Norepinephrine modulates IL-1β-induced catabolic response of human chondrocytes

Background The influence of the sympathetic nervous system (SNS) on metabolism of bone and cartilage expressing β-adrenergic receptors (AR) was suggested. Here, we investigated whether the SNS functions as a modulator of cartilage metabolism induced by interleukin-1beta (IL-1β). Methods Human articular chondrocytes and articular cartilage were collected from patients with osteoarthritis (OA). Chondrocyte monolayer and cartilage explant culture were stimulated with IL-1β. The activity of β-ARs was modulated by an agonist, norepinephrine (NE), and antagonists, including propranolol, atenolol, nebivolol, and nadolol. Results The levels of β1-, β2-, and β3-AR in OA cartilage and IL-1β-treated chondrocytes were lower than normal cartilage and untreated cells. Treatment of chondrocytes with IL-1β and β-blockers, including propranolol, atenolol, nebivolol, and nadolol, for 6 h significantly upregulated IL-1β-induced expression of MMP-1, -3, and − 13, compared to chondrocytes treated with IL-1β alone, indicating that antagonism of β-AR confers catabolic signals. On the other hand, NE antagonized IL-1β-induced catabolic response. In addition, NE significantly inhibited IL-1β-induced release of glycosaminoglycan (GAG) from cartilage explant culture. In addition, β-AR activity significantly affected IL-1β-stimulated phosphorylation of JNK and ERK. These results indicate that β-AR signal is associated with cartilage metabolism. Conclusions Our findings showed that β-ARs is a regulator of cartilage catabolism induced with IL-1β.

Decreased Sulfate Content and Zeta Potential Distinguish Glycosaminoglycans of the Extracellular Matrix of Osteoarthritis Cartilage

We aimed to determine the characteristics that distinguish glycosaminoglycans (GAGs) from osteoarthritis (OA) and normal cartilage and from men and women. Cartilage samples from 30 patients subjected to total joint arthroplasty secondary to OA or fracture (control) were evaluated, and the GAG content (μg/mg dry cartilage) after proteolysis was determined by densitometry, using agarose-gel electrophoresis. Relative percentages of carbon (C), nitrogen (N), and sulfur (S) in GAGs were determined by elemental microanalysis, as well as the zeta potential. Seventeen samples (56.6%) were from patients >70 years old, with 20 (66.6%) from women, and most [20 (66.6%)] were from the hip. The GAG content was similar regardless of patients being >/≤ 70 years old with 96.5 ± 63.5 and 78.5 ± 38.5 μg/mg (P = 0.1917), respectively. GAG content was higher in women as compared to men, with 89.5 ± 34.3 and 51.8 ± 13.3 μg/mg, respectively (P = 0.0022), as well as in OA than fracture samples, with 98.4 ± 63.5 and 63.6 ± 19.6 μg/mg, respectively (P = 0.0355). The GAG extracted from the cartilage of patients >70 years old had increase in N, and there were no gender differences regarding GAG elemental analysis. GAG from OA had a highly significant (P = 0.0005) decrease in S% (1.79% ± 0.25%), as compared to fracture samples (2.3% ± 0.19%), with an associated and significant (P = 0.0001) reduction of the zeta potential in the OA group. This is the first report of a reduced S content in GAG from OA patients, which is associated with a reduced zeta potential.

MiR-486-5p represses chondrocyte multiplication but facilitates their deaths in congenital microtia through aiming at H3F3B

Background MicroRNAs have been reported to play important roles in the development of external ear. The present study was designed to explore the expression patterns and biological function of miR-486-5p in congenital microtia. Methods Cartilage tissue specimens were collected from congenital microtia cases who received reconstructive ear surgeries, while the normal cartilage tissues from other otology surgeries were adopted as normal controls. The relative expression of miR-486-5p was investigated by quantitative-real time polymerase chain reaction. The potential target of miR-486-5p was explored by TargetScanHuman 7.2 and dual-Luciferase reporter assay. MTT and flow cytometry assays were adopted to investigate cell proliferation and apoptosis, respectively. Results The expression of miR-486-5p was significantly up-regulated in microtia cartilage tissues. Knockdown of miR-486-5p in microtia chondrocytes enhanced proliferation and inhibited apoptosis. H3F3B might be a target of miR-486-5p, and miR-486-5p could regulate proliferation and apoptosis of microtia chondrocytes by targeting H3F3B. Moreover, the enforced expression of H3F3B could re-turned the function of miR-486-5p in microtia chondrocytes. Conclusion Up-regulation of miR-486-5p may inhibit microtia chondrocytes proliferation and promote apoptosis by targeting H3F3B.

Norepinephrine Modulates IL-1β-induced Catabolic Response of Human Chondrocytes

BackgroundThe influence of the sympathetic nervous system (SNS) on metabolism of bone and cartilage expressing β-adrenergic receptors (AR) was suggested. Here, we investigated the relation between SNS and interleukin-1beta (IL-1β)-induced cartilage metabolism.MethodsHuman articular chondrocytes and articular cartilage were collected from patients with osteoarthritis (OA). Chondrocyte monolayer and cartilage explant culture were stimulated with IL-1β. The activity of β-ARs was modulated by an agonist, norepinephrine (NE), and antagonists, including propranolol, atenolol, nebivolol, and nadolol.ResultsThe levels of β1-, β2-, and β3-AR in OA cartilage and IL-1β-treated chondrocytes were lower than normal cartilage and untreated cells. Treatment of chondrocytes with IL-1β and β-blockers, including propranolol, atenolol, nebivolol, and nadolol, for 6 h significantly upregulated IL-1β-induced expression of MMP-1, -3, and -13, compared to chondrocytes treated with IL-1β alone, indicating that antagonism of β-AR confers catabolic signals. On the other hand, NE antagonized IL-1β-induced catabolic response. In addition, NE significantly inhibited IL-1β-induced release of glycosaminoglycan (GAG) from cartilage explant culture. In addition, β-AR activity significantly affected IL-1β-stimulated phosphorylation of JNK and ERK. These results indicate that β-AR signal is associated with cartilage metabolism.ConclusionsOur findings showed that β-ARs is a regulator of cartilage catabolism induced with IL-1β.

Perlecan in the Natural and Cell Therapy Repair of Human Adult Articular Cartilage: Can Modifications in This Proteoglycan Be a Novel Therapeutic Approach?

Articular cartilage is considered to have limited regenerative capacity, which has led to the search for therapies to limit or halt the progression of its destruction. Perlecan, a multifunctional heparan sulphate (HS) proteoglycan, promotes embryonic cartilage development and stabilises the mature tissue. We investigated the immunolocalisation of perlecan and collagen between donor-matched biopsies of human articular cartilage defects (n = 10 × 2) that were repaired either naturally or using autologous cell therapy, and with age-matched normal cartilage. We explored how the removal of HS from perlecan affects human chondrocytes in vitro. Immunohistochemistry showed both a pericellular and diffuse matrix staining pattern for perlecan in both natural and cell therapy repaired cartilage, which related to whether the morphology of the newly formed tissue was hyaline cartilage or fibrocartilage. Immunostaining for perlecan was significantly greater in both these repair tissues compared to normal age-matched controls. The immunolocalisation of collagens type III and VI was also dependent on tissue morphology. Heparanase treatment of chondrocytes in vitro resulted in significantly increased proliferation, while the expression of key chondrogenic surface and genetic markers was unaffected. Perlecan was more prominent in chondrocyte clusters than in individual cells after heparanase treatment. Heparanase treatment could be a means of increasing chondrocyte responsiveness to cartilage injury and perhaps to improve repair of defects.

The Role of Autophagy in Osteoarthritis

Chondrocytes are the only cell type in normal cartilage. The pathological changes of osteoarthritis (OA) mostly revolve around the apoptosis and dysfunction of chondrocytes. Autophagy, as an intracellular degradation system that maintains the steady state of energy metabolism in cells, has been shown to restore the function of damaged chondrocytes, alleviating the occurrence and progression of OA. In this review, we explored the relationship between autophagy and OA and the key molecules of autophagy pathway that regulate the progression of OA, providing new ideas for OA treatment by targeting autophagy.

Efficacy of a Thermally Triggered Injectable Hydrogel CS/Col /PLA/GP and BMSCs for Cartilage Tissue Engineering

BackgroundArticular cartilage has limited self-repair ability. Tissue engineering is considered to be one of the most promising therapeutic approaches. Chitosan (CS) based hydrogels are the most widely used scaffolds which still need improvement. The purpose of this study was to investigate the efficacy of a thermally triggered injectable chitosan / type II collagen / polylactic acid / sodium β-glycerophosphate (CS/Col/PLA/GP) hydrogel and bone marrow mesenchymal stem cells (BMSCs) for the treatment of cartilage defects in rabbit knee joints. Material/MethodsThe CS-based hydrogels consisting of CS, Col II, PLA and GP were fabricated by chemical cross-linking method. The gel forming time and elastic modulus of these hydrogels were measured. We tested the viability, proliferation and differentiation of rabbit BMSCs cultured in the hydrogels by fluorescence staining, CCK-8 and PCR method. The hydrogels combined with or without BMSCs were injected into cartilage defects in rabbit knee joints and the materials were collected at 8 weeks after surgery. The repair effect of cartilage defects was evaluated based on gross observation, HE, safranin O and immunohistochemical staining. ResultsThe CS/Col/PLA/GP hydrogel was liquid at room temperature and gelled after 7.5±0.41min at 37°C. CS/Col /PLA/GP hydrogel had a modulus of 8.90 ± 0.12 kPa while CS/GP and CS/Col/GP hydrogels had the modulus of 4.07 ± 0.24 kPa and 4.93 ± 0.09 kPa. The results of Live/Dead cell viability assay reveal that most of BMSCs remained alive in the hydrogels. CCK-8 assay shows that the number of cells in CS/Col /PLA/GP hydrogel was significantly higher in comparison to the other groups on days 2 and 3 of cell culture (p<0.05). Aggrecan mRNA expression in the CS/Col /PLA/GP gel was the highest (p<0.05). Sox9 mRNA expression in the CS/Col /GP group was the highest, in which CS/Col /PLA/GP hydrogel was higher than the CS/GP hydrogel(p<0.05). Furthermore, CS/Col/PLA/GP and CS/Col /GP hydrogels showed higher COL2A1 mRNA expression in comparison to CS/GP constructs (p<0.05). In vivo studies showed that approximately 90% of the cartilage defects of rabbits treated by the hydrogel and BMSCs were repaired with hyaline-like tissue without obvious inflammation response. HE, safranin O, and immunohistochemical staining showed that the hyaline like cartilage was formed in cartilage defects, and the collagen content in the new generated cartilage was similar to the normal cartilage. The neocartilage was thinner than the surrounding normal cartilage, but it exhibited integration with adjacent healthy tissue. The abundant well-defined chondrocytes were aligned in several apparent chondrocyte clusters in the new generated cartilage.ConclusionsThe thermo-sensitive injectable CS/Col/PLA/GP composite hydrogel has better ability to promote survive, proliferation and chondrogenic differentiation of seeded BMSCs as compared against CS/Col/GP and CS/GP hydrogels. Combined with BMSCs to repair cartilage defects of rabbit knee joints, they can effectively reduce the cartilage defect area, and the new generated cartilage is comparable to normal cartilage structure. In addition, abundant availability and simple fabrication process also make CS/Col/PLA/GP composite hydrogel a suitable candidate scaffold in cartilage tissue engineering.

SOX9 Knockout Induces Polyploidy and Changes Sensitivity to Tumor Treatment Strategies in a Chondrosarcoma Cell Line

As most chemotherapeutic drugs are ineffective in the treatment of chondrosarcoma, we studied the expression pattern and function of SOX9, the master transcription factor for chondrogenesis, in chondrosarcoma, to understand the basic molecular principles needed for engineering new targeted therapies. Our study shows an increase in SOX9 expression in chondrosarcoma compared to normal cartilage, but a decrease when the tumors are finally defined as dedifferentiated chondrosarcoma (DDCS). In DDCS, SOX9 is almost completely absent in the non-chondroid, dedifferentiated compartments. CRISPR/Cas9-mediated knockout of SOX9 in a human chondrosarcoma cell line (HTB94) results in reduced proliferation, clonogenicity and migration, accompanied by an inability to activate MMP13. In contrast, adhesion, apoptosis and polyploidy formation are favored after SOX9 deletion, probably involving BCL2 and survivin. The siRNA-mediated SOX9 knockdown partially confirmed these results, suggesting the need for a certain SOX9 threshold for particular cancer-related events. To increase the efficacy of chondrosarcoma therapies, potential therapeutic approaches were analyzed in SOX9 knockout cells. Here, we found an increased impact of doxorubicin, but a reduced sensitivity for oncolytic virus treatment. Our observations present novel insight into the role of SOX9 in chondrosarcoma biology and could thereby help to overcome the obstacle of drug resistance and limited therapy options.

MicroRNA-197 regulates chondrocyte proliferation, migration, and inflammation in pathogenesis of osteoarthritis by targeting EIF4G2

Recent studies have demonstrated that microRNAs (miRNAs) are involved in many pathological conditions including osteoarthritis (OA). In the present study, we aimed to investigate the role of miR-197 in OA and the potential molecular mechanism. The expression levels of miR-197 were detected by quantitative real-time PCR analysis. Cell proliferation and migration abilities were performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide and transwell assays. The concentrations of inflammatory cytokines, including IL-1β, IL-6, and TNF-α, were detect using ELISA assay. Furthermore, luciferase reporter and rescue assays were applied to identify the functional target gene of miR-197 in OA. The results showed that miR-197 expression was significantly down-regulated in the OA cartilage tissues compared with normal cartilage tissues, accompanied by up-regulation of EIF4G2 expression. An inverse correlation was found between EIF4G2 and miR-197 expressions in OA cartilage tissues. Treatment with miR-197 mimics promoted the growth and migration abilities of chondrocytes, while miR-197 inhibitors induced the opposite effects. Furthermore, restoration of miR-197 significantly decreased IL-1β, IL-6, and TNF-α expression, whereas knockdown of miR-197 led to a induction in these inflammatory mediators. Moreover, EIF4G2 was predicted and confirmed as a directly target of miR-197. Overexpressed miR-197 could down-regulate EIF4G2 expression in chondrocytes, while miR-197 knockdown could elevate EIF4G2 expression. Additionally, EIF4G2 overexpression reversed the effects of miR-197 mimics on chondrocytes proliferation, migration, and inflammation. Taken together, our study demonstrated that miR-197 promotes chondrocyte proliferation, increases migration, and inhibits inflammation in the pathogenesis of OA by targeting EIF4G2, indicating the potential therapeutic targets of the miR-197/EIF4G2 axis for OA treatment.

Exosomes from normal cartilage endplate stem cells ameliorate intervertebral disc degeneration more effectively than exosomes from degenerated cartilage endplate stem cell by activating the AKT/autophagy pathway

Background Nucleus pulposus cells (NPCs) apoptosis is an important factor in exacerbating intervertebral disc degeneration (IVDD) that can be effectively suppressed by exosomes. The aim of this study was to reaearch whether normal cartilage endplate stem cells (CESCs) derived exosomes (N-Exos) were more conducive to activation of autophagy and inhibition of NPCs apoptosis and IVDD than degenerated CESCs derived exosomes (D-Exos) or not. Methods Rat CESCs were isolated and identified, and the exosomes produced by normal CESCs and degenerated CESCs were extracted. The bioinformatics differences between normal CESCs derived exosomes (N-Exos) and degenerated CESCs derived exosome (D-Exos) were analyzed by mass spectrometry, heat map and KEGG enrichment analysis biology. The effects of N-Exos and D-Exos on the inhibition of NPCs apoptosis were examined by TUNEL staining, flow cytometry and western blotting. The involvement of the AKT and autophagy signaling pathways was investigated using the signaling inhibitor LY294002. Magnetic resonance imaging, western blotting and immunofluorescence staining were used to evaluate the therapeutic effects of N-Exos in vivo. Results CESCs in the cartilage endplate (CEP) could secrete a large amount of exosomes. N-Exos were more conducive to activation of autophagy than D-Exos. The apoptotic rate of NPCs was decreased obviously after treatment with N-Exos than after D-Exos treatment. N-Exos inhibited NPCs apoptosis or attenuated IVDD in a rat tail model by activating the AKT and autophagy signaling pathways. Conclusions It was the first to confirm that CEP could delay the progression of IVDD through exosomes secreted by normal CESCs. The therapeutic effects of N-Exos on inhibiting NPCs apoptosis and slowing IVDD progression was more effective than D-Exos by activating the PI3K/AKT/autophagy pathway, which explained the reason that the incidence of IVDD was increased after inflammation of the CEP.