Human Osteoarthritis Cartilage Explants Research Articles

Atractylenolide-III alleviates osteoarthritis and chondrocyte senescence by targeting NF-κB signaling.

Atractylenolide-III (AT-III) is well known as its role in antioxidant and anti-inflammatory. Present study was aimed to figure out its effects on osteoarthritis and potential mechanisms. Rat model, human osteoarthritis cartilage explants as well as rat/human chondrocyte cultures were prepared to test AT-III's effects on osteoarthritis progression and chondrocyte senescence. Potential targeted molecules of AT-III were predicted using network pharmacology and molecular docking, assessed by Western blotting and then verified with rescue experiments. AT-III treatment alleviated osteoarthritis severity (shown by OARSI grading score and micro-CT) and chondrocyte senescence (indexed by levels of SA-β-gal, P16, P53, MMP13, ROS and ratio of healthy/collapsed mitochondrial membrane potentials). Network pharmacology and molecular docking suggested that AT-III might play role through NF-κB pathway. Further experiments revealed that AT-III reduced phosphorylation of IKKα/β, IκBα and P65 in NF-κB pathway. As well as nuclear translocation of p65. Both in vivo and in vitro experiments indicated that AT-III's effects on osteoarthritis and anti-senescence were reversed by an NF-κB agonist. AT-III could alleviate osteoarthritis by inhibiting chondrocyte senescence through NF-κB pathway, which indicated that AT-III is a prospective drug for osteoarthritis treatment.

The recombinant Link module of human TSG-6 suppresses cartilage damage in models of osteoarthritis: A potential disease-modifying OA drug

To investigate the role of endogenous TSG-6 in human osteoarthritis (OA) and assess the disease-modifying potential of a TSG-6-based biological treatment in cell, explant and animal models of OA. Knee articular cartilages from OA patients were analyzed for TSG-6 protein and mRNA expression using immunohistochemistry and RNAscope, respectively. The inhibitory activities of TSG-6 and its isolated Link module (Link_TSG6) on cytokine-induced degradation of OA cartilage explants were compared. Human mesenchymal stem/stromal cell-derived chondrocyte pellet cultures were used to determine the effects of Link_TSG6 and full-length TSG-6 on IL-1α-, IL-1β-, or TNF-stimulated ADAMTS4, ADAMTS5, and MMP13 mRNA expression. Link_TSG6 was administered i.a. to the rat ACLTpMMx model; cartilage damage and tactile allodynia were assessed. TSG-6 is predominantly associated with chondrocytes in regions of cartilage damage where high TSG-6 expression aligns with low MMP13, the major collagenase implicated in OA progression. Link_TSG6 is more potent than full-length TSG-6 at inhibiting cytokine-mediated matrix breakdown in human OA cartilage explants;>50% of donor cartilages, from 59 tested, were responsive to Link_TSG6 treatment. Link_TSG6 also displayed more potent effects in 3D pellet cultures, suppressing ADAMTS4, ADAMTS5, and MMP13 gene expression, which was consistent with reduced aggrecanase and collagenase activities in explant cultures. Link_TSG6 treatment reduced touch-evoked pain behavior and dose-dependently inhibited cartilage damage in a rodent model of surgically-induced OA. Link_TSG6 has enhanced chondroprotective activity compared to the full-length TSG-6 protein and shows potential as a disease modifying OA drug via its inhibition of aggrecanase and collagenase activity.

Novel strategy of senescence elimination via toxicity-exempted kinome perturbations by nanoliposome-based thermosensitive hydrogel for osteoarthritis therapy

Cellular senescence and the senescence-associated secretory phenotype (SASP) have been implicated in osteoarthritis (OA). This study aims to determine whether multi-kinase inhibitor YKL-05-099 (Y099) has potential in senescence elimination and OA therapy and whether delivering Y099 by nanoliposmal hydrogel improves the performance of the kinase inhibitor. Y099 inhibited IL-1β-induced inflammation and catabolism and promoted anabolism of chondrocytes. To attenuate the inhibition of cell viability, nanoliposomal Y099-loaded thermosensitive hydrogel (Y099-Lip-Gel) was developed for sustained release and toxicity exemption. Notably, Y099-Lip-Gel exhibited a pronounced effect on promoting anabolism and suppressing catabolism and inflammation without causing the inhibition of chondrocyte viability. Moreover, Y099-Lip-Gel remarkably increased the master regulator of chondrocyte phenotype Sox9 expression. After four intra-articular injections of Y099-Lip-Gel in the OA murine model, the histological lesions of cartilage were attenuated by Y099-Lip-Gel with subchondral bone loss and osteoclast formation inhibited. Transcriptomic analysis and experimental validations revealed that Y099-Lip-Gel suppressed cellular senescence by inhibiting the expression of senescence inducers and SASP factors. Furthermore, the phosphoproteomic analysis showed that Y099-Lip-Gel exerted a significant influence on kinome phosphorylation, inhibiting the MAPK and NF-κB signaling activations. The protective effects of Y099-Lip-Gel were also validated in cultured human OA cartilage explants. In conclusion, nanoliposomal Y099-loaded thermosensitive hydrogel has considerable potential in OA therapy. Nanoliposome-based hydrogel system has strength in reducing kinase inhibition-induced cytotoxicity, enhancing cellular tolerance to kinome perturbation, and improving the performance of protein kinase inhibitors. Senescence elimination via toxicity-exempted kinome perturbations achieved by advanced nanotechnology is a promising strategy for OA.Graphical

Chondroitin Sulfate-Tyramine-Based Hydrogels for Cartilage Tissue Repair.

The degradation of cartilage, due to trauma, mechanical load or diseases, results in abundant loss of extracellular matrix (ECM) integrity and development of osteoarthritis (OA). Chondroitin sulfate (CS) is a member of the highly sulfated glycosaminoglycans (GAGs) and a primary component of cartilage tissue ECM. In this study, we aimed to investigate the effect of mechanical load on the chondrogenic differentiation of bone marrow mesenchymal stem cells (BM-MCSs) encapsulated into CS-tyramine-gelatin (CS-Tyr/Gel) hydrogel in order to evaluate the suitability of this composite for OA cartilage regeneration studies in vitro. The CS-Tyr/Gel/BM-MSCs composite showed excellent biointegration on cartilage explants. The applied mild mechanical load stimulated the chondrogenic differentiation of BM-MSCs in CS-Tyr/Gel hydrogel (immunohistochemical collagen II staining). However, the stronger mechanical load had a negative effect on the human OA cartilage explants evaluated by the higher release of ECM components, such as the cartilage oligomeric matrix protein (COMP) and GAGs, compared to the not-compressed explants. Finally, the application of the CS-Tyr/Gel/BM-MSCs composite on the top of the OA cartilage explants decreased the release of COMP and GAGs from the cartilage explants. Data suggest that the CS-Tyr/Gel/BM-MSCs composite can protect the OA cartilage explants from the damaging effects of external mechanical stimuli. Therefore, it can be used for investigation of OA cartilage regenerative potential and mechanisms under the mechanical load in vitro with further perspectives of therapeutic application in vivo.

Galangin ameliorates osteoarthritis progression by attenuating extracellular matrix degradation in chondrocytes via the activation of PRELP expression

Osteoarthritis (OA) is primarily characterized by progressive degeneration and destruction of articular cartilage. Currently, there is no effective method to treat OA. The metabolic disturbance of cartilage extracellular matrix (ECM) and oxidative stress are critical to promote OA progression. Galangin (Gal) is a small molecule compound that pertains to flavonoids. To determine the protective effect and mechanism of Gal against OA progression, various experiments were performed in vitro and in vivo. In vitro, Gal promoted ECM production and attenuated ECM degradation in human OA chondrocytes. The expression of ECM components from human OA cartilage explants was also stimulated by Gal treatment. As demonstrated from the in vivo study, the intra-articular injection of Gal delayed OA progression in rat models. Moreover, RNA sequencing analysis showed that proline/arginine-rich end leucine repeat protein (PRELP) was a molecular target of Gal activity. Gal inhibited oxidative stress and attenuated ECM degradation by activating PRELP expression. The study demonstrated that Gal could attenuate ECM degradation and ameliorate OA progression, and PRELP may be a potential candidate drug of Gal for treating OA.

Fatty acid sensing GPCR (GPR84) signaling safeguards cartilage homeostasis and protects against osteoarthritis

It is well known that free fatty acids (FFAs) have beneficial effects on the skeletal system, however, which fatty acid sensing GPCR(s) and how the GPCR(s) regulating cartilage development and osteoarthritis (OA) pathogenesis is largely unknown. In this study, we found Gpr84, a receptor for medium-chain FFAs (MCFA), was the only FFA-sensing GPCR in human and mouse chondrocytes that exhibited elevated expression when stimulated by interleukin (IL)-1β. Gpr84-deficiency upregulated cartilage catabolic regulator expression and downregulated anabolic factor expression in the IL-1β-induced cell model and the destabilization of the medial meniscus (DMM)-induced OA mouse model. Gpr84−/− mice exhibited an aggravated OA phenotype characterized by severe cartilage degradation, osteophyte formation and subchondral bone sclerosis. Moreover, activating Gpr84 directly enhanced cartilage extracellular matrix (ECM) generation while knockout of Gpr84 suppressed ECM-related gene expression. Especially, the agonists of GPR84 protected human OA cartilage explants against degeneration by inducing cartilage anabolic factor expression. At the molecular level, GPR84 activation inhibited IL-1β-induced NF-κB signaling pathway. Furthermore, deletion of Gpr84 had little effect on articular and spine cartilaginous tissues during skeletal growth. Together, all of our results demonstrated that fatty acid sensing GPCR (Gpr84) signaling played a critical role in OA pathogenesis, and activation of GPR84 or MCFA supplementation has potential in preventing the pathogenesis and progression of OA without severe cartilaginous side effect.

Imperatorin suppresses IL-1β-induced iNOS expression via inhibiting ERK-MAPK/AP1 signaling in primary human OA chondrocytes

Joint inflammation is a key player in the pathogenesis of osteoarthritis (OA). Imperatorin, a plant-derived small molecule has been reported to have anti-inflammatory properties; however, its effect on chondrocytes is not known. Here, we investigated the effects of Imperatorin on interleukin-1β (IL-1β) induced expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in primary human OA chondrocytes and cartilage explants culture under pathological conditions and explored the associated signaling pathways. We pretreated chondrocytes or explants with Imperatorin (50 μM) followed by IL-1β (1 ng/ml), and the culture supernatant was used to determine the levels of nitrite production by Griess assay and chondrocytes were harvested to prepare cell lysate or RNA for gene expression analysis of iNOS by Western blot or qPCR and in explants by immunohistochemistry (IHC). Pretreatment of primary chondrocytes and cartilage explants with Imperatorin suppressed IL-1β induced expression of iNOS and NO production. Imperatorin blocked the IL-1β-induced phosphorylation of ERK-MAPK/AP1 signaling pathway to suppress iNOS expression. The role of ERK in the regulation of iNOS expression was verified by using ERK inhibitor. Interestingly, we also found that Imperatorin binds to iNOS protein and inhibits its activity in vitro. Our data demonstrated that Imperatorin possess strong anti-inflammatory activity and may be developed as a therapeutic agent for the management of OA.

Osteoarthritis treatment with a novel nutraceutical acetylated ligstroside aglycone, a chemically modified extra-virgin olive oil polyphenol

Recent studies have shown that dietary patterns confer protection from certain chronic diseases related to oxidative stress, the immune system and chronic low-grade inflammatory diseases. The aim of this study was to evaluate the anti-inflammatory potential and the capacity to attenuate cartilage degradation using extra-virgin olive oil–derived polyphenols for the treatment of osteoarthritis. Results show that both nutraceuticals ligstroside aglycone and acetylated ligstroside aglycone showed an anti-inflammatory profile. Acetylated ligstroside aglycone significantly reduced the expression of pro-inflammatory genes including NOS2 and MMP13 at both RNA and protein levels; decreased nitric oxide release; and, importantly, reduced proteoglycan loss in human osteoarthritis cartilage explants. Our study demonstrated that a new synthetic acetylated ligstroside aglycone derivative offers enhanced anti-inflammatory profile than the natural nutraceutical compound in osteoarthritis. These results substantiate the role of nutraceuticals in osteoarthritis with implications for therapeutic intervention and our understanding of osteoarthritis pathophysiology.

Cartilage tissue engineering combining microspheroid building blocks and microneedle arrays

ABSTRACTPurpose: Scaffold-free cartilage tissue engineering circumvents issues with scaffold seeding, potential toxicity response, and impaired host integration. However, precisely controlling and maintaining a scaffold-free construct shape have been challenging. We explored the feasibility of microneedle arrays to print tissue using cellular microspheroids as building blocks.Materials and Methods: Human embryonic-derived mesenchymal stem cells or infrapatellar fat pad mesenchymal stem cells were used to create microspheroids of 500 µm in diameter, which were assembled on microneedle arrays in a predefined arrangement using a robotic system under computer vision. Microspheroids on microneedles were cultured to permit fusion into a tissue construct. Infrapatellar fat pad mesenchymal stem cell constructs were either implanted into chondral defects created in human osteoarthritic cartilage explants or maintained on the microneedle array for 3 weeks. Embryonic-derived mesenchymal stem cell constructs were designed to be press-fit into 3 mm subchondral defects in New Zealand White rabbits and maintained for up to 8 weeks to assess retention, early tissue repair, and more mature cartilage regeneration.Results: Microspheroids of both cell types fused together in culture to form neotissues of predefined shape and size. Infrapatellar fat pad mesenchymal stem cell neotissues expressed high levels of chondrogenic genes and integrated with the surrounding osteoarthritic host cartilage. Embryonic-derived mesenchymal stem cell constructs generated chondrogenic neotissue in vivo as early as 2 weeks and more mature tissue by 8 weeks with increased glycosaminoglycan deposition.Conclusions: We constructed defined scaffold-free shapes by bioprinting and fusing microspheroids. Proof of concept was shown in the repair of ex vivo osteoarthritic human cartilage and in vivo rabbit osteochondral (OC) defects.

Biomimetic proteoglycans diffuse throughout articular cartilage and localize within the pericellular matrix.

Biomimetic proteoglycan (BPG) diffusion into articular cartilage has the potential to restore the lost proteoglycan content in osteoarthritic cartilage given these molecules mimic the structure and properties of natural proteoglycans. We examined the diffusion characteristics of BPGs through cartilage with the use of a custom-made in vitro cartilage diffusion model in both normal bovine and human osteoarthritic cartilage explants. BPGs were introduced into the cartilage through essentially one-dimensional diffusion using osteochondral plugs. The molecular diffusion was shown to be size and concentration dependent. Diffusion profiles were found over different diffusion time intervals and the profiles were fit to a nonlinear Fickian diffusion model. Steady state 011012-7diffusion coefficients for BPGs were found to be 4.01 and 3.53 μm2 /s for 180 and 1600 kDa BPGs, respectfully, and these values are similar to other large molecule diffusion in cartilage. In both bovine and osteoarthritic human cartilage, BPGs were found localized around the chondrocytes. BPG localization was examined by labeling collagen type VI and soaking 5 μm thick sections of cartilage with BPG solutions demonstrating that the BPGs diffused into the cartilage and preferentially localized alongside collagen type VI in the pericellular matrix.

A small molecule promotes cartilage extracellular matrix generation and inhibits osteoarthritis development

Degradation of extracellular matrix (ECM) underlies loss of cartilage tissue in osteoarthritis, a common disease for which no effective disease-modifying therapy currently exists. Here we describe BNTA, a small molecule with ECM modulatory properties. BNTA promotes generation of ECM components in cultured chondrocytes isolated from individuals with osteoarthritis. In human osteoarthritic cartilage explants, BNTA treatment stimulates expression of ECM components while suppressing inflammatory mediators. Intra-articular injection of BNTA delays the disease progression in a trauma-induced rat model of osteoarthritis. Furthermore, we identify superoxide dismutase 3 (SOD3) as a mediator of BNTA activity. BNTA induces SOD3 expression and superoxide anion elimination in osteoarthritic chondrocyte culture, and ectopic SOD3 expression recapitulates the effect of BNTA on ECM biosynthesis. These observations identify SOD3 as a relevant drug target, and BNTA as a potential therapeutic agent in osteoarthritis.

IL37 ameliorates experimental murine osteoarthritis

Purpose: Osteoarthritis (OA) is the world’s most common degenerative joint disorder and leads to pain and disability. Currently, no disease-modifying drugs are available, resulting in a large unmet clinical need. Recently, we have shown that the anti-inflammatory cytokine IL37 lowers pro-inflammatory cytokine production, MMP3 expression and sulfated glycosaminoglycan loss in ex vivo cultured chondrocytes and human OA cartilage explants. In this study, we further explored the ability of IL37 to ameliorate OA pathology by studying its effects both in vitro on synovial fibroblasts and in vivo in experimental OA in mice. Methods: Primary human synovial fibroblasts were obtained by enzymatic digestion of synovial biopsies of 10 patients undergoing total joint replacement surgery. IL37 was overexpressed in these fibroblasts using an adenovirus (Ad-IL37) and subsequently cells were challenged with IL1β or OA-synovium conditioned medium (OAS-cm). Experimental OA was induced by a single intra-articular injection of 3 units of collagenase type VII in the right knee joint of 10 weeks old female C57BL/6J mice, i.e. collagenase-induced OA (CiOA). Four days after collagenase injection, Ad-IL37 or Ad-Luc (= control) was injected in the same joint, and after two weeks this injection was repeated. Mice were sacrificed on day 7, 28 and 42 after collagenase injection. Synovial thickening, cartilage damage (OARSI score) and osteophyte formation (size) were measured histologically. IL37, MMP3 and VDIPEN expression were investigated by immunohistochemistry. Results: In primary synovial fibroblasts, IL1β- and OAS-cm-induced expression of MMP1, MMP3, IL1B and IL6 was reduced by more than 50% by IL37. Prolonged (28 days) overexpression of IL37 in naïve knee joints did not lead to synovial thickening, cartilage damage or osteophyte formation and no overt immune response was detected. Strikingly, at day 7 of CiOA, IL37 overexpression significantly reduced synovial thickness, whereas in cartilage MMP3 protein and the presence of the metalloproteinase-generated aggrecan-degradation neoepitope VDIPEN was reduced. At day 28 of CiOA, cartilage damage was significantly reduced by IL37 treatment at both the lateral and medial side of the femur by 46% and 41%, respectively. Furthermore, osteophyte size at the medial femur was reduced by 70%. At day 42 of CiOA, IL37 overexpression could no longer be detected, in contrast to day 7 and 28. Osteophyte size formation at the medial femur was still significantly reduced by 46%, but only a trend (p = 0.06) towards reduced cartilage damage was observed on both femur and tibia at this time point. Conclusions: Our results show that IL37 inhibits the production of catabolic mediators in OA synovial fibroblasts, similar to what we previously observed in cartilage and chondrocytes. Furthermore, IL37 clearly lowered synovial thickening and ameliorated cartilage damage and osteophyte formation in murine experimental OA. These results indicate that IL37 acts on multiple OA-related processes in multiple joint tissues, which supports IL37 as a potential therapeutic agent for OA.

MicroRNA-181a-5p antisense oligonucleotides attenuate osteoarthritis in facet and knee joints

ObjectivesWe recently identified microRNA-181a-5p (miR-181a-5p) as a critical mediator involved in the destruction of lumbar facet joint (FJ) cartilage. In this study, we tested if locked nucleic acid (LNA) miR-181a-5p...

BMP-7 enhances ribosomal RNA levels in chondrocytes via BAPX1-NKX3.2

Purpose: During osteoarthritis (OA) progression the articular chondrocyte undergoes a phenotypic switch in which the chondrocyte acquires a catabolic and hypertrophy-like state. Bone morphogenetic protein (BMP)-7 (or OP-1) is known for its anti-catabolic and pro-anabolic properties in cartilage repair and in OA chondrocytes. In its anabolic state the chondrocyte’s metabolism and protein synthesis are up-regulated. In order to meet this higher demand of protein synthesis, it is expected that the translational capacity of the chondrocyte is increased after exposure to BMP-7. The cellular availability of maturated ribosomal RNAs (rRNA) is rate-limiting in the assembly of ribosomes and previously it has been shown that BMP-7 treatment resulted in increased expression levels of bagpipe homeobox homolog 1 (BAPX-1/NKX3.2). We therefore hypothesize that BMP-7 enhances the translational capacity of articular chondrocytes via BAPX-1/NKX3.2-dependent synthesis of rRNAs. Methods: OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty. SW1353 cells and OA HACs were exposed to BMP-7 (1 nM) and expression levels of rRNAs (18S, 5.8S, 28S) rRNA processing snoRNAs (RMRP and U3), a crucial co-factor in rRNA transcription (UBF-1) and BAPX-1/NKX3.2 were determined by RT-qPCR (and immunoblotting for BAPX-1/NKX3.2). BAPX-1/NKX3.2 overexpression and knockdown were achieved via transfection of a FLAG-BAPX-1/NKX3.2 plasmid or a BAPX-1/NKX3.2 siRNA duplex. Human OA cartilage explants from total knee arthroplasty were exposed to BMP-7 (1 nM) and gene expression levels of rRNAs were measured via qPCR. Alterations in rRNA gene transcription were determined by transfecting SW1353 cells and OA HACs with a 47S promoter reporter plasmid (Cypridina luciferase) to determine transcriptional activity of the 47S pre-rRNA gene. Translational capacity of the cells was directly determined via puromycilation assays. Results: BMP-7 treatment resulted in increased 18S and 5.8S rRNA levels, increased UBF-1, RMRP and U3 expression. This correlated with increased BAPX-1/NKX3.2 mRNA and protein expression. Overexpression of BAPX-1/NKX3.2 resulted in increased rRNA expression levels and the reciprocal knockdown of BAPX-1/NKX3.2 resulted in decreased rRNA expression levels. Exposure of OA cartilage explants to BMP-7 confirmed our in vitro data (increase of 18S, 5.8S, UBF-1, RMRP, U3 and BAPX-1 expression levels). Treatment of chondrocytic cells with BMP-7 increased 47S pre-rRNA transcription as well as overall translational capacity. Conclusions: Here we show that BMP-7 induces increased cellular levels of maturated rRNAs, with concomitant induction of factors involved in the transcription and maturation of rRNAs. This process is directly influenced by BAPX-1/NKX3.2 in similar ways as BMP-7. In line with these results, 47S pre-rRNA transcription is increased after exposure to BMP-7, as well as the overall translational capacity. In future research the functionality of increased transcriptional activity of the 47S pre-rRNA gene will be determined by quantification of ribosome levels after BMP-7 exposure. Our data provide important novel insight into the mechanism behind the anabolic properties of BMP-7 and may provide a new molecular cue to target the chondrocyte phenotype in OA.

Targeting β-catenin dependent Wnt signaling via peptidomimetic inhibitors in murine chondrocytes and OA cartilage

The canonical Wnt signaling pathway has been shown to be involved in regulating chondrocyte hypertrophic differentiation during Osteoarthritis (OA). The aim of this study was to test the therapeutic potential of two stapled peptide canonical Wnt inhibitors - SAH-Bcl9 and StAx-35R - in preventing Wnt induced cartilage changes in OA. Primary neonatal murine chondrocytes and cartilage explants from OA patients undergoing total joint replacement for knee OA, were used for microscopy to determine matrix and cell penetrating capacity of fluorescein isothiocyanate FITC-tagged SAH-Bcl9 and StAx-35R peptides. T cell factor/lymphoid enhancer-binding factor (TCF/LEF) reporter assays were used to monitor the inhibition of Wnt3a induced β-catenin signaling by each peptide. Changes in chondrocyte phenotypic marker gene expression were analyzed by qRT PCR. Both peptides localized intercellular in primary murine chondrocytes and cartilage explants. They inhibited Wnt3a induced TCF/LEF promoter activity in primary murine chondrocytes. Both inhibitors did not rescue Wnt3a altered expression of chondrocyte phenotypic genes (Sox9, Col2a1, Acan) and hypertrophy marker gene (Col10a1) at high doses (100ng/ml). Upon application of 10ng/ml Wnt3a, StAx-35R partially reversed the Wnt effect on Sox9 and Col2a1 gene expression. Both peptides, however, reversed the downregulation of SOX9 and aggrecan (ACAN), and decrease of COL10A1 gene expression in preserved human OA cartilage explants. These data indicate that blockade of canonical Wnt signaling might be a therapeutic strategy to treat early OA cases and protect further cartilage degradation by preventing chondrocyte hypertrophic differentiation.

Hyaluronic Acid-Based Optical Probe for the Diagnosis of Human Osteoarthritic Cartilage.

Osteoarthritis is typically caused by cartilage injury, followed by localized inflammatory responses and tissue deterioration. Early treatment of osteoarthritis is often impossible due to the lack of diagnostic options. Recent studies have supported that different imaging probes can be used for arthritis detection in mice. However, none of these diagnostic tools have been tested on human articular cartilage. To fill this gap, an optical imaging probe was developed to target activated macrophages and the accumulation of imaging probes on tissue was used to assess the severity of human osteoarthritis.Methods: The probe was fabricated using hyaluronic acid (HA) particles conjugated with near-infrared dye and folic acid (FA). The ability of the FA-HA probes to detect activated macrophages and quantify cartilage injury was evaluated using a cell culture model in vitro and human osteoarthritic cartilage explants ex vivo.Results: Our cell study results supported that the FA-HA probes are cell compatible (up to 0.5mg/mL) and can detect activated macrophages in 30 minutes. Using human articular cartilage, we verified the existence of activated macrophages on osteoarthritic cartilage with highly up-regulated expression of folate receptors (~13 folds by comparison with healthy control). In addition, we found that FA-HA probes had higher binding amounts (~3 folds) to osteoarthritic tissue than healthy ones. Histological analyses confirmed that there was a strong linear relationship (R=0.933) between the fluorescent intensity of tissue-associated probe and the extent of folate receptors on osteoarthritic cartilage. Finally, the co-localization of the imaging probe, folate receptors and cartilage degeneration on the tissue sections indicated the extraordinary accuracy and efficiency of this osteoarthritis diagnostic probe.Conclusions: Our results support the probe as an effective diagnostic tool to detect the area and severity of osteoarthritic human articular cartilage.

Matriptase Induction of Metalloproteinase-Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms.

ObjectiveTo assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA.MethodsAggrecan release from human OA cartilage explants and human stem cell–derived cartilage discs was evaluated, and cartilage‐conditioned media were used for Western blotting. Gene expression was analyzed by real‐time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies.ResultsThe addition of soluble recombinant matriptase promoted a time‐dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease‐activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase– and aggrecanase‐mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low‐density lipoprotein receptor–related protein 1 (LRP‐1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase‐mediated aggrecan cleavage.ConclusionMatriptase potently induces the release of metalloproteinase‐generated aggrecan fragments as well as soluble LRP‐1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease‐modifying therapy in OA.

Wogonin, a plant derived small molecule, exerts potent anti-inflammatory and chondroprotective effects through the activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes

Osteoarthritis (OA), characterized by progressive destruction of articular cartilage, is the most common form of human arthritis. Here, we evaluated the potential chondroprotective and anti-inflammatory effects of Wogonin, a naturally occurring flavonoid, in IL-1β-stimulated human OA chondrocytes and cartilage explants. Wogonin completely suppressed the expression and production of inflammatory mediators including IL-6, COX-2, PGE2, iNOS and NO in IL-1β-stimulated OA chondrocytes. Further, Wogonin exhibits potent chondroprotective potential by switching the signaling axis of matrix degradation from catabolic towards anabolic ends and inhibited the expression, production and activities of matrix degrading proteases including MMP-13, MMP-3, MMP-9, and ADAMTS-4 in OA chondrocytes, and blocked the release of s-GAG and COL2A1 in IL-1β-stimulated OA cartilage explants. Wogonin also elevated the expression of cartilage anabolic factors COL2A1 and ACAN in chondrocytes and inhibited the IL-1β-mediated depletion of COL2A1 and proteoglycan content in the matrix of cartilage explants. The suppressive effect of Wogonin was not mediated through the inhibition of MAPKs or NF-κB activation. Instead, Wogonin induced mild oxidative stress through the generation of ROS and depletion of cellular GSH, thereby modulating the cellular redox leading to the induction of Nrf2/ARE pathways through activation of ROS/ERK/Nrf2/HO-1-SOD2-NQO1-GCLC signaling axis in OA chondrocytes. Molecular docking studies revealed that Wogonin can disrupt KEAP-1/Nrf-2 interaction by directly blocking the binding site of Nrf-2 in the KEAP-1 protein. Genetic ablation of Nrf2 using specific siRNA, significantly abrogated the anti-inflammatory and chondroprotective potential of Wogonin in IL-1β-stimulated OA chondrocytes. Our data indicates that Wogonin exerts chondroprotective effects through the suppression of molecular events involved in oxidative stress, inflammation and matrix degradation in OA chondrocytes and cartilage explants. The study provides novel insights into the development of Nrf2 as a promising candidate and Wogonin as a therapeutic agent for the management of OA.

Cartilage inflammation and degeneration is enhanced by pro-inflammatory (M1) macrophages in vitro, but not inhibited directly by anti-inflammatory (M2) macrophages

Macrophages play a crucial role in the progression of osteoarthritis (OA). Their phenotype may range from pro-inflammatory to anti-inflammatory. The aim of this study was to evaluate the direct effects of macrophage subtypes on cartilage by culturing macrophage conditioned medium (MCM) on human articular cartilage. Human OA cartilage explants were cultured with MCM of pro-inflammatory M(IFNγ+TNFα), or anti-inflammatory M(IL-4) or M(IL-10) human monocyte-derived macrophages. To assess effects of anti-inflammatory macrophages, the cartilage was cultured with a combination of MCM phenotypes as well as pre-stimulated with IFNγ+TNFα cartilage before culture with MCM. The reactions of the explants were assessed by gene expression, nitric oxide (NO) production and release of glycosaminoglycans (GAGs). M(IFNγ+TNFα) MCM affected OA cartilage by upregulation of IL1B (Interleukin 1β), IL6, MMP13 (Matrix Metalloproteinase-13) and ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-5), while inhibiting ACAN (aggrecan) and COL2A1 (collagen type II). M(IL-10) upregulated IL1B and Suppressor of cytokine signaling 1 (SOCS1). NO production and GAG release by the cartilage was increased when cultured with M(IFNγ+TNFα) MCM. M(IL-4) and M(IL-10) did not inhibit the effects of M(IFNγ+TNFα) MCM of neither phenotype affected IFNγ+TNFα pre-stimulated cartilage, in which an inflammatory gene response was deliberately induced. M(IFNγ+TNFα) macrophages have a prominent direct effect on OA cartilage, while M(IL-4) and M(IL-10) do not inhibit the effects of M(IFNγ+TNFα), or IFNγ+TNFα induced inflammation of the cartilage. Therapies aiming at inhibiting cartilage degeneration may take this into account by directing suppression of pro-inflammatory macrophages or stimulation of anti-inflammatory macrophages.

RAAV-mediated combined gene transfer and overexpression of TGF-β and SOX9 remodels human osteoarthritic articular cartilage

Direct administration of therapeutic candidate gene sequences using the safe and effective recombinant adeno-associated virus (rAAV) vectors is a promising strategy to stimulate the biologic activities of articular chondrocytes as an adapted tool to treat human osteoarthritic (OA) cartilage. In the present study, we developed a combined gene transfer approach based on the co-delivery of the pleiotropic transformation growth factor beta (TGF-β) with the specific transcription factor SOX9 via rAAV to human normal and OA chondrocytes in vitro and cartilage explants in situ in light of the mitogenic and pro-anabolic properties of these factors. Effective, durable co-overexpression of TGF-β and SOX9 significantly enhanced the levels of cell proliferation both in human normal and OA chondrocytes and cartilage explants over an extended period of time (21 days), while stimulating the biosynthesis of key matrix components (proteoglycans, type-II collagen) compared with control conditions (reporter lacZ gene transfer, absence of vector treatment). Of further note, expression of hypertrophic type-X collagen significantly decreased following co-treatment by the candidate vectors. The present findings show the value of combining the transfer and expression of potent candidate factors in human OA cartilage as a means to re-establish essential features of normal cartilage and counteract the pathological shift of homeostasis. These observations support the concept of developing dual therapeutic rAAV gene transfer strategies as future, adapted tools for the direct treatment of human OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2181-2190, 2016.