures indicated significant chondroprotective effects. Therapeutically, CD10High sEV treatment resulted in robust chondroprotective effects by retaining articular cartilage structure/composition and PRG4 (lubricin)-expressing cartilage cells in the animal model of acute synovitis/IFP fibrosis. Our study suggests that CD10High sEVs possess immunomodulatory miRNA attributes with strong chondroprotective/anabolic effects for articular cartilage in vivo. The results could serve as a foundation for sEV-based therapeutics for the resolution of detrimental aspects of immune-mediated inflammatory joint changes associated with conditions such as osteoarthritis (OA).

Characterization of miR-335-5p and miR-335-3p in human osteoarthritic tissues.

OBJECTIVE: We aimed to characterize the expression patterns, gene targets, and functional effects of miR-335-5p and miR-335-3p among seven primary human knee and hip osteoarthritic tissue types. METHODS: We collected synovial fluid, subchondral bone, articular cartilage, synovium, meniscus/labrum, infrapatellar/acetabular fat, anterior cruciate ligament/ligamentum teres, and vastus medialis oblique/quadratus femoris muscle (n = 7-20) from surgical patients with early- or late-stage osteoarthritis (OA) and quantified miR-335-5p and miR-335-3p expression by real-time PCR. Predicted gene targets were measured in knee OA infrapatellar fat following miRNA inhibitor transfection (n = 3), and prioritized gene targets were validated following miRNA inhibitor and mimic transfection (n = 6). Following pathway analyses, we performed Oil-Red-O staining to assess changes in total lipid content in infrapatellar fat. RESULTS: Showing a 227-fold increase in knee OA infrapatellar fat (the highest expressing tissue) versus meniscus (the lowest expressing tissue), miR-335-5p was more abundant than miR-335-3p (92-fold increase). MiR-335-5p showed higher expression across knee tissues versus hip tissues, and in late-stage versus early-stage knee OA fat. Exploring candidate genes, VCAM1 and MMP13 were identified as putative direct targets of miR-335-5p and miR-335-3p, respectively, showing downregulation with miRNA mimic transfection. Exploring candidate pathways, predicted miR-335-5p gene targets were enriched in a canonical adipogenesis network (p = 2.1e - 5). Modulation of miR-335-5p in late-stage knee OA fat showed an inverse relationship to total lipid content. CONCLUSION: Our data suggest both miR-335-5p and miR-335-3p regulate gene targets in late-stage knee OA infrapatellar fat, though miR-335-5p appears to be more prominent, with tissue-, joint-, and stage-specific effects.

An Atlas of the Knee Joint Proteins and Their Role in Osteoarthritis Defined by Literature Mining.

Osteoarthritis (OA) is the most prevalent rheumatic pathology. However, OA is not simply a process of wear and tear affecting articular cartilage but rather a disease of the entire joint. One of the most common locations of OA is the knee. Knee tissues have been studied using molecular strategies, generating a large amount of complex data. As one of the goals of the Rheumatic and Autoimmune Diseases initiative of the Human Proteome Project, we applied a text-mining strategy to publicly available literature to collect relevant information and generate a systematically organized overview of the proteins most closely related to the different knee components. To this end, the PubPular literature-mining software was employed to identify protein-topic relationships and extract the most frequently cited proteins associated with the different knee joint components and OA. The text-mining approach searched over eight million articles in PubMed up to November 2022. Proteins associated with the six most representative knee components (articular cartilage, subchondral bone, synovial membrane, synovial fluid, meniscus, and cruciate ligament) were retrieved and ranked by their relevance to the tissue and OA. Gene ontology analyses showed the biological functions of these proteins. This study provided a systematic and prioritized description of knee-component proteins most frequently cited as associated with OA. The study also explored the relationship of these proteins to OA and identified the processes most relevant to proper knee function and OA pathophysiology.

Proteomic and N-glycoproteomic analyses of total subchondral bone protein in patients with primary knee osteoarthritis.

N-glycosylation is an important post-translational modification necessary to maintain the structural and functional properties of proteins. Impaired N-glycosylation has been observed in several diseases. It is significantly modified by the state of cells and is used as a diagnostic or prognostic indicator for multiple human diseases, including cancer and osteoarthritis (OA). Aim of the study was to explore the N-glycosylation levels of subchondral bone proteins in patients with primary knee OA (KOA) and screen for potential biological markers for the diagnosis and treatment of primary KOA. A comparative analysis of total protein N-glycosylation under the cartilage was performed in medial subchondral bone (MSB, N = 5) and lateral subchondral bone (LSB, N = 5) specimens from female patients with primary KOA. To analyse the N-glycosylation sites of the proteins, non-labelled quantitative proteomic and N-glycoproteomic analyses were performed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) data. Parallel reaction monitoring (PRM) validation experiments were carried out on differential N-glycosylation sites of proteins in selected specimens, including MSB (N = 5) and LSB (N = 5), from patients with primary KOA. In total, 1149 proteins with 1369 unique N-chain glycopeptides were detected, and 1215 N-glycosylation sites were found, in which ptmRS scores for 1163 N-glycosylation sites were ≥ 0.9. In addition, N-glycosylation of the total protein in MSB compared to that in LSB was identified, in which 295 N-glycosylation sites were significantly different, including 75 upregulated and 220 downregulated N-glycosylation sites in MSB samples. Importantly, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses of proteins with differential N-glycosylation sites showed that they were primarily associated with metabolic pathways including ECM-receptor interactions, focal adhesion, protein digestion and absorption, amoebiasis, and complement and coagulation cascades. Finally, PRM experiments confirmed the N-glycosylation sites of collagen type VI, alpha 3 (COL6A3, VAVVQHAPSESVDN[+3]ASMPPVK), aggrecan core protein (ACAN, FTFQEAAN[+3]EC[+57]R, TVYVHAN[+3]QTGYPDPSSR), laminin subunit gamma-1 (LAMC1, IPAIN[+3]QTITEANEK), matrix-remodelling-associated protein 5 (MXRA5, ITLHEN[+3]R), cDNA, FLJ92775, highly similar to Homo sapiens melanoma cell adhesion molecule (MCAM), mRNA(B2R642, C[+57]VASVPSIPGLN[+3]R), and aminopeptidase fragment (Q59E93, AEFN[+3]ITLIHPK) in the array data of the top 20 N-glycosylation sites. These abnormal N-glycosylation patterns provide reliable insights for the development of diagnostic and therapeutic methods for primary KOA.

Formononetin improves the inflammatory response and bone destruction in knee joint lesions by regulating the NF-kB and MAPK signaling pathways.

Formononetin (FMN) is a phytoestrogen that belongs to the isoflavone family. It has antioxidant and anti-inflammatory effects, as well as, many other biological activities. Existing evidence has aroused interest in its ability to protect against osteoarthritis (OA) and promote bone remodeling. To date, research on this topic has not been thorough and many issues remain controversial. Therefore, the purpose of our study was to explore the protective effect of FMN against knee injury and clarify the possible molecular mechanisms. We found that FMN inhibited osteoclast formation induced by receptor activator of NF-κB ligand (RANKL). Inhibition of the phosphorylation and nuclear translocation of p65 in the NF-κB signaling pathway plays a role in this effect. Similarly, during the inflammatory response of primary knee cartilage cells activated by IL-1ß, FMN inhibited the NF-κB signaling pathway and the phosphorylation of the ERK and JNK proteins in the MAPK signaling pathway to suppress the inflammatory response. In addition, in vivo experiments showed that both low- and high-dose FMN had a clear protective effect against knee injury in the DMM (destabilization of the medial meniscus) model, and the therapeutic effect of high-dose FMN was stronger. In conclusion, these studies provide evidence of the protective effect of FMN against knee injury.

Metabolic and inflammatory profiles define phenotypes with clinical relevance in female knee osteoarthritis patients with joint effusion.

OBJECTIVES: Osteoarthritis has been the subject of abundant research in the last years with limited translation to the clinical practice, probably due to the disease's high heterogeneity. In this study, we aimed to identify different phenotypes in knee osteoarthritis (KOA) patients with joint effusion based on their metabolic and inflammatory profiles. METHODS: A non-supervised strategy based on statistical and machine learning methods was applied to 45 parameters measured on 168 female KOA patients with persistent joint effusion, consecutively recruited at our hospital after a monographic OA outpatient visit. Data comprised anthropometric and metabolic factors and a panel of systemic and local inflammatory markers. The resulting clusters were compared regarding their clinical, radiographic and ultrasound severity at baseline and their radiographic progression at two years. RESULTS: Our analyses identified four KOA inflammatory phenotypes (KOIP): a group characterized by metabolic syndrome, probably driven by body fat and obesity, and by high local and systemic inflammation (KOIP-1); a metabolically healthy phenotype with mild overall inflammation (KOIP-2); a non-metabolic phenotype with high inflammation levels (KOIP-3); and a metabolic phenotype with low inflammation and cardiovascular risk factors not associated with obesity (KOIP-4). Of interest, these groups exhibited differences regarding pain, functional disability and radiographic progression, pointing to a clinical relevance of the uncovered phenotypes. CONCLUSION: Our results support the existence of different KOA phenotypes with clinical relevance and differing pathways regarding their pathophysiology and disease evolution, which entails implications in patients' stratification, treatment tailoring and the search of novel and personalized therapies.