The alterations in nerve growth factor concentration in plasma and synovial fluid before and after total knee arthroplasty.

Total knee arthroplasty (TKA) is an effective procedure for pain relief; however, the emergence of postsurgical pain remains a concern. In this study, we investigated the production of nerve growth factor (NGF) and mediators that affect NGF production and their function in the synovial fluid and plasma after TKA. This study included 19 patients (20 knees) who had rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and knee osteoarthritis (OA) who underwent TKA, categorized into OA and non-OA groups. The levels of NGF, inflammatory cytokines, and lipid mediators were analyzed before and after surgery. The intraoperative synovial fluid NGF concentration was more than seven times higher in the non-OA group than in the OA group. The intra-articular NGF levels increased significantly by more than threefold postoperatively in the OA group but not in the non-OA group. Moreover, the levels of inflammatory cytokines and lipid mediators were increased in the synovial fluid of both groups. The intra-articular cytokines or NGF concentrations positively correlated with postoperative pain. Targeted NGF control has the potential to alleviate postsurgical pain in TKA, especially in patients with OA, emphasizing the importance of understanding NGF dynamics under different knee conditions.

Analysis of the Level of Adiponectin and Selected Cytokines in Patients with Knee Osteoarthritis.

Background and Objectives: Knee osteoarthritis (KOA) is a degenerative disease that is continuously targeting people of different ages, but especially the elderly population, the number of which tends to increase continuously at the global level. Apart from age, excess weight can influence the evolution of the disease, with obesity being associated with a weak inflammation stage and an imbalance between pro-inflammatory and anti-inflammatory cytokines. The present work aimed to analyze specific biomarkers, namely ACRP-30, IL-10, TNF-α, and IL-6, in knee synovial fluid, and correlate them with KOA patients' clinical data, radiographic changes, and functional and pain scores. Materials and Methods: 24 subjects with KOA and over 50 years of age participate in the present study. Synovial fluid was harvested using ultrasound guidance from the target knees of the enrolled KOA patients, and the levels of ACRP-30, IL-10, TNF-α, and IL-6 were measured using enzyme-linked immunosorbent assays (ELISA). All patients underwent a supine X-ray at the target knee and were classified using Kellgren-Lawrence (K-L) grading. The Western Ontario and McMaster University Osteoarthritis Index (WOMAC) was used to assess self-reported physical function, pain, and stiffness. Results: The obtained results highlighted a significant correlation between age and adiponectin level (p = 0.0451, r = -0.412). Also, the IL-10 values are lower in cases where the intensity of the pain is more pronounced (p = 0.0405, r = -0.421). In addition, analyzing the data by gender, it was observed that in the case of males, stiffness is more related to age (p = 0.0079, r = 0.7993), compared to women (p = 0.0203, r = 0.6223). In the case of women, the progression of the disease tends to increase more intensively the WOMAC score's total values (p = 0.00031, r = 0.8342), compared with men (p = 0.0289, r = 7013). Regarding interleukins and BMI, significant correlations were observed only in the case of men. Conclusions: A significant correlation between age and adiponectin, and adiponectin and IL-6, suggests that advanced age may contribute to adiponectin reduction. Comparing men with women, it was observed that men's age is more related to rigidity, and IL-6 and IL-10 are directly correlated to BMI; in addition, women seem to be more sensitive to pain and stiffness.

Intraoperative calprotectin lateral flow immunoassay can assist decision-making between one- and two-stage revision total hip arthroplasty for patients with suspected periprosthetic joint infection.

Aims: Accurate diagnosis of chronic periprosthetic joint infection (PJI) presents a significant challenge for hip surgeons. Preoperative diagnosis is not always easy to establish, making the intraoperative decision-making process crucial in deciding between one- and two-stage revision total hip arthroplasty (THA). Calprotectin is a promising point-of-care novel biomarker that has displayed high accuracy in detecting PJI. We aimed to evaluate the utility of intraoperative calprotectin lateral flow immunoassay (LFI) in THA patients with suspected chronic PJI. Methods: The study included 48 THAs in 48 patients with a clinical suspicion of PJI, but who did not meet European Bone and Joint Infection Society (EBJIS) PJI criteria preoperatively, out of 105 patients undergoing revision THA at our institution for possible PJI between November 2020 and December 2022. Intraoperatively, synovial fluid calprotectin was measured with LFI. Cases with calprotectin levels ≥ 50 mg/l were considered infected and treated with two-stage revision THA; in negative cases, one-stage revision was performed. At least five tissue cultures were obtained; the implants removed were sent for sonication. Results: Calprotectin was positive (≥ 50 mg/l) in 27 cases; out of these, 25 had positive tissue cultures and/or sonication. Calprotectin was negative in 21 cases. There was one false negative case, which had positive tissue cultures. Calprotectin showed an area under the curve of 0.917, sensitivity of 96.2%, specificity of 90.9%, positive predictive value of 92.6%, negative predictive value of 95.2%, positive likelihood ratio of 10.6, and negative likelihood ratio of 0.04. Overall, 45/48 patients were correctly diagnosed and treated by our algorithm, which included intraoperative calprotectin measurement. This yielded a 93.8% concordance with postoperatively assessed EBJIS criteria. Conclusion: Calprotectin can be a valuable tool in facilitating the intraoperative decision-making process for cases in which chronic PJI is suspected and diagnosis cannot be established preoperatively.

Fibroblast expression of neurotransmitter receptor HTR2A associates with inflammation in rheumatoid arthritis joint.

The study of neuroimmune crosstalk and the involvement of neurotransmitters in inflammation and bone health has illustrated their significance in joint-related conditions. One important mode of cell-to-cell communication in the synovial fluid (SF) is through extracellular vesicles (EVs) carrying microRNAs (miRNAs). The role of neurotransmitter receptors in the pathogenesis of inflammatory joint diseases, and whether there are specific miRNAs regulating differentially expressed HTR2A, contributing to the inflammatory processes and bone metabolism is unclear. Expression of neurotransmitter receptors and their correlated inflammatory molecules were identified in rheumatoid arthritis (RA) and osteoarthritis (OA) synovium from a scRNA-seq dataset. Immunohistochemistry staining of synovial tissue (ST) from RA and OA patients was performed for validation. Expression of miRNAs targeting HTR2A carried by SF EVs was screened in low- and high-grade inflammation RA from a public dataset and validated by qPCR. HTR2A reduction by target miRNAs was verified by miRNAs mimics transfection into RA fibroblasts. HTR2A was found to be highly expressed in fibroblasts derived from RA synovial tissue. Its expression showed a positive correlation with the degree of inflammation observed. 5 miRNAs targeting HTR2A were decreased in RA SF EVs compared to OA, three of which, miR-214-3p, miR-3120-5p and miR-615-3p, mainly derived from monocytes in the SF, were validated as regulators of HTR2A expression. The findings suggest that fibroblast HTR2A may play a contributory role in inflammation and the pathogenesis of RA. Additionally, targeting miRNAs that act upon HTR2A could present novel therapeutic strategies for alleviating inflammation in RA.

Self-actuating inflammation responsive hydrogel microsphere formulation for controlled drug release in rheumatoid arthritis (RA): Animal trials and study in human fibroblast like synoviocytes (hFLS) of RA patients.

Patients with rheumatoid arthritis (RA) often have one or more painfuljoints despite adequate medicine. Local drug delivery to the synovial cavity bids for high drug concentration with minimal systemic adverse effects. However, anti-RA drugs show short half-lives in inflamed joints after intra-articular delivery. To improve the therapeutic efficacy, it is essential to ensure that a drug is only released from the formulation when it is needed. In this work, we developed an intelligent "Self-actuating" drug delivery system where Disease-modifying anti-rheumatic Drug (DMARD) methotrexate is incorporated within a matrix intended to be injected directly into joints. This formulation has the property to sense the need and release medication only when joints are inflamed in response to inflammatory enzyme Matrix metalloproteinases (MMP). These enzymes are important proteases in RA pathology, and several MMP are present in augmented levels in synovial fluid and tissues. A high level of MMP present in synovial tissues of RA patients would facilitate the release of drugs in response and ascertain controlled drug release. The formulation is designed to be stable within the joint environment, but to dis-assemble in response to inflammation. The synthesized enzyme-responsive methotrexate (Mtx) encapsulated micron-sized polymer-lipid hybrid hydrogel microspheres (Mtx-PLHM) was physiochemically characterized and tested in synovial fluid, Human Fibroblast like synoviocytes (h-FLS) (derived from RA patients) and a rat arthritic animal model. Mtx-PLHM can self-actuate and augment the release of Mtx drug upon contact with either exogenously added MMP or endogenous MMP present in the synovial fluid of patients with RA. The drug release from the prepared formulation is significantly amplified to several folds in the presence of MMP-2 and MMP-9 enzymes. In the rat arthritic model, Mtx-PLHM showed promising therapeutic results with the significant alleviation of RA symptoms through decrease in joint inflammation, swelling, bone erosion, and joint damage examined by X-ray analysis, histopathology and immune-histology. This drug delivery system would be nontoxic as it releases more drug only during the period of exacerbation of inflammation. This will simultaneously protect patients from unwanted side effects when the disease is inactive and lower the need for repeated joint injections.

Lactic Acid-Induced Colloidal Microrheology of Synovial Fluids.

The presence of colloidal scaffolds composed of proteins and hyaluronic acid engenders unique viscous and elastic properties to the synovial fluid (SF). While the elastic resistance of SF due to the presence of such nanoscale structures provides the load-bearing capacity, the viscous nature enables fluidity of the joints during the movements to minimize the wear and tear of the adjacent muscle, cartilage, or bone tissues. It is well-known that the hypoxic conditions at the bone joints often increase the lactic acid (LA) concentration due to the occurrence of excess anaerobic respiration during either hyperactivity or arthritic conditions. The present study uncovers that in such a scenario, beyond a critical loading of LA, the colloidal nanoscaffolds of SF break down to precipitate higher molecular weight (MW) proteins and hyaluronic acid (HA). Subsequently, the viscosity and elasticity of SF reduce drastically to manifest a fluid that has reduced load bearing and wear and tear resistance capacity. Interestingly, the study also suggests that a heathy SF is a viscoelastic fluid with a mild Hookean elasticity and non-Newtonian fluidity, which eventually transforms into a viscous watery liquid in the presence of a higher loading of LA. We employ this knowledge to biosynthesize an artificial SF that emulates the characteristics of the real one. Remarkably, the spatiotemporal microscopic images uncover that even for the artificial SF, a dynamic cross-linking of the high MW proteins and HA takes place before precipitating out of the same from the artificial SF matrix, emulating the real one. Control experiments suggest that this phenomenon is absent in the case when LA is mixed with either pure HA or proteins. The experiments unfold the specific role of LA in the destruction of colloidal nanoscaffolds of synovia, which is an extremely important requirement for the biosynthesis and translation of artificial synovial fluid.

The Relationship Between Intra-articular Fracture Energy and a Patient's Inflammatory Response.

OBJECTIVES: Prior studies have demonstrated elevated inflammatory cytokine concentrations in the synovial fluid of articular fracture patients postinjury. Similarly, CT-based fracture energy measurements have been correlated with posttraumatic osteoarthritis risk after pilon fracture. The purpose of this study was to determine the associations between synovial fluid cytokine levels, fracture energy, and overall trauma to the body in articular fracture patients. METHODS: Acute tibial plateau, tibial plafond, and rotational ankle fracture patients were prospectively enrolled from December 2011 through January 1, 2019. Synovial fluid concentrations of interleukin-1 beta, interleukin-1 receptor antagonist, IL-6, IL-8, IL-10, matrix metallopeptidase-1, MMP-3, and MMP-13 were quantified. Patient CT scans were used to calculate fracture energy. The Injury Severity Score (ISS) was used to relate cytokine levels to whole-body injury severity. Spearman rho correlation coefficients were calculated to assess the relationship between injury severity metrics and synovial fluid cytokine, chemokine, and matrix metallopeptidase concentrations. RESULTS: Eighty-seven patients were enrolled with 42 had a tibial plateau fractures (OTA/AO 41B1-2, 41B2-14, 41B3-3, 41C1-3, 41C2-4, 41C3-16), 24 patients had a tibial plafond fracture (OTA/AO 43B1-2, 43B2-4, 43B3-5, 43C1-2, 43C2-3, 43C3-8), and 21 had a rotational ankle fracture (OTA/AO 44B1-3, 44B2-3, 44B3-6, 44C1-4, 44C2-5). Fracture energy significantly differed between fracture patterns, with ankle fractures involving substantially less fracture energy (median = 2.92 J) than plafond (10.85 J, P < 0.001) and plateau fractures (13.05 J, P < 0.001). After adjustment for multiple comparisons, MMP-3 was significantly correlated with transformed fracture energy (r = 0.41, 95% confidence interval [CI], 0.22-0.58, P < 0.001), while IL-1ß was significantly correlated with the Injury Severity Score (Spearman ρ = 0.31, 95% CI, 0.08-0.49, P = 0.004). CONCLUSIONS: Synovial fluid MMP-3 concentration was significantly correlated with CT-quantified fracture energy in intra-articular fracture patients. Given that in clinical practice fracture energy tends to correlate with posttraumatic osteoarthritis risk, MMP-3 may warrant further investigation for its role in posttraumatic osteoarthritis development after articular fracture. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Endoplasmic reticulum stress-related protein GRP78 and CHOP levels in synovial fluid correlate with disease progression of primary knee osteoarthritis: A cross-sectional study.

BACKGROUND: Endoplasmic reticulum (ER) stress has been shown to play an important role in osteoarthritis (OA). OBJECTIVE: This study was aimed at assessing the relationship of endoplasmic reticulum (ER) stress-related glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) concentrations in the serum/synovial fluid (SF) with disease severity of primary knee osteoarthritis (pkOA). METHODS: Patients with pkOA together with healthy individuals were consecutively recruited from our hospital. The levels of GRP78 and CHOP in serum / SF were detected using enzyme-linked immunosorbent assay. The levels of IL-6 and MMP-3 were also examined. Radiographic progression of pkOA was evaluated based on Kellgren-Lawrence (K-L) grades. Receiver Operating Characteristic (ROC) curves were used to assess the diagnostic value of GRP78/CHOP levels with regard to K-L grades. The assessment of clinical severity was conducted using the visual analogue scale (VAS), Oxford knee score (OKS), and Lequesne algofunctional index (LAI). RESULTS: A total of 140 pkOA patients and 140 healthy individuals were included. Serum GRP78 and CHOP levels in pkOA patients were not significantly different from those in healthy individuals. The SF GRP78 and CHOP levels in healthy controls were not detected due to ethical reasons. Compared to those with K-L grade 2 and 3, the pkOA patients with K-L grade 4 had higher GRP78 and CHOP levels in the SF with statistical significance. In addition, the pkOA patients with K-L grade 3 exhibited drastically upregulated GRP78 and CHOP concentrations in the SF compared to those with K-L grade 2. Positive correlations of GRP78 and CHOP levels with K-L grades, IL-6, and MMP-3 levels in the SF were observed. ROC curve analysis indicated that both GRP78 and CHOP levels may act as decent indicators with regard to OA. GRP78 and CHOP concentrations in the SF were positively correlated with VAS/LAI score and negatively associated with OKS score. CONCLUSION: The study indicated that GRP78 and CHOP levels in the SF but not the serum were positively correlated with disease severity of pkOA.

Metabolic profiling of synovial fluid in human temporomandibular joint osteoarthritis.

Introduction: Temporomandibular joint (TMJ) osteoarthritis (OA) is a common TMJ degenerative disease with an unclear mechanism. Synovial fluid (SF), an important component of TMJ, contains various proteins and metabolites that may directly contribute to OA. The present study aimed to investigate the influence of SF in TMJOA at the metabolite level. Methods: Untargeted and widely targeted metabolic profiling were employed to identify metabolic changes in SF of 90 patients with different TMJOA grades according to TMJ magnetic resonance imaging. Results: A total 1498 metabolites were detected. Most of the metabolites were amino acids and associated metabolites, benzene and substituted derivatives, and lipids. Among patients with mild, moderate and severe TMJOA, 164 gradually increasing and 176 gradually decreasing metabolites were identified, indicating that biosynthesis of cofactors, choline metabolism, mineral absorption and selenocompound metabolism are closely related to TMJOA grade. Combined metabolomics and clinical examination revealed 37 upregulated metabolites and 16 downregulated metabolites in patients with pain, of which 19 and 26 metabolites were positively and negatively correlated, respectively, with maximum interincisal opening. A model was constructed to diagnose TMJOA grade and nine biomarkers were identified. The identified metabolites are key to exploring the mechanism of TMJOA. Discussion: In the present study, a metabolic profile was constructed and assessed using a much larger number of human SF samples from patients with TMJOA, and a model was established to contribute to the diagnosis of TMJOA grade. The findings expand our knowledge of metabolites in human SF of TMJOA patients, and provide an important basis for further research on the pathogenesis and treatment of TMJOA.

The acute phase reactant orosomucoid-2 directly promotes rheumatoid inflammation.

Acute phase proteins involved in chronic inflammatory diseases have not been systematically analyzed. Here, global proteome profiling of serum and urine revealed that orosomucoid-2 (ORM2), an acute phase reactant, was differentially expressed in rheumatoid arthritis (RA) patients and showed the highest fold change. Therefore, we questioned the extent to which ORM2, which is produced mainly in the liver, actively participates in rheumatoid inflammation. Surprisingly, ORM2 expression was upregulated in the synovial fluids and synovial membranes of RA patients. The major cell types producing ORM2 were synovial macrophages and fibroblast-like synoviocytes (FLSs) from RA patients. Recombinant ORM2 robustly increased IL-6, TNF-α, CXCL8 (IL-8), and CCL2 production by RA macrophages and FLSs via the NF-κB and p38 MAPK pathways. Interestingly, glycophorin C, a membrane protein for determining erythrocyte shape, was the receptor for ORM2. Intra-articular injection of ORM2 increased the severity of arthritis in mice and accelerated the infiltration of macrophages into the affected joints. Moreover, circulating ORM2 levels correlated with RA activity and radiographic progression. In conclusion, the acute phase protein ORM2 can directly increase the production of proinflammatory mediators and promote chronic arthritis in mice, suggesting that ORM2 could be a new therapeutic target for RA.

Extracellular microRNAs induce dendritic cell-dependent joint inflammation and potentiate osteoclast differentiation via TLR7/8 engagement.

OBJECTIVES: Monocyte-derived dendritic cells (DCs) are key players in the induction of inflammation, autoreactive T cell activation and loss of tolerance in rheumatoid arthritis (RA), but the precise mechanisms underlying their activation remain elusive. Here, we hypothesized that extracellular microRNAs released in RA synovial fluids may represent a novel, physiological stimulus triggering unwanted immune response via TLR8-expressing DC stimulation. METHODS: Human monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors. RESULTS: Ex-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts. CONCLUSIONS: This work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.

LncRNA expression profiling in exosomes derived from synovial fluid of patients with rheumatoid arthritis.

OBJECTIVE: To identify the long non-coding RNA (lncRNA) expression profiling in exosomes derived from synovial fluid of rheumatoid arthritis (RA) patients, and carry out bioinformatics analysis on target genes of differentially expressed lncRNAs. METHODS: Exosomes were isolated from synovial fluid via ultracentrifugation. RNAs were extracted from exosomes by using HiPure Liquid RNA/miRNA kits, followed by lncRNA sequencing. Differentially expressed lncRNAs in RA were screened, and bioinformatics analysis of their target genes was carried out. qRT-PCR was used to verify the lncRNA expression levels. RESULTS: Compared with osteoarthritis (OA), 347 lncRNAs were found differentially expressed in RA. Compared with gout, 805 lncRNAs were found differentially expressed in RA. Compared with both OA and gout, 85 lncRNAs were found specially expressed in RA (65 were upregulated (including ENST00000433825.1)). Functional analysis of target genes of the specially expressed lncRNAs revealed significant enrichment of "autophagy" and "mTOR signaling pathway". The qRT-PCR results indicated that ENST00000433825.1 was highly expressed in RA, compared with both OA and gout (P < 0.05), which matched the lncRNA sequencing results. Correlation analysis showed that the level of ENST00000433825.1 in RA patients was significantly and positively correlated with the level of C-reactive protein (CRP) (P < 0.001). CONCLUSIONS: The lncRNA expression profiling in exosomes derived from synovial fluid of RA was significantly different from OA and gout. ENST00000433825.1 was highly and uniquely expressed in RA and significantly and positively correlated with CRP, which might provide a diagnostic and therapeutic biomarker for RA.

Soluble signal inhibitory receptor on leukocytes-1 reflects disease activity and assists diagnosis of patients with rheumatoid arthritis.

BACKGROUND: SIRL-1, an immunosuppressive receptor encoded by the VSTM1 gene, has recently been linked to rheumatoid arthritis (RA) due to its association with activated polymorphonuclear neutrophils (PMNs). Considering that the activated PMNs play a crucial role in the pathogenesis of rheumatoid arthritis (RA), we aimed to measure the levels of soluble SIRL-1, investigating whether they add value to RA in the clinical diagnosis. METHODS: Utilizing an enzyme-linked immunosorbent assay, the concentration of sSIRL-1 was measured in serum samples from cohort 1 diagnosed with RA (n = 96), gout (n = 54), osteoarthritis (n = 47), healthy controls (n = 86) and synovial fluid samples from OA (n = 8) and RA (n = 8) patients, respectively. Additionally, an external validation in cohort 2 (n = 156) comprising various inflammatory diseases was employed. RESULTS: The study revealed a distinctive upregulation of sSIRL-1 in the serum of RA compared to HC and other arthralgia diseases (p < 0.0001), which also displayed a significant elevation in synovial fluid from RA compared to OA (p < 0.05). Notably, sSIRL-1 levels exhibited a significant decrease in patients who achieved disease remission (p < 0.05). Furthermore, the diagnostic accuracy of RA was enhanced when sSIRL-1 was combined with anti-CCP and RF, yielding an impressive AUC value of 0.950. CONCLUSION: The expression pattern of sSIRL-1 in RA, coupled with its correlation with disease activity, underscores its potential clinical utility for both diagnosis and disease monitoring in RA patients. This study offers valuable insights into the evolving diagnostic landscape of RA.

Synovia tissue-specific exosomes participate in the dual variation of the osteoarthritis microenvironment via miR-182.

Osteoarthritis (OA) is the most common type of joint disease and the leading cause of chronic disability among older adults. As an important component of the joint, synovium influences the inflammatory and degenerative process of OA. This study found that miRNA 182 (miR-182) in synovium-specific exosomes can modulate inflammation and apoptotic signaling. It also regulated different biological functions to promote the progression of OA. Experiments based on rat OA model and synovium samples from OA patients, we found that synovium-derived miR-182 regulates inflammatory response in the early stage of OA by regulating the expression level of forkhead box O-3 (FOXO3). However, the expression of miR-182 was significantly increased in synovial tissue of advanced OA, which was involved in the apoptotic signal of severe OA. These findings suggest that miR-182 may directly regulate OA progression by modulating FOXO3 production inflammation, and apoptosis.

Comparative study of 1H-NMR metabolomic profile of canine synovial fluid in patients affected by four progressive stages of spontaneous osteoarthritis.

The study aimed to assess the metabolomic profile of the synovial fluid (SF) of dogs affected by spontaneous osteoarthritis (OA) and compare any differences based on disease progression. Sixty client-owned dogs affected by spontaneous OA underwent clinical, radiographic, and cytologic evaluations to confirm the diagnosis. The affected joints were divided into four study groups based on the Kallgreen-Lawrence classification: OA1 (mild), OA2 (moderate), OA3 (severe), and OA4 (extremely severe/deforming). The osteoarthritic joint's SF was subjected to cytologic examination and 1H-NMR analysis. The metabolomic profiles of the study groups' SF samples were statistically compared using one-way ANOVA. Sixty osteoarthritic joints (45 stifles, 10 shoulders and 5 elbows) were included in the study. Fourteen, 28, and 18 joints were included in the OA1, OA2, and OA3 groups, respectively (0 joints in the OA4 group). Metabolomic analysis identified 48 metabolites, five of which were significantly different between study groups: Mannose and betaine were elevated in the OA1 group compared with the OA2 group, and the 2-hydroxyisobutyrate concentration decreased with OA progression; in contrast, isoleucine was less concentrated in mild vs. moderate OA, and lactate increased in severe OA. This study identified different 1H-NMR metabolomic profiles of canine SF in patients with progressive degrees of spontaneous OA, suggesting 1H-NMR metabolomic analysis as a potential alternative method for monitoring OA progression. In addition, the results suggest the therapeutic potentials of the metabolomic pathways that involve mannose, betaine, 2-hydroxyisobutyrate, isoleucine, and lactate.

Does Chronic Pancreatitis in Growing Pigs Lead to Articular Cartilage Degradation and Alterations in Subchondral Bone?

Chronic pancreatitis (CP), a progressive inflammatory disease, poses diagnostic challenges due to its initially asymptomatic nature. While CP's impact on exocrine and endocrine functions is well-recognized, its potential influence on other body systems, particularly in young individuals, remains underexplored. This study investigates the hypothesis that CP in growing pigs leads to alterations in articular cartilage and subchondral bone, potentially contributing to osteoarthritis (OA) development. Utilizing a pig model of cerulein-induced CP, we examined the structural and compositional changes in subchondral bone, articular cartilage, and synovial fluid. Histological analyses, including Picrosirius Red and Safranin-O staining, were employed alongside immuno-histochemistry and Western blotting techniques. Our findings reveal significant changes in the subchondral bone, including reduced bone volume and alterations in collagen fiber composition. Articular cartilage in CP pigs exhibited decreased proteoglycan content and alterations in key proteins such as MMP-13 and TGF-ß1, indicative of early cartilage degradation. These changes suggest a link between CP and musculoskeletal alterations, underscoring the need for further research into CP's systemic effects. Our study provides foundational insights into the relationship between CP and skeletal health, potentially guiding future pediatric healthcare strategies for early CP diagnosis and management.

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.

Patients with higher numbers of allergies have lower levels of anti-inflammatory cytokines and increased pain at the time of ACL reconstruction.

BACKGROUND: Synovial fluid biomarkers are well studied indicators of inflammation and healing in the setting of orthopedic injuries. However, it has not been studied if patients with one or more allergies have a difference in the concentrations of synovial fluid inflammatory cytokines compared to patients without allergies. The purpose of the current study is to analyze the concentration of 10 pro- and anti-inflammatory cytokines in the synovial fluid of isolated ACL injury patients with and without at least one allergy. STUDY DESIGN: Retrospective Case-Control. METHODS: A database of patients who underwent surgery for isolated ACL injury between September 2011 and July 2023 was analyzed. All patients had SF aspirated from the operative knee prior to the surgical incision and the concentrations of pre- and anti-inflammatory biomarkers were quantified. From this cohort, 24 patients were identified to have allergies by chart review. These patients were matched 1:1 to 24 patients without allergies based on age and sex. RESULTS: There were no significant differences between the allergy and no allergy cohorts with respect to age (28.5 ± 10.3 vs. 29.5 ± 8.9, p = 0.76) and sex (70.8 % female vs. 70.8 % female, p = 1.00). The allergy cohort had a decreased concentration of TIMP-1 (492.41 ± 616.20 ng/mL vs. 1041.48 ± 942.04 ng/mL, p = 0.03) and IL-1Ra (101.70 ± 93.37 pg/mL vs. 359.94 ± 399.77 pg/mL, p = 0.01) compared to patients without allergies. A linear regression analysis found a significant association between increasing number of patients reported allergies and decreasing concentration of TIMP-1 (ß = -231.59, p = 0.03) and IL-1Ra (ß = -71.69p = 0.03) concentrations when controlling for age and sex. Finally, the allergy cohort was found to have a significantly higher value for the VAS pain scale at the time of surgery (26.84 ± 24.73 vs. 7.37 ± 10.98, p < 0.01) compared to those without an allergy. CONCLUSION: Patients undergoing ACL reconstruction with at least one allergy were found to have decreased concentrations of the anti-inflammatory cytokines TIMP-1 and IL-1Ra in their synovial fluid compared to those without allergies on the day of surgery. Furthermore, an increase in total number of allergies was found to be an associated with a decrease in TIMP-1 and IL-1Ra levels. Finally, the allergy cohort also had a higher value for the VAS pain scale at the time of surgery, implicating the role of a patient's innate immune system to their biologic and symptomatic response to injury.

Association of synovial fluid and urinary C2C-HUSA levels with surgical outcomes post-total knee arthroplasty.

OBJECTIVES: After total knee arthroplasty (TKA), ∼30% of knee osteoarthritis (KOA) patients show little symptomatic improvement. Earlier studies have correlated urinary (u) type 2 collagen C terminal cleavage peptide assay (C2C-HUSA), which detects a fragment of cartilage collagen breakdown, with KOA progression. This study determines whether C2C levels in urine, synovial fluid, or their ratio, are associated with post-surgical outcomes. METHODS: From a large sample of 489 subjects, diagnosed with primary KOA undergoing TKA, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain and function scores were collected at baseline (time of surgery) and one-year post-TKA. Baseline urine (u) and synovial fluid (sf) were analysed using the IBEX-C2C-HUSA assay, with higher values indicating higher amounts of cartilage degradation. For urine, results were normalised to creatinine. Furthermore, subjects' changes in WOMAC scores were categorised based on percent reduction in pain or improvement in function, compared to baseline, such that >66.7%, >33.3 to ≤66.7%, and ≤33.3% denoted "strong", "moderate" and "mild/worse" responses, respectively. Associations of individual biofluid C2C-HUSA levels, or their ratio, with change in WOMAC pain and function scores up to one-year post-TKA, or category of change, were analysed by linear, logistic, or cumulative odds models. RESULTS: Higher baseline uC2C-HUSA levels or a lower ratio of baseline sfC2C-HUSA to uC2C-HUSA were associated with improvements in WOMAC pain by linear multivariable modelling [odds ratio -0.40 (95% confidence interval -0.76, -0.05) p = 0.03; 0.36 (0.01, 0.71), p = 0.04, respectively], while sfC2C-HUSA alone was not. However, lower ratios of sfC2C-HUSA to uC2C-HUSA were associated with improvements in WOMAC function [1.37 (0.18, 2.55), p = 0.02], while sfC2C-HUSA and uC2C-HUSA alone were not. Lower ratios of sfC2C-HUSA to uC2C-HUSA were also associated with an increased likelihood of a subject being categorised in a group where TKA was beneficial in both univariable [pain, 0.81 (0.68, 0.96), p = 0.02; function, 0.92 (0.85, 0.99), p = 0.035] and multivariable [pain, 0.81 (0.68, 0.97) p = 0.02; function, 0.92 (0.85, 1.00), p = 0.043] ordinal modelling, while sfC2C-HUSA and uC2C-HUSA alone were not. CONCLUSIONS: Overall, ratios of baseline sfC2C-HUSA to uC2C-HUSA, and baseline uC2C-HUSA, may play an important role in studying post-TKA surgical outcomes.

Definition of follicular helper T cell and cytokines expression in synovial fluid of rheumatoid arthritis.

OBJECTIVE: This study aimed to assess the role of synovial fluid (SF) CD4+T, CD19+B, follicular helper cells (Tfh), and cytokines in the pathogenesis of rheumatoid arthritis (RA). METHODS: This study enrolled 16 patients with RA and 8 patients with osteoarthritis (OA). The frequencies of the SF CD4+ T, CD19+ B, Tfh cells, and Tfh subsets were assessed using flow cytometry. The medical condition in patients with RA was evaluated using The Disease Activity Score 28 (DAS28), the Clinical Disease Activity Index (CDAI), and the Simplified Disease Activity Index (SDAI). Levels of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), anti-cyclic citrullinated peptide (anti-CCP), and rheumatoid factor (RF) were measured. The cytokines IL-4, IL-13, IL-21, and BLyS were measured by ELISA test. RESULTS: The percentages of SF CD4+T, CD19+B, and PD-1+CXCR5+ Tfh in RA patients were higher than those in OA patients. And the Tfh2 was the main subset among Tfh subsets. In addition, levels of IL-21 and BLyS were higher in patients with RA compared to patients with OA. Furthermore, the treatment of TNF-α inhibitors may be associated with decreased levels of SF Tfh. CONCLUSIONS: Elevated SF Tfh, B cell, and cytokines expression profiles were observed in RA patients. Tfh2 was the major subset of the Tfh, and IL-21 and BLyS were significantly enhanced. Additionally, TNF-α inhibitors reduced Tfh in SF. Therefore, Tfh, B, and Tfh2 cells could play a significant role in the progression of RA. Key Points •Tfh cells in the synovial fluid are significantly higher in RA patients and are dominated by the Tfh2 subpopulation. •Synovial fluid Tfh cells decrease in RA patients after anti-TNF-α treatment.