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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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Periprosthetic joint infection (PJI) represents a devastating complication of total joint arthroplasty associated with significant morbidity and mortality. Literature suggests a possible higher incidence of periprosthetic joint infection (PJI) in patients with rheumatoid arthritis (RA). There is, however, no consensus on this purported risk nor a well-defined mechanism. This study investigates how collagen-induced arthritis (CIA), a validated animal model of RA, impacts infectious burden in a well-established model of PJI., Methods: Control mice were compared against CIA mice. Whole blood samples were collected to quantify systemic IgG levels via ELISA. Ex vivo respiratory burst function was measured via dihydrorhodamine assay. Ex vivo Staphylococcus aureus Xen36 burden was measured directly via colony forming unit (CFU) counts and crystal violet assay to assess biofilm formation. In vivo, surgical placement of a titanium implant through the knee joint and inoculation with S. aureus Xen36 was performed. Bacterial burden was then quantified by longitudinal bioluminescent imaging., Results: Mice with CIA demonstrated significantly higher levels of systemic IgG compared with control mice (p = 0.003). Ex vivo, there was no significant difference in respiratory burst function (p = 0.89) or S. aureus bacterial burden as measured by CFU counts (p = 0.91) and crystal violet assay (p = 0.96). In vivo, no significant difference in bacterial bioluminescence between groups was found at all postoperative time points. CFU counts of both the implant and the peri-implant tissue were not significantly different between groups (p = 0.82 and 0.80, respectively)., Conclusion: This study demonstrated no significant difference in S. aureus infectious burden between mice with CIA and control mice. These results suggest that untreated, active RA may not represent a significant intrinsic risk factor for PJI, however further mechanistic translational and clinical studies are warranted., Competing Interests: The authors have declared that no competing interests exist.

Conditions that resemble osteoarthritis (OA) were produced by injection of sodium monoiodoacetate (MIA) into the knee joints of mice. Bone marrow derived mast cells (BMMCs) injected into the OA knee joints enhanced spontaneous pain. Since no spontaneous pain was observed when BMMCs were injected into the knee joints of control mice that had not been treated with MIA, BMMCs should be activated within the OA knee joints and release some pain-inducible factors. Protease activated receptor-2 (PAR2) antagonist (FSLLRY-NH2) almost abolished the pain-enhancing effects of BMMCs injected into the OA knee joints, suggesting that tryptase, a mast cell protease that is capable of activating PAR2, should be released from the injected BMMCs and enhance pain through activation of PAR2. When PAR2 agonist (SLIGKV-NH2) instead of BMMCs was injected into the OA knee joints, it was also enhanced pain. Apyrase, an ATP degrading enzyme, injected into the OA knee joints before BMMCs suppressed the pain enhanced by BMMCs. We showed that purinoceptors (P2X4 and P2X7) were expressed in BMMCs and that extracellular ATP stimulated the release of tryptase from BMMCs. These observations suggest that ATP may stimulate degranulation of BMMCs and thereby enhanced pain. BMMCs injected into the OA knee joints stimulated expression of IL-1β, IL-6, TNF-α, CCL2, and MMP9 genes in the infrapatellar fat pads, and PAR2 antagonist suppressed the stimulatory effects of BMMCs. Our study suggests that intermittent pain frequently observed in OA knee joints may be due, at least partly, to mast cells through activation of PAR2 and action of ATP, and that intraarticular injection of BMMCs into the OA knee joints may provide a useful experimental system for investigating molecular mechanisms by which pain is induced in OA knee joints., Competing Interests: The authors have declared that no competing interests exist.

Objective: Psoriatic arthritis (PsA) is an inflammatory rheumatic disease, mediated in part by TNFα and associated with bone loss. Anti-TNFα treatment should inhibit this phenomenon and reduce the systemic bone loss. Ultra-high field MRI (UHF MRI) may be used to quantify bone microarchitecture (BM) in-vivo. In this study, we quantified BM using UHF MRI in a PsA patient and followed up the changes related to anti-TNFα treatment., Subjects and Methods: A non-treated PsA patient with knee arthritis and 7 gender-matched controls were scanned using a gradient re-echo sequence at UHF MRI. After a year of Adalimumab treatment, the patient underwent a second UHF MRI. A PET-FNa imaging was performed before and after treatment to identify and localize the abnormal metabolic areas. BM was characterized using typical morphological parameters quantified in 32 regions of interest (ROIs) located in the patella, proximal tibia, and distal femur., Results: Before treatment, the BM parameters were statistically different from controls in 24/32 ROIs with differences reaching up to 38%. After treatment, BM parameters were normalized for 15 out of 24 ROIs. The hypermetabolic areas disclosed by PET-FNa before the treatment partly resumed after the treatment., Conclusion: Thanks to UHF MRI, we quantified in vivo BM anomalies in a PsA patient and we illustrated a major reversion after one year of treatment. Moreover, BM results highlighted that the abnormalities were not only localized in hypermetabolic regions identified by PET-FNa, suggesting that the bone loss was global and not related to inflammation., Competing Interests: The authors have declared that no competing interests exist.

Versican is a large proteoglycan in the extracellular matrix. During embryonic stages, it plays a crucial role in the development of cartilage, heart, and dermis. Previously, we reported that Prx1-Vcan conditional knockout mice, lacking Vcan expression in mesenchymal condensation areas of the limb bud, show the impaired joint formation and delayed cartilage development. Here, we investigated their phenotype in adults and found that they develop swelling of the knee joint. Histologically, their newborn joint exhibited impaired formation of both anterior and posterior cruciate ligaments. Immunostaining revealed a decrease in scleraxis-positive cells in both articular cartilage and ligament of Prx1-Vcan knee joint, spotty patterns of type I collagen, and the presence of type II collagen concomitant with the absence of versican expression. These results suggest that versican expression during the perinatal period is required for cruciate ligaments' formation and that its depletion affects joint function in later ages., Competing Interests: The authors have declared that no competing interests exist.

The expression of some chemokines and chemokine receptors is induced during the development of post-traumatic osteoarthritis (PTOA), but their involvement in the pathogenesis of the disease is unclear. The goal of this study was to test whether CCL21 and CXCL13 play a role in PTOA development. For this purpose, we evaluated the expression profiles of the chemokines Ccl21 and Cxcl13, matrix metalloproteinase enzymes Mmp3 and Mmp13, and inflammatory cell markers in response to partial medial meniscectomy and destabilization (MMD). We then assessed the effect of local administration of CCL21 neutralizing antibody on PTOA development and post-knee injury inflammation. The mRNA expression of both Ccl21 and Cxcl13 was induced early post-surgery, but only Ccl21 mRNA levels remained elevated 4 weeks post-surgery in rat MMD-operated knees compared to controls. This suggests that while both CXCL13 and CCL21 are involved in post-surgery inflammation, CCL21 is necessary for development of PTOA. A significant increase in the mRNA levels of Cd4, Cd8 and Cd20 was observed during the first 3 days post-surgery. Significantly, treatment with CCL21 antibody reduced post-surgical inflammation that was accompanied by a reduction in the expression of Mmp3 and Mmp13 and post-MMD cartilage degradation. Our findings are consistent with a role for CCL21 in mediating changes in early inflammation and subsequent cartilage degeneration in response to knee injury. Our results suggest that targeting CCL21 signaling pathways may yield new therapeutic approaches effective in delaying or preventing PTOA development following injury., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Increased intake of dietary saturated fatty acids has been linked to obesity and the development of Osteoarthritis (OA). However, the mechanism by which these fats promote cartilage degradation and the development of OA is not clearly understood. Here, we report the effects of consumption of common dietary saturated and unsaturated fatty acids, palmitate and oleate, respectively, on body weight, metabolic factors, and knee articular cartilage in a mouse model of diet-induced obesity. Mice fed on a diet rich in saturated or unsaturated fatty acid gained an equal amount of weight; however, mice fed a palmitate diet, but not a control or oleate diet, exhibited more cartilage lesions and increased expression of 1) unfolded protein response (UPR)/endoplasmic reticulum (ER) stress markers including BIP, P-IRE1α, XBP1, ATF4, and CHOP; 2) apoptosis markers CC3 and C-PARP; and 3) negative cell survival regulators Nupr1 and TRB3, in knee articular cartilage. Palmitate-induced apoptosis was confirmed by TUNEL staining. Likewise, dietary palmitate was also increased the circulatory levels of classic proinflammatory cytokines, including IL-6 and TNF-α. Taken together, our results demonstrate that increased weight gain is not sufficient for the development of obesity-linked OA and suggest that dietary palmitate promotes UPR/ER stress and cartilage lesions in mouse knee joints. This study validates our previous in vitro findings and suggests that ER stress could be the critical metabolic factor contributing to the development of diet/obesity induced OA., Competing Interests: The authors declare no competing interests.

Whether osteoarthritis (OA) is a systemic metabolic disorder remains controversial. The aim of this study was to investigate the metabolic characteristics between plasma and knee joint fluid (JF) of patients with advanced OA using a differential correlation metabolic (DCM) networks approach. Plasma and JF were collected during the joint replacement surgery of patients with knee OA. The biological samples were pretreated with standard procedures for metabolite analysis. The metabolic profiling was conducted by means of liquid mass spectrometry coupled with a AbsoluteIDQ kit. A DCM network approach was adopted for analyzing the metabolomics data between the plasma and JF. The variation in the correlation of the pairwise metabolites was quantified across the plasma and JF samples, and networks analysis was used to characterize the difference in the correlations of the metabolites from the two sample types. Core metabolites that played an important role in the DCM networks were identified via topological analysis. One hundred advanced OA patients (50 men and 50 women) were included in this study, with an average age of 65.0 ± 7.6 years (65.6 ± 7.1 years for females and 64.4 ± 8.1 years for males) and a mean BMI of 32.6 ± 5.8 kg/m2 (33.4 ± 6.3 kg/m2 for females and 31.7 ± 5.3 kg/m2 for males). Age and BMI matched between the male and female groups. One hundred and forty-five nodes, 567 edges, and 131 nodes, 407 edges were found in the DCM networks (p < 0.05) of the female and male groups, respectively. Six metabolites in the female group and 5 metabolites in the male group were identified as key nodes in the network. There was a significant difference in the differential correlation metabolism networks of plasma and JF that may be related to local joint metabolism. Focusing on these key metabolites may help uncover the pathogenesis of knee OA. In addition, the differential metabolic correlation between plasma and JF mostly overlapped, indicating that these common correlations of pairwise metabolites may be a reflection of systemic characteristics of JF and that most significant correlation variations were just a result of "housekeeping" biological reactions., Competing Interests: The authors have read the journal’s policy and have the following competing interests: BYX receives a salary from Hangzhou Heze Pharmaceutical Technology CO., LTD (http://www.hezepharm.com/index.php?lang=en). There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Osteoarthritis (OA) is the most common arthropathy, characterized by progressive degeneration of the articular cartilage. Currently, there are no disease-modifying approaches for OA treatment. Adeno-associated virus (AAV)-mediated gene therapy has recently become a potential treatment for OA due to its exceptional characteristics; however, the tropism and transduction efficiency of different AAV serotypes to articular joints and the safety profile of AAV applications are still unknown. The present study aims to screen an ideal AAV serotype to efficiently transfer genes to arthritic cartilage. AAV vectors of different serotypes expressing eGFP protein were injected into the knee joint cavities of mice, with all joint tissues collected 30 days after AAV injection. The transduction efficiency of AAVs was quantified by assessing the fluorescent intensities of eGFP in the cartilage of knee joints. Structural and morphological changes were analyzed by toluidine blue staining. Changes to ECM metabolism and pyroptosis of chondrocytes were determined by immunohistochemical staining. Fluorescence analysis of eGFP showed that eGFP was expressed in the cartilage of knee joints injected with each AAV vector. Quantification of eGFP intensity indicated that AAV2, 7 and 8 had the highest transduction efficiencies. Both toluidine blue staining and Mankin score showed that AAV6 aggravated cartilage degeneration. The analysis of key molecules in ECM metabolism suggested that AAV5 and 7 significantly reduced collagen type II, while AAV9 increased ADAMTS-4 but decreased MMP-19. In addition, transduction with AAV2, 5, 7 and 8 had no obvious effect on pyroptosis of chondrocytes. Comprehensive score analysis also showed that AAV2 had the highest score in intra-articular gene transfer. Collectively, our findings point to AAV2 as the best AAV serotype candidate for gene transfer on arthritic cartilage, resulting in minimal impact to ECM metabolism and pyroptosis of chondrocytes., Competing Interests: The authors have declared that no competing interests exist.

The relationship between osteoarthritis (OA) structural change and pain is complex. Surgical models of OA in rodents are often rapid in onset, limiting mechanistic utility and translational validity. We aimed to investigate the effect of refining surgical small rodent models of OA on both joint pathology and pain behaviour. Adult male C57BL/6 mice (n = 76, 10-11 weeks of age at time of surgery) underwent either traditional (transection of the medial meniscotibial ligament [MMTL]) or modified (MMTL left intact, transection of the coronary ligaments) DMM surgery, or sham surgery. Adult male Sprague Dawley rats (n = 76, weight 175-199g) underwent either modified meniscal transection (MMNX) surgery (transection of the medial meniscus whilst the medial collateral ligament is left intact) or sham surgery. Pain behaviours (weight bearing asymmetry [in mice and rats] and paw withdrawal thresholds [in rats]) were measured pre-surgery and weekly up to 16 weeks post-surgery. Post-mortem knee joints were scored for cartilage damage, synovitis, and osteophyte size. There was a significant increase in weight bearing asymmetry from 13 weeks following traditional, but not modified, DMM surgery when compared to sham operated mice. Both traditional and modified DMM surgery led to similar joint pathology. There was significant pain behaviour from 6 weeks following MMNX model compared to sham operated control rats. Synovitis was significant 4 weeks after MMNX surgery, whereas significant chondropathy was first evident 8 weeks post-surgery, compared to sham controls. Pain behaviour is not always present despite significant changes in medial tibial plateau cartilage damage and synovitis, reflecting the heterogeneity seen in human OA. The development of a slowly progressing surgical model of OA pain in the rat suggests that synovitis precedes pain behaviour and that chondropathy is evident later, providing the foundations for future mechanistic studies into the disease., Competing Interests: The authors have declared that no competing interests exist.

This study was designed to evaluate the anti-inflammatory effects of a curcumin treatment on the knee of rats with induced osteoarthritis. Fifteen adult rats were used and divided in three groups: the osteoarthritis group (OAG), control group (CG-without induction of osteoarthritis), and curcumin-treated osteoarthritis group (COAG). Osteoarthritis was induced in the right knee of rats in the OAG and COAG by administering an intra-articular injection of 1 mg of zymosan. Fourteen days after induction, 50 mg/kg curcumin was administered by gavage daily for 60 days to the COAG. After the treatment period, rats from all groups were euthanized. Medial femoral condyles were collected for light microscopy and immunohistochemical staining. The expression of SOX-5, IHH, MMP-8, MMP-13, and collagen 2 (Col2) was analyzed. The COAG exhibited an increase in the number of chondrocytes in the surface and middle layers compared with that of the OAG and CG, respectively. The COAG also showed a decrease in the thicknesses of the middle and deep layers compared with those of the OAG, and an increase in Col2 expression was observed in all articular layers (surface, middle, and deep) in the COAG compared with that in the OAG. SOX-5 expression was increased in the surface and deep layers of the COAG compared with those in the OAG and CG. Based on the results of this study, the curcumin treatment appeared to exert a protective effect on cartilage, as it did not result in an increase in cartilage thickness or in MMP-8 and MMP-13 expression but led to increased IHH, Col2, and SOX-5 expression and the number of chondrocytes., Competing Interests: The authors have declared that no competing interests exist.

Osteoarthritis (OA) is a progressive disease associated with cartilage injury and its inherently limited repair capability. Synovium-based cellular constructs (sConstructs) are proposed as possible treatments. Equine sConstructs were produced from decellularized synovium-based extracellular matrix scaffolds (sECM) seeded with synovium-derived mesenchymal stem cells (sMSC), and engineered to express green fluorescent protein (GFP), or bone morphogenetic protein-2 (BMP-2). Survival, distribution, and chondrogenic potential of the sConstructs in vitro and in vivo were assessed. sConstructs in co-culture with chondrocytes increased chondrocyte proliferation, viability, and Col II production, greatest in BMP-2-sConstructs. Chondrocyte presence increased the production of hyaluronic acid (HA), proteoglycan (PG), and BMP-2 by the sConstructs in a positive feedback loop. sECM alone, or GFP- or BMP-2-sConstructs were implanted in synovium adjacent to clinically created full-thickness rat-knee cartilage lesions. At 5 weeks, the lesion area and implants were resected. Gross anatomy, adjacent articulate cartilage growth and subchondral bone repair were scored; and peripheral, central and cartilage lesion measurements taken. For all scores and measurements, sConstruct implants were significantly greater than controls, greatest with the BMP-2-sConstructs. Immunohistochemistry demonstrated migration of endogenous cells into the sECM, with greater cellularity in the constructs with intense positive GFP staining confirming engraftment of implanted sMSC and continued gene expression. In summary, exposing cartilage to sConstructs was chondrogenic in vitro and in vivo, and resulted in substantially increased growth in vivo. This effect was mediated, in part, by soluble ECM and cell factors and upregulation of anabolic growth proteins, such as BMP-2. This work is "proof of concept" that sConstructs surgically implanted adjacent to cartilage damage can significantly improve cartilage and subchondral bone repair, and potentially prevent the progression of OA., Competing Interests: The authors have declared that no competing interests exist.

Surgical transection of the anterior cruciate ligament (ACL) in the porcine model leads to posttraumatic osteoarthritis if left untreated. However, a recently developed surgical treatment, bridge-enhanced ACL repair, prevents further cartilage damage. Since the synovial fluid bathes all the intrinsic structures of knee, we reasoned that a comparative analysis of synovial fluid protein contents could help to better understand the observed chondroprotective effects of the bridge-enhanced ACL repair. We hypothesized that post-surgical changes in the synovial fluid proteome would be different in the untreated and repaired knees, and those changes would correlate with the degree of cartilage damage. Thirty adolescent Yucatan mini-pigs underwent unilateral ACL transection and were randomly assigned to either no further treatment (ACLT, n = 14) or bridge-enhanced ACL repair (BEAR, n = 16). We used an isotopically labeled high resolution LC MS/MS-based proteomics approach to analyze the protein profile of synovial fluid at 6 and 12 months after ACL transection in untreated and repaired porcine knees. A linear mixed effect model was used to compare the normalized protein abundance levels between the groups at each time point. Bivariate linear regression analyses were used to assess the correlations between the macroscopic cartilage damage (total lesion area) and normalized abundance levels of each of the identified secreted proteins. There were no significant differences in cartilage lesion area or quantitative abundance levels of the secreted proteins between the ACLT and BEAR groups at 6 months. However, by 12 months, greater cartilage damage was seen in the ACLT group compared to the BEAR group (p = 0.005). This damage was accompanied by differences in the abundance levels of secreted proteins, with higher levels of Vitamin K-dependent protein C (p = 0.001), and lower levels of Apolipoprotein A4 (p = 0.021) and Cartilage intermediate layer protein 1 (p = 0.049) in the ACLT group compared to the BEAR group. There were also group differences in the secreted proteins that significantly changed in abundance between 6 and 12 months in ACLT and BEAR knees. Increased concentration of Ig lambda-1 chain C regions and decreased concentration of Hemopexin, Clusterin, Coagulation factor 12 and Cartilage intermediate layer protein 1 were associated with greater cartilage lesion area. In general, ACLT knees had higher concentrations of pro-inflammatory proteins and lower concentrations of anti-inflammatory proteins than BEAR group. In addition, the ACLT group had a lower and declining synovial concentrations of CILP, in contrast to a consistently high abundance of CILP in repaired knees. These differences suggest that the knees treated with bridge-enhanced ACL repair may be maintaining an environment that is more protective of the extracellular matrix, a function which is not seen in the ACLT knees., Competing Interests: The authors have no competing interests directly related to this study. However, it should be noted that Dr. Murray holds several patents related to the BEAR surgical procedure, and that Drs. Murray and Fleming recently founded a company (Miach Orthopaedics Inc) to translate the BEAR surgical procedure to clinical use. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Patents: - Biologic replacement for fibrin clot (EP US WO Application US20040059416A1 & US WO Application US20020123805A1) - Device for mixing and delivering fluids for tissue repair (CA EP JP US WO Grant US8308681B2) - Methods and procedures for ligament repair (CA EP JP US WO Application WO2007087353A2) - Activation and delivery devices for therapeutic compositions(US Application US20120116300A1) - Methods and collagen products for tissue repair (US EP JP CA WO Application WO2008060361A2) - Biologic replacement for fibrin clot for intra-articular use (WO Application WO2000078370A1 & EP CA Application CA2377595A1) - Indirect method of articular tissue repair (CA WO Application CA3024196A1)

Sclerostin is a potent inhibitor of the canonical Wnt signaling pathway. Wnt signaling pathways have multiple roles in the regulation of cartilage development, growth, and maintenance. This study focused on the role of sclerostin in the process of chondrogenic differentiation. We hypothesized that sclerostin is essential to induce chondrogenic differentiation and regulate endochondral ossification. ATDC5 cells were used to investigate chondrogenic differentiation and terminal calcification. During chondrogenic differentiation, intrinsic sclerostin was upregulated in the early stage, but downregulated in the late stage. Addition of sclerostin elevated expressions of Sox9 and Col2a1 (P<0.05) and reduced expressions of Runx2, Col10a1, MMP-3, MMP-13, and ADAMTS5 (P<0.05) through inhibition of the Wnt-β-catenin signaling pathway (P<0.05). Terminal calcification was significantly inhibited by sclerostin (P<0.05). In contrast, deletion of sclerostin decreased expressions of Sox9 and Col2a1 (P<0.05), increased expressions of Runx2, Col10a1, MMP-3, and MMP-13 (P<0.05), and promoted terminal calcification (P<0.05). This study provides insights into the possible role of sclerostin in the regulation of chondrogenic differentiation. Sclerostin is upregulated in the early stage of chondrogenic differentiation, but is not required in endochondral ossification. Sclerostin is a candidate modulator for chondrogenic differentiation., Competing Interests: The authors have declared that no competing interests exist.

The balance between synthesis and degradation of the cartilage extracellular matrix is severely altered in osteoarthritis, where degradation predominates. One reason for this imbalance is believed to be due to the ligation of the α5β1 integrin, the classic fibronectin (FN) receptor, with soluble FN fragments instead of insoluble FN fibrils, which induces matrix metalloproteinase (MMP) expression. Our objective was to determine whether the lack of α5β1-FN binding influences cartilage morphogenesis in vivo and whether non-ligated α5β1 protects or aggravates the course of osteoarthritis in mice. We engineered mice (Col2a-Cre;Fn1RGE/fl), whose chondrocytes express an α5β1 binding-deficient FN, by substituting the aspartic acid of the RGD cell-binding motif with a glutamic acid (FN-RGE). At an age of 5 months the knee joints were stressed either by forced exercise (moderate mechanical load) or by partially resecting the meniscus followed by forced exercise (high mechanical load). Sections of femoral articular knees were analysed by Safranin-O staining and by immunofluorescence to determine tissue morphology, extracellular matrix proteins and matrix metalloproteinase expression. The articular cartilage from untrained control and Col2a-Cre;Fn1RGE/fl mice was normal, while the exposure to high mechanical load induced osteoarthritis characterized by proteoglycan and collagen type II loss. In the Col2a-Cre;Fn1RGE/fl articular cartilage osteoarthritis progressed significantly faster than in wild type mice. Mechanistically, we observed increased expression of MMP-13 and MMP-3 metalloproteinases in FN-RGE expressing articular cartilage, which severely affected matrix remodelling. Our results underscore the critical role of FN-α5β1 adhesion as ECM sensor in circumstances of articular cartilage regeneration., Competing Interests: The authors have declared that no competing interests exist.

Cartilage has limited self-repair ability. The purpose of this study was to investigate the effects of different species of collagen-engineered neocartilage for the treatment of critical-size defects in the articular joint in a rabbit model. Type II and I collagen obtained from rabbits and rats was mixed to form a scaffold. The type II/I collagen scaffold was then mixed with rabbit chondrocytes to biofabricate neocartilage constructs using a rotating cell culture system [three-dimensional (3D)-bioreactor]. The rabbit chondrocytes were mixed with rabbit collagen scaffold and rat collagen scaffold to form neoRBT (neo-rabbit cartilage) and neoRAT (neo-rat cartilage) constructs, respectively. The neocartilage matrix constructs were implanted into surgically created defects in rabbit knee chondyles, and histological examinations were performed after 2 and 3 months. Cartilage-like lacunae formation surrounding the chondrocytes was noted in the cell cultures. After 3 months, both the neoRBT and neoRAT groups showed cartilage-like repair tissue covering the 5-mm circular, 4-mm-deep defects that were created in the rabbit condyle and filled with neocartilage plugs. Reparative chondrocytes were aligned as apparent clusters in both the neoRAT and neoRBT groups. Both neoRBT and neoRAT cartilage repair demonstrated integration with healthy adjacent tissue; however, more integration was obtained using the neoRAT cartilage. Our data indicate that different species of type II/I collagen matrix and 3D bioreactor cultivation can facilitate cartilage engineering in vitro for the repair of critical-size defect.

Introduction: One of the characteristics of early osteoarthritis (OA) is the loss of fixed charged density (FCD) of glycosaminoglycans in the superficial zone of articular cartilage. However, possible local changes in the FCD content of the pericellular matrix (PCM) are not fully understood. Hence, our aim was to investigate the effect of unilateral anterior cruciate ligament transection (ACLT) in rabbit knees on estimated FCD in the PCM compared to that in the ECM, and relate these results with cell morphology., Methods: Articular cartilage samples were collected from ACLT, contralateral and intact control knee joints from lateral and medial femoral condyles and tibial plateaus, and from the femoral groove and patella. Histological samples were prepared and stained with Safranin-O to estimate the FCD content around the chondrocytes in the PCM and the ECM with digital densitometry., Results: As a result of ACLT, the greatest decreases in the FCD content of the PCM were observed in the superficial zone of the lateral femoral condyle (p = 0.02), medial tibial plateau (p = 0.002) and patellar (p < 0.001) cartilage. The normalized FCD content of the PCM compared to the surrounding ECM was increased most in the femoral condyles (p < 0.01) and medial tibial plateau (p = 0.02) cartilage. The high normalized FCD content of the PCM in the superficial zone of lateral femoral condyle cartilage was consistent with the round cell morphology in that location., Conclusions: In conclusion, we suggest that certain sites in the knee joint, particularly the lateral femoral condyle cartilage, experience less FCD loss in the PCM than in the ECM in early post-traumatic OA, which could lead to altered cell shape.

Objective: Bioactive oxidised lipids (oxylipins) are important signalling mediators, capable of modulating the inflammatory state of the joint and anticipated to be of importance in joint homeostasis and status of osteoarthritis. The aim of this study was to quantify oxylipin levels in plasma and synovial fluid from rats with experimentally induced osteoarthritis to investigate the potential role of oxylipins as a marker in the disease process of early osteoarthritis., Design: Forty rats were randomly allocated to a standard or high-fat diet group. After 12 weeks, local cartilage damage was induced in one knee joint in 14 rats of each diet group. The remaining 6 rats per group served as controls. At week 24, samples were collected. Oxylipin levels were quantified by liquid chromatography-mass spectrometry., Results: Overall, 31 lipid-derived inflammatory mediators were detected in fasted plasma and synovial fluid. Principal component analysis identified four distinct clusters associated with histopathological changes. Diet induced differences were evident for 13 individual plasma oxylipins, as well as 5,6-EET in synovial fluid. Surgical-model induced differences were evident for three oxylipins in synovial fluid (15-HETE, 8,9-DHET and 17R-ResolvinD1) with a different response in lipid concentrations for synovial fluid and plasma., Conclusions: We demonstrate the quantification of oxidised lipids in rat plasma and synovial fluid in a model of early experimental osteoarthritis. Oxylipins in the synovial fluid that were altered as consequence of the surgically induced osteoarthritis were not represented in the plasma. Our findings suggest differential roles of the oxylipins in the local versus peripheral compartment.

Background: Anterior cruciate ligaments (ACL) injuries represent a major risk factor for early osteoarthritis (OA)., Purpose: To evaluate the prevalence and 4-year progression of knee OA measured with 3T MR-imaging in individuals with ruptured, reconstructed or normal ACL and to assess the impact of thigh muscle characteristics., Methods: A total of 54 knees (23/54 male, 31/54 female) were recruited from the Osteoarthritis Initiative (OAI). At baseline, 15/54 subjects had prevalent ACL ruptures and 15/54 subjects had prevalent ACL reconstruction (24/54 normal ACL). Western Ontario and McMasters Universities Arthritis Index (WOMAC) scores, Physical Activity Scores of the Elderly (PASE) and thigh muscle characteristics including strength, fat infiltration (Goutallier score) and thigh muscle cross-sectional area (CSA) MR measurements were obtained at baseline. Whole-organ MR-imaging Scores (WORMS) were obtained at baseline and at a 4-year follow-up time-point. Multivariate regression models, adjusting for covariates (age, gender, body mass index), were used for statistical analysis., Results: At baseline, subjects with prevalent ACL ruptures had worse WORMS total scores (mean±SEM, 44.1±3.5) than subjects with ACL reconstruction (30.8±4.0; P = 0.015) and worse than subjects with normal ACL (21.3±3.0; P<0.001). Cartilage scores were worse in both femorotibial compartments in ACL injured knees than in knees with normal ACL (P<0.05). Knees with ACL reconstruction showed an increased degeneration of the medial meniscus (P = 0.036), cartilage degeneration at the medial femoral condyle (P = 0.011). In a multivariate regression model, including both ACL groups and total muscle characteristics as influence parameters, high thigh muscle CSA, high muscle/ fat ratio and low Goutallier scores were associated with less degenerative changes at the knee, independent of ACL status. Knees with ACL reconstruction showed an increased progression of cartilage degeneration at the medial tibia compared to the normal ACL group (P = 0.027)., Conclusions: High thigh muscle CSA is associated with less degenerative changes at the knee, independent of the ACL status and may potentially be advantageous in the prevention of early OA., Competing Interests: The funding does not alter our adherence to PLOS ONE policies on sharing data and materials.

Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage., Competing Interests: The authors have declared that no competing interests exist.

The changes in knee laxity and relaxin receptor expression at different phases of rodent estrous cycle are not known. Here, changes in the parameter were investigated in rats at different phases of the estrous cycle. Estrous cycle phases of intact female rats were determined by cytological examination of the vaginal smear. Following phase identification, blood was collected for serum hormone analyses. Knee passive range of motion (ROM) was determined by using a digital miniature goniometer. The animals were then sacrificed and patellar tendon, collateral ligaments and hamstring muscles were harvested for relaxin/insulin-like family peptide receptor 1 and 2 (RXFP1/RXFP2) analyses. Knee passive ROM was the highest at proestrus followed by diestrus and the lowest at estrus. Estrogen level was the highest at proestrus while progesterone and relaxin levels were the highest at diestrus. A strong correlation was observed between relaxin and progesterone levels. At proestrus, expression of RXFP1 and RXFP2 proteins and mRNAs were the highest at proestrus followed by diestrus and estrus. The finding shows that higher level of progesterone and relaxin in diestrus might be responsible for higher laxity of knee joint in rats.

Introduction: The presence of leukocytes in platelet concentrates is deemed to cause deleterious effects when injected intra articularly. The aim of this study is to analyse both local and systemic effects induced by leukocyte-rich Platelet-rich Plasma (PRP) injections through a proteomic characterization of serial synovial fluid and blood samples obtained from subjects treated for knee OA. Secondary aim was to compare the effects on knee homeostasis and systemic response with those obtained with visco-supplementation., Methods: Thirty-six OA patients treated either by autologous L-PRP or HA intra-articular knee injections, administered in series of three at one-week intervals, were analyzed. Just before the injection, 1 ml of synovial fluid was collected through the same needle way. In the same time, a peripheral blood sample was obtained and plasma separated. A further peripheral blood sample was collected at 2, 6, and 12 months. L-PRP, plasma and synovial fluid were tested by multiplex bead-based sandwich immunoassay by means of the Bio-Plex suspension array system (Bio-Rad Laboratories) for the presence of pro- and anti-inflammatory cytokines (IL-1beta, IL-6, IL-8, IL-17 and IL-4, IL-10, IL-13) and growth factors (FGF-b, HGF, PDGF-AB/BB)., Results: In general, pro-inflammatory cytokine levels were similar at basal condition and after treatment whereas anti-inflammatory ones were nearly undetectable. L-PRP administration did not modulate significant changes of cytokine concentrations either in synovial fluid or plasma, whatever the time points analyzed. No different trend was observed between L-PRP and HA administration in terms of pro- and anti-inflammatory cytokines, as well as growth factors., Conclusions: In contrast with the evidence reported by "in vitro" studies, where a cellular pro-inflammatory response appears to be induced by the presence of leukocytes, these results suggest that the presence leukocyte-rich PRP doesn't induce a relevant in vivo up regulation of pro-inflammatory mediators.

Background: Osteoarthritis (OA) is a degenerative joint disease with poorly understood etiology and pathobiology. Mitogen activated protein kinases (MAPKs) including ERK and p38 play important roles in the mediation of downstream pathways involved in cartilage degenerative processes. Dual specificity phosphatase 1 (DUSP1) dephosphorylates the threonine/serine and tyrosine sites on ERK and p38, causing deactivation of downstream signalling. In this study we examined the role of DUSP1 in spontaneous OA development at 21 months of age using a genetically modified mouse model deficient in Dusp1 (DUSP1 knockout mouse)., Results: Utilizing histochemical stains of paraffin embedded knee joint sections in DUSP1 knockout and wild type female and male mice, we showed similar structural progression of cartilage degeneration associated with OA at 21 months of age. A semi-quantitative cartilage degeneration scoring system also demonstrated similar scores in the various aspects of the knee joint articular cartilage in DUSP1 knockout and control mice. Examination of overall articular cartilage thickness in the knee joint demonstrated similar results between DUSP1 knockout and wild type mice. Immunostaining for cartilage neoepitopes DIPEN, TEGE and C1,2C was similar in the cartilage lesion sites and chondrocyte pericellular matrix of both experimental groups. Likewise, immunostaining for phosphoERK and MMP13 showed similar intensity and localization between groups. SOX9 immunostaining demonstrated a decreased number of positive cells in DUSP1 knockout mice, with correspondingly decreased staining intensity. Analysis of animal walking patterns (gait) did not show a discernable difference between groups., Conclusion: Loss of DUSP1 does not cause changes in cartilage degeneration and gait in a mouse model of spontaneous OA at 21 months of age. Altered staining was observed in SOX9 immunostaining which may prove promising for future studies examining the role of DUSPs in cartilage and OA, as well as models of post-traumatic OA.

Objectives: To explore the pathogenesis of rheumatoid arthritis (RA), the different metabolites were screened in synovial fluid by metabolomics., Methods: Synovial fluid from 25 RA patients and 10 normal subjects were analyzed by GC/TOF MS analysis so as to give a broad overview of synovial fluid metabolites. The metabolic profiles of RA patients and normal subjects were compared using multivariate statistical analysis. Different proteins were verified by qPCR and western blot. Different metabolites were verified by colorimetric assay kit in 25 inactive RA patients, 25 active RA patients and 20 normal subjects. The influence of hypoxia-inducible factor (HIF)-1α pathway on catabolism was detected by HIF-1α knockdown., Results: A subset of 58 metabolites was identified, in which the concentrations of 7 metabolites related to energy metabolism were significantly different as shown by importance in the projection (VIP) (VIP ≥ 1) and Student's t-test (p<0.05). In the 7 metabolites, the concentration of glucose was decreased, and the concentration of lactic acid was increased in the synovial fluid of RA patients than normal subjects verified by colorimetric assay Kit. Receiver operator characteristic (ROC) analysis shows that the concentration of glucose and lactic acid in synovial fluid could be used as dependable biomarkers for the diagnosis of active RA, provided an AUC of 0.906 and 0.922. Sensitivity and specificity, which were determined by cut-off points, reached 84% and 96% in sensitivity and 95% and 85% in specificity, respectively. The verification of different proteins identified in our previous proteomic study shows that the enzymes of anaerobic catabolism were up-regulated (PFKP and LDHA), and the enzymes of aerobic oxidation and fatty acid oxidation were down-regulated (CS, DLST, PGD, ACSL4, ACADVL and HADHA) in RA patients. The expression of HIF-1α and the enzymes of aerobic oxidation and fatty acid oxidation were decreased and the enzymes of anaerobic catabolism were increased in FLS cells after HIF-1α knockdown., Conclusion: It was found that enhanced anaerobic catabolism and reduced aerobic oxidation regulated by HIF pathway are newly recognized factors contributing to the progression of RA, and low glucose and high lactic acid concentration in synovial fluid may be the potential biomarker of RA.

Objectives: Emerging data suggest that several metabolic factors, released mainly by white adipose tissue (WAT) and joint tissues, and collectively named adipokines, might have a role in the pathophysiology of OA. Recently, novel adipokines such as SERPINE2, WISP2, GPNMB and ITIH5 have been identified in WAT. The main goal of this study was to analyse the expression of these novel adipokines in synovium, infrapatellar fat pad and chondrocytes and to compare the expression of these molecules in healthy and OA tissues., Methods: Synovial tissues, infrapatellar fat pad and chondrocytes were obtained from 36 OA patients (age 52-85; mean BMI 28.9) who underwent total knee replacement surgery. Healthy synovial tissues and infrapatellar fat pad were obtained from 15 traumatic knee patients (age 23-53; mean BMI 23.5). mRNA and protein expression were determined by qRT-PCR and western blot analysis respectively., Results: All the novel adipokines, matter of our study, are expressed in OA synovium, infrapatellar fat pad and chondrocytes. Moreover, we detected a differential expression of SERPINE2 and ITIH5 in OA synovial tissues as compared to healthy samples. Finally, we also observed an increased expression of WISP2 in OA infrapatellar fat pad in comparison to healthy controls., Conclusions: In this study we demonstrated for the first time the expression of four novel adipokines in different joint tissues and how these molecules are differentially expressed in healthy and OA joint tissues.

Steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR), are nuclear receptors that are expressed at high levels in the liver and the intestine where they function as xenobiotic sensors that induce expression of genes involved in detoxification and drug excretion. Recent evidence showed that SXR and PXR are also expressed in bone tissue where they mediate bone metabolism. Here we report that systemic deletion of PXR results in aging-dependent wearing of articular cartilage of knee joints. Histomorphometrical analysis showed remarkable reduction of width and an enlarged gap between femoral and tibial articular cartilage in PXR knockout mice. We hypothesized that genes induced by SXR in chondrocytes have a protective effect on articular cartilage and identified Fam20a (family with sequence similarity 20a) as an SXR-dependent gene induced by the known SXR ligands, rifampicin and vitamin K2. Lastly, we demonstrated the biological significance of Fam20a expression in chondrocytes by evaluating osteoarthritis-related gene expression of primary articular chondrocytes. Consistent with epidemiological findings, our results indicate that SXR/PXR protects against aging-dependent wearing of articular cartilage and that ligands for SXR/PXR have potential role in preventing osteoarthritis caused by aging.

Proopiomelanocortin-derived peptides exert pleiotropic effects via binding to melanocortin receptors (MCR). MCR-subtypes have been detected in cartilage and bone and mediate an increasing number of effects in diathrodial joints. This study aims to determine the role of MC1-receptors (MC1) in joint physiology and pathogenesis of osteoarthritis (OA) using MC1-signaling deficient mice (Mc1re/e). OA was surgically induced in Mc1re/e and wild-type (WT) mice by transection of the medial meniscotibial ligament. Histomorphometry of Safranin O stained articular cartilage was performed with non-operated controls (11 weeks and 6 months) and 4/8 weeks past surgery. µCT-analysis for assessing epiphyseal bone architecture was performed as a longitudinal study at 4/8 weeks after OA-induction. Collagen II, ICAM-1 and MC1 expression was analysed by immunohistochemistry. Mc1re/e mice display less Safranin O and collagen II stained articular cartilage area compared to WT prior to OA-induction without signs of spontaneous cartilage surface erosion. This MC1-signaling deficiency related cartilage phenotype persisted in 6 month animals. At 4/8 weeks after OA-induction cartilage erosions were increased in Mc1re/e knees paralleled by weaker collagen II staining. Prior to OA-induction, Mc1re/e mice do not differ from WT with respect to bone parameters. During OA, Mc1re/e mice developed more osteophytes and had higher epiphyseal bone density and mass. Trabecular thickness was increased while concomitantly trabecular separation was decreased in Mc1re/e mice. Numbers of ICAM-positive chondrocytes were equal in non-operated 11 weeks Mc1re/e and WT whereas number of positive chondrocytes decreased during OA-progression. Unchallenged Mc1re/e mice display smaller articular cartilage covered area without OA-related surface erosions indicating that MC1-signaling is critical for proper cartilage matrix integrity and formation. When challenged with OA, Mc1re/e mice develop a more severe OA-pathology. Our data suggest that MC1-signaling protects against cartilage degradation and subchondral bone sclerosis in OA indicating a beneficial role of the POMC system in joint pathophysiology.

Introduction: Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines., Methods: To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays., Results: Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays., Conclusion: Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.

Osteoarthritis (OA) is characterized by degradation of the cartilage matrix, leading to pathologic changes in the joints. However, the pathogenic effects of synovial tissue inflammation on OA knees are not clear. To investigate whether the inflammation caused by the medial plica is involved in the pathogenesis of osteoarthritis, we examined the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the medial plica and pannus-like tissue in the knees of patients with medial compartment OA who underwent either arthroscopic medial release (stage II; 15 knee joints from 15 patients) or total knee replacement (stage IV; 18 knee joints from 18 patients). MMP-2, MMP-3, MMP-9, IL-1β, and TNF-α mRNA and protein levels measured, respectively, by quantitative real-time PCR and Quantibody human MMP arrays, were highly expressed in extracts of medial plica and pannus-like tissue from stage IV knee joints. Immunohistochemical staining also demonstrated high expression of MMP-2, MMP-3, and MMP-9 in plica and pannus-like tissue of stage IV OA knees and not in normal cartilage. Some TIMP/MMP ratios decreased significantly in both medial plica and pannus-like tissue as disease progressed from stage II to stage IV. Furthermore, the migration of cells from the pannus-like tissue was enhanced by IL-1β, while plica cell migration was enhanced by TNF-α. The results suggest that medial plica and pannus-like tissue may be involved in the process of cartilage degradation in medial compartment OA of the knee.

Background: Chronic pain is the most prominent and disabling symptom of osteoarthritis (OA). Clinical data suggest that subchondral bone lesions contribute to the occurrence of joint pain. The present study investigated the effect of the inhibition of subchondral bone lesions on joint pain., Methods: Osteoarthritic pain was induced by an injection of monosodium iodoacetate (MIA) into the rat knee joint. Zoledronic acid (ZOL), a third generation of bisphosphonate, was used to inhibit subchondral bone lesions. Joint histomorphology was evaluated using X-ray micro computed tomography scanning and hematoxylin-eosin staining. The activity of osteoclast in subchondral bone was evaluated using tartrate-resistant acid phosphatase staining. Joint pain was evaluated using weight-bearing asymmetry, the expression of calcitonin gene-related peptide (CGRP) in the dorsal root ganglion (DRG), and spinal glial activation status using glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule-1 (Iba-1) immunofluorescence. Afferent neurons in the DRGs that innervated the joints were identified using retrograde fluorogold labeling., Results: MIA injections induced significant histomorphological alterations and joint pain. The inhibition of subchondral bone lesions by ZOL significantly reduced the MIA-induced weight-bearing deficit and overexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal horn at 3 and 6 weeks after MIA injection; however, joint swelling and synovial reaction were unaffected., Conclusions: The inhibition of subchondral bone lesions alleviated joint pain. Subchondral bone lesions should be a key target in the management of osteoarthritic joint pain.

Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic "expanding" phase of the disease, and then suddenly "collapse" during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITY®). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITY® injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.553 ± 0.007 vs. 0.008 ± 0.003; p<0.05). Moreover, we define the upper (>0.030) and lower (<0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost-effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare.

Obesity and high body mass index are associated with a higher incidence of osteoarthritis (OA). The aim of this study is to investigate the involvement of the infrapatellar fat pad (IPFP) in the sub-acute effect of a high fat diet (HFD) on the development of knee-OA. C57BL/6J male mice were fed either a HFD or a normal diet beginning at seven weeks of age. Tissue sections were evaluated with immunohistological analysis. The IPFP was excised, and mRNA expression profiles were compared using real-time RT-PCR analysis. Osteoarthritic changes were initiated in the HFD group after eight weeks of the HFD. Increased synovial cell number and angiogenesis at the anterior edge of the tibial plateau were exhibited prior to osteophyte formation. Quantitative histological analysis indicated that osteophyte volume was significantly increased in the HFD group after eight weeks, along with an increase in the IPFP volume, the size of individual adipocytes and the number of vessels in the IPFP. Histomorphometrical analysis revealed osteophyte area was significantly associated with IPFP area, individual adipocyte area and vascular area. Real-time RT-PCR analysis demonstrated elevated mRNA expression of inflammatory cytokines, growth factor, and adipokines in the IPFP after eight weeks of the HFD. These findings are in parallel with increased expression of the CD68 macrophage marker after eight weeks of the HFD. Expression levels of the adipokines were significantly correlated with expression of TNF-α, VEGF and TGF-β. Immunohistological analysis revealed that the Nampt protein was highly expressed in the IPFP especially around the site of osteophyte formation. Apoptosis and proliferation of chondrocytes were both enhanced at the site of osteophyte formation, indicating higher cell turnover at this region. These observations suggest the IPFP plays a pivotal role in the formation of osteophytes and functions as a secretory organ in response to a HFD.

Background: Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation., Methods: Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163(+) chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated., Results: In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P<0.05). However, the levels of ACP-1, NO and ROS, which relate to cellular digestion capability were unchanged (P>0.05). CD163(+) chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163(+) chondrocytes with enhanced phagocytic activity were present in Col-II(+) chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163(+) chondrocytes were also found in isolated Col-II(+) chondrocytes stimulated with TNF-α (P<0.05). Mid-zone distribution of CD163(+) cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II(+) chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both P<0.05)., Conclusions: An increased number of CD163(+) chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a role in eliminating degraded tissues. Targeting these cells provides a new strategy for the treatment of arthritis.

Osteoarthritis (OA) is a common disease in the elderly due to an imbalance in cartilage degradation and synthesis. Heterotopic ossification (HO) occurs when ectopic masses of endochondral bone form within the soft tissues around the joints and is triggered by inflammation of the soft tissues. Procyanidin B3 (B3) is a procyanidin dimer that is widely studied due to its high abundance in the human diet and antioxidant activity. Here, we evaluated the role of B3 isolated from grape seeds in the maintenance of chondrocytes in vitro and in vivo. We observed that B3 inhibited H(2)O(2)-induced apoptosis in primary chondrocytes, suppressed H(2)O(2)- or IL-1ß-induced nitric oxide synthase (iNOS) production, and prevented IL-1ß-induced suppression of chondrocyte differentiation marker gene expression in primary chondrocytes. Moreover, B3 treatment enhanced the early differentiation of ATDC5 cells. To examine whether B3 prevents cartilage destruction in vivo, OA was surgically induced in C57BL/6J mice followed by oral administration of B3 or vehicle control. Daily oral B3 administration protected articular cartilage from OA and prevented chondrocyte apoptosis in surgically-induced OA joints. Furthermore, B3 administration prevented heterotopic cartilage formation near the surgical region. iNOS protein expression was enhanced in the synovial tissues and the pseudocapsule around the surgical region in OA mice fed a control diet, but was reduced in mice that received B3. Together, these data indicated that in the OA model, B3 prevented OA progression and heterotopic cartilage formation, at least in a part through the suppression of iNOS. These results support the potential therapeutic benefits of B3 for treatment of human OA and heterotopic ossification.

Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint.