Chondrocyte Gene Research Articles

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.

Cartilage injury suppresses endogenous retinoic acid through activation of TGFβ-activated kinase 1 (TAK1)

Purpose: A recent Genome Wide Association Study (GWAS) in hand OA has identified the association of hypomorphic variants in aldehyde dehydrogenase 1 family member A2 (ALDH1A2) with severe hand OA. This gene encodes the enzyme that irreversibly catalyzes the production of all-trans retinoic acid (atRA) from retinaldehyde. atRA has an essential role in embryonic limb development but its role in adult cartilage and in OA is not clear. Our group has demonstrated that endogenous retinoic acid is strongly suppressed by cartilage injury and this affects the regulation of inflammatory response genes. Maintaining cellular retinoic acid levels at time of injury, with a CYP 26 inhibitor, restrained inflammatory gene regulation. Previous work from our group has found that cartilage injury activates several signaling pathways involving release of transforming growth factor beta (TGFβ) and fibroblast growth factor (FGF2), and activation of TGFβ-activated kinase 1 (TAK1). Here we investigated the mechanism by which cartilage injury suppresses endogenous retinoic acid signaling. Methods: Articular cartilage was obtained from porcine metacarpophalangeal joints of 3-6 months old pigs. Cartilage injury was performed by explanting cartilage from the joint surface. To examine the role of specific injury pathways, cartilage injury was also performed on joints that had been injected with selective inhibitors (TGFβR (SB431542), FGF2R (SB402451) and TAK1 ((5Z)-7-oxozeanol) 1 hour prior to injury. RT-PCR was performed for known atRA-dependent genes. To identify injury-induced regulators of retinoic acid, a murine cartilage injury microarray was interrogated for molecules involved in the synthesis and degradation of atRA. Results: Cartilage injury led to rapid down-regulation of retinoic acid dependent genes (CYP26a, CYP26b and retinoic acid receptors: RARα, RARβ and RARγ) between 40 and 80% by 4h. For most genes this was sustained for up to 24h. This apparent fall in atRA dependent genes was not affected by blocking TGFβ or FGF2, but was inhibited by the TAK1 inhibitor, indicating that TAK1 controls atRA levels or signaling. Our cartilage injury microarray identified RDH12 as the most strongly regulated gene on the atRA pathway (12.5 fold, p=1.5x10-9). RDH12 is an important upstream enzyme, which converts retinaldehyde to retinol, and its overexpression will lead to reduction of cellular atRA level. ALDH1A2 protein was also suppressed 4 hours after porcine cartilage injury, predicting a further reduction of cellular atRA. Conclusions: Our study demonstrates that atRA dependent genes in chondrocytes are strongly suppressed by mechanical injury through TAK1 activation, indicating a reduction of endogenous atRA level. The strong induction of RDH12 and suppression of ALDH1A2 after injury may be responsible for driving these changes. We conclude that maintaining physiological levels of atRA is anti-inflammatory, and targeting the atRA pathway could reduce the catabolic response of cartilage to injury.

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.

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

Methylation of 4-aminobutyrate aminotransferase (ABAT) by dnmy3b regulates chondrocytes metabolism and the development of osteoarthritis (OA)

Purpose: Our recent work established that loss of Dnmt3b in chondrocytes leads to OA progression, at least in part, due to accelerated TCA cycle and elevated mitochondrial respiration. The goal of this study is to determine the downstream targets of Dnmt3b potentially involved in regulating chondrocyte energy metabolism and in the pathogenesis of OA. Methods: Primary articular chondrocyte (AC) isolation and culture: ACs were isolated from 2-month-old Dnmt3b f/f mice following with Ad-Cre virus to delete Dnmt3b gene. Abat siRNA, Lenti Abat and Vigabatrin were used to regulate Abat expression and activity. Whole genome bisulfite sequencing (Methyl-seq) and RNA-seq: Genomic DNA and RNA were isolated from Dnmt3b loss-of-function (LOF) ACs for detailed gene expression and methylation profile analyses. HPLC: TCA metabolites were measured by HPLC, including succinate and fumarate. Mito stress assay: Mitochondrial function was assessed by seahorse mito stress test to measure basal and maximal oxygen consumption rate (OCR) as well as ATP production. Meniscal Ligament Injury (MLI): MLI surgeries were performed on 10-week-old wild type mice to induce OA progression. Vigabatrin was given by i.p injection every other day for 6 or 10 weeks. Results: Increased Abat expression in OA chondrocytes. RNA-seq and Methyl-seq were performed to investigate the downstream targets of Dnmt3b and integrative analysis revealed an enrichment in genes and pathways that regulate cellular energy metabolism in Dnmt3b LOF cells. Among these gene targets was 4-aminobutyrate aminotransferase (Abat). Direct analysis of the Abat promoter region confirmed that Dnmt3b LOF resulted in reduced methylation in ACs (50% methylation in Control; and 3% methylation in Dnmt3b LOF. qPCR and Western blot further showed that both Abat gene and protein expression were induced in Dnmt3b LOF ACs and OA patients. Abat enhances mitochondrial oxidative phosphorylation (OXPHOS) and chondrocyte hypertrophy. Since Abat catalyzes the metabolism of GABA to succinate, HPLC mass spectroscopy validated an increase in both succinate (300%) and fumarate (150%) levels in Dnmt3b LOF cells. We further evaluated if Abat is involved in regulation of OXPHOS and AC hypertrophy and found that Abat siRNA resulted in a decrease in OXPHOS as reduced basal OCR (50%), maximal OCR (60%), and ATP production (50%). Abat inhibition in Dnmt3b LOF ACs by Vigabatrin blocked the increase in the expression of Col10a1, Mmp13, and Runx2 observed in the Dnmt3b LOF chondrocytes. In contrast, Abat overexpression led to a marked increase in basal OCR (300%), maximal OCR (500%), and ATP production (500%). Over-expression of Abat stimulated chondrocyte hypertrophy as reflected by upregulation of Runx2 (250%) and Mmp13 (600%). To link the Abat mediated increase of TCA metabolites as a mechanism regulating OXPHOS, we added 1mM succinate to WT AC cultures and found an increase in hypertrophic chondrocyte genes. In addition, succinate treatment increased the basal OCR (150%), maximal OCR (150%), and ATP production (200%). Abat inhibition attenuates OA progression in mice following knee injury: MLI surgeries were performed on 10-week-old male WT mice and vehicle treated mice developed advanced osteoarthritic changes with loss of cartilage, development of osteophytes, and subchondral bone thickening. However, mice treated with Vigabatrin displayed a protective effect against injury-induced OA progression at 6 and 10 weeks post MLI surgery. OARSI scoring showed marked lower scores in Vigabatrin treated mice. Conclusions: Our previous work has established that loss of Dnmt3b stimulated chondrocyte hypertrophy and resulted in increased OXPHOS, with increases in TCA metabolites, NADH, and basal respiration and maximal O2 capacity. Through integration of RNA-seq and Methyl-seq data, we now show that Abat is a target of Dnmt3b, and further establish a critical role for energy metabolism in the development of OA.

Development of novel lubrication vectors to reduce surface friction and inhibit pathological chondrocyte gene regulation in vivo

Development of novel lubrication vectors to reduce surface friction and inhibit pathological chondrocyte gene regulation in vivo

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.

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.

Expression of Pluripotency Genes in Chondrocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) constitute an important breakthrough in regenerative medicine, particularly in orthopedics, where more effective treatments are urgently needed. Despite the promise of hiPSCs only limited data on in vitro chondrogenic differentiation of hiPSCs are available. Therefore, we compared the gene expression profile of pluripotent genes in hiPSC-derived chondrocytes (ChiPS) to that of an hiPSC cell line created by our group (GPCCi001-A). The results are shown on heatmaps and plots and confirmed by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. Unlike the ChiPS, our GPCCi001-A cells maintained their pluripotency state during long-term culture, thus demonstrating that this cell line was comprised of stable, fully pluripotent hiPSCs. Moreover, these chondrocyte-like cells not only presented features that are characteristic of chondrocytes, but they also lost their pluripotency, which is an important advantage in favor of using this cell line in future clinical studies.

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.

Intracellular biosynthesis of lipids and cholesterol by Scap and Insig in mesenchymal cells regulates long bone growth and chondrocyte homeostasis.

ABSTRACTDuring enchondral ossification, mesenchymal cells express genes regulating the intracellular biosynthesis of cholesterol and lipids. Here, we have investigated conditional deletion of Scap or of Insig1 and Insig2 (Scap inhibits intracellular biosynthesis and Insig proteins activate intracellular biosynthesis). Mesenchymal condensation and chondrogenesis was disrupted in mice lacking Scap in mesenchymal progenitors, whereas mice lacking the Insig genes in mesenchymal progenitors had short limbs, but normal chondrogenesis. Mice lacking Scap in chondrocytes showed severe dwarfism, with ectopic hypertrophic cells, whereas deletion of Insig genes in chondrocytes caused a mild dwarfism and shortening of the hypertrophic zone. In vitro studies showed that intracellular cholesterol in chondrocytes can derive from exogenous and endogenous sources, but that exogenous sources cannot completely overcome the phenotypic effect of Scap deficiency. Genes encoding cholesterol biosynthetic proteins are regulated by Hedgehog (Hh) signaling, and Hh signaling is also regulated by intracellular cholesterol in chondrocytes, suggesting a feedback loop in chondrocyte differentiation. Precise regulation of intracellular biosynthesis is required for chondrocyte homeostasis and long bone growth, and these data support pharmacological modulation of cholesterol biosynthesis as a therapy for select cartilage pathologies.

Systematic Analysis of Differential Expression Profile in Rheumatoid Arthritis Chondrocytes Using Next-Generation Sequencing and Bioinformatics Approaches.

Cartilage destruction in rheumatoid arthritis (RA) occurs primarily in the pannus-cartilage interface. The close contact of the synovium-cartilage interface implicates crosstalk between synovial fibroblasts and chondrocytes. The aim of this study is to explore the differentially expressed genes and novel microRNA regulations potentially implicated in the dysregulated cartilage homeostasis in joint destruction of RA. Total RNAs were extracted from human primary cultured normal and RA chondrocytes for RNA and small RNA expression profiling using next-generation sequencing. Using systematic bioinformatics analyses, we identified 463 differentially expressed genes in RA chondrocytes were enriched in biological functions related to altered cell cycle process, inflammatory response and hypoxic stimulation. Moreover, fibroblast growth factor 9 (FGF9), kynureninase (KYNU), and regulator of cell cycle (RGCC) were among the top dysregulated genes identified to be potentially affected in the RA joint microenvironment, having similar expression patterns observed in arrays of clinical RA synovial tissues from the Gene Expression Omnibus database. Additionally, among the 31 differentially expressed microRNAs and 10 candidate genes with potential microRNA-mRNA interactions in RA chondrocytes, the novel miR-140-3p-FGF9 interaction was validated in different microRNA prediction databases, and proposed to participate in the pathogenesis of joint destruction through dysregulated cell growth in RA. The findings provide new perspectives for target genes in the management of cartilage destruction in RA.

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.

Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis

Osteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood. We investigated genes and pathways that mark OA progression in isolated primary chondrocytes taken from paired intact versus degraded articular cartilage samples across 38 patients undergoing joint replacement surgery (discovery cohort: 12 knee OA, replication cohorts: 17 knee OA, 9 hip OA patients). We combined genome-wide DNA methylation, RNA sequencing, and quantitative proteomics data. We identified 49 genes differentially regulated between intact and degraded cartilage in at least two –omics levels, 16 of which have not previously been implicated in OA progression. Integrated pathway analysis implicated the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Using independent replication datasets, we showed that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes. AQP1, COL1A1 and CLEC3B were significantly differentially regulated across all three –omics levels, confirming their differential expression in human disease. Through integration of genome-wide methylation, gene and protein expression data in human primary chondrocytes, we identified consistent molecular players in OA progression that replicated across independent datasets and that have translational potential.

Effect of electromagnetic fields on human osteoarthritic and non-osteoarthritic chondrocytes

BackgroundStudies of the effects of electromagnetic fields (EMFs) on cartilaginous cells show a broad range of outcomes. However EMFs are not yet clinically applied as standard treatment of osteoarthritis, as EMF effects are showing varying outcomes in the literature. The aim of this study was to examine effects of EMFs (5 mT or 8 mT) on osteoarthritic (OA) and non-OA chondrocytes in order to investigate whether EMF effects are related to chondrocyte and EMF quality.MethodsPellets of human OA and non-OA chondrocytes were exposed to a sinusoidal 15 Hz EMF produced by a solenoid. Control groups were cultivated without EMF under standard conditions for 7 days. Cultures were examined by staining, immunohistochemistry and quantitative real-time PCR for RNA corresponding to cartilage specific proteins (COL2A1, ACAN, SOX9).ResultsOA chondrocytes increased the expression of COL2A1 and ACAN under 5 mT EMF compared to control. In contrast no changes in gene expression were observed in non-OA chondrocytes. OA and non-OA chondrocytes showed no significant changes in gene expression under 8 mT EMF.ConclusionA 5 mT EMF increased the expression of cartilage specific genes in OA chondrocytes whereas in non-OA chondrocytes no changes in gene expression were observed. An 8 mT EMF however showed no effect altogether. This suggests that EMF effects are related to EMF but also to chondrocyte quality. Further studies about the clinical relevance of this effect are necessary.

Downregulation of DEC1 contributes to the neurotoxicity induced by MPP+ by suppressing PI3K/Akt/GSK3β pathway.

Differentiated embryonic chondrocyte gene 1 (DEC1) is involved in the neuronal differentiation and development. The aim of this study is to investigate the role of DEC1 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP+ )-induced PD model. The location of DEC1 and tyrosine hydroxylase (TH)-positive neurons were detected by immunofluorescence. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse subacute model of PD was established to evaluate the change of DEC1 expression in midbrain. Then, SH-SY5Y cells were used to investigate the role of DEC1 in MPP+ -induced neurotoxicity. We showed that the co-expressed DEC1 and TH neurons took up more than 80% of the expressed TH neurons in the midbrain of mice. DEC1/TH double-positive neurons decreased by 40.6% in SNpc and 28.8% in VTA of MPTP-injured mice. Consistently, DEC1, TH and dopamine transporter (DAT) expression decreased in the midbrain of MPTP mice. In SY-SY5Y cells, MPP+ significantly suppressed DEC1 expression and increased the cleaved caspase 3/caspase 3 and Bax/Bcl-2. DEC1 overexpression relieved, whereas DEC1 knockdown aggravated MPP+ -induced cytotoxicity. Likewise, DEC1 overexpression and knockdown inversely regulated the expression of β-catenin and PI3Kp110α (PIK3CA), an essential role in Wnt/β-catenin and PI3K/Akt signaling pathways. Interestingly, LY294002, an inhibitor of PI3K/Akt signaling, aggravated, whereas LiCl, an activator of Wnt/β-catenin signaling, abolished the reduction in DEC1 by MPP+ . It is established that these two pathways are interconnected by the phosphorylation status of GSK3β. DEC1 overexpression increased but MPP+ and DEC1 knockdown decreased GSK3β phosphorylation. Downregulation of DEC1 contributes to MPP+ -induced neurotoxicity by suppressing PI3K/Akt/GSK3β pathway.

Effect of alginate concentration on chondrogenesis of co-cultured human adipose-derived stem cells and nasal chondrocytes: a biological study

BackgroundThe three-dimensional (3D) system is one of the important factors to engineer a biocompatible and functional scaffold for the applications of cell-based therapies for cartilage repair. The 3D alginate hydrogels system has previously been shown to potentially promote chondrogenesis. The chondrocytic differentiation of co-cultured adipose-derived stem cells (ADSCs) and nasal chondrocytes (NCs) within alginate constructs are hypothesized to be influenced by concentration of alginate hydrogel. In this study, we evaluated the effects of alginate concentration on chondrogenic differentiation of ADSCs and NCs co-cultured in a biological approach.MethodThe co-cultured cells of 2:1 ADSCs-to-NCs ratio were encapsulated in alginate constructs in one of three concentrations (1.0%, 1.2% and 1.5%) and cultured under serum free conditions for 7 days. Cell viability, cell proliferation, immunohistochemical, gycosaminogylycans (GAG) synthesis, and gene expression were examined.ResultsOverall, the 1.2% alginate concentration group was relatively effective in chondrocytic differentiation in comparable to other groups. The cell morphology, cell viability, and cell proliferation revealed initial chondrogenic differentiation by the formation of cell clusters as well as the high permeability for exchange of solutes. The formation of newly synthesis cartilage-specific extracellular matrix in 1.2% group was demonstrated by positive immunohistochemical staining of collagen type II. The co-cultured cells in 1.2% group highly expressed COL II, ACP and SOX-9, compared to 1.0% and 1.5% groups, denote the retention of cartilaginous-specific phenotype by suppressing the undifferentiation stem cell markers of SOX-2 and OCT-4. The study showed 1.2% group was less likely to differentiate towards osteogenesis by downregulating hyperthrophy chondrocytic gene of COL X and osseous marker genes of OSC and OSP.ConclusionThis study suggests that variations in the alginate concentration of co-cultured ADSCs and NCs influenced the chondrogenesis. The remarkable biological performance on chondrogenic differentiation in regulating the concentration of alginate 3D culture provides new insights into the cell cross-talk and demonstrates the effectiveness in regenerative therapies of cartilage defects in tissue engineering.

Global chondrocyte gene expression after a single anabolic loading period: Time evolution and re-inducibility of mechano-responses.

Aim of this study was a genome-wide identification of mechano-regulated genes and candidate pathways in human chondrocytes subjected to a single anabolic loading episode and characterization of time evolution and re-inducibility of the response. Osteochondral constructs consisting of a chondrocyte-seeded collagen-scaffold connected to β-tricalcium-phosphate were pre-cultured for 35 days and subjected to dynamic compression (25% strain, 1 Hz, 9 × 10 min over 3 hr) before microarray-profiling was performed. Proteoglycan synthesis was determined by 35 S-sulfate-incorporation over 24 hr. Cell viability and hardness of constructs were unaltered by dynamic compression while proteoglycan synthesis was significantly stimulated (1.45-fold, p = 0.016). Among 115 significantly regulated genes, 114 were up-regulated, 48 of them ≥ twofold. AP-1-relevant transcription factors FOSB and FOS strongly increased in line with elevated ERK1/2-phosphorylation and rising MAP3K4 expression. Expression of proteoglycan-synthesizing enzymes CHSY1 and GALNT4 was load-responsive as were factors associated with the MAPK-, TGF-β-, calcium-, retinoic-acid-, Wnt-, and Notch-signaling pathway which were significantly upregulated SOX9, and BMP6 levels rose significantly also after multiple loading episodes at daily intervals even at the 14th cycle with no indication for desensitation. Canonical pSmad2/3 and pSmad1/5/9-signaling showed no consistent regulation. This study associates novel genes with mechanoregulation in chondrocytes, raising SOX9 protein levels with anabolic loading and suggests that more pathways than so far anticipated apparently work together in a complex network of stimulators and feedback-regulators. Upregulation of mechanosensitive indicators extending differentially into the resting time provides crucial knowledge to maximize cartilage matrix deposition for the generation of high-level cartilage replacement tissue.