BackgroundPlain radiography is the first choice for diagnosis and monitoring of knee-osteoarthritis (OA) while, Kellgren–Lawrence score (KL) is most widely used to grade OA severity. However, incompetency for reproducibility of joint space measurement in longitudinal assessment and non-linearity of KL-score system, limits radiography-based early diagnosis of the disease. Glycosaminoglycan (GAG) is direct cartilage-degradation product, which can be measured biochemically. We strived to correlate KL-score and GAG from OA patients to compliment KL-system.MethodsWe obtained 34 synovial-fluid (SF) samples from 28 OA patients (few bilateral) with different disease severity using arthrocetesis. All patients were categorised using radiographic KL-score-system. SFs were further analysed for GAG estimation using 1,2-dimethylmethylene blue (DMMB) assay.ResultsA substantial increase in GAG was noted in KL-grade-II and III, comparing grade-I patients, indicating amplified cartilage-degradation. KL-grade-IV patients revealed further rise in GAG reflecting more cartilage-loss. Another category of grade-IV patients with lower GAG were also detected, indicating close to total cartilage-loss.ConclusionsAccurate diagnosis of cartilage-loss remains a challenge with OA due to limitations of KL-system; thus no target intervention is available to arrest active cartilage-loss. We propose, GAG-estimation in OA patients, characterizes accurate biochemical depiction of cartilage degeneration. General Significance: Radiology often fails to reveal an accurate cartilage loss, associated with OA. GAG levels from the SFs of OA patients' serve as a useful marker, which parallels cartilage degeneration and strengthen radiographic grading system, ultimately

Failure of conventional anti-inflammatory therapies in osteoarthritis (OA) underlines the insufficient knowledge about inflammatory mechanisms, patterns and their relationship with cartilage degradation. Considering non-linear nature of cartilage loss in OA, a better understanding of inflammatory milieu and MMP status at different stages of OA is required to design early-stage therapies or personalized disease management. For this, an investigation based on a synovium-synovial fluid (SF) axis was planned to study OA associated changes in synovium and SF along the progressive grades of OA. Gene expressions in synovial-biopsies from different grades OA patients (N = 26) revealed a peak of IL-1β, IL-15, PGE2 and NGF in early OA (Kellgren–Lawrence (KL) grade-I and II); the highest MMP levels were found in advanced stages (KL grade-III and IV). MMPs (MMP-1, 13, 2 and 9) abundance and FALGPA activity estimated in forty SFs of progressive grades showed the maximum protein levels and activity in KL grade-II and III. In an SF challenge test, SW982 and THP1 cells were treated with progressive grade SFs to study the dynamics of MMPs modulation in inflammatory microenvironment; the test yielded a result pattern, which matched with FALGPA and the protein-levels estimation. Inflammatory mediators in SFs served as steering factor for MMP up-regulation. A correlation-matrix of IL-1β and MMPs revealed expressional negative correlation.

The inflammatory nature of synovial fluid (SF) of varying grade osteoarthritis (OA) patients was estimated by measuring pro-inflammatory factors and through a unique cell-challenge experiment. SF samples were collected from six OA and one non-OA patient; spanning Kellgren-Lawrence (KL) grades were analyzed for interlukin-1-beta (IL-1β), nitric oxide (NO) and its derivatives, and glycosaminoglycan (GAG). Levels of IL-1β, NO, and GAG in SF did not correlate with KL grades of the patients studied. In the cell-challenge experiment, cultured rat synoviocyte fibroblasts (RSFs) were challenged by the patient's SFs with and without pre-treatment of IL-1β and lipopolysaccharide (LPS). NO released by the cells was taken as an indicator of inflammation. SFs from KL grades 2 and 3 induced maximum inflammation in cultured RSFs (grade 2 64.61 ± 4.8 and 89.51 ± 5.6 μM/ml after 48 and 72 h, grade 3 58.27 ± 2.7 and 64.22 ± 2.8 μM/ml after 48 and 72 h, respectively). Similar trend was observed in RSF pretreated with either recombinant IL-1β or LPS suggesting that SF from patients KL grades 2 and 3 accumulates more pro-inflammatory factors. IL-1β-pre-treated RSFs challenged by SF for 72 h showed 234.41 ± 17.6 μM/ml increase (patient 3, grade 3), whereas higher NO after LPS pre-treatment was recorded (118.92 ± 6.2 μM/ml; patient 3, grade 3). Interestingly, SFs from grade 1 and non-OA patient could reduce released NO to 27.10 ± 2.2 μM/ml showing potency to alleviate inflammation. These interesting findings, however, need to be confirmed on a wider number of patients, which may offer significant therapeutic application in treatment of OA.

OBJECTIVES:Current osteoarthritis (OA) research experiences an incline toward Ayurveda to attain a complete cure without notable adverse effects. Ayurveda uses natural products, which are known to perform the multi-faceted role, a much demanding approach for OA management. However, lack of scientific evidence is a major drawback hindering their wider use. The present work investigated the anti-arthritic potential of Ashwagandharishta, Balarishta, Dashmoolarishta, and Triphala-extract to establish molecular-evidence for their clinical use.MATERIALS AND METHODS:Rabbit synoviocytes were induced using interleukin-1 beta (IL-1 β) and lipopolysaccharide (LPS) separately and were further treated with study formulations to test anti-inflammatory and anti-oxidant potential, using nitric oxide (NO) and malondialdehyde (MDA) assays. Collagenase inhibition activity was estimated with N-(3-[2-Furyl] acryloyl)-Leu-Gly-Pro-Ala (FALGPA)-substrate and gelatinase spot assays. Data were analyzed with GraphPad Prism using one-way ANOVA followed by Bonferroni's multiple comparison.RESULTS:The study formulations were effective against synovitis, oxidative-stress, and inhibiting collagenase. They caused NO reduction in selected concentrations. DA showed the maximum NO decline of 0.02 ± 0 and 0.97 ± 0.62 μM/ml with IL-1 β and LPS induction at 5 and 20 μg/ml concentrations, respectively. Estimated by FALGPA assay, increasing collagenase inhibition was observed as the function of concentration. All formulations showed a significant MDA decline, in dose-dependent manner.CONCLUSION:We assessed the anti-OA efficacy of conventionally prescribed Ayurvedic drugs using relevant biochemical assays. The studied formulations revealed potential to restrain synovitis, cartilage degeneration and to reduce oxidative stress, and the signature OA features. With established molecular authenticity, Ayurvedic drugs can offer a safer and affordable therapeutic option for OA.

Purpose: Synovial fluid is an interstitial fluid secreted by fibroblastic cells in the synovial membrane. The physiological functions of synovial fluid may include reduction of friction, shock absorption, nutrient, and waste transportation in the joint. In addition, previous studies showed various types of cells reside in synovial fluid. Those are inflammatory cells such as macrophages and T cells, and mesenchymal stem cells (MSCs), those are considered to contribute the tissue homeostasis and regeneration. We have reported that pathological conditions such as joint injury or joint inflammation increased both cellular components and inflammatory cytokine levels in synovial fluid. We predict that both of them may play important roles during the recovery process after joint injury or surgery. In this study, we aimed to analyze the dynamic changes of cellular components in synovial fluid after anterior cruciate ligament reconstruction surgery (ACL-R) and compared them with the biochemical parameters such as inflammatory cytokine levels in synovial fluid from each patient. Here we report that the population of CD66b-positive Granulocytes surged in the early stage after ACL-R and positively correlated with IL1 beta and IL8 levels in synovial fluid. Methods: This study was approved by the Ethics Committee of this institute. All patients included in this study gave their full, written, informed consent for participation prior to the operative procedure. Synovial fluid was obtained from patients who underwent ACL-R from February 2013 till March 2014 in our University's Hospital (just before surgery, day 3e4, day 19e21, and day 35 post surgery: n¼58). Cellular components in synovial fluid were analyzed by flow cytometry (BD FACS Verse). In this study, we quantitated the population of T-cells (CD3, CD4, CD8), Granulocytes (CD66b), Macrophages (CD11b, CD14), and MSCs (CD73, CD90, CD105, CD44) in each time point. Inflammatory cytokine levels in synovial fluid (IL1 beta, IL2, IL6, IL8, IL10, TNF alpha, and IFN gamma) were quantitated by ELISA. Kruskal-Wallis test and Peasons correlation analysis were employed for statistical analysis and probability values less than 0.05 were considered as significant. Results: Flow cytometric analyses detected T cells, Granulocytes, Macrophages, and MSCs in synovial fluid. Dynamic analysis indicated that T cell and Macrophage populations in synovial fluid surged at day 19e21 post surgery (1.3-fold and 7.8-fold respectively if compared with that before surgery). Population of MSC gradually increased with time post surgery (1.2-fold at day 35; compared to the population before surgery). Granulocyte population significantly increased at day 3e4 post surgery. We observed the highest individual variation in Granulocyte population between the patients (minimum 0.1% and maximum 96.5% at day 3e4). To examine if the individual variation of Granulocyte population correlates clinical conditions of each patient, we compared it with cytokine levels in synovial fluid at day 3e4 post ACL-R. Among the cytok...