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Objective: Chronic inflammation plays an important role in the osteoarthritis (OA) pathology but how this influence OA disease progression is unclear. Leukotriene B4 (LTB 4 ) is a potent proinflammatory lipid mediator generated from arachidonic acid through the sequential activities of 5-lipoxygenase, 5-lipoxygenase-activating protein, Leukotriene A 4 hydrolase (LTA 4 H) and its downstream product LTB 4 . The aim of this study is to investigate the involvement and the potential therapeutic target of the LTB 4 pathway in OA disease progression., Design: Both clinical human cartilage samples ( n = 7) and mice experimental OA models ( n = 6) were used. The levels of LTA 4 H and leukotriene B4 receptor 1 were first examined using immunostaining in human OA/non-OA cartilage and mice experimental OA models. We also determined whether the LTA 4 H pathway was associated with cartilage degeneration and synovitis inflammation in OA mice models and human articular chondrocytes., Results: We found that both LTA 4 H and LTB 4 receptor (BLT1) were highly expressed in human and mice OA cartilage. Inhibition of LTA 4 H suppressed cartilage degeneration and synovitis in OA mice model. Furthermore, inhibition of LTA 4 H promoted cartilage regeneration by upregulating chondrogenic genes expression such as aggrecan ( ACAN ), collagen 2A1 ( COL2A1 ), and SRY-Box transcription factor 9 ( SOX9 )., Conclusions: Our results indicate that the LTA 4 H pathway is a crucial regulator of OA pathogenesis and suggest that LTA 4 H could be a therapeutic target in combat OA., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Altered subchondral bone and articular cartilage interactions have been implicated in the pathogenesis of osteoarthritis (OA); however, the mechanisms remain unknown. Exosomes are membrane-derived vesicles that have recently been recognized as important mediators of intercellular communication. Herein, we investigated if OA subchondral bone derived exosomes alter transcriptional and bioenergetic signatures of chondrocytes. Exosomes were isolated and purified from osteoblasts of nonsclerotic or sclerotic zones of human OA subchondral bone and their role on the articular cartilage chondrocytes was evaluated by measuring the extent of extracellular matrix production, cellular bioenergetics, and the expression of chondrocyte activity associated marker genes. Exosomal microRNAs were analyzed using RNA sequencing and validated by quantitative real-time PCR and loss-of-function. In coculture studies, chondrocytes internalized OA sclerotic subchondral bone osteoblast derived exosomes and triggered catabolic gene expression and reduced chondrocyte-specific marker expression a phenomenon that is often observed in OA cartilage. RNA sequencing and miRNA profiling have identified miR-210-5p, which is highly enriched in OA sclerotic subchondral bone osteoblast exosomes, triggered the catabolic gene expression in articular cartilage chondrocytes. Importantly, we demonstrate that miR-210-5p suppresses the oxygen consumption rate of chondrocytes, altering their bioenergetic state that is often observed in OA conditions. These effects were markedly inhibited by the addition of a miR-210-5p inhibitor. Our study indicates that exosomes released by OA sclerotic subchondral bone osteoblasts plays a critical role in progression of cartilage degeneration and might be a potential target for therapeutic intervention in OA.

Virtually all bone marrow-derived stromal cell (BMSC) chondrogenic induction cultures include greater than 2 weeks exposure to transforming growth factor-β (TGF-β), but fail to generate cartilage-like tissue suitable for joint repair. Herein we used a micro-pellet model (5 × 10 3 BMSC each) to determine the duration of TGF-β1 exposure required to initiate differentiation machinery, and to characterize the role of intrinsic programming. We found that a single day of TGF-β1 exposure was sufficient to trigger BMSC chondrogenic differentiation and tissue formation, similar to 21 days of TGF-β1 exposure. Despite cessation of TGF-β1 exposure following 24 hours, intrinsic programming mediated further chondrogenic and hypertrophic BMSC differentiation. These important behaviors are obfuscated by diffusion gradients and heterogeneity in commonly used macro-pellet models (2 × 10 5 BMSC each). Use of more homogenous micro-pellet models will enable identification of the critical differentiation cues required, likely in the first 24-hours, to generate high quality cartilage-like tissue from BMSC.

Objectives: Obesity is a known strong risk factor for the onset of knee osteoarthritis and is often accompanied by dysregulated lipid metabolism with elevated levels of free fatty acids such as saturated fatty acids (SFAs). The purpose of this study was to determine how autophagy varies in chondrocytes in response to predominant SFAs such as lauric, myristic, palmitic, and stearic acids., Methods: Normal human articular cartilage chondrocytes and C28/I2 chondrocyte cell lines were stimulated with different SFAs in both the absence and presence of interleukin-1β to study the effects of SFA and inflammatory cytokines in mediating the activation of autophagy. The effects of rapamycin and LY290042 on autophagy in response to different SFAs were also assessed., Results: Palmitic and stearic acid stimulation of chondrocytes resulted in increased activation of both autophagy and the canonical NFκB pathway as evidenced by increased expressions of the key autophagy markers microtubule-associated protein-1 light chain 3, autophagy-related 5, beclin-1, and NFκB p65. In contrast, lauric acid stimulation resulted in decreased autophagy activation as shown by decreased expressions of microtubule-associated protein-1 light chain 3, autophagy-related 5, and beclin-1, which suggests decreased cellular stress., Conclusions: These results represent a novel mechanism by which various SFAs activate autophagy and simultaneously modulate NFκB signaling pathways and the expression of chondrocyte regulatory genes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Of the many cell-based treatments that have been tested in an effort to regenerate osteoarthritic articular cartilage, none have ever produced cartilage that compare with native hyaline cartilage. Studies show that different cell types lead to inconsistent results and for cartilage regeneration to be considered successful, there must be an absence of fibrotic tissue. Here we report of a series of experiments in which bone marrow-derived stem cells (BMSCs) and articular cartilage chondrocytes (ACCs) were mixed in a 1:1 ratio and tested for their ability to enhance cartilage regeneration in three different conditions: (1) in an in vitro differentiation model; (2) in an ex vivo cartilage defect model implanted subcutaneously in mice; and (3) as an intra-articular injection in a meniscectomy-induced OA model in rats. The mixed cells were compared with monocultures of BMSCs and ACCs. In all three experimental models there was significantly enhanced cartilage regeneration and decreased fibrosis in the mixed BMSCs+ACCs group compared with the monocultures. Molecular analysis showed a reduction in vascularization and hypertrophy, coupled with higher chondrogenic gene expression resulting from the BMSCs+ACCs treatment. Together, our data suggest that mixed BMSCs+ACCs treatment is highly chondro-protective and is more effective in regenerating damaged cartilage in both the ex vivo cartilage defect and post-trauma OA disease models. The results from this approach could potentially be used for regeneration of cartilage in OA patients.

The predominant saturated fatty acids (SFA) in human diets are lauric acid (LA, C12:0), myristic acid (MA, C14:0), palmitic acid (PA, C16:0) and stearic acid (SA, C18:0). The aim of this study was to investigate whether diets containing individual SFA together with excess simple carbohydrates induce osteoarthritis (OA)-like changes in knee joints and signs of metabolic syndrome in rats. Rats were given either a corn starch diet or a diet composed of simple carbohydrates together with 20% LA, MA, PA, SA or beef tallow for 16 weeks. Rats fed beef tallow, SA, MA or PA diets developed signs of metabolic syndrome, and also exhibited cartilage degradation and subchondral bone changes similar to OA. In contrast, replacement of beef tallow with LA decreased signs of metabolic syndrome together with decreased cartilage degradation. Furthermore, PA and SA but not LA increased release of matrix sulphated proteoglycans in cultures of bovine cartilage explants or human chondrocytes. In conclusion, we have shown that longer-chain dietary SFA in rats induce both metabolic syndrome and OA-like knee changes. Thus, diets containing SFA are strongly relevant to the development or prevention of both OA and metabolic syndrome.

Objective: The aim of this study was to test the possible involvement, relevance and significance of dentin matrix protein 1 (DMP1) in chondrocyte redifferentiation and OA., Methods: To examine the function of DMP1 in vitro, bone marrow stromal cells (BMSCs) and articular chondrocytes (ACs) were isolated and differentiated in micromasses in the presence or absence of DMP1 small interfering RNA and analysed for chondrogenic phenotype. The association of DMP1 expression with OA progression was analysed time dependently in the OA menisectomy rat model and in grade-specific OA human samples., Results: It was found that DMP1 was strongly related to chondrogenesis, which was evidenced by the strong expression of DMP1 in the 14.5-day mouse embryonic cartilage development stage and in femoral heads of post-natal days 0 and 4. In vitro chondrogenesis in BMSCs and ACs was accompanied by a gradual increase in DMP1 expression at both the gene and protein levels. In addition, knockdown of DMP1 expression led to decreased chondrocyte marker genes, such as COL2A1, ACAN and SOX9, and an increase in the expression of COL10A and MMP13 in ACs. Moreover, treatment with IL-1β, a well-known catabolic culprit of proteoglycan matrix loss, significantly reduced the expression of DMP1. Furthermore, we also observed the suppression of DMP1 protein in a grade-specific manner in knee joint samples from patients with OA. In the menisectomy-induced OA model, an increase in the Mankin score was accompanied by the gradual loss of DMP1 expression., Conclusion: Observations from this study suggest that DMP1 may play an important role in maintaining the chondrogenic phenotype and its possible involvement in altered cartilage matrix remodelling and degradation in disease conditions like OA., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×10(5)-5×10(5) chondrocytes are aggregated, resulting in "macro" pellets having diameters ranging from 1-2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1-2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×10(5) human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.

Articular cartilage is organized in depth zones with phenotypically distinct subpopulations of chondrocytes that are exposed to different oxygen tensions. Despite growing evidence of the critical role for oxygen in chondrogenesis, little is known about its effect on chondrocytes from different zones. This study evaluates zonal marker expression of human articular chondrocytes from different zones under various oxygen tensions. Chondrocytes isolated from full-thickness, superficial, and middle/deep cartilage from knee replacement surgeries were expanded and redifferentiated under hypoxic (5% O(2)) or normoxic (20% O(2)) conditions. Differentiation under hypoxia increased expression of hypoxia-inducible factors 1alpha and 2alpha and accumulation of extracellular matrix, particularly in middle/deep chondrocytes, and favored re-expression of proteoglycan 4 by superficial chondrocytes compared with middle/deep cells. Zone-dependent expression of clusterin varied with culture duration. These results demonstrate that zonal chondrocytes retain important phenotypic differences during in vitro cultivation, and that these characteristics can be improved by altering the oxygen environment. However, transcript levels for pleiotrophin, cartilage intermediate layer protein, and collagen type X were similar between zones, challenging their reliability as zonal markers for tissue-engineered cartilage from osteoarthritis patients. Key factors including oxygen tension and cell source should be considered to prescribe zone-specific properties to tissue-engineered cartilage.

Objectives: The p38 mitogen-activated protein kinase (MAPK) signal transduction pathway is involved in a variety of inflammatory responses, including cytokine generation, cell differentiation proliferation and apoptosis. Here, we examined the effects of systemic p38 MAPK inhibition on cartilage cells and OA disease progression by both in vitro and in vivo approaches., Methods: p38 kinase activity was evaluated in normal and OA cartilage cells by measuring the amount of phosphorylated protein. To examine the function of p38 signalling pathway in vitro, normal chondrocytes were isolated and differentiated in the presence or absence of p38 inhibitor SB203580 and analysed for chondrogenic phenotype. Effect of systemic p38 MAPK inhibition in normal and OA (induced by menisectomy) rats were analysed by treating animals with vehicle alone [dimethylsulphoxide (DMSO)] or p38 inhibitor (SB203580). Damage to the femur and tibial plateau was evaluated by modified Mankin score, histology and immunohistochemistry., Results: Our in vitro studies have revealed that a down-regulation of chondrogenic and an increase of hypertrophic gene expression occurs in the normal chondrocytes when p38 is neutralized by a pharmacological inhibitor. We further observed that the basal levels of p38 phosphorylation were decreased in OA chondrocytes compared with normal chondrocytes. These findings together indicate the importance of this pathway in the regulation of cartilage physiology and its relevance to OA pathogenesis. At the in vivo level, systematic administration of a specific p38 MAPK inhibitor, SB203580, continuously for more than a month led to a significant loss of proteoglycan, aggrecan and cartilage thickness. On the other hand, SB203580-treated normal rats showed a significant increase in Terminal dUTP nick end-labelling (TUNEL)-positive cells, cartilage hypertrophy markers such as Type 10 collagen, Runt-related transcription factor and MMP-13 and substantially induced OA-like phenotypic changes in the normal rats. In addition, menisectomy-induced OA rat models that were treated with p38 inhibitor showed aggravation of cartilage damage., Conclusions: In summary, this study has provided evidence that the component of the p38 MAPK pathway is important to maintain cartilage health, and its inhibition can lead to severe cartilage degenerative changes. The observations in this study highlight the possibility of using activators of the p38 pathway as an alternative approach in the treatment of OA.

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3α mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.

Objective: Degradative enzymes, such as A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinases (MMP), play key roles in development of osteoarthritis (OA). We investigated if crosstalk between subchondral bone osteoblasts (SBO) and articular cartilage chondrocytes (ACC) in OA alters the expression and regulation of ADAMTS5, ADAMTS4, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-13, and also tested the possible involvement of mitogen-activated protein kinase (MAPK) signaling pathway during this process., Methods: ACC and SBO were isolated from normal and OA patients. An in vitro coculture model was developed to study the regulation of ADAMTS and MMP under normal and OA joint crosstalk conditions. The MAPK-ERK inhibitor PD98059 was applied to delineate the involvement of specific pathways during this interaction process., Results: Indirect coculture of OA SBO with normal ACC resulted in significantly increased expression of ADAMTS5, ADAMTS4, MMP-2, MMP-3, and MMP-9 in ACC, whereas coculture of OA ACC led to increased MMP-1 and MMP-2 expression in normal SBO. Upregulation of ADAMTS and MMP under these conditions was correlated with activation of the MAPK-ERK1/2 signaling pathway, and addition of the MAPK-ERK inhibitor PD98059 reversed the overexpression of ADAMTS and MMP in cocultures., Conclusion: These results add to the evidence that in human OA, altered bidirectional signals between SBO and ACC significantly influence the critical features of both cartilage and bone by producing abnormal levels of ADAMTS and MMP. We have demonstrated for the first time that this altered crosstalk was mediated by the phosphorylation of MAPK-ERK1/2 signaling pathway.

Objective: Previous studies have shown the influence of subchondral bone osteoblasts (SBOs) on phenotypical changes of articular cartilage chondrocytes (ACCs) during the development of osteoarthritis (OA). The molecular mechanisms involved during this process remain elusive, in particular, the signal transduction pathways. The aim of this study was to investigate the in vitro effects of OA SBOs on the phenotypical changes in normal ACCs and to unveil the potential involvement of MAPK signaling pathways during this process., Methods: Normal and arthritic cartilage and bone samples were collected for isolation of ACCs and SBOs. Direct and indirect coculture models were applied to study chondrocyte hypertrophy under the influence of OA SBOs. MAPKs in the regulation of the cell-cell interactions were monitored by phosphorylated antibodies and relevant inhibitors., Results: OA SBOs led to increased hypertrophic gene expression and matrix calcification in ACCs by means of both direct and indirect cell-cell interactions. In this study, we demonstrated for the first time that OA SBOs suppressed p38 phosphorylation and induced ERK-1/2 signal phosphorylation in cocultured ACCs. The ERK-1/2 pathway inhibitor PD98059 significantly attenuated the hypertrophic changes induced by conditioned medium from OA SBOs, and the p38 inhibitor SB203580 resulted in the up-regulation of hypertrophic genes in ACCs., Conclusion: The findings of this study suggest that the pathologic interaction of OA SBOs and ACCs is mediated via the activation of ERK-1/2 phosphorylation and deactivation of p38 phosphorylation, resulting in hypertrophic differentiation of ACCs.

The demand for treatment strategies for damaged musculoskeletal tissue is continuously growing, especially with the increasing number of older people with degenerative diseases of the skeletal system such as osteoarthritis (OA). Because depletion of multipotent cells has been implicated in degenerative joint diseases, cell-based therapies have been proposed for tissue regeneration, especially for cartilage repair. The aim of the present study is to focus on the possibility of deriving and expanding multipotential mesenchymal stem cells (MSCs) from bone marrow samples of patients with OA by characterizing MSCs at the single cell level. Single-cell clonal cultures were established in 96-well plates by limiting dilution of bone marrow stromal cells (BMSCs) from three patients with OA. Fourteen clones were established for subsequent characterization. There was a wide variation in cell doubling times, with the time taken to reach 20 population doublings ranging from 37 days to more than 100 days. The clones were grouped into fast-growing and slow-growing clones. All except one of the fast-growing stem cell clones were tripotential. However, the slow-growing clones showed limited differentiation potential and morphological changes associated with cellular senescence with extended duration in culture. Flow cytometric analysis indicated a strong need to investigate for novel cell-surface characteristic markers of BMSCs because there was no obvious difference in the expression of the selected characteristic BMSC cell surface markers CD29, CD44, CD90, CD105, and CD166 between fast-growing and slow-growing clones. This study has demonstrated the existence of a fast-growing multipotential MSC population from bone marrow samples of patients with OA. Therefore, despite a supposedly smaller stem cell compartment in these patients, we demonstrate here that they can still yield a potentially therapeutically useful source of syngeneic MSCs.

Objectives: Epidemiological and experimental studies have established obesity to be an important risk factor for osteoarthritis (OA), however, the mechanisms underlying this link remains largely unknown. Here, we studied local inflammatory responses in metabolic-OA. Methods: Wistar rats were fed with control diet (CD) and high-carbohydrate, high-fat diet (HCHF) for period of 8 and 16 weeks. After euthanasia, the knees were examined to assess the articular cartilage changes and inflammation in synovial membrane. Further IHC was conducted to determine the macrophage-polarization status of the synovium. In addition, CD and HCHF synovial fluid was co-cultured with bone marrow-derived macrophages to assess the effect of synovial fluid inflammation on macrophage polarisation. Results: Our study showed that, obesity induced by a high-carbohydrate, high-fat (HCHF) diet is associated with spontaneous and local inflammation of the synovial membranes in rats even before the cartilage degradation. This was followed by increased synovitis and increased macrophage infiltration into the synovium and a predominant elevation of pro-inflammatory M1 macrophages. In addition, bone marrow derived macrophages, cultured with synovial fluid collected from the knees of obese rats exhibited a pro-inflammatory M1 macrophage phenotype. Conclusion: Our study demonstrate a strong association between obesity and a dynamic immune response locally within synovial tissues. Furthermore, we have also identified synovial resident macrophages to play a vital role in the inflammation caused by the HCHF diet. Therefore, future therapeutic strategies targeted at the synovial macrophage phenotype may be the key to break the link between obesity and OA. [ABSTRACT FROM AUTHOR]