Primary Human Chondrocytes Research Articles

MiR-199a-5p Reduces Chondrocyte Hypertrophy and Attenuates Osteoarthritis Progression via the Indian Hedgehog Signal Pathway.

Osteoarthritis (OA), the most common type of arthritis, is an age-associated disease, characterized by the progressive degradation of articular cartilage, synovial inflammation, and degeneration of subchondral bone. Chondrocyte proliferation is regulated by the Indian hedgehog (IHH in humans, Ihh in animals) signaling molecule, which regulates hypertrophy and endochondral ossification in the development of the skeletal system. microRNAs (miRNAs, miRs) are a family of about 22-nucleotide endogenous non-coding RNAs, which negatively regulate gene expression. In this study, the expression level of IHH was upregulated in the damaged articular cartilage tissues among OA patients and OA cell cultures, while that of miR-199a-5p was the opposite. Further investigations demonstrated that miR-199a-5p could directly regulate IHH expression and reduce chondrocyte hypertrophy and matrix degradation via the IHH signal pathway in the primary human chondrocytes. The intra-articular injection of synthetic miR-199a-5p agomir attenuated OA symptoms in rats, including the alleviation of articular cartilage destruction, subchondral bone degradation, and synovial inflammation. The miR-199a-5p agomir could also inhibit the Ihh signaling pathway in vivo. This study might help in understanding the role of miR-199a-5p in the pathophysiology and molecular mechanisms of OA and indicate a potential novel therapeutic strategy for OA patients.

Cellular adhesion and chondrogenic differentiation inside an alginate-based bioink in response to tailorable artificial matrices and tannic acid treatment

Many established bioinks fulfill important requirements regarding fabrication standards and cytocompatibility. Current research focuses on development of functionalized bioinks with an improved support of tissue-specific cell differentiation. Many approaches primarily depend on decellularized extracellular matrices or blood components. In this study, we investigated the combination of a highly viscous alginate-methylcellulose (algMC) bioink with collagen-based artificial extracellular matrix (aECM) as a finely controllable and tailorable system composed of collagen type I (col) with and without chondroitin sulfate (CS) or sulfated hyaluronan (sHA). As an additional stabilizer, the polyphenol tannic acid (TA) was integrated into the inks. The assessment of rheological properties and printability as well as hydrogel microstructure revealed no adverse effect of the integrated components on the inks. Viability, adhesion, and proliferation of bioprinted immortalized human mesenchymal stem cells (hTERT-MSC) was improved indicating enhanced interaction with the designed microenvironment. Furthermore, chondrogenic matrix production (collagen type II and sulfated glycosaminoglycans) by primary human chondrocytes (hChon) was enhanced by aECM. Supplementing the inks with TA was required for these positive effects but caused cytotoxicity as soon as TA concentrations exceeded a certain amount. Thus, combining tailorable aECM with algMC and balanced TA addition proved to be a promising approach for promoting adhesion of immortalized stem cells and differentiation of chondrocytes in bioprinted scaffolds.

Individual immune cell and cytokine profiles determine platelet-rich plasma composition

ObjectivePlatelet-rich plasma (PRP) therapy is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune cell and cytokine profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples and assessed the effect of pro-inflammatory cytokines on chondrocytes.DesignFor each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using whole blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Primary human chondrocyte 2D and 3D in vitro cultures were exposed to recombinant IFN-γ and TNF-α. Proliferation and chondrogenic differentiation were quantitatively assessed.ResultsAll three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood. Furthermore, the pro-inflammatory cytokines IFN-γ and TNF-α were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products. Chondrocyte proliferation was impaired by IFN-γ and enhanced by TNF-α treatment. Differentiation and cartilage formation were compromised upon treatment with both cytokines, resulting in altered messenger ribonucleic acid (mRNA) expression of collagen type 1A1 (COL1A1), COL2A1, and aggrecan (ACAN) as well as reduced proteoglycan content.ConclusionsIndividuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy, especially as IFN-γ and TNF-α impacted primary chondrocyte proliferation and their characteristic gene expression profile. Both the individual’s immune profile and the concentration method appear to impact the final PRP product.Trial registrationThis study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4 November 2021 (registration number DRKS00026175).

Ribosomal RNA-based epitranscriptomic regulation of chondrocyte translation and proteome in osteoarthritis

Osteoarthritis-related cartilage extracellular matrix remodelling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n=5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analysed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9% to 14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33% to 64%]). This resulted in an altered ribosome translation modus (e.g CrPV IRES, FC 3, 95% CI [2.2 to 4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3 to 2.0]). Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.

Generation of human immortalized chondrocytes from osteoarthritic and healthy cartilage

After a few passages of in vitro culture, primary human articular chondrocytes undergo senescence and loss of their phenotype. Most of the available chondrocyte cell lines have been obtained from cartilage tissues different from diarthrodial joints, and their utility for osteoarthritis (OA) research is reduced. Thus, the goal of this research was the development of immortalized chondrocyte cell lines proceeded from the articular cartilage of patients with and without OA. Using telomerase reverse transcriptase (hTERT) and SV40 large T antigen (SV40LT), we transduced primary OA articular chondrocytes. Proliferative capacity, degree of senescence, and chondrocyte surface antigen expression in transduced chondrocytes were evaluated. In addition, the capacity of transduced chondrocytes to synthesize a tissue similar to cartilage and to respond to interleukin (IL)-1β was assessed. Coexpression of both transgenes (SV40 and hTERT) were observed in the nuclei of transduced chondrocytes. Generated chondrocyte cell lines showed a high proliferation capacity and less than 2% of senescent cells. These cell lines were able to form 3D aggregates analogous to those generated by primary articular chondrocytes, but were unsuccessful in synthesizing cartilage-like tissue when seeded on type I collagen sponges. However, generated chondrocyte cell lines maintained the potential to respond to IL-1β stimulation. Through SV40LT and hTERT transduction, we successfully immortalized chondrocytes. These immortalized chondrocytes were able to overcome senescence in vitro, but were incapable of synthesizing cartilage-like tissue under the experimental conditions. Nonetheless, these chondrocyte cell lines could be advantageous for OA investigation since, similarly to primary articular chondrocytes, they showed capacity to upregulate inflammatory mediators in response to the IL-1β cytokine.Cite this article: Bone Joint Res2023;12(1):46-57.

RUNX2 stabilization by long non-coding RNAs contributes to hypertrophic changes in human chondrocytes.

Background: Chondrocyte hypertrophy has been implicated in endochondral ossification and osteoarthritis (OA). In OA, hypertrophic chondrocytes contribute to the destruction and focal calcification of the joint cartilage. Although studies in this field have remarkably developed the modulation of joint inflammation using gene therapy and regeneration of damaged articular cartilage using cell therapy, studies that can modulate or prevent hypertrophic changes in articular chondrocytes are still lacking. Methods: In vitro hypertrophic differentiation and inflammation assays were conducted using human normal chondrocyte cell lines, TC28a2 cells. Human cartilage tissues and primary articular chondrocytes were obtained from OA patients undergoing total knee arthroplasty. Long non-coding RNAs (lncRNAs), LINC02035 and LOC100130207, were selected through RNA-sequencing analysis using RNAs extracted from TC28a2 cells cultured in hypertrophic medium. The regulatory mechanism was evaluated using western blotting, real-time quantitative polymerase chain reaction, osteocalcin reporter assay, RNA-immunoprecipitation (RNA-IP), RNA-in situ hybridization, and IP. Results: LncRNAs are crucial regulators of various biological processes. In this study, we identified two important lncRNAs, LINC02035 and LOC100130207, which play important roles in hypertrophic changes in normal chondrocytes, through RNA sequencing. Interestingly, the expression level of RUNX2, a master regulator of chondrocyte hypertrophy, was regulated at the post-translational level during hypertrophic differentiation of the normal human chondrocyte cell line, TC28a2. RNA-immunoprecipitation proved the potential interaction between RUNX2 protein and both lncRNAs. Knockdown (KD) of LINC02035 or LOC100130207 promoted ubiquitin-mediated proteasomal degradation of RUNX2 and prevented hypertrophic differentiation of normal chondrocyte cell lines, whereas overexpression of both lncRNAs stabilized RUNX2 protein and generated hypertrophic changes. Furthermore, the KD of the two lncRNAs mitigated the destruction of important cartilage matrix proteins, COL2A1 and ACAN, by hypertrophic differentiation or inflammatory conditions. We also confirmed that the phenotypic changes raised by the two lncRNAs could be rescued by modulating RUNX2 expression. In addition, the KD of these two lncRNAs suppressed hypertrophic changes during chondrogenic differentiation of mesenchymal stem cells. Conclusion: Therefore, this study suggests that LINC02035 and LOC100130207 contribute to hypertrophic changes in normal chondrocytes by regulating RUNX2, suggesting that these two novel lncRNAs could be potential therapeutic targets for delaying or preventing OA development, especially for preventing chondrocyte hypertrophy.

Therapeutic effect of induced pluripotent stem cell -derived extracellular vesicles in an in vitro and in vivo osteoarthritis model.

Osteoarthritis (OA) is a multifactorial joint disease associated with the deterioration of chondrocytes and inflammation. Treatment of OA is only aimed at reducing pain and improving joint function. Recently, extracellular vesicles (EVs) secreted from stem cells have emerged as a cell regenerative tool in several degenerative diseases, including OA. We hypothesised that induced pluripotent stem cell (iPSC)-derived EVs would be beneficial for regenerating chondrocytes and OA therapy. Therefore, we aimed to investigate iPSC-EVs' effects on chondrocyte behaviour in an interleukin 1 beta (IL-1β)-induced in vitro OA model and anterior cruciate ligament transection (ACLT)-induced in vivo OA model of rabbit articular cartilage. The iPSC-EVs were isolated by sequential ultracentrifugation from a 48-h-incubated conditional medium of iPSC. The isolated iPSC-EVs were characterised by transmission electron microscopy, western blot analyses, and dynamic light scatter. The effects of iPSC-EVs on the viability of human primary chondrocytes and cell senescence were analysed. Premature senescence of cells was induced by long-term incubation with low doses of hydrogen peroxide. To investigate the therapeutic effect of iPSC-EVs on OA chondrocytes in vitro, IL-1β was used to induce chondrocyte damage. Inflammatory macrophages were activated from THP-1 monocytes to observe the impact of iPSC-EV on macrophage polarisation. The phenotypes of the macrophages exposed to iPSC-EVs were evaluated by ELISA and western blot analyses. The primary chondrocytes were co-cultured with different phenotypes of macrophages to observe the expression of collagen II and catabolic enzymes in chondrocytes. iPSC-EVs were injected intraarticularly into the rabbit with an ACLT-induced OA model. The progression of lesions was assessed through macroscopic and histopathological studies. We showed that iPSC-EVs significantly stimulated the proliferation of primary human chondrocytes and suppressed cell senescence by regulating the expression of p21 and collagen II. iPSC-EVs reduced matrix degradation enzymes and IL-6 expression and attenuated IL-1β-mediated cell death of chondrocytes. Furthermore, iPSC-EVs modulated macrophage polarisation, resulting in the rescue of damaged chondrocytes in an inflammatory microenvironment. In the rabbit ACLT model, the OA-like lesions, including inflammation, subchondral bone protrusion, and articular cartilage destruction, were ameliorated by iPSC-EV. A histopathological study consistently revealed that iPSC-EVs attenuated ACLT-mediated alteration of MMP13 and ADAMTS5 and collagen II expression. iPSC-EVs protected chondrocytes by enhancing cell proliferation, suppressing premature senescence, and maintaining homeostasis of collagen II synthesis and matrix degradation enzymes such as matrix metalloproteinases (MMPs) and ADAMTS5. iPSC-EVs also reduced cell death in IL-1β-mediated chondrocyte cell damage. In the rabbit ACLT-induced OA model, iPSC-EV injection reduced cartilage destruction, as indicated by the upregulation of collagen II and down-regulation of MMP13 and ADAMTS5. Overall, our results suggest that iPSC-EVs possess therapeutic potential and may be used as an OA treatment option. This study highlights the potential of iPSC-EVs as a therapeutic option for chondrocyte regeneration and OA treatment.

CCN4/WISP1 Promotes Migration of Human Primary Osteoarthritic Chondrocytes.

Previously, we have shown the involvement of cellular communication network factor 4/Wnt-activated protein Wnt-1-induced signaling protein 1 (CCN4/WISP1) in osteoarthritic (OA) cartilage and its detrimental effects on cartilage. Here, we investigated characteristics of CCN4 in chondrocyte biology by exploring correlations of CCN4 with genes expressed in human OA cartilage with functional follow-up. Spearman correlation analysis was performed for genes correlating with CCN4 using our previously established RNA sequencing dataset of human preserved OA cartilage of the RAAK study, followed by a pathway enrichment analysis for genes with ρ ≥|0.6.| Chondrocyte migration in the absence or presence of CCN4 was determined in a scratch assay, measuring scratch size using a live cell imager for up to 36 h. Changes in expression levels of 12 genes, correlating with CCN4 and involved in migratory processes, were determined with reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Correlation of CCN4 with ρ ≥|0.6| was found for 58 genes in preserved human OA cartilage. Pathway analysis revealed "neural crest cell migration" as most significant enriched pathway, containing among others CORO1C, SEMA3C, and SMO. Addition of CCN4 to primary chondrocytes significantly enhance chondrocyte migration as demonstrated by reduced scratch size over the course of 36 h, but at the timepoints measured no effect was observed on mRNA expression of the 12 genes. CCN4 increases cell migration of human primary OA chondrocytes. Since WISP1 expression is known to be increased in OA cartilage, this may serve to direct chondrocytes toward cartilage defects and orchestrate repair.

Theaflavin-3,3'-Digallate Inhibits Erastin-Induced Chondrocytes Ferroptosis via the Nrf2/GPX4 Signaling Pathway in Osteoarthritis.

There is evidence that osteoarthritis (OA) is associated with ferroptosis which is a kind of lipid peroxidation-related cell death. Theaflavin-3,3'-digallate(TF3), a polyphenol compound extracted from black tea, possesses antioxidative and anti-inflammatory properties, but its effects on chondrocyte ferroptosis in osteoarthritis (OA) remain unclear. Our present study aims at exploring the protective role and underlying mechanisms of TF3 against erastin-induced chondrocyte ferroptosis in OA. In human primary chondrocytes treated with erastin alone or combined with different doses of TF3, cell viability was assessed by MTS. Ferroptosis-related proteins, including Gpx4, HO-1, and FTH1, were detected by western blot. The levels of lipid peroxidation and Fe2+ were determined by fluorescence staining. Meanwhile, the change of related proteins in the Nrf2/Gpx4 signaling pathway was determined by western blot. siRNA-mediated Nrf2 knockdown and the Gpx4 inhibitor RSL3 were used to explore molecular mechanisms for TF3-induced ferroptosis in OA chondrocyte. The magnetic resonance imaging (MRI), HE staining, Masson's staining, and immunohistochemistry were used to evaluate articular cartilage damages in the rat OA model. The results showed that Gpx4 expression was markedly downregulated in the chondrocytes of OA patients. TF3 reversed erastin-induced ferroptosis of human cultured chondrocytes, lipid ROS, and Fe2+ production in mitochondria. Moreover, the expression of Gpx4, HO-1, FTH1, and Nrf2 was markedly induced by TF3 in the erastin-treated chondrocytes. The antiferroptotic effect of TF3 was related to enhance Nrf2/Gpx4 signaling pathway. Finally, TF3 inhibited OA progression by alleviating in vivo cartilage damage related to chondrocyte ferroptosis. Thus, TF3 significantly inhibits chondrocyte ferroptosis by activating the Nrf2/Gpx4 signaling pathway, suggesting that TF3 serves as a potential therapeutic supplement for OA treatment.

3,3'-diindolylmethane inhibits LPS-induced human chondrocytes apoptosis and extracellular matrix degradation by activating PI3K-Akt-mTOR-mediated autophagy.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by articular cartilage destruction. The pathological mechanisms are complex; in particular, inflammation, autophagy, and apoptosis are often involved. 3,3-Diindolylmethane (DIM), a phytoconstituent extracted from cruciferous vegetables, has various effects such as anti-inflammatory, antioxidant and anti-apoptotic. However, the effects of DIM on osteoarthritic chondrocytes remain undetermined. In this study, we simulated a lipopolysaccharide (LPS)-induced osteoarthritis model in human primary chondrocytes. We found that LPS stimulation significantly inhibited autophagy, induced chondrocyte apoptosis and extracellular matrix (ECM) degradation, which could be ameliorated by DIM. DIM inhibited the expression of a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS-5), matrix metalloproteinase 13 (MMP13), cleaved caspase-3, Bax, and p62, and increased the expression level of collagen II, aggrecan, Bcl-2, light chain 3 Ⅱ (LC3 Ⅱ), and beclin-1. Mechanistic studies showed that DIM increased chondrocyte autophagy levels by inhibiting the activation of PI3K/AKT/mTOR pathway. In mice destabilization of the medial meniscus (DMM) model, immunohistochemical analysis showed that DIM inhibited the expression of p-PI3K and cleaved caspase-3, increased the expression of LC3 Ⅱ. Furthermore, DIM relieved joint cartilage degeneration. In conclusion, our findings demonstrate for the first time that DIM inhibits LPS-induced chondrocyte apoptosis and ECM degradation by regulating the PI3K/AKT/mTOR-autophagy axis and delays OA progression in vivo.

Hypoxia and Wnt signaling inversely regulate expression of chondroprotective molecule ANP32A in articular cartilage

ANP32A is a key protector of cartilage health, via preventing oxidative stress and Wnt hyper-activation. We aimed to unravel how ANP32A is regulated in cartilage. A bioinformatics pipeline was applied to identify regulators of ANP32A. Pathways of interest were targeted to study their impact on ANP32A in in vitro cultures of the human chondrocyte C28/I2 cell-line and primary human articular chondrocytes (hACs) from up to five different donors, using Wnt-activator CHIR99021, hypoxia-mimetic IOX2 and a hypoxia chamber. ANP32A was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In vivo, the effect of hypoxia was examined by immunohistochemistry in mice injected intra-articularly with IOX2 after destabilization of the medial meniscus. Effects of Wnt hyper-activation were investigated using Frzb-knockout mice and wild-type mice treated intra-articularly with CHIR99021. Wnt inhibition effects were assessed upon intra-articular injection of XAV939. The hypoxia and Wnt signaling pathways were identified as networks controlling ANP32A expression. In vitro and in vivo experiments demonstrated increases in ANP32A upon hypoxic conditions (1.3-fold in hypoxia in C28/I2 cells with 95% confidence interval (CI) [1.11-1.54] and 1.90-fold in hACs [95% CI: 1.56-2] and 1.67-fold in ANP32A protein levels after DMM surgery with IOX2 injections [95% CI: 1.33-2.08]). Wnt hyper-activation decreased ANP32A in chondrocytes in vitro (1.23-fold decrease [95% CI: 1.02-1.49]) and in mice (1.45-fold decrease after CHIR99021 injection [95% CI: 1.22-1.72] and 1.41-fold decrease in Frzb-knockout mice [95% CI: 1.00-1.96]). Hypoxia and Wnt modulated ataxia-telangiectasia mutated serine/threonine kinase (ATM), an ANP32A target gene, in hACs (1.89-fold increase [95% CI: 1.38-2.60] and 1.41-fold decrease [95% CI: 1.02-1.96]). Maintaining hypoxia and limiting Wnt activation sustain ANP32A and protect against osteoarthritis.

An integrated in silico-in vitro approach for identifying therapeutic targets against osteoarthritis

BackgroundWithout the availability of disease-modifying drugs, there is an unmet therapeutic need for osteoarthritic patients. During osteoarthritis, the homeostasis of articular chondrocytes is dysregulated and a phenotypical transition called hypertrophy occurs, leading to cartilage degeneration. Targeting this phenotypic transition has emerged as a potential therapeutic strategy. Chondrocyte phenotype maintenance and switch are controlled by an intricate network of intracellular factors, each influenced by a myriad of feedback mechanisms, making it challenging to intuitively predict treatment outcomes, while in silico modeling can help unravel that complexity. In this study, we aim to develop a virtual articular chondrocyte to guide experiments in order to rationalize the identification of potential drug targets via screening of combination therapies through computational modeling and simulations.ResultsWe developed a signal transduction network model using knowledge-based and data-driven (machine learning) modeling technologies. The in silico high-throughput screening of (pairwise) perturbations operated with that network model highlighted conditions potentially affecting the hypertrophic switch. A selection of promising combinations was further tested in a murine cell line and primary human chondrocytes, which notably highlighted a previously unreported synergistic effect between the protein kinase A and the fibroblast growth factor receptor 1.ConclusionsHere, we provide a virtual articular chondrocyte in the form of a signal transduction interactive knowledge base and of an executable computational model. Our in silico-in vitro strategy opens new routes for developing osteoarthritis targeting therapies by refining the early stages of drug target discovery.Graphical

The Impact of JWH-133 on Articular Cartilage Regeneration in Osteoarthritis Via Metalloproteinase 13-Dependent Mechanism.

Objective: Osteoarthritis (OA) is common degenerative joint disease, mostly characterized by gradual cartilage breakdown. Currently there are no disease-modifying drugs available, therefore, there is an increasing need for basic research to focus on cartilage function in OA. Changes in cannabinoid receptor 2 (CB2) expression were observed in the OA-affected joints, although its action on cartilage chondrocytes remain unclear. We studied the action of dimethylbutyl-deoxy-delta-8-THC (JWH-133), selective CB2 agonist, on chondrocytes metabolism using both in vitro and in vivo studies. Design: Intraarticular (i.a.) injection of monoiodoacetate (MIA) was used to induce OA in rats. OA-related pain symptoms were assessed by pressure application measurements (PAMs). Primary human chondrocytes treated with MIA were used to investigate action of JWH-133 on chondrocytes viability, proliferation, and motility. Cannabinoid system components, inflammatory cytokines and metalloproteinases (MMPs) expression was measured on messenger RNA and protein levels in chondrocytes and animal cartilage. Results: Repeated, i.a. administration of JWH-133 showed antinociceptive potential in PAM, as well as decreased levels of MMPs, which suggests that CB2 agonism may modify degradation of cartilage. JWH-133 administration partially reduced toxicity, increased proliferation, and chondrocytes' migration. Moreover, our data suggest that CB2 agonism leads to alleviation of MMPs expression both in vitro and in vivo. Conclusions: In this study, we demonstrate modifying effect of JWH-133 local administration on cartilage metabolism and MMP13 expression that was shown to be involved in cartilage degradation. CB2 receptors' activation may be of benefit for chondrocytes' proliferation, therefore delaying disease progression. Our results propose direction of studies on OA-modifying treatment that can benefit in management of human OA.

WWP2 confers risk to osteoarthritis by affecting cartilage matrix deposition via hypoxia associated genes

To explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele rs1052429-A on WWP2 expression in a human 3D in vitro model of cartilage. Co-expression behavior of WWP2 with genes expressed in lesioned OA articular cartilage (N=35 samples) was explored. By applying lentiviral particle mediated WWP2 upregulation in 3D in vitro pellet cultures of human primary chondrocytes (N=8 donors) the effects of upregulation on cartilage matrix deposition was evaluated. Finally, we transfected primary chondrocytes with miR-140 mimics to evaluate whether miR-140 and WWP2 are involved in similar pathways. Upon performing Spearman correlations in lesioned OA cartilage, 98 highly correlating genes (|ρ|>0.7) were identified. Among these genes, we identified GJA1, GDF10, STC2, WDR1, and WNK4. Subsequent upregulation of WWP2 on 3D chondrocyte pellet cultures resulted in a decreased expression of COL2A1 and ACAN and an increase in EPAS1 expression. Additionally, we observed a decreased expression of GDF10, STC2, and GJA1. Proteomics analysis identified 42 proteins being differentially expressed with WWP2 upregulation, which were enriched for ubiquitin conjugating enzyme activity. Finally, upregulation of miR-140 in 2D chondrocytes resulted in significant upregulation of WWP2 and WDR1. Mimicking the effect of OA risk allele rs1052429-A on WWP2 expression initiates detrimental processes in the cartilage shown by a response in hypoxia associated genes EPAS1, GDF10, and GJA1 and a decrease in anabolic markers, COL2A1 and ACAN.

Biomimetic hydrogel blanket for conserving and recovering intrinsic cell properties

BackgroundCells in the human body experience different growth environments and conditions, such as compressive pressure and oxygen concentrations, depending on the type and location of the tissue. Thus, a culture device that emulates the environment inside the body is required to study cells outside the body.MethodsA blanket-type cell culture device (Direct Contact Pressing: DCP) was fabricated with an alginate-based hydrogel. Changes in cell morphology due to DCP pressure were observed using a phase contrast microscope. The changes in the oxygen permeability and pressure according to the hydrogel concentration of DCP were analyzed. To compare the effects of DCP with normal or artificial hypoxic cultures, cells were divided based on the culture technique: normal culture, DCP culture device, and artificial hypoxic environment. Changes in phenotype, genes, and glycosaminoglycan amounts according to each environment were evaluated. Based on this, the mechanism of each culture environment on the intrinsic properties of conserving chondrocytes was suggested.ResultsChondrocytes live under pressure from the surrounding collagen tissue and experience a hypoxic environment because collagen inhibits oxygen permeability. By culturing the chondrocytes in a DCP environment, the capability of DCP to produce a low-oxygen and physical pressure environment was verified. When human primary chondrocytes, which require pressure and a low-oxygen environment during culture to maintain their innate properties, were cultured using the hydrogel blanket, the original shapes and properties of the chondrocytes were maintained. The intrinsic properties could be recovered even in aged cells that had lost their original cell properties.ConclusionsA DCP culture method using a biomimetic hydrogel blanket provides cells with an adjustable physical pressure and a low-oxygen environment. Through this technique, we could maintain the original cellular phenotypes and intrinsic properties of human primary chondrocytes. The results of this study can be applied to other cells that require special pressure and oxygen concentration control to maintain their intrinsic properties. Additionally, this technique has the potential to be applied to the re-differentiation of cells that have lost their original properties.

3D chromatin structure in chondrocytes identifies putative osteoarthritis risk genes.

Genome-wide association studies have identified over 100 loci associated with osteoarthritis risk, but the majority of osteoarthritis risk variants are noncoding, making it difficult to identify the impacted genes for further study and therapeutic development. To address this need, we used a multiomic approach and genome editing to identify and functionally characterize potential osteoarthritis risk genes. Computational analysis of genome-wide association studies and ChIP-seq data revealed that chondrocyte regulatory loci are enriched for osteoarthritis risk variants. We constructed a chondrocyte-specific regulatory network by mapping 3D chromatin structure and active enhancers in human chondrocytes. We then intersected these data with our previously collected RNA-seq dataset of chondrocytes responding to fibronectin fragment, a known osteoarthritis trigger. Integration of the 3 genomic datasets with recently reported osteoarthritis genome-wide association study variants revealed a refined set of putative causal osteoarthritis variants and their potential target genes. One of the putative target genes identified was SOCS2, which was connected to a putative causal variant by a 170-kb loop and is differentially regulated in response to fibronectin fragment. CRISPR-Cas9-mediated deletion of SOCS2 in primary human chondrocytes from 3 independent donors led to heightened expression of inflammatory markers after fibronectin fragment treatment. These data suggest that SOCS2 plays a role in resolving inflammation in response to cartilage matrix damage and provides a possible mechanistic explanation for its influence on osteoarthritis risk. In total, we identified 56 unique putative osteoarthritis risk genes for further research and potential therapeutic development.

Pneumatic piston hydrostatic bioreactor for cartilage tissue engineering

During exercise, mechanical loads from the body are transduced into interstitial fluid pressure changes which are sensed as dynamic hydrostatic forces by cells in cartilage. The effects of these loading forces in health and disease are of interest to biologists, but the availability of affordable equipment for in vitro experimentation is an obstacle to research progress. Here, we report the development of a cost-effective hydropneumatic bioreactor system for research in mechanobiology. The bioreactor was assembled from readily available components (a closed-loop stepped motor and pneumatic actuator) and a minimal number of easily-machined crankshaft parts, whilst the cell culture chambers were custom designed by the biologists using CAD and entirely 3 D printed in PLA. The bioreactor system was shown to be capable of providing cyclic pulsed pressure waves at a user-defined amplitude and frequency ranging from 0 to 400 kPa and up to 3.5 Hz, which are physiologically relevant for cartilage. Tissue engineered cartilage was created from primary human chondrocytes and cultured in the bioreactor for five days with three hours/day cyclic pressure (300 kPa at 1 Hz), simulating moderate physical exercise. Bioreactor-stimulated chondrocytes significantly increased their metabolic activity (by 21%) and glycosaminoglycan synthesis (by 24%), demonstrating effective cellular transduction of mechanosensing. Our Open Design approach focused on using ‘off-the-shelf’ pneumatic hardware and connectors, open source software and in-house 3 D printing of bespoke cell culture containers to resolve long-standing problems in the availability of affordable bioreactors for laboratory research.

Repurposing drugs to inhibit innate immune responses associated with TLR4, IL1, and NLRP3 signaling in joint cells

Osteoarthritis (OA) affects more than 300 million people worldwide and it is about to become the first disabling disease. OA is characterized by the progressive degradation of the articular cartilage but is a disease of the whole joint. Articular innate immune responses (IIR) associated with tissue degradation contribute to its progression. However, no treatment is available to block these IIRs. Through data text mining and computational pharmacology, we identified two clinical available drugs, naloxone, and thalidomide, with potential inhibitory properties on toll-like receptor 4 (TLR4), a major activator of these IIR. Proteome analysis confirmed that activation of this receptor or the IL1 receptor generated OA-like and gout-like proteomic changes in human primary chondrocytes. Both compounds were found to block TLR4 complex and inhibit TLR4 and IL1R-mediated IIR in OA chondrocytes, osteoblasts, and synoviocytes. Furthermore, naloxone and thalidomide inhibitory effects involved the downregulation of the NLRP3 inflammasome pathway, which is downstream of TLR4/IL1R signaling. We demonstrated that these compounds, within a therapeutic range of concentrations, exhibited anti-inflammatory and anti-catabolic properties in joint primary OA cells without any toxic effect. This data underpins naloxone & thalidomide repurpose to treat OA-associated inflammatory responses.

WISP-2 modulates the induction of inflammatory mediators and cartilage catabolism in chondrocytes

AbstractWnt-1 inducible signaling pathway protein 2 (WISP-2/CCN5) is a recently identified adipokine that has been described as an important mediator of canonical Wnt activation in adipogenic precursor cells. In osteoarthritis (OA), the most common form of arthritis, chondrocytes exhibit aberrant and increased production of pro-inflammatory mediators and matrix degrading enzymes such as IL-1β and MMP-13. Although recent evidence suggests a role for Wnt signaling in OA physiopathology, little is known about the involvement of WISP-2 in cartilage degradation. In the present study, we determined the expression of WISP-2 in healthy and OA human chondrocytes. WISP-2 expression is modulated along chondrocyte differentiation and downregulated at the onset of hypertrophy by inflammatory mediators. We also investigated the effect of WISP-2 on cartilage catabolism and performed WISP-2 loss-of-function experiments using RNA interference technology in human T/C-28a2 immortalized chondrocytes. We demonstrated that recombinant human WISP-2 protein reduced IL-1β-mediated chondrocyte catabolism, that IL-1β and WNT/b-catenin signaling pathways are involved in rhWISP-2 protein and IL-1β effects in human chondrocytes, and that WISP-2 has a regulatory role in attenuating the catabolic effects of IL-1β in chondrocytes. Gene silencing of WISP-2 increased the induction of the catabolic markers MMP-13 and ADAMTS-5 and the inflammatory mediators IL-6 and IL-8 triggered by IL-1β in human primary OA chondrocytes in a Wnt/β-catenin dependent manner. In conclusion, here we have shown for the first time that WISP-2 may have relevant roles in modulating the turnover of extracellular matrix in the cartilage and that its downregulation may detrimentally alter the inflammatory environment in OA cartilage. We also proved the participation of Wnt/β-catenin signaling pathway in these processes. Thus, targeting WISP-2 might represent a potential therapeutical approach for degenerative and/or inflammatory diseases of musculoskeletal system, such as osteoarthritis.

Biomaterial composition and stiffness as decisive properties of 3D bioprinted constructs for type II collagen stimulation

Gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) are frequently used biomaterials for 3D bioprinting, with individual well-established material characteristics.To identify an ideal combination of GelMA and HAMA for chondrogenesis, a novel, primary human chondrocyte COL2A1-Gaussia luciferase reporter system (HuCol2gLuc) was developed. With this non-destructive, high-throughput temporal assay, Gaussia luciferase is secreted from the cells and used as a proxy for measuring type II collagen production. GelMA:HAMA ratios were screened using the reporter system before proceeding to 3D bioprinting. This method is efficient, saving on time and materials, resulting in a streamlined process of biomaterial optimization. The screen revealed that the addition of HAMA to GelMA improved chondrogenesis over GelMA (15%) alone. Storage moduli were measured using dynamic mechanical analysis of the same GelMA:HAMA ratios and established an initial threshold for chondrogenesis of ∼30kPa. To determine if biomaterial storage moduli impact cell mobility, human primary chondrocytes transduced with green fluorescent protein (GFP) were 3D bioprinted in either 1:1 or 2:1 ratios with storage moduli of 32kPa and 57.9kPa, respectively. We found that reduced cell mobility, in the stiffer biomaterial, had higher type II collagen expression, than the softer material with more cell mobility. Finally, after 3D bioprinting with HuCol2gLuc cells we successfully identified an optimal combination (2:1) of GelMA:HAMA and photo-crosslinking time (38s) for chondrogenesis. Statement of significanceOne challenge of 3D bioprinting is identifying ideal biomaterials that stimulate articular cartilage development. To identify an optimal combination of gelatin methacrylate and hyaluronic acid methacrylate for chondrogenesis we developed a primary human chondrocyte type II collagen Gaussia luciferase reporter cell (HuCol2gLuc). This non-destructive, high-throughput assay uses a secreted Gaussia luciferase as a proxy for temporal type II collagen production. This reporter system streamlines the biomaterial optimization process before 3D bioprinting. We also used it to determine the level of stiffness required for chondrogenesis. And for the first time, we quantified chondrocyte mobility in a 3D bioprinted construct. Together these results indicate that a biomaterial with a higher storage modulus and less cell mobility, improves chondrogenesis.