Chondrocyte Matrix Research Articles

Circ0083429 Regulates Osteoarthritis Progression via the Mir-346/SMAD3 Axis.

Osteoarthritis (OA) is a degenerative joint disease. Currently, apart from symptomatic treatment or joint replacement, no other effective treatments for OA exist. The mechanisms underlying OA remain elusive and require further research. Circular RNAs (circRNAs) are known to be involved in many diseases; however, their function in OA is not yet fully understood. Here, we identified a novel circRNA, Circ0083429. The role of Circ0083429 in OA was confirmed via western blot (WB), quantitative real-time PCR (qRT-PCR), and immunofluorescence (IF) through knockdown and overexpression experiments. The binding of Circ0083429 to downstream miR-346 and its target gene SMAD3 was predicted via bioinformatics analysis and verified using a luciferase reporter assay and RNA pulldown experiments. Finally, the function of Circ0083429 was evaluated in mouse OA models. In our study, we found that Circ0083429 regulates the homeostasis of the extracellular matrix (ECM) in human chondrocytes. Mechanistically, Circ0083429 affects OA by regulating the mRNA level of SMAD3 through the sponging of microRNA (miRNA)-346. Injecting adeno-associated virus Circ0083429 into the intra-junction of the mouse knee alleviated OA. In conclusion, Circ0083429 regulates the ECM via the regulation of the downstream miRNA-346/SMAD3 in human chondrocytes, which provides a new therapeutic strategy for OA.

MicroRNA-130b downregulation potentiates chondrogenic differentiation of bone marrow mesenchymal stem cells by targeting SOX9.

Osteoarthritis (OA) is a chronic health condition. MicroRNAs (miRs) are critical in chondrocyte apoptosis in OA. We aimed to investigate the mechanism of miR-130b in OA progression. Bone marrow mesenchymal stem cells (BMSCs) and chondrocytes were first extracted. Chondrogenic differentiation of BMSCs was carried out and verified. Chondrocytes were stimulated with interleukin (IL)-1β to imitate OA condition in vitro. The effect of miR-130b on the viability, inflammation, apoptosis, and extracellular matrix of OA chondrocytes was studied. The target gene of miR-130b was predicted and verified. Rescue experiments were performed to further study the underlying downstream mechanism of miR-130b in OA. miR-130b first increased and drastically reduced during chondrogenic differentiation of BMSCs and in OA chondrocytes, respectively, while IL-1β stimulation resulted in increased miR-130b expression in chondrocytes. miR-130b inhibitor promoted chondrogenic differentiation of BMSCs and chondrocyte growth and inhibited the levels of inflammatory factors. miR-130b targeted SOX9. Overexpression of SOX9 facilitated BMSC chondrogenic differentiation and chondrocyte growth, while siRNA-SOX9 contributed to the opposite trends. Silencing of SOX9 significantly attenuated the pro-chondrogenic effects of miR-130b inhibitor on BMSCs. Overall, miR-130b inhibitor induced chondrogenic differentiation of BMSCs and chondrocyte growth by targeting SOX9.

Curcumin Supplementation Enhances Bone Marrow Mesenchymal Stem Cells to Promote the Anabolism of Articular Chondrocytes and Cartilage Repair

Mesenchymal stem cells derived from bone marrows (BMSCs) and curcumin derived from turmeric were used for osteoarthritis (OA) treatment, respectively. We invested the effects of curcumin supplementation for BMSC therapeutic effects. In vitro, rat BMSCs were identified by dual-immunofluorescent staining of CD44 and CD90, and flow cytometry. Primary articular chondrocytes were identified by toluidine blue staining and immunofluorescent staining of Col2a1. EdU incorporation, migration assay, real-time quantitative polymerase chain reaction, and Western blot analyses were performed to evaluate the alterations of chondrocytes cocultured with BMSCs. In vivo, the rat model of OA was established by monoiodoacetic acid. After intra-articular injection of allogeneic BMSCs, articular cartilage damage and OA progression were evaluated by histological staining, and Osteoarthritis Research Society International and Mankin score evaluation. Although curcumin alone did not improve cell viability of primary articular chondrocytes, it promoted proliferation and migration of chondrocytes when cocultured with BMSCs. Meanwhile, the expression of anabolic genes in chondrocytes was remarkably increased both at mRNA and protein levels. In OA rats, curcumin and BMSCs cooperated to greatly promote articular cartilage repair and retard OA progression. Therefore, curcumin supplementation enhanced the BMSC function for the proliferation and migration of articular chondrocytes, and anabolic gene expression of extracellular matrix in articular chondrocytes in vitro, and the generation of articular cartilage in vivo. Our study shed light on the potential clinical application of curcumin cooperated with BMSCs in cartilage repair for OA treatment.

Development of fibrocartilage layers in Achilles tendon enthesis in rabbits

Objective: The details regarding the development of fibrocartilage layers in Achilles tendon (AT) enthesis are unknown. Therefore, we evaluated the development of fibrocartilage layers in AT enthesis using a rabbit model.Materials and Methods: Forty-eight male Japanese white rabbits were used in this study. Six of them were euthanized at different stages (day 1, and 1, 2, 4, 6, 8, 12, and 24 weeks of age). The proliferation, apoptosis, Sox9-positivity rates, and chondrocyte number were evaluated. Additionally, safranin O-stained glycosaminoglycan (GAG) areas, width of AT enthesis, and calcaneus length were assessed. All parameters were compared to those at 24 weeks of age.Results: The level of chondrocyte apoptosis was high from 1 to 8 weeks of age, and high expression level of Sox9 was maintained from day 1 to 6 weeks of age, which decreased gradually. Safranin O-stained GAG areas increased up to 12 weeks, calcaneus length increased up to 6 weeks, and the width of AT enthesis increased up to 1 week of age.Conclusion: The changes in chondrocyte and extracellular matrix were completed by 8 and 12 weeks of age, respectively. The development of fibrocartilage layers in AT enthesis was completed by 12 weeks of age. Our results contribute to the administration of appropriate treatments based on age and aid in the development of novel methods for regenerating AT enthesis.

Long term usage of dexamethasone accelerating accelerates the initiation of osteoarthritis via enhancing chondrocyte apoptosis and the extracellular matrix calcification and apoptosis of chondrocytes.

Systemic application of glucocorticoids is an essential anti-inflammatory and immune-modulating therapy for severe inflammatory or autoimmunity conditions. However, its long-term effects on articular cartilage of patients' health need to be further investigated. In this study, we studied the effects of dexamethasone (Dex) on the homeostasis of articular cartilage and the progress of destabilization of medial meniscus (DMM)-induced osteoarthritis (OA) in adult mice. Long-term administration of Dex aggravates the proteoglycan loss of articular cartilage and drastically accelerates cartilage degeneration under surgically induced OA conditions. In addition, Dex increases calcium content in calcified cartilage layer of mice and the samples from OA patients with a history of long-term Dex treatment. Moreover, long term usage of Dex results in decrease subchondral bone mass and bone density. Further studies showed that Dex leads to calcification of extracellular matrix of chondrocytes partially through activation of AKT, as well as promotes apoptosis of chondrocytes in calcified cartilage layer. Besides, Dex weakens the stress-response autophagy with the passage of time. Taken together, our data indicate that long-term application of Dex may predispose patients to OA and or even accelerate the OA disease progression development of OA patients.

Melatonin Prevents Cartilage Degradation in Early-Stage Osteoarthritis Through Activation of miR-146a/NRF2/HO-1 Axis.

Reactive oxygen species (ROS) are implicated in induction of inflammatory response and cartilage degradation in osteoarthritis (OA). Melatonin has been shown to improve the chondrogenic differentiation and promote cartilage matrix synthesis in mesenchymal stem cells. However, the underlying mechanisms of melatonin-regulated antioxidant activity in OA cartilage are not known. The aim of this study was to explore the effect of melatonin on nuclear factor-erythroid 2-related factor 2 (NRF2), a key antioxidant transcription factor, and its target antioxidant genes in early-stage OA cartilage. Primary chondrocytes were isolated from rats with surgically induced OA. In vitro treatment of melatonin significantly increased cartilage matrix synthesis and upregulated antioxidant enzymes, mainly heme oxygenase 1 (HO-1), while decreasing matrix degradation enzymes and intracellular ROS. In vivo intraarticular injection of melatonin effectively ameliorated cartilage degeneration in an experimental rat OA model. Inhibition of melatonin membrane receptors by Luzindole or 4-P-PDOT reversed the beneficial effects of melatonin on cartilage matrix synthesis, implying that melatonin receptor-mediated pathway is involved in its anti-arthritic effects. Interestingly, melatonin showed no significant effect on the mRNA level of Nrf2 but significantly increased its protein level. Silencing of Nrf2 or HO-1 expression abolished the protective effects of melatonin, as shown by increased ROS levels and matrix degradation enzyme expression. Microarray assays revealed that miR-146a, a predicted target for Nrf2, was significantly upregulated in OA chondrocytes but was markedly reduced by melatonin treatment. Overexpression of miR-146a diminished the protective effects of melatonin by inhibiting NRF2 expression and aggravating OA-induced cartilage degradation. These findings demonstrate that melatonin supports the anabolic metabolism of cartilage matrix in OA chondrocytes by enhancing the protein levels of NRF2 via suppressing miR-146a. Melatonin-mediated activation of the NRF2/HO-1 axis prevents cartilage degeneration and represents a promising therapeutic target for treatment of early-stage OA. © 2022 American Society for Bone and Mineral Research (ASBMR).

Baicalin promotes extracellular matrix synthesis in chondrocytes via the activation of hypoxia-inducible factor-1α.

Chinese herbal extracts are being used increasingly to treat osteoarthritis (OA) in recent years. Baicalin (BA) is an active component of Scutellaria baicalensis Georgi extracts and protects chondrocytes against damage. The aim of the present study was to examine the mechanism of action of BA on chondrocytes from mouse articular cartilage. In total, 44 µM BA and 10 µM hypoxia-inducible-factor-1α (HIF-1α) inhibitor BAY-87-2243 were screened by the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] method. Alcian blue and Safran O staining were used to investigate the synthesis of extracellular matrix (ECM) in chondrocytes treated with BA. The expression of HIF-1α and chondrogenic marker genes including SOX9, AGG and Col2α was detected by western blotting or reverse-transcription quantitative (RT-qPCR), the expression of PHD1,2,3 and catabolic genes including ADAMTS5, MMP9 and MMP13 were detected by RT-qPCR. To investigate the effect of BA on the ECM synthesis of chondrocytes, 44 µM BA and 10 µM BAY were chosen for further experimentation. It was confirmed that BA at a concentration of 44 µM could significantly promote the secretion of ECM. The expressions of genes including HIF-1α, SOX9, collagen type 2 (Col2α) and aggrecan (AGG) were elevated following BA pretreatment and decreased by subsequent BAY-87-2243 stimulation for 24 h. Compared with untreated chondrocytes, the expressions of genes including ADAMTS5, MMP9, MMP13, PHD1, PHD2 and PHD3 in chondrocytes treated by BA were downregulated, however, BAY-87-2243 reversed the effect of BA on the genes including ADAMTS5, MMP9, MMP13, PHD1, PHD2 and PHD3 in chondrocytes. The findings of the present study suggest that BA may promote ECM synthesis and marker gene expression in chondrocytes by activating HIF-1α. Therefore, BA may represent a novel clinical drug for OA.

A modified staining protocol to study early development of Rainbow trout Oncorhynchus mykiss (Walbaum, 1792) skull

Alcian blue binds to the proteoglycans components of the extracellular matrix in chondrocytes and turns the cartilage color to blue. Alizarin red stains free calcium and certain calcium compound to a red or light purple color. This procedure is a useful tool to study the skeletal development in several fish species and then to precociously diagnose malformations in cartilages and bones development in small larvae and eventually to better characterize the effect of different environmental and/or nutritional factors on the ossification status of specific skeletal components. It should be noted that according to different species, the modifications in the protocol are still reported. In this experiment 520 specimens of rainbow trout in different life stages from one-day post hatching (dph) to 30 days, 1.5±0.1 and 3.5±0.5 cm in length have been studied. The double staining protocol for developing rainbow trout has been defined based on diverse published protocols. For an optimal staining protocol design, the incubation times of the different solutions were adjusted. Then the photos of stained specimens have been used to study and compare. The detection of cartilages and bones helps to understand the ontogeny of fish skeletal structure and the effects of environmental factors on bone density and plasticity. In addition, given the importance of skeletal and muscle's interactions, it is possible to conduct a survey on specific factors to change the skeletal tissues.

Genome-Wide Occupancy Profiling Reveals Critical Roles of FoxO1 in Regulating Extracellular Matrix and Circadian Rhythm Genes in Human Chondrocytes.

Osteoarthritis (OA) is the most common age-related joint disease. With aging and in OA, the expression of FoxO transcription factors is reduced, diminishing their chondroprotective actions. In order to elucidate the molecular mechanisms by which FoxO1 protects chondrocytes, we sought to identify the genome-wide occupancy profile of FoxO1. We performed FoxO1 chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) on human primary chondrocytes. ChIP-Seq data were integrated with RNA sequencing (RNA-Seq) data sets. Bioinformatics results were confirmed in primary chondrocytes that were treated with a FoxO1 inhibitor. Analysis of FoxO1 ChIP-Seq on human primary chondrocytes showed that pathways implicated in OA pathogenesis are mainly regulated by FoxO1 binding to tissue-specific enhancers with suboptimal binding sites (20% of the peaks), while more ubiquitous FoxO1 pathways are regulated at the promoter level through interaction with its canonical binding motif (7% of the peaks). Integrating FoxO1 occupancy data with RNA-Seq data comparing OA and healthy human cartilage revealed 428 putative FoxO1 target genes that are dysregulated in OA. Pathway analysis showed enrichment for genes belonging to the senescence pathway (logP = -6.73), extracellular matrix (ECM) pathway (logP = -12.97), and circadian clock pathway (logP = -6.30), which suggests that FoxO1 dysregulation plays an important role in their abnormal expression in OA. Using an inhibitor of FoxO1, we confirmed that FoxO1 regulates these pathways in cultured human chondrocytes. FoxO1 regulates ubiquitous and cartilage-specific genes in chondrocytes by using different mechanisms. The FoxO1 transcriptional network is a key player in regulating homeostasis, ECM, and circadian clock genes and plays an important role in the abnormal expression of these pathways observed in OA pathogenesis.

Integrin α2β1 is involved in T-2 toxin-induced decrease of type II collagen in C28/I2 chondrocytes

The T-2 toxin exerts a variety of toxic effects on both experimental animals and humans. The integrin family plays a major role in mediating cell-ECM interactions. Therefore, the present study aimed to investigate the involvement of integrin α2β1 in T-2 toxin-induced C28/I2 chondrocyte damage. The pathological damage of articular cartilage injury induced by T-2 toxin was observed by H&E staining. The expression levels of collagen 2 and MMP-13 (Matrix metalloproteinases 13) were detected using immunohistochemistry in articular cartilage tissues and western blotting in the cells. The blocking effect of integrin α2β1 inhibitor on T-2 toxin-induced chondrocyte matrix degradation was examined by western blotting. Further, the effect of integrin α2β1 inhibitor on T-2 toxin-induced chondrocyte apoptosis was analyzed. About 100 ng/g body weight (BW)/day T-2 toxin was fed to Sprague-Dawley (SD) rats, T-2 toxin treatment (0, 6, 12, and 24 ng/mL) induced C28/I2 chondrocytes. Both in vivo and in vitro, chondrocyte survival was inhibited, and the production of type II collagen was significantly reduced (p < 0.05). However, the level of MMP-13 was up-regulated (p < 0.05). Matrix degradation was effectively blocked after the pre-treatment by integrin α2β1 inhibitor (p < 0.05). Conclusively, Integrin α2β1 is a critical signaling pathway for communication between cells and the extracellular matrix, the present study provides a new clue to elucidate the mechanism of T-2 toxin-induced chondrocyte damage.

MicroRNA-1 promotes cartilage matrix synthesis and regulates chondrocyte differentiation via post-transcriptional suppression of Ihh expression.

Indian hedgehog signaling molecule (Ihh) is known to play critical roles in chondrogenesis and cartilage development. However, it remains largely unknown how Ihh is regulated during the process. Previous studies suggest that Ihh plays an important regulatory role in the growth and development of articular cartilage, but whether it is regulated by miRNAs is unclear. The present study aimed to investigate the effects of miR-1 on chondrocyte differentiation and matrix synthesis, and to determine whether miR-1 can regulate the Ihh signaling pathway. In the present study, the expression level of miR-1 was altered via transfection of the miR-1 mimic or inhibitor in mouse thorax chondrocytes, and the impact on chondrocyte phenotypes and Ihh expression was examined. Overexpression of miR-1 promoted the expression of the matrix synthesis-associated molecules collagen (Col)-II and aggrecan, two key components in cartilage matrix. Conversely, overexpression of miR-1 significantly downregulated the expression of chondrocyte differentiation markers Col-X and matrix metallopeptidase 13. Moreover, overexpression of miR-1 dose-dependently inhibited endogenous Ihh expression, and an association was observed between miR-1 and Ihh expression. The 3′ untranslated region (UTR) of Ihh from various species contains two miR-1 binding sites. Luciferase reporter assays indicated that miR-1 post-transcriptionally suppressed Ihh expression, which was dependent on the binding of miR-1 to one of the two putative binding sites of the Ihh 3′UTR. Furthermore, via inhibition of Ihh expression, miR-1 decreased the expression of molecules downstream of Ihh in the Hedgehog signaling pathway in mouse thorax chondrocytes. This study provided new insight into the molecular mechanisms of miR-1 in regulating chondrocyte phenotypes via targeting the Ihh pathway.

AB0067 CHONDROCALCINOSIS IS ASSOCIATED WITH A SPECIFIC EFFECT ON THE CHONDROCYTE PHENOTYPE THAT MARKEDLY DIFFERS FROM OA

Background:Calcification of cartilage with BCP crystals is a common finding during osteoarthritis (OA) and is directly linked to the severity of the disease and hypertrophic differentiation of chondrocytes. Chondrocalcinosis (CC) is associated with CPPD crystal formation. There is only little knowledge about the effect of CPPD crystals on chondrocytes.Objectives:The aim of this study was to investigate the chondrocyte phenotype in CC cartilage and the effect of CPPD crystals on chondrocytes.Methods:Cartilage samples of patients with CC were used and compared with samples of severe OA patients without chondrocalcinosis and healthy cartilage samples served as control. Radiological presence of chondrocalcinosis was evaluated using standard X-ray pictures, as well as macroscopically inspection. The cartilage samples were stained using von Kossa/Safranin-orange staining. These stainings were used for OA severity scoring using the Chambers-Score. FTIR analyses was performed to distinguish CPPD and BCP crystals in cartilage. Chondrocyte differentiation markers were evaluated using Collagen 2 and X, as well as Sox9 and aggrecan as markers for chondrocyte hypertrophic differentiation in immunohistochemistry and qRT-PCR. TUNEL staining was performed to investigate cell death. In vivo results were validated using qRT-PCR for the expression of the respective genes after stimulation of C28 chondrocytes with CPPD and BCP crystals.Results:Radiologically detectable cartilage calcifications were evident in chondrocalcinosis patients, but absent in OA patients without CC. CPPD crystals were detected on the cartilage surface, whereas BCP crystals were detected in the pericellular matrix of hypertrophic chondrocytes. CC cartilage exhibited an increased collagen X expression compared to healthy cartilage, as well as to severe OA cartilage containing BCP calcification. Interestingly, aggrecan and collagen 2 were not reduced in CC cartilage, but markedly reduced in OA cartilage. TUNEL positive cells were significantly increased in CPPD cartilage compared to OA cartilage, although the histological OA severity was lower. qRT-PCR indicated no relevant influence of CPPD crystals on hypertrophic marker genes, whereas BCP crystals significantly induced hypertrophic differentiation.Conclusion:BCP and CPPD crystals seem to trigger differential effects on the chondrocyte phenotype. BCP crystals induce hypertrophic differentiation, which is not induced by CPPD crystals.Acknowledgments:The project was funded by theDeutsche Rheumastifung by the sponsor Dr. Sigrid Schuler.Disclosure of Interests:Franziska Meyer: None declared, Miriam Bollmann: None declared, Uwe Kornak: None declared, Jessica Bertrand Grant/research support from: Pfizer, Speakers bureau: Pfizer

Glutamine Metabolism Controls Chondrocyte Identity and Function.

Correct functioning of chondrocytes is crucial for long bone growth and fracture repair. These cells are highly anabolic but survive and function in an avascular environment, implying specific metabolic requirements that are, however, poorly characterized. Here, we show that chondrocyte identity and function are closely linked with glutamine metabolism in a feedforward process. The master chondrogenic transcription factor SOX9 stimulates glutamine metabolism by increasing glutamine consumption and levels of glutaminase 1 (GLS1), a rate-controlling enzyme in this pathway. Consecutively, GLS1 action is critical for chondrocyte properties and function via a tripartite mechanism. First, glutamine controls chondrogenic gene expression epigenetically through glutamate dehydrogenase-dependent acetyl-CoA synthesis, necessary for histone acetylation. Second, transaminase-mediated aspartate synthesis supports chondrocyte proliferation and matrix synthesis. Third, glutamine-derived glutathione synthesis avoids harmful reactive oxygen species accumulation and allows chondrocyte survival in the avascular growth plate. Collectively, our study identifies glutamine as a metabolic regulator of cartilage fitness during bone development.

Osteochondral Autograft Plugs versus Paste Graft: Ex Vivo Morselization Increases Chondral Matrix Production.

Patients undergoing articular cartilage paste grafting have been shown in studies to have significant improvement in pain and function in long-term follow-ups. We hypothesized that ex vivo impacting of osteochondral autografts results in higher chondrocyte matrix production versus intact osteochondral autograft plugs. This institutional review board-approved study characterizes the effects of impacting osteochondral plugs harvested from the intercondylar notch of 16 patients into a paste, leaving one graft intact as a control. Cell viability/proliferation, collagen type I/II, SOX-9, and aggrecan gene expression via qRT-PCR (quantitative reverse transcription-polymerase chain reaction) were analyzed at 24 and 48 hours. Matrix production and cell morphology were evaluated using histology. Paste samples from patients (mean age 39.7) with moderate (19%) to severe (81%) cartilage lesions displayed 34% and 80% greater cell proliferation compared to plugs at 24 and 48 hours post processing, respectively (P = 0.015 and P = 0.021). qRT-PCR analysis yielded a significant (P = 0.000) increase of aggrecan, SOX-9, collagen type I and II at both 24 and 48 hours. Histological examination displayed cell division throughout paste samples, with accumulation of aggrecan around multiple chondrocyte lacunae. Paste graft preparation resulted in increased mobility of chondrocytes by matrix disruption without loss of cell viability. The impaction procedure stimulated chondrocyte proliferation resulting in a cellular response to reestablish native extracellular matrix. Analysis of gene expression supports a regenerative process of cartilage tissue formation and contradicts long-held beliefs that impaction trauma leads to immediate cell death. This mechanism of action translates into clinical benefit for patients with moderate to severe cartilage damage.

High-Throughput Identification of MiR-145 Targets in Human Articular Chondrocytes.

MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3′UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

BCP crystals promote chondrocyte hypertrophic differentiation in OA cartilage by sequestering Wnt3a

ObjectiveCalcification of cartilage with basic calcium phosphate (BCP) crystals is a common phenomenon during osteoarthritis (OA). It is directly linked to the severity of the disease and known to be...

Metformin pretreatment suppresses alterations to the articular cartilage ultrastructure and knee joint tissue damage secondary to type 2 diabetes mellitus in rats

ABSTRACT Osteoarthritis (OA) secondary to diabetes affects millions of people worldwide and can lead to disability. The protective effect of metformin pretreatment against alterations to the articular cartilage ultrastructure induced by type 2 diabetes mellitus (T2DM) associated with the inhibition of oxidative stress and inflammation has not been investigated before. Therefore, we induced T2DM in rats (the model group) using high carbohydrate and fat diet and a single injection of streptozotocin (50 mg/kg body weight). The protective group of rats started metformin (200 mg/kg body weight) treatment 14 days before diabetic induction and continued on metformin until the end of the experiment at week 12. Harvested tissues obtained from knee joints were prepared for staining with hematoxylin and eosin (H&E), safranin o staining, and electron microscopy. Histology images showed that OA was developed in the T2DM rats as demonstrated by a substantial damage to the articular cartilage and profound chondrocyte and territorial matrix ultrastructural alterations, which were partially protected by metformin. In addition, metformin significantly (p < .05) reduced hyperglycemia, glycated hemoglobin (HbA1 c), malondialdehyde (MDA), high sensitivity C-reactive protein (hs-CRP), and interleukin-6 blood levels induced by diabetes. Furthermore, a significant (p ≤ 0.015) correlation between either OA cartilage grade score or the thickness of the articular cartilage and the blood levels of HbA1 c, hs-CRP, MDA, superoxide dismutase (SOD) were observed. These findings demonstrate effective protection of the articular cartilage by metformin against damage induced secondary to T2DM in rats, possibly due to the inhibition of hyperglycemia and biomarkers of oxidative stress and inflammation.

Fibulin-5 protects the extracellular matrix of chondrocytes by inhibiting the Wnt/β-catenin signaling pathway and relieves osteoarthritis.

Osteoarthritis (OA) is a common disease in the elderly and seriously affects the quality of life of patients. The purpose of this study was to explore the protective effect of Fibulin-5 on articular chondrocytes and its mechanism of action. Articular cartilage tissues from patients with OA and normal people were selected and tested for differences in Fibulin-5 expression. In addition, human chondrocytes were cultured, and the effects of Fibulin-5 on the extracellular matrix (ECM) of chondrocytes and the level of inflammation were examined by means of cell transfection and cytokine intervention. SKL2001, an agonist of the Wnt/β-catenin signaling pathway, was used to validate the mechanism of action of Fibulin-5 to protect chondrocytes. Fibulin-5 was lowly expressed in the cartilage tissue of patients with OA. Overexpression of Fibulin-5 significantly increased the expressions of ECM collagen II and aggrecan in chondrocytes, while decreasing the expressions of MMP-3 and MMP-13. In addition, Fibulin-5 reduced IL-1β-induced inflammation of chondrocytes, as well as expressions of IL-6, IL-8, and TNF-α. Overexpression of Fibulin-5 also reduced the activity of Wnt/β-catenin signaling pathway, and activation of Wnt/β-catenin signaling pathway attenuated the protective effects of Fibulin-5 on the ECM of chondrocytes. Fibulin-5 can protect the ECM of chondrocytes and reduce the inflammatory response of chondrocytes by inhibiting the Wnt/β-catenin signaling pathway.

Exosomes derived from umbilical cord mesenchymal stem cells in mechanical environment show improved osteochondral activity via upregulation of LncRNA H19.

BackgroundExosomes derived from stem cells have been demonstrated to be good candidates for the treatment of osteochondral injury. Our previous studies have demonstrated that mechanical stimulation could be crucial for the secretion of exosomes derived from umbilical cord mesenchymal stem cells (U-MSCs). Therefore, we explore whether mechanical stimulation caused by a rotary cell culture system (RCCS) has a beneficial effect on exosome yield and biological function.MethodsU-MSCs were subjected to an RCCS at different rotational speeds and exosomes were characterised by transmission electron microscopy, nanoparticle tracking analysis and western blotting. small-interfering RNAs of Rab27a (siRNA-Rab27a) was used to reduce exosome production. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of mechanically sensitive long non-coding RNA H19 (LncRNA H19). The effects of exosomes on chondrocyte proliferation were examined using cell counting kit-8 (CCK-8), toluidine blue staining and a series of related genes. Annexin V-FITC and PI (V-FITC/PI) flow cytometry was used to detect the effect of exosomes on the inhibition of chondrocyte apoptosis. Macroscopic evaluation, MRI quantification and immunohistochemical staining were conducted to investigate the in vivo effects of exosomal LncRNA H19 through SD rat cartilage defect models.ResultsRCCS significantly promoted exosome production at 36 rpm/min within 196 h. Mechanical stimulation was able to increase the expression level of exosomes. The exosomal LncRNA H19 was found to promote chondrocyte proliferation and matrix synthesis and inhibit apoptosis in vitro. Chondral regeneration activity was lost in LncRNA H19-defective exosomes. The injection of exosomal LncRNA H19 in vivo resulted in improved macroscopic assessment, MRI quantification and histological analysis. Moreover, exosomal LncRNA H19 was able to relieve pain levels during the early stages of cartilage repair in an animal experiment.ConclusionOur findings confirmed that mechanical stimulation can enhance exosome yield as well as biological function for the repair of cartilage defects. The underlying mechanism may be related to the high expression of LncRNA H19 in exosomes. The translational potential of this article: This study provides a theoretical support of optimizing exosome production. It advances the yield of mesenchymal stem cell exosome and facilitate the clinical application to repair of osteochondral damage.

Norepinephrine modulates IL-1β-induced metabolism of cartilage matrix in human chondrocytes

Purpose: The sympathetic nervous system (SNS) was reported to be involved in bone and cartilage metabolism via β-adrenergic receptors. In this study, we examined whether SNS plays a role in regulation of cartilage metabolism driven by IL-1β.