Primary Osteoarthritis Chondrocytes Research Articles

OA30 Primary osteoarthritis chondrocyte map of chromatin conformation reveals novel effector genes

Abstract Background/Aims Osteoarthritis is a complex disease with a huge public health burden. Genome-wide association studies (GWAS) have identified hundreds of osteoarthritis-associated sequence variants, but the effector genes underpinning these signals remain largely elusive. Understanding chromosome organisation in 3D space is essential for identifying long-range contacts between distant genomic features (e.g., between genes and regulatory elements), in a tissue-specific manner. Here, we generate the first whole genome chromosome conformation analysis (Hi-C) map of primary osteoarthritis chondrocytes and identify novel effector genes for the disease. Methods Primary chondrocytes collected from eight knee osteoarthritis patients underwent Hi-C analysis to link chromosomal structure to genomic sequence. The identified loops were then combined with osteoarthritis GWAS results to identify variants involved in gene regulation via enhancer-promoter interactions. Results We identified 36 genetic variants residing within chromatin loop anchors that are associated with 13 osteoarthritis GWAS signals. Five of these variants reside directly in enhancer regions of 3 enhancer-promoter loops newly described in chondrocytes, pointing to high-confidence effector genes: PAPPA, NSD2 and NCOA6. PAPPA is directly associated with the turnover of IGF-1 transport proteins, and IGF-1 is an important factor in the repair of damaged chondrocytes. Conclusion We have constructed the first Hi-C map of primary human chondrocytes and have made it available as a resource for the scientific community. By integrating 3D-genomics with large-scale genetic association data, we identify high-confidence osteoarthritis effector genes which enhance our understanding of disease and can serve as putative high-value novel targets. Disclosure N. Bittner: None. C. Shi: None. D. Zhao: None. J. Ding: None. L. Southam: None. D. Swift: None. P. Kreitmaier: None. M. Tutino: None. J. Hankinson: None. M. Wilkinson: None. G. Orozco: None. E. Zeggini: None.

Primary osteoarthritis chondrocyte map of chromatin conformation reveals novel candidate effector genes

ObjectivesOsteoarthritis is a complex disease with a huge public health burden. Genome-wide association studies (GWAS) have identified hundreds of osteoarthritis-associated sequence variants, but the effector genes underpinning these signals remain...

Synergistic roles of CBX4 chromo and SIM domains in regulating senescence of primary human osteoarthritic chondrocytes

BackgroundCellular senescence is a critical factor contributing to osteoarthritis (OA). Overexpression of chromobox homolog 4 (CBX4) in a mouse system was demonstrated to alleviate post-traumatic osteoarthritis (PTOA) by reducing cellular senescence. Additionally, replicative cellular senescence of WI-38 fibroblasts can be attenuated by CBX4. However, the mechanisms underlying this senomorphic function of CBX4 are not fully understood. In this study, we aimed to investigate the role of CBX4 in cellular senescence in human primary osteoarthritic chondrocytes and to identify the functional domains of CBX4 necessary for its function in modulating senescence.MethodsChondrocytes, isolated from 6 individuals undergoing total knee replacement for OA, were transduced with wild-type CBX4, mutant CBX4, and control lentiviral constructs. Senescence-related phenotypic outcomes included the following: multiple flow cytometry-measured markers (p16INK4A, senescence-associated β-galactosidase [SA-β-gal] activity and dipeptidyl peptidase-4 [DPP4], and proliferation marker EdU), multiplex ELISA-measured markers in chondrocyte culture media (senescence-associated secretory phenotypes [SASPs], including IL-1β, IL-6, IL-8, TNF-α, MMP-1, MMP-3, and MMP-9), and PCR array-evaluated senescence-related genes.ResultsCompared with control, CBX4 overexpression in OA chondrocytes decreased DPP4 expression and SASP secretion and increased chondrocyte proliferation confirming CBX4 senomorphic effects on primary human chondrocytes. Point mutations of the chromodomain domain (CDM, involved in chromatin modification) alone were sufficient to partially block the senomorphic activity of CBX4 (p16INK4A and DPP4 increased, and EdU decreased) but had minimal effect on SASP secretion. Although having no effect on p16INK4A, DPP4, and EdU, deletion of two small-ubiquitin-like-modifier-interaction motifs (CBX4 ΔSIMs) led to increased SASP secretion (IL-1β, TNF-α, IL-8). The combination CBX4 CDMΔSIMs altered all these measures adversely and to a greater degree than the single domain mutants. Deletion of the C-terminal (CBX4 ΔC-box) involved with transcriptional silencing of polycomb group proteins increased IL-1β slightly but significantly but altered none of the other senescence outcome measures.ConclusionsCBX4 has a senomorphic effect on human osteoarthritic chondrocytes. CDM is critical for CBX4-mediated regulation of senescence. The SIMs are supportive but not indispensable for CBX4 senomorphic function while the C-box is dispensable.

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.

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.

Basal and IL-1\u03b2 enhanced chondrocyte chemotactic activity on monocytes are co-dependent on both IKK\u03b1 and IKK\u03b2 NF-\u03baB activating kinases

IKKα and IKKβ are essential kinases for activating NF-κB transcription factors that regulate cellular differentiation and inflammation. By virtue of their small size, chemokines support the crosstalk between cartilage and other joint compartments and contribute to immune cell chemotaxis in osteoarthritis (OA). Here we employed shRNA retroviruses to stably and efficiently ablate the expression of each IKK in primary OA chondrocytes to determine their individual contributions for monocyte chemotaxis in response to chondrocyte conditioned media. Both IKKα and IKKβ KDs blunted both the monocyte chemotactic potential and the protein levels of CCL2/MCP-1, the chemokine with the highest concentration and the strongest association with monocyte chemotaxis. These findings were mirrored by gene expression analysis indicating that the lowest levels of CCL2/MCP-1 and other monocyte-active chemokines were in IKKαKD cells under both basal and IL-1β stimulated conditions. We find that in their response to IL-1β stimulation IKKαKD primary OA chondrocytes have reduced levels of phosphorylated NFkappaB p65pSer536 and H3pSer10. Confocal microscopy analysis revealed co-localized p65 and H3pSer10 nuclear signals in agreement with our findings that IKKαKD effectively blunts their basal level and IL-1β dependent increases. Our results suggest that IKKα could be a novel OA disease target.

Effects of Extracellular Vesicles from Blood-Derived Products on Osteoarthritic Chondrocytes within an Inflammation Model.

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. One major driver of OA is inflammation, in which cytokines such as IL-6, TNF-α and IL-1β are secreted by activated chondrocytes, as well as synovial cells—including macrophages. Intra-articular injection of blood products—such as citrate-anticoagulated plasma (CPRP), hyperacute serum (hypACT), and extracellular vesicles (EVs) isolated from blood products—is gaining increasing importance in regenerative medicine for the treatment of OA. A co-culture system of primary OA chondrocytes and activated M1 macrophages was developed to model an OA joint in order to observe the effects of EVs in modulating the inflammatory environment. Primary OA chondrocytes were obtained from patients undergoing total knee replacement. Primary monocytes obtained from voluntary healthy donors and the monocytic cell line THP-1 were differentiated and activated into proinflammatory M1 macrophages. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and Western blot. Gene expression analysis of chondrocytes by RT-qPCR revealed increased type II collagen expression, while cytokine profiling via ELISA showed lower TNF-α and IL-1β levels associated with EV treatment. In conclusion, the inflammation model provides an accessible tool to investigate the effects of blood products and EVs in the inflammatory context of OA.

The miR-302c/transforming growth factor-β receptor type-2 axis modulates interleukin-1β-induced degenerative changes in osteoarthritic chondrocytes.

Chondrocyte production of catabolic and inflammatory mediators participating in extracellular matrix degradation has been regarded as a central event in osteoarthritis (OA) development. During OA pathogenesis, interleukin-1β (IL-1β) decreases the mRNA expression and protein levels of transforming growth factor-β receptor type-2 (TGFBR2), thus disrupting transforming growth factor-β signaling and promoting OA development. In the present study, we attempted to identify the differentially expressed genes in OA chondrocytes upon IL-1β treatment, investigate their specific roles in OA development, and reveal the underlying mechanism. As shown by online data analysis and experimental results, TGFBR2 expression was significantly downregulated in IL-1β-treated human primary OA chondrocytes. IL-1β treatment induced degenerative changes in OA chondrocytes, as manifested by increased matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 proteins, decreased Aggrecan and Collagen II proteins, and suppressed OA chondrocyte proliferation. These degenerative changes were significantly reversed by TGFBR2 overexpression. miR-302c expression was markedly induced by IL-1β treatment in OA chondrocytes. miR-302c suppressed the expression of TGFBR2 via direct binding to its 3'- untranslated region. Similar to TGFBR2 overexpression, miR-302c inhibition significantly improved IL-1β-induced degenerative changes in OA chondrocytes. Conversely, TGFBR2 silencing enhanced IL-1β-induced degenerative changes and significantly reversed the effects of miR-302c inhibition in response to IL-1β treatment. In conclusion, the miR-302c/TGFBR2 axis could modulate IL-1β-induced degenerative changes in OA chondrocytes and might become a novel target for OA treatment.

Nuclear Magnetic Resonance Therapy Modulates the miRNA Profile in Human Primary OA Chondrocytes and Antagonizes Inflammation in Tc28/2a Cells.

Nuclear magnetic resonance therapy (NMRT) is discussed as a participant in repair processes regarding cartilage and as an influence in pain signaling. To substantiate the application of NMRT, the underlying mechanisms at the cellular level were studied. In this study microRNA (miR) was extracted from human primary healthy and osteoarthritis (OA) chondrocytes after NMR treatment and was sequenced by the Ion PI Hi-Q™ Sequencing 200 system. In addition, T/C-28a2 chondrocytes grown under hypoxic conditions were studied for IL-1β induced changes in expression on RNA and protein level. HDAC activity an NAD(+)/NADH was measured by luminescence detection. In OA chondrocytes miR-106a, miR-27a, miR-34b, miR-365a and miR-424 were downregulated. This downregulation was reversed by NMRT. miR-365a-5p is known to directly target HDAC and NF-ĸB, and a decrease in HDAC activity by NMRT was detected. NAD+/NADH was reduced by NMR treatment in OA chondrocytes. Under hypoxic conditions NMRT changed the expression profile of HIF1, HIF2, IGF2, MMP3, MMP13, and RUNX1. We conclude that NMRT changes the miR profile and modulates the HDAC and the NAD(+)/NADH signaling in human chondrocytes. These findings underline once more that NMRT counteracts IL-1β induced changes by reducing catabolic effects, thereby decreasing inflammatory mechanisms under OA by changing NF-ĸB signaling.

Chondrocytes from osteoarthritic cartilage of obese patients show altered adiponectin receptors expression and response to adiponectin.

Obesity and osteoarthritis (OA) are well-known comorbidities and their precise molecular interactions are still unidentified. Adiponectin, a major adipokine, known to have an anti-inflammatory effect in atherosclerosis or Type 2 Diabetes Mellitus (T2DM), has also been postulated to be pro-inflammatory in OA. This dual role of adiponectin is still not explained. The precise mechanism by which adiponectin affects cartilage and chondrocytes remains to be elucidated. In the present observational study chondrocytes from 30 patients with OA (18 females and 12 males) undergoing total knee replacement (TKR) were isolated. Expression of adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2) was examined both at gene and protein levels in chondrocytes. The difference in adiponectin receptor expression between lean and obese patients with OA and the role of adiponectin in regulating pro-inflammatory genes (MCP-1, IL-6, and VCAM-1, MMP-1, MMP-2, and TIMP-1) has been investigated. We found that ADIPOR1 represented the most abundant adiponectin receptor in primary OA chondrocytes. ADIPOR1 and ADIPOR2 genes and ADIPOR1 protein were differently expressed in OA chondrocytes obtained from obese compared with lean patients with OA. Adiponectin induced gene expression of MCP-1, IL-6, and MMP-1 in all OA patients' chondrocytes. In contrast, VCAM-1 and MMP-2 were differently regulated by adiponectin depending on the patient's body mass index. This study suggests that adiponectin and ADIPOR1 may have important roles in the pathogenesis of cartilage degeneration in OA of obese subjects.

Boswellia serrata extract and curcumin increase GDF15 production by human primary osteoarthritis chondrocytes: a new mechanism of action

Boswellia serrata extract and curcumin increase GDF15 production by human primary osteoarthritis chondrocytes: a new mechanism of action

Human Primary Osteoarthritic Chondrocytes Revert to a Healthier Extracellular Matrix Composition Following Long‐term, Serum‐free, Three‐dimensional Alginate Culture

Osteoarthritis (OA) is a common condition involving the loss of articular cartilage which is primarily made up of type II collagen, proteoglycans, and water. In OA, there is an increase in collagenase activity that degrades collagens and proteoglycans necessary for healthy cartilage and leads to the abnormal production of type I collagen. Previous studies in our lab have shown that primary cultures of human osteoarthritic chondrocytes (HOACs) can be reared in serum‐free, three‐dimensional alginate culture and components of the extracellular matrix (ECM) can be measured. However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. In this study, we obtained HOACs from the femoral condyles and the tibial plateau of patients undergoing total knee arthroplasty. HOACs of greater or least pathology, determined by gross observation, were isolated and plated in 12‐well cultures at a density of 1.8 × 106 cells/0.5 mL alginate. Collagens I and II degradation and proteoglycan synthesis were measured in conditioned media and alginate‐associated matrix of the cultures at days 2, 5, 8 and 11. During long term HOAC culture, collagen degradation was reduced (p<0.001 for coll I) while proteoglycans were retained in the ECM. This trend suggests that long term, three‐dimensional, serum‐free culture of HOACs may revert to a healthier phenotype. The largest changes in extracellular matrix production were demonstrated between days 2 and 5 in HOAC culture giving insight of a treatment window to test possible therapeutics.Support or Funding InformationSupport was provided by New Jersey Health Foundation Research Award, Cooper Foundation Research Grant and the Division of Research, PCOM.

Human Primary Osteoarthritic Chondrocytes Reared in Long‐term, Serum‐free, Three‐dimensional Alginate Culture Remain Metabolically Stable: A Nucleic Acid Study

The pathogenic process that defines osteoarthritis (OA) is the imbalance between excess degradation of the extracellular matrix (ECM) in articular cartilage and a decreased ability of chondrocytes to remodel the matrix. There are many factors that contribute to this disease including unchecked actions of matrix metalloproteinase 13 (MMP‐13), an enzyme specific for the breakdown of collagen type II which is the main component of cartilage extracellular matrix. We have developed a three‐dimensional, serum‐free culture system to rear primary, human OA chondrocytes (HOACs). However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. Primary human articular chondrocytes were isolated from patients who underwent surgery for total knee arthroplasty (TKA). The medial and lateral aspects of the joint samples were kept separated and labeled as greatest or least pathology. The number of live cells were determined and plated at 1.8 × 106 cells/0.5ml alginate per well of a 12‐well plate. Cells were cultured in complete serum‐free media for 11 days with RNA and DNA collected at days 2, 5, 8 and 11. Total RNA and DNA were isolated via Trizol reagent protocol. Quantitation of nucleic acids was accomplished with the NanoDrop system. Over the eleven days in culture there was no statistically significant difference in the total amount of RNA isolated either in the least pathology samples (day 2=4.3ugRNA/culture versus day 11=2.8ugRNA/culture) or the greatest pathology samples (day 2=5.3ugRNA/culture versus day 11=6.2ugRNA/culture). The same was true for the total DNA isolated from the least pathology samples (day 2=1.9ugDNA/culture versus day 11=1.6ugDNA/culture) or the greatest pathology samples (day 2=8ugDNA/culture versus day 11=1.7ugDNA/culture). Future studies include quantitative, real‐time PCR to measure expression of metabolic agents including MMPs and collagens present in OA chondrocytes. This study is the first of its kind to isolate RNA and DNA from completely serum‐free, three‐dimensional cultures of primary human OA chondrocytes.Support or Funding InformationThis study has been supported in part by the New Jersey Health Foundation and PCOM Division of Research

Primary human chondrocytes respond to compression with phosphoproteomic signatures that include microtubule activation

Chondrocytes are responsible for maintaining the cartilage that helps joints bear load and move smoothly. These cells typically respond to physiological compression with pathways consistent with matrix synthesis, and chondrocyte mechanotransduction is essential for homeostasis. In osteoarthritis (OA), chondrocyte mechanotransduction appears to be dysregulated, yet the mechanisms remain poorly understood. The objective of this study is to document the phosphoproteomic responses of primary osteoarthritic chondrocytes to physiological sinusoidal compression. We show that OA chondrocytes respond to physiological compression by first activating proteins consistent with cytoskeletal remodeling and decreased transcription, and then later activating proteins for transcription. These results show that several microtubule-related proteins respond to compression. Our results demonstrate that compression is a relevant physiological stimulus for osteoarthritic chondrocytes. Future analyses may build on these results to find differences in compression-induced phosphoproteins between normal and OA cells that lead to druggable targets to restore homeostasis to diseased joints.

DNA methylation regulates MiR-140 expression in primary osteoarthritic chondrocytes by altering SMAD-3 binding affinity

Purpose: Osteoarthritis (OA) is a whole-joint disorder that is characterized by the loss of articular cartilage of synovial joints. Abnormal expression of miRNAs, through changes of DNA methylation status of their regulatory regions has been suggested to contribute to pathogenic alterations of an OA joint. MiR-140 is specifically expressed in cartilage and its reduced expression has been associated with abnormal chondrocytes’ metabolism in OA. The aim of the present study was to investigate the role of DNA methylation on miR-140 expression in osteoarthritic chondrocytes. Methods: Articular osteoarthritic and normal cartilage were obtained from 16 patients with primary osteoarthritis and 9 individuals with no history of joint disease, respectively. MiR-140 mRNA expression levels were investigated using real-time PCR. The methylation status of miR-140 regulatory region was analysed using quantitative methylation-specific PCR (qMSP) and bisulfite DNA sequencing assay. The effect of 5’-Aza-2-deoxycytidine (5-AzadC) on miR-140 expression was investigated and SMAD-3 binding to miR-140 regulatory region was assessed by chromatin immunoprecipitation (ChΙP). Statistical analysis was performed using the SPSS software 24. MiR-140 relative expression was assessed using Mann-Whitney. Spearman’s rank correlation coefficient was used to analyze correlative relationships. For all comparisons, p values < 0.05 were considered statistically significant and data were expressed as the mean ± standard error of the mean. Results: We observed that miR-140 expression levels were significantly reduced in OA compared with normal chondrocytes. Downregulation of miR-140 was correlated with increased percentage of methylation of miR-140 regulatory region in OA chondrocytes. Bisulfite DNA sequencing of miR-140 regulatory region revealed 6 CpG sites located near SMAD-3 binding sites to be highly methylated in OA compared to normal chondrocytes. We also observed a weaker binding affinity of SMAD-3 in OA chondrocytes compared to normal, whose miR-140 regulatory region was hypermethylated. Treatment of OA chondrocytes with 5-AzadC resulted in significant upregulation of miR-140 compared to untreated, which was associated with demethylation of miR-140 regulatory region and stronger binding of SMAD-3 in this region. Conclusions: We provide novel findings that DNA methylation regulates miR-140 expression in OA chondrocytes, by changing SMAD-3 binding affinity to miR-140 regulatory region. Our results provide strong evidence that abnormal expression of miRNAs observed in OA is regulated by epigenetics, such as DNA methylation, suggesting a new strategy for treating OA patients, as epigenetic mechanisms are reversible processes.

Heparan Sulfate Proteoglycan Synthesis Is Dysregulated in Human Osteoarthritic Cartilage

Osteoarthritis (OA) is a common degenerative joint disease, characterized by cartilage loss and subchondral bone remodeling in response to abnormal mechanical load. Heparan sulfate (HS) proteoglycans bind to many proteins that regulate cartilage homeostasis, including growth factors, morphogens, proteases, and their inhibitors, and modulate their localization, retention, and biological activity. Changes in HS expression and structure may thus have important consequences for joint health. We analyzed normal and osteoarthritic human knee cartilage, and found HS biosynthesis was markedly disrupted in OA, with 45% of the 38 genes analyzed differentially regulated in diseased cartilage. The expression of several HS core proteins, biosynthesis, and modification enzymes was increased in OA cartilage, whereas the expression of the HS proteoglycans syndecan 4 and betaglycan was reduced. The structure of HS was also altered, with increased levels of 6-O-sulfation in osteoarthritic samples, which correlated with increased expression of HS6ST1, a 6-O-sulfotransferase, and GLCE, an epimerase that promotes 6-O-sulfation. siRNA silencing of HS6ST1 expression in primary OA chondrocytes inhibited extracellular signal-regulated kinase phosphorylation in response to fibroblast growth factor 2, showing that changes in 6-O-sulfation impact a key cartilage signaling pathway. Given the broad range of homeostatic and repair pathways that HS regulates, these changes in proteoglycan expression and HS structure are likely to have significant effects on joint health and progression of OA.

Oleocanthal Inhibits Catabolic and Inflammatory Mediators in LPS-Activated Human Primary Osteoarthritis (OA) Chondrocytes Through MAPKs/NF-κB Pathways

Background/Aims: Oleocanthal (OC), a phenolic compound present in extra virgin olive oil (EVOO), has attracted attention since its discovery for its relevant pharmacological properties in different pathogenic processes, including inflammation. Here, we investigated the involvement of OC in LPS-activated osteoarthritis (OA) human primary chondrocytes. Methods: Human primary chondrocytes were harvested from articular cartilage samples obtained from OA patients. The effects of OC on the viability of chondrocytes were tested by MTT assay. Protein and mRNA expression of several catabolic and pro-inflammatory factors after OC treatment were measured by RT-qPCR and western blot respectively. Moreover, we analysed the NO production by Griess reaction. Finally, several pathways mediators were analysed by western blot. Results: We demonstrated that OC did not have any cytotoxic effect. Oleocanthal inhibited NO production and strongly decreased NOS2 and COX-2 protein and mRNA expression in LPS-activated human primary OA chondrocytes. Interestingly, OC also inhibits MMP-13 and ADAMTS-5. In addition, OC downregulates several pro-inflammatory factors, such as IL-6, IL-8, CCL3, LCN2 and TNF-α induced by LPS in human primary OA chondrocytes. Finally, we demonstrated that OC exerts its effects through the MAPK/P38/NF-kB pathways. Conclusion: These data show that OC is able to block LPS-mediated inflammatory response and MMP-13 and ADAMTS-5 induction in human primary OA chondrocytes via MAPKs/NF-kB pathways, suggesting that OC may be a promising agent for the treatment of inflammation in cartilage and a potential molecule to prevent disease progression by inhibiting metalloproteases and aggrecanases.

Metabolic flux analysis of primary osteoarthritic chondrocytes after dynamic compression

Metabolic flux analysis of primary osteoarthritic chondrocytes after dynamic compression

Platelet Rich Plasma and Hyaluronic Acid Blend for the Treatment of Osteoarthritis: Rheological and Biological Evaluation.

IntroductionOsteoarthritis (OA) is the most common musculoskeletal disease. Current treatments for OA are mainly symptomatic and inadequate since none results in restoration of fully functional cartilage. Hyaluronic Acid (HA) intra-articular injections are widely accepted for the treatment of pain associated to OA. The goal of HA viscosupplementation is to reduce pain and improve viscoelasticity of synovial fluid. Platelet-rich plasma (PRP) has been also employed to treat OA to possibly induce cartilage regeneration. The combination of HA and PRP could supply many advantages for tissue repair. Indeed, it conjugates HA viscosupplementation with PRP regenerative properties. The aim of this study was to evaluate the rheological and biological properties of different HA compositions in combination with PRP in order to identify (i) the viscoelastic features of the HA-PRP blends, (ii) their biological effect on osteoarthritic chondrocytes and (iii) HA formulations suitable for use in combination with PRP.Materials and MethodsHA/PRP blends have been obtained mixing human PRP and three different HA at different concentrations: 1) Sinovial, 0.8% (SN); 2) Sinovial Forte 1.6% (SF); 3) Sinovial HL 3.2% (HL); 4) Hyalubrix 1.5% (HX). Combinations of phosphate buffered saline (PBS) and the four HA types were used as control. Rheological measurements were performed on an Anton PaarMCR-302 rheometer. Amplitude sweep, frequency sweep and rotational measurements were performed and viscoelastic properties were evaluated. The rheological data were validated performing the tests in presence of Bovine Serum Albumin (BSA) up to ultra-physiological concentration (7%). Primary osteoarthritic chondrocytes were cultured in vitro with the HA and PRP blends in the culture medium for one week. Cell viability, proliferation and glycosaminoglycan (GAG) content were assessed.ResultsPRP addition to HA leads to a decrease of viscoelastic shear moduli and increase of the crossover point, due to a pure dilution effect. For viscosupplements with HA concentration below 1% the viscoelasticity is mostly lost. Results were validated also in presence of proteins, which in synovial fluid are more abundant than HA.Chondrocytes proliferated overtime in all different culture conditions. The proliferation rate was higher in chondrocytes cultured in the media containing PRP compared to the cultures with different HA alone. GAG content was significantly higher in chondrocytes cultured in PRP and HL blend.DiscussionWe investigated the rheological and biological properties of four different HA concentrations when combined with PRP giving insights on viscoelastic and biological properties of a promising approach for future OA therapy. Our data demonstrate that PRP addition is not detrimental to the viscosupplementation effect of HA. Viscosupplements containing low HA concentration are not indicated for combination with PRP, as the viscoelastic properties are lost. Although having the same rheological behavior of SF and HX, HL was superior in stimulating extracellular matrix production in vitro.

Long Intergenic Noncoding RNAs Mediate the Human Chondrocyte Inflammatory Response and Are Differentially Expressed in Osteoarthritis Cartilage.

ObjectiveTo identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA.MethodsOA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non‐OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase–polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex.ResultsRNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin‐1β (IL‐1β) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50‐associated cyclooxygenase 2–extragenic RNA (PACER) and 2 novel chondrocyte inflammation–associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non‐OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL‐1–stimulated secretion of proinflammatory cytokines.ConclusionThe inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role in mediating inflammation‐driven cartilage degeneration in OA.