Chondrocyte Gene Expression Research Articles

MiR-497 Is Implicated in the Process of Chondrogenesis and Inhibits IHH Gene Expression in Human Chondrocytes.

The aim of this study was to examine differences in microRNA-497 (miR-497) expression during cartilage tissue formation and to test whether miR-497 directly interferes with Indian hedgehog (IHH) gene and inhibits IHH expression in human chondrocytes. At different cartilage development stages and different time points in bone matrix gelatin-induced endochondral ossification (BMG-ECO) rat models, the expression of miR-497 and the Ihh gene was monitored at the mRNA level. Bioinformatic analysis, gene mutation, dual luciferase reporter gene assays and gene expression assays at both the mRNA and protein levels in human chondrocytes were subsequently performed to validate the interaction between miR-497 and the IHH gene. The mRNA expression of miR-497 or the Ihh gene in BMG-ECO rats showed significant differences between the cartilage development stages and between different time points, and the trends in the expression of miR-497 and Ihh were reversed. Bioinformatic and dual luciferase reporter gene assays demonstrated a direct interaction between miR-497 and the IHH gene. Differential mRNA and protein expression profiles of the IHH gene in human chondrocytes after 48 hours of transfection with miR-497 mimics and a negative control indicated that miR-497 inhibited IHH expression. Our study provided new clues for further functional and molecular mechanism studies of miR-497 in chondrogenesis and demonstrated a potential target for clinical therapy for cartilage degenerative disease.

Surface chemistry, substrate, and topography guide the behavior of human articular chondrocytes cultured in vitro.

Understanding the behavior of chondrocytes in contact with artificial culture surfaces is becoming increasingly important in attaining appropriate ex vivo culture conditions of chondrocytes in cartilage regeneration. Chondrocyte transplantation-based cartilage repair requires efficiently expanded chondrocytes, and the culture surface plays an important role in guiding the behavior of the cell. Micro- and nano-engineered surfaces make it possible to modulate cell behavior. We hypothesized that the combined influence of topography, substrate, and surface chemistry may affect the chondrocyte culturing in terms of proliferation and phenotypic means. Human chondrocytes were cultured on polystyrene fabricated microstructures, flat polydimethylsiloxane (PDMS), or polystyrene treated with fibronectin or oxygen plasma and cultured for 1, 4, 7, and 10 days. The behavior of chondrocytes was evaluated by proliferation, viability, chondrogenic gene expression, and cell morphology. Contrary to our hypothesis, microstructures in polystyrene did not significantly influence the behavior of chondrocytes neither under normoxic- nor hypoxic conditions. However, changes in the substrate stiffness and surface chemistry were found to influence cell viability, gene expression, and morphology of human chondrocytes. Oxygen plasma treatment was the most important parameter followed by the softer substrate type PDMS. The findings indicate the culture of human chondrocytes on softer substratum and surface activation by oxygen plasma may prevent dedifferentiation and may improve chondrocyte transplantation-based cartilage repair. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2805-2816, 2018.

Exosomal miR-95-5p regulates chondrogenesis and cartilage degradation via histone deacetylase 2/8.

MicroRNAs play critical roles in the pathogenesis of osteoarthritis, the most common chronic degenerative joint disease. Exosomes derived from miR‐95‐5p‐overexpressing primary chondrocytes (AC‐miR‐95‐5p) may be effective in treating osteoarthritis. Increased expression of HDAC2/8 occurs in the tissues and chondrocyte‐secreted exosomes of patients with osteoarthritis and mediates cartilage‐specific gene expression in chondrocytes. We have been suggested that exosomes derived from AC‐miR‐95‐5p (AC‐miR‐95‐5p‐Exos) would enhance chondrogenesis and prevent the development of osteoarthritis by directly targeting HDAC2/8. Our in vitro experiments showed that miR‐95‐5p expression was significantly lower in osteoarthritic chondrocyte‐secreted exosomes than in normal cartilage. Treatment with AC‐miR‐95‐5p‐Exos promoted cartilage development and cartilage matrix expression in mesenchymal stem cells induced to undergo chondrogenesis and chondrocytes, respectively. In contrast, co‐culture with exosomes derived from chondrocytes transfected with an antisense inhibitor of miR‐95‐5p (AC‐anti‐miR‐95‐5p‐Exos) prevented chondrogenic differentiation and reduced cartilage matrix synthesis by enhancing the expression of HDAC2/8. MiR‐95‐5p suppressed the activity of reporter constructs containing the 3ʹ‐untranslated region of HDAC2/8, inhibited HDAC2/8 expression and promoted cartilage matrix expression. Our results suggest that AC‐miR‐95‐5p‐Exos regulate cartilage development and homoeostasis by directly targeting HDAC2/8. Thus, AC‐miR‐95‐5p‐Exos may act as an HDAC2/8 inhibitor and exhibit potential as a disease‐modifying osteoarthritis drug.

Corrigendum to: Effects of Arthrocen, an avocado/soy unsaponifiables agent, on inflammatory mediators and gene expression in human chondrocytes.

[This corrects the article DOI: 10.1002/2211-5463.12176.].

Nonlinear effects of caffeine on the viability, synthesis and gene expression of chondrocytes from the offspring of rats treated during pregnancy

ObjectiveEvaluate the effects of doses of caffeine administered to pregnant rats on the articular cartilage chondrocytes of their offspring. MethodsTwenty-four adult Wistar rats were randomly assigned to four groups, with one control group and three groups being treated with caffeine at doses of 25, 50 and 100 mg/kg throughout pregnancy. At birth, three offspring/females were euthanized so that the chondrocytes could be extracted. At 7, 14 and 21 days of culture, the chondrocytes were subjected to the MTT cell viability assay and an evaluation of their alkaline phosphatase activity and collagen synthesis. Chondrocytes were also stained by Hematoxylin-eosin, PAS, Safranin-O and Alcian Blue. The Sox-9, Runx-2, aggrecan, collagen-II and alkaline phosphatase gene transcript levels were also evaluated. Mean comparisons were performed by the Student–Newman–Keuls test. ResultsChondrocyte cultures from the 25 mg/kg group had the lowest results, as chondrocytes from this group had reduced viability, percentage of cells, alkaline phosphatase activity and collagen and chondrogenic matrix synthesis. A reduced expression of Sox-9, alkaline phosphatase and collagen-II was also detected in the 25 mg/kg group. Chondrocyte cultures of the group treated with 50 mg/kg caffeine showed reduced collagen synthesis and Sox-9 expression. The caffeine dose of 100 mg/kg also reduced collagen and Sox-9 and alkaline phosphatase expression. ConclusionCaffeine administered to pregnant rats negatively alters the articular cartilage chondrocytes of their offspring, reducing the synthesis of collagen and Sox-9 expression regardless of the dose. This study also concluded that the effects of caffeine are not linear or dose-dependent.

Changes in osteogenic gene expression in hypertrophic chondrocytes induced by SIN-1.

The molecular mechanisms underlying osteoarthritis (OA) and Kashin-Beck disease (KBD) remain poorly understood. Hypertrophic chondrocytes serve an important role in the development of both OA and KBD, whereas oxidative stress can contribute to the pathological progression of cartilage damage. Therefore, the aim of the present study was to detect altered expression of osteogenesis-related genes in hypertrophic chondrocytes, following treatment with 3-morpholinosydnonimine (SIN-1). ATDC5 cells were induced to develop into hypertrophic chondrocytes via Insulin-Transferrin-Selenium. The appropriate concentration and time of SIN-1 treatment was determined via MTT assay. Following hypertrophic chondrocyte stimulation with SIN-1, a liquid chip was analyzed using a polymerase chain reaction (PCR) array. Reverse transcription-quantitative PCR was conducted on individual genes to validate the array-based data. Analyses of protein-protein interactions, gene ontology functions and Kyoto Encyclopedia of Genes and Genomes pathway enrichment of the differentially expressed genes were also performed. A total of 6 upregulated and 34 downregulated genes were identified, including the mothers against decapentaplegic homolog (Smad) family (Smad1-4), bone morphogenetic proteins and their receptors (Bmp2, Bmp3, Bmpr1α and Bmpr1β), and matrix metalloproteinases (MMP2,−9 and−10). These genes are associated with collagen biology, transcriptional control, skeletal development, bone mineral metabolism, and cell adhesion. SIN-1 induced death of hypertrophic chondrocytes likely through TGF-β/Smad or BMP/Smad pathways. Oxidative-stress-dependent induction of abnormal gene expression may be associated with chondronecrosis in the cartilage of patients with OA or KBD.

Effects of simulated muscle exercise on chondrocyte gene expression in a 3D-alginate bead model system

Purpose: The joint organ is a complex system composed of bone, muscle, tendon, ligament, synovial and cartilage tissues. Myokines produced and released by muscle cells (myocytes) in response to muscular contractions have autocrine, paracrine and/or endocrine effects. Exercise is one of the recommended core treatments for all osteoarthritis patient; exercise-induced myokines appear to be involved in mediating both systemic as well as local anti-inflammatory effects. We hypothesized that myokines, produced by muscle stimulation mimicking exercise, will have anti-inflammatory and anabolic paracrine effects on chondrocytes. We tested this hypothesis by analyzing the effects of conditioned media from skeletal muscle myobundles (with and without electrical stimulation) on gene expression of chondrocytes in a 3D-alginate culture system. Methods: Electrically responsive, contractile human skeletal muscle myobundles were made from primary myogenic cells isolated from standard muscle biopsies obtained under IRB approval. After four-weeks in culture, when these human myobundles matured, electrical pulse simulation of myobundles was performed to simulate different exercise conditions: 1) Non-stimulated, 2) 1 Hz stimulation (simulated slow-twitch exercise), 3) 10 Hz stimulation (simulated fast-twitch exercise). Conditioned media (CM) from all 3 groups were collected each day for 7 days. Primary chondrocytes, collected as surgical waste, were isolated from normal appearing articular cartilage of the lateral femoral condyle from patients undergoing knee replacement for osteoarthritis (OA). Cartilage was diced, digested in 0.1% (weight/volume) collagenase type II for 16-24 h at 37 °C, and the cells were cultured in F12 media in a T-75 cm2 flask in 5% CO2 until confluent. Passage 1-4 chondrocytes were encapsulated in 1.2% alginate solution (2x10e6 cells/ml) by drop solidification into 102mM CaCl2 solution, then cultured for up to 7 days in DMEM/F12 culture media. Chondrocytes (3 biological replicates) in alginate were cultured for 48 hr in non-stimulated myobundle CM or the stimulated myobundle CM at a ratio of 1:1 to chondrocyte growth media. Quantitative PCR was performed on RNA isolated from alginate bead cultured chondrocytes for analysis of gene expression of MMP13, ADAMTS4, ADAMTS5, COL2A1, COL10A1 and ACAN and YWHAZ (housekeeping control). Results: Compared to non-stimulated CM, stimulated CM tended to reduce chondrocyte gene expression (normalized to the housekeeping control) of ACAN (p=0.086), ADAMTS5 (p=0.081) and MMP13 (p=0.111). Compared to 1 Hz stimulated CM, chondrocytes exposed for 48 hours to 10 Hz stimulated CM exhibited elevated gene expression (normalized to the control and the non-stimulated level of gene expression) of collagens II and X (p=0.045, p=0.01, Figure 1). Conclusions: Our study suggests that myokines produced by muscle stimulation mimicking exercise have anti-catabolic effects. Compared with low frequency stimulation, myokines produced in response to high frequency stimulation induced markers of chondrogenesis (collagen II) and late-stage chondrocyte hypertrophy (collagen X). This pilot study provides evidence for a paracrine effect of muscle on cartilage. Factors from muscle responsible for these effects warrant further investigation and identification to understand the physiologic interactions of muscle and cartilage.Table 1Chondrocyte gene expression in response to muscle conditioned media (CM).GeneChondrocyte gene expression (fold change in stimulated vs non-stimulated muscle CM)P valueChondrocyte gene expression (fold change in 10 Hz vs 1 Hz stimulated muscle CM)P valueCOL21.130.3961.420.045COL100.640.1731.120.010ACAN0.670.0861.040.387ADAMTS40.740.2831.040.442ADAMTS50.630.0811.090.210MMP130.630.1111.050.259 Open table in a new tab

Implication of 14-3-3epsilon as a potential alarmin in bone/cartilage communication in osteoarthritis

Purpose: Osteoarthritis (OA) is the most frequent and disabling joint disease. OA is now considered as a whole-joint disease characterized by cartilage degradation, synovial inflammation and bone remodeling. Our team identified 14-3-3ε as a novel soluble mediator critical in the communication between subchondral bone and cartilage in OA. This protein acts as a potent MMP-3 and MMP-13-stimulatory factor in chondrocytes leading to catabolic phenotype. 14-3-3ε seems to share common characteristics with alarmins, endogenous molecules with intracellular functions that are released in the extracellular media after infection or tissue damage. Alarmins (such as S100 and HMGB1 involved in cartilage degradation) bind to receptors expressed on the surface of cells engaged in host defense and tissue repair. These receptors include the Toll-like receptors, TLR2 and TLR4, as well as RAGE. Moreover, innate alarmins can stimulate the activation of resident immune cells, particularly macrophages. Our aim is to investigate the interactions between 14-3-3ε and its potential receptors, TLR2, TLR4 and RAGE and his role as an alarmin particularly as a new molecular mediator determining macrophage polarization in OA joint. Methods: Primary cultures of chondrocytes were performed from murine cartilage of the C57BL/6J mice (5 days old), and from human cartilage samples, obtained from OA patients undergoing total joint replacement surgery. The receptors TLR2, TLR4 and RAGE were blocked by monoclonal antibodies and pharmacologic inhibitors in chondrocytes (15mn) and then stimulated by recombinant 14-3-3ε during 24h. Immunocytochemistry was realized on murine chondrocytes with receptors antibodies. Chondrocyte gene expression and release of MMP-3, MMP-13 and IL-6 were evaluated by RT-PCR and ELISA. The direct interaction of 14-3-3ε and TLR2 was measured using SPR on a Biacore 3000 instrument. The potential 14-3-3ε-binding sites on receptors were investigated by in silico computational analysis PyMOL. Polarisation M1/M2 of marcrophages was performed by activating THP1 cells (ATCC) with PMA during 24h and stimulating them 24h hours with IL-4, LPS or 14-3-3ε. Different markers of the M1/M2 types were analysed by ELISA and RT-qPCR to identify the polarisation of macrophages (M1: TNF, IL-6, IL-1b, MCP1, Nos2, M2: IL-10, TGFb, Arg-1, FIZZ1, YM2). Results: TLR2, TLR4 and RAGE receptors were found on murine articular chondrocytes by immunocytochemistry using monoclonal antibodies. Invalidation of TLR2, TLR4 or RAGE receptors by blocking antibody in articular chondrocytes reduced significantly mRNA expression and protein release of MMP-3, MMP-13 and IL-6 induced by 14-3-3ε. For MMP3 mRNA expression, the inhibition rate reached 78% with blocking antibodies and 98% with OxPAPC, a TLR2/TLR4 inhibitor. The inhibition rate reached 65% (blocking antibodies), 98% (OxPAPC) for MMP13 and 74% (blocking antibodies) and 93% (OxPAPC) for IL-6. ELISA experiments showed an inhibition rate of 84% (blocking antibodies) and 97% (OxPAPC) for MMP3. Similar rates are observed for IL-6 secretion (83% and 97%). These results should be confirmed on cultures of human chondrocytes. Human recombinant TLR2 and TLR4 were covalently immobilized on CM5 chip. Results showed a direct interaction between recombinant TLR2 and 14-3-3ε (83 RU) and between TLR4 and 14-3-3ε (41 RU). Modeling experiments seems to show a direct interaction between 14-3-3ε and TLR2 receptor through a phosphorylated residue (TYR). Polarization of macrophages stimulated by recombinant 14-3-3ε is ongoing and expression of M1 type is expected. Conclusions: We have discovered 14-3-3ε, a new catabolic factor, produced by the subchondral bone and able to induce a catabolic phenotype of chondrocytes. Taking together, our results could designate 14-3-3ε as a novel alarmin to be explored in OA either for therapeutic or for prognostic purposes and its interaction with receptors could be a novel target in OA.

Identification of distinctive DNA methylation patterns correlated with differential gene expression in murine hypertrophic chondrocytes in vitro

Purpose: Osteoarthritis (OA) is joint disorder in which the articular cartilage is permanently damaged. Articular chondrocytes undergo profound phenotypic changes in OA, part of which mimic developmental steps observed in hypertrophic growth plate chondrocytes, including increased Runx2, Mmp13 and collagen type X expression. Hypertrophy is a normal step during endochondral ossification processes, which are required for normal chondrogenesis, long bone growth and ossification. Thus, to better understand the molecular mechanisms that trigger a hypertrophy-like reprogramming of articular chondocytes in osteoarthritis, we require a better understanding of gene regulation during hypertrophy. DNA methylation is one of the principal mechanisms by which cells maintain dominant and stable phenotypes, and altered DNA methylation is associated with abnormal gene expression in different pathologies, including OA. This study aims to identify genes that are differentially expressed during hypertrophic differentiation in vitro, and that are correlated with changes in DNA methylation patterns. Methods: Chondrocytes were isolated from articular cartilage obtained from 5 to 6 days old C57BL/6J mice. We used 3D/pellet culture systems to drive chondrocyte hypertrophy in vitro. Pellets were harvested at 7 and 21 days for RNA or DNA isolation. Total RNA was used for RNA sequencing, and DNA was used for Reduced Representation Oxidative Bisulfite Sequencing (RRoxBS) at the Epigenomics Core at Weill Cornell Medicine (EC-WCM). After sequencing, the RNAseq reads were processed using a dedicated RNAseq pipeline, and the expression of selected genes was further validated using SYBR-green-based real-time PCR analyses (RTqPCR). For methylation profiling, libraries were made using the ERRBS protocol. Per sample, 50-60 million ERRBS reads were aligned and processed using the EC-WCM’s in-house bioinformatics pipeline to yield methylation values for each CpG. Oxidative bisulfite (oxBS) technology was applied to distinguish between 5mC and 5hmC. Differential DNA methylation was analyzed using Methylkit and eDMR, and subsequent assessment and annotation of differentially methylated regions was done using R Core Team. Results: Hypertrophic differentiation in vitro was confirmed by RTqPCR analyses, which showed upregulation of Mmp13, collagen X and Runx2 and down-regulation of Sox9 and collagen II. RNAseq analyses uncovered more than 3000 differentially expressed genes between 7 (non hypertrophic) and 21 (hypertrophic) days (p less than 0.01). Among these genes, we identified novel targets with previously unreported roles in chondrocyte hypertrophy. Analysis of RRoxBS sequencing data also revealed changes in DNA methylation patterns accompanying hypertrophic differentiation and differential expression of hypertophic-related genes, highlighting the contribution of DNA methylation to gene expression during chondrocyte hypertrophy. Conclusions: Our results suggest that DNA methylation significantly contributes to changes in phenotype and gene expression during chondrocyte hypertrophy. In addition, we found a number of differentially expressed genes with previously unreported roles in hypertrophic chondrocytes, and with potential relevance to developmental and pathological processes. Ongoing studies are aimed to verify the results obtained in vitro using in vivo systems of chondrocyte hypertrophy during developmental processes (i.e., growth plate chondrocytes) and of hypertrophic-like conversion during OA, using surgical murine models of OA and human samples obtained from OA patients. We believe that our novel integrative approaches have the potential of uncovering new targets for therapeutic intervention.

EZH2 inhibition reduces IL-1β-mediated inflammation and increases anabolic gene expression in human chondrocytes

EZH2 inhibition reduces IL-1β-mediated inflammation and increases anabolic gene expression in human chondrocytes

ZCCHC6 (TUT7) deletion inhibit the expression of IL-6 and MMP-13 and reduces the severity of post-traumatic osteoarthritis in a murine model

Purpose: Approximately 20-50% of patients diagnosed with osteoarthritis (OA) develop the disease in joints that previously sustained trauma. RNA terminal uridyltransferases (TUTs), including Zcchc6 (zinc-finger CCHC-domain containing protein 6/TUT7), have been implicated in the post-transcriptional regulation of inflammatory mediators. However, role of Zcchc6 (TUT7) in the pathogenesis of OA or post-traumatic OA has not been explored. Methods: All murine studies were approved by the IACUC and the studies with deidentified, discarded human cartilage were approved by the IRB. Zcchc6 knockout mice were generated by gene targeting (KOMP, UC-Davis). Human or murine chondrocytes were prepared by the enzymatic digestion of the OA cartilage or knee cartilage respective and treated with recombinant human or mouse IL-1β (5ng/ml, R&D Systems, #: 201-LB-025 and #:401-ML-005 respectively) for in vitro studies. IL-6 and MMP13 gene and protein expression in chondrocytes was analyzed by qPCR and immunoblotting respectively. IL-6 mRNA stability was determined by Actinomycin-D chase experiments. For in vivo studies, post-traumatic OA in the knee joints of Zcchc6 deficient male mice and wild type littermates was induced by the surgical destabilization of medial meniscus (DMM) and mice were sacrificed after eight weeks of DMM and the severity of OA was determined per OARSI guidelines for murine OA. Contra lateral knee joints with sham surgery served as controls. Expression of MMP13 (Santa Cruz Biotechnology, #sc-30071), Type-II Collagen cleavage (IBEX Pharmaceuticals, #C1,2C) and Zcchc6 (Santa Cruz Biotechnology, #sc-137947) in vivo were analyzed by immunohistochemistry. Fluorescence in situ hybridization (FISH) was performed using the RNAScope technology. RNA immunoprecipitation (RIP) was performed using validated antibodies. siRNAs were used to deplete the expression of ZCCHC6. Results: Knee cartilage chondrocytes prepared from Zcchc6 KO mice showed increased levels of IL-6 and MMP13 mRNA expression compared to WT chondrocytes in response to IL-1β stimulation. Of interest was the observation that the protein levels of IL-6 and MMP13 were significantly downregulated in IL-1β stimulated Zcchc6 KO chondrocytes lysate and culture supernatants in comparison to WT chondrocytes. FISH and RIP analysis revealed that ZCCHC6 protein binds to IL-6 mRNAs in primary human OA chondrocytes under pathological conditions. Furthermore, IL-6 mRNA half-life was reduced by 50% in Zcchc6 KO murine chondrocytes and ZCCHC6 depleted human primary chondrocytes. The expression of Zcchc6 in the knee joints of wild type mice with DMM surgery was highly upregulated in comparison to control joints. Zcchc6 KO mice expressed low levels of MMP13 and showed lesser matrix degradation in the joints with DMM surgery compared to joints of the WT mice and the control contralateral joints and developed less severe OA as determined by Safranin-O-Fast green staining followed by OARSI scoring. Synovitis was also found to be decreased in the KO mice DMM joints in comparison to WT DMM joints. Conclusions: Our data demonstrate that Zcchc6 is upregulated in DMM joints and functions as a positive regulator of IL-6 and MMP13 expression in murine chondrocytes and cartilage in vivo. Severity of the posttraumatic OA and cartilage degradation was significantly low in Zcchc6 KO mice and correlated with reduced expression of MMP-13. These results identify ZCCHC6 as a potential therapeutic target for the treatment of OA.

Bromodomain inhibitors are potent epigenetic regulators of catabolic gene expression in human osteoarthritic chondrocytes

Bromodomain inhibitors are potent epigenetic regulators of catabolic gene expression in human osteoarthritic chondrocytes

Effect of dexamethasone, interleukin-1 receptor antagonist protein, and toll-like receptor antagonist 242 (resatorvid) on gene expression in equine chondrocytes in a chronic inflammation model in vitro

Effect of dexamethasone, interleukin-1 receptor antagonist protein, and toll-like receptor antagonist 242 (resatorvid) on gene expression in equine chondrocytes in a chronic inflammation model in vitro

Effect of Hyperbaric Oxygen on Proliferation and Gene Expression of Human Chondrocytes: An In Vitro Study.

The present study investigated the effects of hyperbaric oxygen (HBO) on human chondrocyte proliferation and gene expression patterns. Chondrocyte cultures were transferred to a HBO chamber and exposed to 100% oxygen for 7 consecutive days. Within groups, pressure was varied between 1 and 2 atm and duration of HBO administration was varied among 60, 90, and 120 minutes. Cell counts were performed using the WST-1 assay at 1, 3, 5, and 7 days after initiation of HBO treatment to obtain data to plot a growth curve. Gene expression of apoptosis markers PARP and caspase 3, as well as cartilage specific proteins collagen II and COMP, were detected by reverse transcription polymerase chain reaction. The experiments showed that in vitro administration of HBO inhibit chondrocyte growth. When applied compression was increased up to 2 atm, chondrocyte cell count was reduced by half at days 3 and 7 in association with an upregulation of the apoptosis markers PARP and caspase 3 as well as the cartilage specific proteins collagen II and COMP. No significant differences were monitored from varied duration of daily treatment. Chondrocyte growth was inhibited in vitro by treatment of HBO. This inhibitory effect was even increased by elevating the applied pressure, while molecular testing showed reduced chondrocyte growth. Higher levels of HBO inhibited cell growth even more, but up-regulation of apoptosis specific markers and cartilage specific proteins were seen during administration of high oxygen levels. Thus, it has to be evaluated that there is a critical level of hypo-/hyperoxia required to stimulate or at least maintain chondrocyte cell proliferation.

Effects of stanozolol on normal and IL-1\u03b2-stimulated equine chondrocytes in vitro

BackgroundIntra-articular administration of stanozolol has shown promising results by improving the clinical management of lameness associated with naturally-occurring osteoarthritis (OA) in horses, and by decreasing osteophyte formation and subchondral bone reaction in sheep following surgically induced OA. However, there is limited evidence on the anti-inflammatory and modulatory properties of stanozolol on articular tissues. The objective of the current study was to evaluate the effects of stanozolol on chondrocyte viability and gene expression in normal equine chondrocytes and an inflammatory in vitro system of OA (interleukin-1β (IL-1β) treated chondrocytes).ResultsChondrocytes from normal metacarpophalangeal joints of skeletally mature horses were exposed to four treatment groups: (1) media only (2) media+IL-1β (3) media+IL-1β + stanozolol (4) media+stanozolol. Following exposure, chondrocyte viability and the expression of catabolic, anabolic and structural genes were determined. General linear models with Dunnet’s comparisons with Bonferroni’s adjustment were performed. Cell viability was similar in all groups. Stanozolol treatment reduced gene expression of MMP-13, MMP-1, IL-6 and COX-2 in both normal and IL-1β treated chondrocytes. Stanozolol treatment reduced ADAMTS4 gene expression in normal chondrocytes. Stanozolol reduced the expression of COL2A1.ConclusionsThe current study demonstrates stanozolol has chondroprotective effects through downregulation of genes for pro-inflammatory/catabolic cytokines and enzymes associated with OA. However, there is no evidence of increased cartilage stimulation through upregulation of the anabolic and structural genes tested.

A novel variant in GLIS3 is associated with osteoarthritis

ObjectivesOsteoarthritis (OA) is a complex disease, but its genetic aetiology remains poorly characterised. To identify novel susceptibility loci for OA, we carried out a genome-wide association study (GWAS) in individuals...

Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds.

In this study, we first used gelatin/chondroitin-6-sulfate/hyaluronan/chitosan highly elastic cryogels, which showed total recovery from large strains during repeated compression cycles, as 3D scaffolds to study the effects of cyclic dynamic compressive loading on chondrocyte gene expression and extracellular matrix (ECM) production. Dynamic culture of porcine chondrocytes was studied at 1 Hz, 10% to 40% strain and 1 to 9 h/day stimulation duration, in a mechanical-driven multi-chamber bioreactor for 14 days. From the experimental results, we could identify the optimum dynamic culture condition (20% and 3 h/day) to enhance the chondrocytic phenotype of chondrocytes from the expression of marker (Col I, Col II, Col X, TNF-α, TGF-β1 and IGF-1) genes by quantitative real-time polymerase chain reactions (qRT-PCR) and production of ECM (GAGs and Col II) by biochemical analysis and immunofluorescence staining. With up-regulated growth factor (TGF-β1 and IGF-1) genes, co-culture of chondrocytes with porcine adipose-derived stem cells (ASCs) was employed to facilitate chondrogenic differentiation of ASCs during dynamic culture in cryogel scaffolds. By replacing half of the chondrocytes with ASCs during co-culture, we could obtain similar production of ECM (GAGs and Col II) and expression of Col II, but reduced expression of Col I, Col X and TNF-α. Subcutaneous implantation of cells/scaffold constructs in nude mice after mono-culture (chondrocytes or ASCs) or co-culture (chondrocytes + ASCs) and subject to static or dynamic culture condition in vitro for 14 days was tested for tissue-engineering applications. The constructs were retrieved 8 weeks post-implantation for histological analysis by Alcian blue, Safranin O and Col II immunohistochemical staining. The most abundant ectopic cartilage tissue was found for the chondrocytes and chondrocytes + ASCs groups using dynamic culture, which showed similar neo-cartilage formation capability with half of the chondrocytes replaced by ASCs for co-culture. This combined co-culture/dynamic culture strategy is expected to cut down the amount of donor chondrocytes needed for cartilage-tissue engineering.

MicroRNA-193b-3p regulates chondrogenesis and chondrocyte metabolism by targeting HDAC3.

Histone deacetylase 3 (HDAC3) plays a pivotal role in the repression of cartilage-specific gene expression in human chondrocytes. The aim of this study was to determine whether microRNA-193b-3p (miR-193b-3p) regulates the expression of HDAC3 during chondrogenesis and chondrocyte metabolism.Methods: miR-193b-3p expression was assessed in a human mesenchymal stem cell (hMSC) model of chondrogenesis, in interleukin-1β (IL-1β)-treated primary human chondrocytes (PHCs), and in non-degraded and degraded cartilage. hMSCs and PHCs were transfected with miR-193b-3p or its antisense inhibitor. A direct interaction between miR-193b-3p and its putative binding site in the 3′-untranslated region (3′-UTR) of HDAC3 mRNA was confirmed by performing luciferase reporter assays. Chondrocytes were transfected with miR-193b-3p before performing a chromatin immunoprecipitation assay with an anti-acetylated histone H3 antibody. To investigate miR-193b-3p-transfected PHCs in vivo, they were seeded in tricalcium phosphate-collagen-hyaluronate (TCP-COL-HA) scaffolds, which were then implanted in nude mice. In addition, plasma exosomal miR-193b-3p in samples from normal controls and patients with osteoarthritis (OA) were measured.Results: miR-193b-3p expression was elevated in chondrogenic and hypertrophic hMSCs, while expression was significantly reduced in degraded cartilage compared to non-degraded cartilage. In addition, miR-193b-3p suppressed the activity of reporter constructs containing the 3′-UTR of HDAC3, inhibited HDAC3 expression, and promoted histone H3 acetylation in the COL2A1, AGGRECAN, COMP, and SOX9 promoters. Treatment with the HDAC inhibitor trichostatin A (TSA) increased cartilage-specific gene expression and enhanced hMSCs chondrogenesis. TSA also increased AGGRECAN expression and decreased MMP13 expression in IL-1β-treated PHCs. Further, 8 weeks after implanting PHC-seeded TCP-COL-HA scaffolds subcutaneously in nude mice, we found that miR-193b overexpression strongly enhanced in vivo cartilage formation compared to that found under control conditions. We also found that patients with OA had lower plasma exosomal miR-193b levels than control subjects.Conclusions: These findings indicate that miR-193b-3p directly targets HDAC3, promotes H3 acetylation, and regulates hMSC chondrogenesis and metabolism in PHCs.

Electrospun thermosensitive hydrogel scaffold for enhanced chondrogenesis of human mesenchymal stem cells.

Hydrogels have demonstrated the excellent ability to enhance chondrogenesis of stem cells due to their hydrated fibrous nanostructure providing a cellular environment similar to native cartilage. However, the necessity for multi-step processes, including mixing of hydrogel precursor with cells and subsequent gelation in a mold to form a defined shape, limits their off-the-shelf usage. In this study, we developed a hybrid scaffold by combining a thermosensitive hydrogel with a mechanically stable polymer, which provides a facile means to inoculate cells in a 3D hydrogel with a mold-less, single step cell seeding process. We further showed that the hybrid scaffold enhanced chondrogenesis of mesenchymal stem cells, demonstrating its potential for cartilage tissue engineering.

Kruppel-like factor 4 regulates matrix metalloproteinase and aggrecanase gene expression in chondrocytes.

Kruppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays crucial roles during the development and maintenance of multiple organs. We and others have previously shown that KLF4 is involved in bone modeling and remodeling but roles played by KLF4 during skeletogenesis are still not fully understood. Here, we show that KLF4 is expressed in the epiphyseal growth plate and articular chondrocytes. Most articular chondrocytes expressed KLF4 in embryos but it localized only in a subset of superficial zone cells in postnatal mice. When KLF4 was overexpressed in chondrocytes in vitro, it severely repressed chondrocytic gene expressions. Global gene expression profiling of KLF4-transduced chondrocytes revealed matrix degrading proteinases of the matrix metalloproteinase and disintegrin and metalloproteinase with thrombospondin-1 domain families within the group of upregulated genes. Proteinase induction by KLF4 was alleviated by Trichostatin A treatment suggesting the possible involvement of epigenetic mechanisms on proteinase induction by KLF4. These results indicate the possible involvement of KLF4 in physiological and pathological aspects during cartilage development and maintenance.