Cultures Of Articular Chondrocytes Research Articles

Inhibitory effects of Acanthopanax sessiliflorus Harms extract on the etiology of rheumatoid arthritis in a collagen-induced arthritis mouse model

BackgroundThe biological function of Acanthopanax sessiliflorus Harm (ASH) has been investigated on various diseases; however, the effects of ASH on arthritis have not been investigated so far. This study investigates the effects of ASH on rheumatoid arthritis (RA).MethodsSupercritical carbon dioxide (CO2) was used for ASH extract preparation, and its primary components, pimaric and kaurenoic acids, were identified using gas chromatography-mass spectrometer (GC–MS). Collagenase-induced arthritis (CIA) was used as the RA model, and primary cultures of articular chondrocytes were used to examine the inhibitory effects of ASH extract on arthritis in three synovial joints: ankle, sole, and knee.ResultsPimaric and kaurenoic acids attenuated pro-inflammatory cytokine-mediated increase in the catabolic factors and retrieved pro-inflammatory cytokine-mediated decrease in related anabolic factors in vitro; however, they did not affect pro-inflammatory cytokine (IL-1β, TNF-α, and IL-6)-mediated cytotoxicity. ASH effectively inhibited cartilage degradation in the knee, ankle, and toe in the CIA model and decreased pannus development in the knee. Immunohistochemistry demonstrated that ASH mostly inhibited the IL-6-mediated matrix metalloproteinase. Gene Ontology and pathway studies bridge major gaps in the literature and provide insights into the pathophysiology and in-depth mechanisms of RA-like joint degeneration.ConclusionsTo the best of our knowledge, this is the first study to conduct extensive research on the efficacy of ASH extract in inhibiting the pathogenesis of RA. However, additional animal models and clinical studies are required to validate this hypothesis.

Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes.

Context: Osteoarthritis (OA) is an invalidating articular disease characterized by cartilage degradation and inflammatory events. In horses, OA is associated with up to 60% of lameness and leads to reduced animal welfare along with extensive economic losses; currently, there are no curative therapies to treat OA. The mesenchymal stromal cell (MSC) secretome exhibits anti-inflammatory properties, making it an attractive candidate for improving the management of OA. In this study, we determined the best storage conditions for conditioned media (CMs) and tested whether priming MSCs with cytokines can enhance the properties of the MSC secretome. Methods: First, properties of CMs collected from bone-marrow MSC cultures and stored at -80°C, -20°C, 4°C, 20°C or 37°C were assessed on 3D cultures of equine articular chondrocytes (eACs). Second, we primed MSCs with IL-1β, TNF-α or IFN-γ, and evaluated the MSC transcript levels of immunomodulatory effectors and growth factors. The primed CMs were also harvested for subsequent treatment of eACs, either cultured in monolayers or as 3D cell cultures. Finally, we evaluated the effect of CMs on the proliferation and the phenotype of eACs and the quality of the extracellular matrix of the neosynthesized cartilage. Results: CM storage at -80°C, -20°C, and 4°C improved collagen protein accumulation, cell proliferation and the downregulation of inflammation. The three cytokines chosen for the MSC priming influenced MSC immunomodulator gene expression, although each cytokine led to a different pattern of MSC immunomodulation. The cytokine-primed CM had no major effect on eAC proliferation, with IL-1β and TNF-α slightly increasing collagen (types IIB and I) accumulation in eAC 3D cultures (particularly with the CM derived from MSCs primed with IL-1β), and IFN-γ leading to a marked decrease. IL-1β-primed CMs resulted in increased eAC transcript levels of MMP1, MMP13 and HTRA1, whereas IFNγ-primed CMs decreased the levels of HTRA1 and MMP13. Conclusion: Although the three cytokines differentially affected the expression of immunomodulatory molecules, primed CMs induced a distinct effect on eACs according to the cytokine used for MSC priming. Different mechanisms seemed to be triggered by each priming cytokine, highlighting the need for further investigation. Nevertheless, this study demonstrates the potential of MSC-CMs for improving equine OA management.

Cytotoxic effects of different mouthwash solutions on primary human articular chondrocytes and normal human articular cartilage – an in vitro study

ObjectivesTo compare the cytotoxicity of octenidine dihydrochloride and chlorhexidine gluconate at different concentrations on primary human articular chondrocytes and cartilage.Materials and methodsPrimary cultures of human normal adult articular chondrocytes were exposed to octenidine dihydrochloride (0.001562%, 0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, and 0.1%), chlorhexidine gluconate (0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, 0.1%, and 0.2%), and control (Dulbecco’s modified Eagle medium or phosphate-buffered saline) for 30 s. Normal human articular cartilage explants were exposed to octenidine dihydrochloride (0.1% versus control) and chlorhexidine gluconate (0.1% versus control) for 30 s. The viability of human articular chondrocytes was measured by Trypan blue staining, Cell Proliferation Reagent WST-1, and Live/Dead staining. The proliferation of human chondrocytes was measured using the Cell Proliferation Reagent WST-1. The viability of human articular cartilage explants was measured by using Live/Dead staining.ResultsOctenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability and proliferation in a dose-dependent manner in primary human articular chondrocytes. Octenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability in human articular cartilage explant cultures.ConclusionThe degree of toxicity varied between octenidine dihydrochloride and chlorhexidine gluconate, with chlorhexidine gluconate being less toxic than octenidine dihydrochloride at the same concentration. Additionally, both octenidine dihydrochloride and chlorhexidine gluconate evaluation had cytotoxic effects on human articular cartilage. Therefore, dosing for the antimicrobial mouthwash ingredients administration would ideally be determined to remain below IC50.Clinical relevanceThese data support the in vitro safety of antimicrobial mouthwashes on primary adult human articular chondrocytes.

Hyaluronic acid and multiwalled carbon nanotubes as bioink additives for cartilage tissue engineering

Articular cartilage and meniscus injuries are prevalent disorders with insufficient regeneration responses offered by available treatment methods. In this regard, 3D bioprinting has emerged as one of the most promising new technologies, offering novel treatment options. Additionally, the latest achievements from the fields of biomaterials and tissue engineering research identified constituents facilitating the creation of biocompatible scaffolds. In this study, we looked closer at hyaluronic acid and multi-walled carbon nanotubes as bioink additives. Firstly, we assessed the minimal concentrations that stimulate cell viability, and decrease reactive oxygen species and apoptosis levels in 2D cell cultures of normal human knee articular chondrocytes (NHAC) and human adipose-derived mesenchymal stem cells (hMSC-AT). In this regard, 0.25 mg/ml of hyaluronic acid and 0.0625 mg/ml of carbon nanotubes were selected as the most optimal concentrations. In addition, we investigated the protective influence of 2-phospho-L-ascorbic acid in samples with carbon nanotubes. Tests conducted on 3D bioprinted constructs revealed that only a combination of components positively impacted cell viability throughout the whole experiment. Gene expression analysis of COL1A1, COL6A1, HIF1A, COMP, RUNX2, and POU5F1 showed significant changes in the expression of all analyzed genes with a progressive overall loss of transcriptional activity in most of them.

3D culture of bovine articular chondrocytes in viscous medium encapsulated in agarose hydrogels for investigation of viscosity influence on cell functions.

The mechanical properties of an extracellular microenvironment can affect cell functions. The effects of elasticity and viscoelasticity on cell functions have been extensively studied with hydrogels of tunable mechanical properties. However, investigation of the viscosity effect on cell functions is still very limited and it can be tricky to explore how viscosity affects cells in three-dimensional (3D) culture due to the lack of appropriate tools. In this study, agarose hydrogel containers were prepared and used to encapsulate viscous media for 3D cell culture to investigate the viscosity effect on the functions of bovine articular chondrocytes (BACs). Polyethylene glycol of different molecular weights was used to adjust culture medium viscosity in a large range (72.8-679.2 mPa s). The viscosity affected gene expression and secretion of cartilagenious matrices, while it did not affect BAC proliferation. The BACs cultured in the lower viscosity medium (72.8 mPa s) showed a higher level of cartilaginous gene expression and matrix secretion.

First study on the effect of transforming growth factor beta 1 and insulin-like growth factor 1 on the chondrogenesis of elephant articular chondrocytes in a scaffold-based 3D culture model.

Background and Aim:Osteoarthritis (OA) is recognized as a degenerative joint disease that leads to chronic pain and low quality of life in animals. Captive elephants, the largest land mammals with a long lifespan, are more prone to develop OA due to restricted spaces and insufficient physical activity. This study aimed to investigate the effect of transforming growth factor-β1 (TGF-β1) and insulin-like growth factor 1 (IGF-1) on elephant chondrogenesis in a scaffold culture of articular chondrocytes.Materials and Methods:Elephant chondrocytes-seeded gelatin scaffolds were cultured in chondrogenic media with or without 10 ng/mL of TGF-β1 or IGF-1 alone or 5–10 ng/mL of their combination for up to 21 days. The mRNA expression of cartilage-specific anabolic genes, ACAN and COL2A1, was analyzed using a real-time reverse transcription-polymerase chain reaction. The amounts of sulfated glycosaminoglycans (sGAGs) in conditioned media and contents in cultured scaffolds were determined through dimethylmethylene blue assay. Cell morphology, accumulation of proteoglycans, and details of the cultured scaffolds were determined using hematoxylin-eosin staining, safranin O staining, and scanning electron microscopy (SEM), respectively.Results:TGF-β1 alone significantly upregulated ACAN gene expression but not COL2A1, while IGF-1 alone did not enhance both ACAN and COL2A1 genes. The combination significantly upregulated both mRNA expression levels of ACAN and COL2A1 gene at day 14. The sGAGs accumulation and contents in the treatment groups, except IGF-1 tended to be higher than the controls, concomitantly with the production of the extracellular matrix, showed the formation of a cartilage-like tissue through histological and SEM analyses.Conclusion:Together, our results suggest that the single treatment of TGF-β1 has a selective effect on ACAN gene, while the combined growth factors seem to be an advantage on elephant chondrogenesis. This three-dimensional culture model is probably helpful for developing cartilage regeneration in vitro and is further applied in tissue engineering for OA treatment in vivo.

Inhibitory Effects of IL-6-Mediated Matrix Metalloproteinase-3 and -13 by Achyranthes japonica Nakai Root in Osteoarthritis and Rheumatoid Arthritis Mice Models

Achyranthes japonica Nakai root (AJNR) is used to treat osteoarthritis (OA) and rheumatoid arthritis (RA) owing to its anti-inflammatory and antioxidant effects. This study investigated the inhibitory effects of AJNR on arthritis. AJNR was extracted using supercritical carbon dioxide (CO2), and its main compounds, pimaric and kaurenoic acid, were identified. ANJR’s inhibitory effects against arthritis were evaluated using primary cultures of articular chondrocytes and two in vivo arthritis models: destabilization of the medial meniscus (DMM) as an OA model, and collagenase-induced arthritis (CIA) as an RA model. AJNR did not affect pro-inflammatory cytokine (IL-1β, TNF-α, IL-6)-mediated cytotoxicity, but attenuated pro-inflammatory cytokine-mediated increases in catabolic factors, and recovered pro-inflammatory cytokine-mediated decreases in related anabolic factors related to in vitro. The effect of AJNR is particularly specific to IL-6-mediated catabolic or anabolic alteration. In a DMM model, AJNR decreased cartilage erosion, subchondral plate thickness, osteophyte size, and osteophyte maturity. In a CIA model, AJNR effectively inhibited cartilage degeneration and synovium inflammation in either the ankle or knee and reduced pannus formation in both the knee and ankle. Immunohistochemistry analysis revealed that AJNR mainly acted via the inhibitory effects of IL-6-mediated matrix metalloproteinase-3 and -13 in both arthritis models. Therefore, AJNR is a potential therapeutic agent for relieving arthritis symptoms.

Integrin \u03b12\u03b21 plays an important role in the interaction between human articular cartilage-derived chondrocytes and atelocollagen gel

Although atelocollagen gel is used as a scaffold for culturing human articular cartilage-derived chondrocytes, little is known about cell–gel interactions. In this study, we investigated the mechanism via which atelocollagen gel affects human articular cartilage-derived chondrocytes. Two types of three-dimensional cultures of human articular cartilage-derived chondrocytes (i.e., with and without atelocollagen gel) were compared. While the amount of atelocollagen gel in culture gradually decreased with time, it promoted the expression of matrix metalloproteinases (MMPs) during the early stages of culture. Genome-wide differential gene expression analysis revealed that cell membrane- and extracellular matrix-related genes were highly ranked among up- and down-regulated groups in cells cultured in the presence of atelocollagen gel. Among the integrin family of genes, the expression of integrin subunit alpha 2 and integrin subunit alpha 10 was significantly increased in the presence of atelocollagen gel. Blocking α2β1 integrin with the specific inhibitor BTT 3033 had a significant effect on cell proliferation, MMP expression, and cell shape, as well as on the response to mechanical stimulation. Taken together, our findings indicate that the α2β1 integrin pathway plays an important role in the interaction of atelocollagen gel with human articular cartilage-derived chondrocytes and may be a potential therapeutic target for articular cartilage disorders.

Metabolic Syndrome Predisposes to Osteoarthritis: Lessons from Model System.

The present study aims to assess for temporal changes in tibial subchondral bone and cartilage in WNIN/Gr-Ob rats (portraying obesity, insulin resistance, dyslipidemia, impaired glucose tolerance, hypertension) in comparison with Wistar controls (WNIN) using anthropometry, micro-computed tomography (micro-CT), scanning electron microscopy (SEM), histopathology, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence. Body weight, abdominal circumference, body mass index (BMI), lean/fat mass, serum tumor necrosis factor (TNF)-α levels were measured (ELISA), followed by ultrastructural analysis of tibial subchondral bone (micro-CT) and cartilage architecture (histopathology and SEM) in WNIN/Gr-Ob and WNIN rats with age (3, 6 and 9 months). Additionally, primary cultures of articular chondrocytes isolated from 6-month-old WNIN/Gr-Ob and WNIN rats were assessed for matrix metalloproteinase (MMP)-13 and Collagen type II (COL2A1) by immunofluorescence. WNIN/Gr-Ob rats exhibited frank obesity with increased BMI, lean and fat mass vis-à-vis significantly higher levels of serum TNF-α (6>9>3 months) as compared with the controls. With an increase in BMI, WNIN/Gr-Ob rats presented with tibial cartilage fibrillation, erosion, osteophyte formation (6 months) and subchondral bone cyst (9 months) confirmed by histology and SEM. An increase in subchondral trabecular bone volume (sclerosis with decreased plate porosity) was observed in all ages in WNIN/Gr-Ob rats compared to their Control. Gaining insights, primary cultures of articular chondrocytes complemented with altered cellular expressions of COL2A1 and MMP-13 from WNIN/Gr-Ob rats, indicating osteoarthritis (OA) progression. Multiple metabolic perturbations featured in WNIN/Gr-Ob rats were effective to induce spontaneous OA-like degenerative changes affecting knee joints akin to human OA.

GSK5182, 4-Hydroxytamoxifen Analog, a New Potential Therapeutic Drug for Osteoarthritis.

Estrogen-related receptors (ERRs) are the first identified orphan nuclear receptors. The ERR family consists of ERRα, ERRβ, and ERRγ, regulating diverse isoform-specific functions. We have reported the importance of ERRγ in osteoarthritis (OA) pathogenesis. However, therapeutic approaches with ERRγ against OA associated with inflammatory mechanisms remain limited. Herein, we examined the therapeutic potential of a small-molecule ERRγ inverse agonist, GSK5182 (4-hydroxytamoxifen analog), in OA, to assess the relationship between ERRγ expression and pro-inflammatory cytokines in mouse articular chondrocyte cultures. ERRγ expression increased following chondrocyte exposure to various pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Pro-inflammatory cytokines dose-dependently increased ERRγ protein levels. In mouse articular chondrocytes, adenovirus-mediated ERRγ overexpression upregulated matrix metalloproteinase (MMP)-3 and MMP-13, which participate in cartilage destruction during OA. Adenovirus-mediated ERRγ overexpression in mouse knee joints or ERRγ transgenic mice resulted in OA. In mouse joint tissues, genetic ablation of Esrrg obscured experimental OA. These results indicate that ERRγ is involved in OA pathogenesis. In mouse articular chondrocytes, GSK5182 inhibited pro-inflammatory cytokine-induced catabolic factors. Consistent with the in vitro results, GSK5182 significantly reduced cartilage degeneration in ERRγ-overexpressing mice administered intra-articular Ad-Esrrg. Overall, the ERRγ inverse agonist GSK5182 represents a promising therapeutic small molecule for OA.

Progenitor Cells Activated by Platelet Lysate in Human Articular Cartilage as a Tool for Future Cartilage Engineering and Reparative Strategies.

Regenerative strategies for human articular cartilage are still challenging despite the presence of resident progenitor cell population. Today, many efforts in the field of regenerative medicine focus on the use of platelet derivatives due to their ability to reactivate endogenous mechanisms supporting tissue repair. While their use in orthopedics continues, mechanisms of action and efficacy need further characterization. We describe that the platelet lysate (PL) is able to activate chondro-progenitor cells in a terminally differentiated cartilage tissue. Primary cultures of human articular chondrocytes (ACs) and cartilage explants were set up from donor hip joint biopsies and were treated in vitro with PL. PL recruited a chondro-progenitors (CPCs)-enriched population from ex vivo cartilage culture, that showed high proliferation rate, clonogenicity and nestin expression. CPCs were positive for in vitro tri-lineage differentiation and formed hyaline cartilage-like tissue in vivo without hypertrophic fate. Moreover, the secretory profile of CPCs was analyzed, together with their migratory capabilities. Some CPC-features were also induced in PL-treated ACs compared to fetal bovine serum (FBS)-control ACs. PL treatment of human articular cartilage activates a stem cell niche responsive to injury. These facts can improve the PL therapeutic efficacy in cartilage applications.

Post-treatment of hyaluronan to decrease the apoptotic effects of carprofen in canine articular chondrocyte culture.

A major concern associated with the use of drugs is their adverse side effects. Specific examples of the drugs of concern include antibiotic agents and non-steroidal anti-inflammatory drugs. Despite the presence of a high degree of efficacy for specific conditions, these drugs may deteriorate the surrounding tissues that are exposed to them. Often, carprofen is used for joint inflammation; however, it may stimulate cartilage degradation which can then lead to osteoarthritis progression. In this study, hyaluronan was combined with carprofen treatment in three different applications (pre-treatment, co-treatment and post-treatment) on normal canine chondrocytes to determine whether Hyaluronan (HA) is capable of mitigating the degree of chondrotoxicity of carprofen. Our findings revealed that carprofen at IC20 (0.16 mg/mL) decreased viability and increased nitric oxide (NO) production. Importantly, carprofen induced the apoptosis of canine chondrocytes via the up-regulation of Bax, Casp3, Casp8, Casp9 and NOS2 as compared to the control group. Although the co-treatment of HA and carprofen appeared not to further alleviate the chondrotoxicity of carprofen due to the presence of a high number of apoptotic chondrocytes, post-treatment with HA (carprofen treatment for 24 h and then changed to HA for 24 h) resulted in a decrease in chondrocyte apoptosis by the down-regulation of Bax, Casp3, Casp8, Casp9, NOS2, along with NO production when compared with the treatment of carprofen for 48 h (P < 0.05). These results suggest that HA can be used as a therapeutic agent to mitigate the degree of chondrotoxicity of carprofen.

Rapid Photocrosslinking of Silk Hydrogels with High Cell Density and Enhanced Shape Fidelity.

Silk fibroin hydrogels crosslinked through di-tyrosine bonds are clear, elastomeric constructs with immense potential in regenerative medicine applications. In this study, demonstrated is a new visible light-mediated photoredox system for di-tyrosine bond formation in silk fibroin that overcomes major limitations of current conventional enzymatic-based crosslinking. This photomediated system rapidly crosslinks silk fibroin (<1 min), allowing encapsulation of cells at significantly higher cell densities (15 million cells mL-1 ) while retaining high cell viability (>80%). The photocrosslinked silk hydrogels present more stable mechanical properties which do not undergo spontaneous transition to stiff, β-sheet-rich networks typically seen for enzymatically crosslinked systems. These hydrogels also support long-term culture of human articular chondrocytes, with excellent cartilage tissue formation. This system also facilitates the first demonstration of biofabrication of silk fibroin constructs in the absence of chemical modification of the protein structure or rheological additives. Cell-laden constructs with complex, ordered, graduated architectures, and high resolution (40 µm) are fabricated using the photocrosslinking system, which cannot be achieved using the enzymatic crosslinking system. Taken together, this work demonstrates the immense potential of a new crosslinking approach for fabrication of elastomeric silk hydrogels with applications in biofabrication and tissue regeneration.

Intracellular iron uptake is favored in Hfe‐KO mouse primary chondrocytes mimicking an osteoarthritis‐related phenotype

HFE-hemochromatosis is a disease characterized by a systemic iron overload phenotype mainly associated with mutations in the HFE protein (HFE) gene. Osteoarthritis (OA) has been reported as one of the most prevalent complications in HFE-hemochromatosis patients, but the mechanisms associated with its onset and progression remain incompletely understood. In this study, we have characterized the response to high iron concentrations of a primary culture of articular chondrocytes isolated from newborn Hfe-KO mice and compared the results with that of a similar experiment developed in cells from C57BL/6 wild-type (wt) mice. Our data provide evidence that both wt- and Hfe-KO-derived chondrocytes, when exposed to 50 μM iron, develop characteristics of an OA-related phenotype, such as an increased expression of metalloproteases, a decreased extracellular matrix production, and a lower expression level of aggrecan. In addition, Hfe-KO cells also showed an increased expression of iron metabolism markers and MMP3, indicating an increased susceptibility to intracellular iron accumulation and higher levels of chondrocyte catabolism. Accordingly, upon treatment with 50 μM iron, these chondrocytes were found to preferentially differentiate toward hypertrophy with increased expression of collagen I and transferrin and downregulation of SRY (sex-determining region Y)-box containing gene 9 (Sox9). In conclusion, high iron exposure can compromise chondrocyte metabolism, which, when simultaneously affected by an Hfe loss of function, appears to be more susceptible to the establishment of an OA-related phenotype.

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.

A Human Chondrocyte-Derived In Vitro Model of Alcohol-Induced and Steroid-Induced Femoral Head Necrosis.

BackgroundWorldwide, femoral head necrosis (FHN), which is also known as avascular necrosis of the femoral head or osteonecrosis of the femoral head, affects millions of people. Excess alcohol intake and steroid use are two common associations with FHN, but their pathogenesis remains unknown. The aim of this study was to develop an in vitro model using human chondrocytes to study alcohol-induced and steroid-induced FHN.Material/MethodsIn this study, the in vitro model used a monolayer culture of articular chondrocytes derived from patients with non-traumatic FHN (Ficat and Arlet, Stage III). Normal chondrocytes were obtained from patients with femoral neck fracture resulting from road traffic accident (Garden, Stage IV). Alcohol-stimulated and steroid-stimulated articular chondrocytes were evaluated by a cell proliferation assay, measurement of calcium levels (alizarin red), measurement of alkaline phosphatase (ALP) levels, detection of glycosaminoglycan (GAG) secretion using safranin O histochemical staining, and analysis of cartilage-specific genes, ACAN, SOX9, OPG, TGF-β, RANKL, and RUNX2, using quantitative real-time polymerase chain reaction (qRT-PCR).ResultsBoth alcohol and steroids, but especially steroids, accelerated the degradation of cartilage by suppression of chondrogenesis while promoting chondrocyte hypertrophy and activating osteogenic differentiation, as assessed by cell proliferation assay, detection of glycosaminoglycan (GAG) secretion, and analysis of cartilage-specific genes.ConclusionsA human chondrocyte-derived in vitro model of alcohol-induced and steroid-induced FHN demonstrated chondrocyte hypertrophy and activated osteogenic differentiation.

Suramin increases cartilage proteoglycan accumulation in vitro and protects against joint damage triggered by papain injection in mouse knees in vivo

ObjectivesSuramin is an old drug used for the treatment of African sleeping sickness. We investigated therapeutic repositioning of suramin to protect against cartilage damage, as suramin may interact with tissue...

Selective Role of Discoidin Domain Receptor 2 in Murine Temporomandibular Joint Development and Aging

Temporomandibular joint (TMJ) disorders are often associated with development of osteoarthritis-like changes in the mandibular condyle. Discoidin domain receptor 2 (DDR2), a collagen receptor preferentially activated by type I and III collagen found in the TMJ and other fibrocartilages, has been associated with TMJ degeneration, but its role in normal joint development has not been previously examined. Using Ddr2 LacZ-tagged mice and immunohistochemistry, we found that DDR2 is preferentially expressed and activated in the articular zone of TMJs but not knee joints. To assess the requirement for Ddr2 in TMJ development, studies were undertaken to compare wild-type and smallie (slie) mice, which contain a spontaneous deletion in Ddr2 to produce an effective null allele. Analysis of TMJs from newborn Ddr2slie/slie mice revealed a developmental delay in condyle mineralization, as measured by micro–computed tomography and histologic analysis. In marked contrast, knee joints of Ddr2slie/slie mice were normal. Analysis of older Ddr2slie/slie mice (3 and 10 mo) revealed that the early developmental delay led to a dramatic and progressive loss of TMJ articular integrity and osteoarthritis-like changes. Mutant condyles had a rough and flattened bone surface, accompanied by a dramatic loss of bone mineral density. Mankin scores showed significantly greater degenerative changes in the TMJs of 3- and 10-mo-old Ddr2slie/slie mice as compared with wild-type controls. No DDR2-dependent degenerative changes were seen in knees. Analysis of primary cultures of TMJ articular chondrocytes from wild-type and Ddr2slie/slie mice showed defects in chondrocyte maturation and mineralization in the absence of Ddr2. These studies demonstrate that DDR2 is necessary for normal TMJ condyle development and homeostasis and that these DDR2 functions are restricted to TMJ fibrocartilage and not seen in the hyaline cartilage of the knee.

Glycogen Synthase Kinase-3\u03b2 Inhibition Links Mitochondrial Dysfunction, Extracellular Matrix Remodelling and Terminal Differentiation in Chondrocytes

Following inflammatory stimuli, GSK3 inhibition functions as a hub with pleiotropic effects leading to cartilage degradation. However, little is known about the effects triggered by its direct inhibition as well as the effects on mitochondrial pathology, that contributes to osteoarthritis pathogenesis. To this aim we assessed the molecular mechanisms triggered by GSK3β inactivating stimuli on 3-D (micromass) cultures of human articular chondrocytes. Stimuli were delivered either at micromass seeding (long term) or after maturation (short term) to explore “late” effects on terminal differentiation or “early” mitochondrial effects, respectively. GSK3β inhibition significantly enhanced mitochondrial oxidative stress and damage and endochondral ossification based on increased nuclear translocation of Runx-2 and β-catenin, calcium deposition, cell death and enhanced remodelling of the extracellular matrix as demonstrated by the increased collagenolytic activity of supernatants, despite unmodified (MMP-1) or even reduced (MMP-13) collagenase gene/protein expression. Molecular dissection of the underlying mechanisms showed that GSK3β inhibition achieved with pharmacological/silencing strategies impacted on the control of collagenolytic activity, via both decreased inhibition (reduced TIMP-3) and increased activation (increased MMP-10 and MMP-14). To conclude, the inhibition of GSK3β enhances terminal differentiation via concerted effects on ECM and therefore its activity represents a tool to keep articular cartilage homeostasis.

Fabrication of Highly Crosslinked Gelatin Hydrogel and Its Influence on Chondrocyte Proliferation and Phenotype.

Gelatin methacrylate (GelMA) hydrogels have been widely studied for biomedical applications, such as tissue engineering and drug delivery, because of their good biocompatibility and injectability. However, the quick degradation and low mechanical property of GelMA hydrogels need to be improved for further applications, especially for long-term implantation. In this study, a sequential double modification of gelatin was used to achieve high density of photocrosslinkable double bonds in gelatin derivatives. The amino groups in gelatin were first reacted with methacrylic anhydride. After this, the hydroxyl and carboxyl groups in gelatin were reacted with glycidyl methacrylate to obtain the double modified gelatin macromer. The double modified gelatin macromer was used to prepare gelatin hydrogels with high crosslinking density. The hydrogels exhibited high storage modulus and low degradation. Culture of bovine articular chondrocytes in the gelatin hydrogels showed that chondrocytes had round morphology and maintained a cartilaginous phenotype while cell proliferation was hampered. This method for increasing crosslinking density should be useful for preparation of stable hydrogels for cartilage tissue engineering.