The low‐grade, chronic inflammation initiated by TLR4‐triggered innate immune responses has a central role on early osteoarthritis. Amurensin H is a resveratrol dimer with anti‐inflammatory and ...anti‐apoptotic effects, while its effects on TLR‐4 signals to inhibit osteoarthritis are still unclear. In the present study, treatment with amurensin H for 2 weeks in monosodium iodoacetate‐induced mice significantly slows down cartilage degeneration and inflammation using macroscopic evaluation, haematoxylin and eosin (HE) staining and micro‐magnetic resonance imaging. In IL‐1β‐stimulated rat chondrocytes, amurensin H suppresses the production of inflammatory mediators including nitric oxide, IL‐6, IL‐17, PGE2 and TNF‐α using Greiss and ELISA assay. Amurensin H inhibits matrix degradation via decreasing levels of MMP‐9 and MMP‐13 using Western blot assay, promotes synthesis of type II collagen and glycosaminoglycan using immunostaining and safranin O staining, respectively. Amurensin H inhibits intracellular and mitochondrial reactive oxygen species (ROS) generation, and mitochondrial membrane depolarization using DCFH‐DA, MitoSOX Red and JC‐1 assay as well. IL‐1β stimulates TLR4 activation and Syk phosphorylation in chondrocytes, while amurensin H inhibits TLR4/Syk signals and downstream p65 phosphorylation and translocation in a time and dose‐dependent manner. Together, these results suggest that amurensin H exerts chondroprotective effects by attenuating oxidative stress, inflammation and matrix degradation via the TLR4/Syk/NF‐κB pathway.
Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease with the cessation of matrix anabolism and aggravation of inflammation, which results in severe pain and impaired joint ...function. However, the mechanisms are not well understood. Circular RNAs (circRNAs) are reported to have various biological functions and participate in the development, diagnosis, prognosis, and treatment of different diseases. This study aimed to investigate the roles and mechanisms of circ-slain2 in TMJOA. We first established TMJOA mouse models and found circ-slain2 was lowly expressed in the cartilage of TMJOA through sequencing data. We observed that circ-slain2 is predominantly localized in the cytoplasm and downregulated in mouse condylar chondrocytes (mCCs) treated with tumor necrosis factor α (TNFα) and interferon γ (IFNγ). Micro–computed tomography and histological examination showed that intra-articular injection of circ-slain2 overexpressing adeno-associated virus could alleviate cartilage catabolism and synovial inflammation to relieve TMJOA in vivo. In addition, elevated circ-slain2 also showed anticatabolic and anti-inflammatory effects on IFNγ- and TNFα-stimulated mouse condylar chondrocytes (mCCs). Functional enrichment analysis indicated that protein processing in endoplasmic reticulum (ER) was associated with TMJOA, and further functional experiments confirmed that circ-slain2 could suppress ER stress in OA mCCs. RNA binding protein immunoprecipitation assay revealed an overt interaction between activating transcription factor 6 (ATF6) and circ-slain2. Inhibition of the expression of both ATF6 and circ-slain2 resulted in dilation of the ER and enhanced the expression of ER stress markers, whose ER stress level was higher than inhibition of ATF6 but lower than knockdown of circ-slain2 expression. Collectively, our research demonstrated that circ-slain2 could regulate ATF6 to relieve ER stress, reducing temporomandibular joint cartilage degradation and synovial inflammation. These findings provide prospects for developing novel osteoarthritis therapies based on circ-slain2 by focusing on reducing the inflammation of synovium and the imbalance between matrix synthesis and degradation.
In normal articular cartilage, chondrocytes do not readily proliferate or terminally differentiate, and exhibit a low level of metabolism. Hypertrophy-like changes of chondrocytes have been proposed ...to play a role in the pathogenesis of osteoarthritis by inducing protease-mediated cartilage degradation and calcification; however, the molecular mechanisms underlying these changes are unclear. Glycans are located on the outermost cell surface. Dynamic cellular differentiation can be monitored and quantitatively characterized by profiling the glycan structures of total cellular glycoproteins. This study aimed to clarify the alterations in glycans upon late differentiation of chondrocytes, during which hypertrophy-like changes occur. Primary mouse chondrocytes were differentiated using an insulin-induced chondro-osteogenic differentiation model. Comprehensive glycomics, including N-glycans, O-glycans, free oligosaccharides, glycosaminoglycan, and glycosphingolipid, were analyzed for the chondrocytes after 0-, 10- and 20-days cultivation. The comparison and clustering of the alteration of glycans upon hypertrophy-like changes of primary chondrocytes were performed. Comprehensive glycomic analyses provided complementary alterations in the levels of various glycans derived from glycoconjugates during hypertrophic differentiation. In addition, expression of genes related to glycan biosynthesis and metabolic processes was significantly correlated with glycan alterations. Our results indicate that total cellular glycan alterations are closely associated with chondrocyte hypertrophy and help to describe the glycophenotype by chondrocytes and their hypertrophic differentiation. our results will assist the identification of diagnostic and differentiation biomarkers in the future.
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Layer-by-layer bioprinting is a logical choice for the fabrication of stratified tissues like articular cartilage. Printing of viable organ replacements, however, is dependent on ...bioinks with appropriate rheological and cytocompatible properties. In cartilage engineering, photocrosslinkable glycosaminoglycan-based hydrogels are chondrogenic, but alone have generally poor printing properties. By blending the thermoresponsive polymer poly(N-isopropylacrylamide) grafted hyaluronan (HA-pNIPAAM) with methacrylated hyaluronan (HAMA), high-resolution scaffolds with good viability were printed. HA-pNIPAAM provided fast gelation and immediate post-printing structural fidelity, while HAMA ensured long-term mechanical stability upon photocrosslinking. The bioink was evaluated for rheological properties, swelling behavior, printability and biocompatibility of encapsulated bovine chondrocytes. Elution of HA-pNIPAAM from the scaffold was necessary to obtain good viability. HA-pNIPAAM can therefore be used to support extrusion of a range of biopolymers which undergo tandem gelation, thereby facilitating the printing of cell-laden, stratified cartilage constructs with zonally varying composition and stiffness.
Background/Aims: Osteoarthritis is a degenerative joint disease characterized by cartilage degeneration and a chondrocyte inflammatory response that induces an inflammatory environment closely linked ...to extracellular matrix (ECM) degradation. Ligustilide (LIG) is a major component of the herb Radix Angelicae Sinensis, with demonstrated anti-inflammatory effects. To confirm whether LIG has an equally inhibitory effect on inflammation in human osteoarthritis chondrocytes, we performed in vivo and in vitro experiments to validate the above conjectures and determine the relevant mechanisms. Methods: Quantitative realtime PCR and western blotting were performed to evaluate the expression of MMP-3, MMP-13, ADAMTS-5, iNOS, and COX-2 at both gene and protein levels. An enzyme-linked immunosorbent assay was used to evaluate the levels of other inflammatory factors (PGE2, TNF-α, and IL-6). The PI3K/AKT and nuclear factor kappa B (NF-κB) signaling pathways were also analyzed by western blotting, whereas immunofluorescence was used to assess the expression of collagen II and aggrecan. The in vitro effect of LIG was evaluated by intraperitoneal injection into a mouse osteoarthritis model induced by destabilization of the medial meniscus. Results: LIG lowered the phosphorylation levels of p65, IκBα, and IKKα/β and suppressed the IL-1β-induced expression of MMP-3, ADAMTS-5, iNOS, and COX-2 and the inflammatory factors PGE2, TNF-α, and IL-6. LIG markedly decreased IL-1β-induced degradation of collagen II and aggrecan. In vivo results showed that LIG-treated mouse cartilage showed less damage than the control group; the Osteoarthritis Research Society International (OARSI) score was also lower. LIG further reduced the thickness of the subchondral bone plate and alleviated the synovitis. Conclusion: LIG may act as a promising therapeutic agent for osteoarthritis by attenuating IL-1β-induced inflammation in chondrocytes and ECM degradation via suppression of NF-κB activation by the PI3K/AKT pathway.
Osteoarthritis (OA), the most prevalent degenerative joint disease, still lacks a true disease-modifying therapy. The involvement of the NF-κB pathway and its upstream activating kinases in OA ...pathogenesis has been recognized for many years. The ability of the N-acetyl phenylalanine glucosamine derivative (NAPA) to increase anabolism and reduce catabolism via inhibition of IKKα kinase has been previously observed in vitro and in vivo. The present study aims to confirm the chondroprotective effects of NAPA in an in vitro model of joint OA established with primary cells, respecting both the crosstalk between chondrocytes and synoviocytes and their phenotypes. This model satisfactorily reproduces some features of the previously investigated DMM model, such as the prominent induction of ADAMTS-5 upon inflammatory stimulation. Both gene and protein expression analysis indicated the ability of NAPA to counteract key cartilage catabolic enzymes (ADAMTS-5) and effectors (MCP-1). Molecular analysis showed the ability of NAPA to reduce IKKα nuclear translocation and H3Ser10 phosphorylation, thus inhibiting IKKα transactivation of NF-κB signalling, a pivotal step in the NF-κB-dependent gene expression of some of its targets. In conclusion, our data confirm that NAPA could truly act as a disease-modifying drug in OA.
Colorectal cancer (CRC) is the second deadly and the third most common malignancy worldwide. It has been projected that annual new cases of CRC will increase by 63% in 2040, constituting an even ...greater health challenge for decades to come. This study has linked DEC1 (differentiated embryonic chondrocyte expressed gene 1) to the pathogenesis of CRC. Based on the analysis of patient samples and database data, DEC1 is expressed much higher in CRC than the adjacent normal tissues. CRC patients with higher DEC1 expression have a shorter survival time. The carcinogenesis protocol with azoxymethane/dextran sulfate induces a higher number of tumors with larger sizes in DEC1+/+ than DEC1−/− mice. Overexpression of DEC1 increases the expression of proliferation- and antiapoptosis-related genes, but decreases the level of proapoptotic genes. Mechanistically, this study has shown that DEC1 is functionally looped to the IL-6/STAT3 signaling pathway (interleukin-6/signal transducer and activator of transcription 3). IL-6 induces DEC1, and DEC1 enhances the phosphorylation of STAT3, resulting in increased pSTAT3/STAT3 ratio. DEC1 and STAT3 are present in reciprocal immunocomplexes, pointing to physical interactions (presumably with pSTAT3). These findings establish that DEC1 is a CRC enhancer. The enhancement is achieved largely through the IL-6/STAT3 pathway. The potential of the physical interaction between DEC1 and STAT3 will likely serve as a foundation to develop intervention strategies for CRC prevention and therapy.
Tissue cells sense and respond to differences in substrate stiffness. In chondrocytes, it has been shown that substrate stiffness regulates cell spreading, proliferation, chondrogenic gene ...expression, and TGF-β signaling. But how the substrate stiffness together with soluble factors influences the mechanical properties of chondrocyte is still unclear. In this study, we cultured goat articular chondrocytes on polyacrylamide gels of 1, 11, and 90 kPa (Young’s modulus), and measured cellular stiffness, traction force, and response to stretch in the presence of TGF-β1 or IL-1β. We found that TGF-β1 increased cellular stiffness and traction force and enhanced the response to stretch, while IL-1β increased cellular stiffness, but lowered traction force and weakened the response to stretch. Importantly, the effects of TGF-β1 on chondrocyte mechanics were potent in cells cultured on 90 kPa substrates, while the effects of IL-1β were potent on 1 kPa substrates. We also demonstrated that such changes of chondrocyte mechanoresponse were due to not only the changes of actin cytoskeleton and focal adhesion, but also the alteration of chondrocyte extracellular matrix synthesis. Taken together, these results provide insights into how chondrocytes integrate physical and biochemical cues to regulate their biomechanical behavior, and thus have implications for the design of optimized mechanical and biochemical microenvironments for engineered cartilage.
Changes in extracellular osmolality have been shown to alter gene expression patterns and metabolic activity of various cell types, including chondrocytes. However, mechanisms by which physiological ...or pathological changes in osmolality impact chondrocyte function remain unclear. Here we use quantitative image analysis, electron microscopy, and a DNase I assay to show that hyperosmotic conditions (>400 mOsm/kg) induce chromatin condensation, while hypoosmotic conditions (100 mOsm/kg) cause decondensation. Large density changes (p < 0.001) occur over a very narrow range of physiological osmolalities, which suggests that chondrocytes likely experience chromatin condensation and decondensation during a daily loading cycle. The effect of changes in osmolality on nuclear morphology (p < 0.01) and chromatin condensation (p < 0.001) also differed between chondrocytes in monolayer culture and three-dimensional agarose, suggesting a role for cell adhesion. The relationship between condensation and osmolality was accurately modeled by a polymer gel model which, along with the rapid nature of the chromatin condensation (<20 s), reveals the basic physicochemical nature of the process. Alterations in chromatin structure are expected to influence gene expression and thereby regulate chondrocyte activity in response to osmotic changes.
Background:
To avoid complicated procedures requiring in vitro chondrocyte expansion for cartilage repair, the development of a culture-free, 1-stage approach combining platelet-rich fibrin (PRF) and ...autologous cartilage grafts may be the solution.
Purpose:
To develop a feasible 1-step procedure to combine PRF and autologous cartilage grafts for articular chondral defects.
Study Design:
Controlled laboratory study
Methods:
The chemotactic effects of PRF on chondrocytes harvested from the primary culture of rabbit cartilage were evaluated in vitro and ex vivo. The rabbit chondrocytes were cultured with different concentrations of PRF media and evaluated for their cell proliferation, chondrogenic gene expression, cell viability, and extracellular matrix synthesis abilities. For the in vivo study, the chondral defects were created on established animal models of rabbits. The gross anatomy, histology, and objective scores were evaluated to validate the treatment results.
Results:
PRF improved the chemotaxis, proliferation, and viability of the cultured chondrocytes. The gene expression of the chondrogenic markers, including type II collagen and aggrecan, revealed that PRF induced the chondrogenic differentiation of cultured chondrocytes. PRF increased the formation and deposition of the cartilaginous matrix produced by cultured chondrocytes. The efficacy of PRF on cell viability was comparable with that of fetal bovine serum. In animal disease models, morphologic, histological, and objectively quantitative evaluation demonstrated that PRF combined with cartilage granules was feasible in facilitating chondral repair.
Conclusion:
PRF enhances the migration, proliferation, viability, and differentiation of chondrocytes, thus showing an appealing capacity for cartilage repair. The data altogether provide evidence to confirm the feasibility of 1-stage, culture-free method of combining PRF and autologous cartilage graft for repairing articular chondral defects.
Clinical Relevance:
The single-stage, culture-free method of combining PRF and autologous cartilage is useful for repairing articular chondral defects. These advantages benefit clinical translation by simplifying and potentiating the efficacy of autologous cartilage transplantation.
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