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BACKGROUND: Circular RNAs (circRNAs) are demonstrated to play vital roles in human diseases, including rheumatoid arthritis (RA). Therefore, this research aimed to explore the effects of hsa_circRNA_0025908 (circ_0025908) on RA. METHODS: RNA expression of circ_0025908, microRNA-650 (miR-650), and Signal peptide-CUBepidermal growth factor-like containing protein 2 (SCUBE2) were assessed by real-time quantitative polymerase chain reaction; protein expression of SCUBE2, apoptosis- and invasion-related proteins was evaluated by western blot assay. Functional assays were performed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide, 5-ethynyl-2'-deoxyuridine, transwell, flow cytometry, and enzyme linked immunosorbent assay assays. Dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays confirmed the interaction relationship among circ_0025908, miR-650, and SCUBE2. RESULTS: Circ_0025908 was overexpressed in synovial tissues and fibroblast-like synoviocytes (FLS) from RA patients. Inhibition of circ_0025908 repressed proliferation, migration, invasion, inflammation, and cell cycle progression, while induced apoptosis in the FLS isolated from RA patients (FLS-RA), accompanied with increased Bax, cleaved caspase-3 and E-cadherin, but declined Bcl-2, N-cadherin and Vimentin. MiR-650 was a target of circ_0025908, and SCUBE2 was a target for miR-650. Silencing of miR-650 could overturned above effects of circ_0025908 knockdown in FLS-RA, whereas its overexpression could mimic those effects by downregulating SCUBE2. Additionally, SCUBE2 expression could be positively regulated by circ_0025908 and inversely regulated by miR-650. Notably, Pearson's correlation analysis confirmed the linear correlation among circ_0025908, miR-650 and SCUBE2 in these RA tissues. CONCLUSION: Circ_0025908 inhibition can suppress FLS-RA dysfunctions through targeting miR-650/SCUBE2 axis, suggesting a new potential therapeutic clue for RA patients.
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Proteínas Adaptadoras Transductoras de Señales , Artritis Reumatoide , Proteínas de Unión al Calcio , MicroARNs , ARN Circular , Sinoviocitos , Proteínas Adaptadoras Transductoras de Señales/genética , Apoptosis/genética , Artritis Reumatoide/metabolismo , Bromuros/metabolismo , Bromuros/uso terapéutico , Cadherinas/metabolismo , Cadherinas/uso terapéutico , Proteínas de Unión al Calcio/genética , Caspasa 3/metabolismo , Caspasa 3/uso terapéutico , Movimiento Celular/genética , Proliferación Celular/genética , Fibroblastos/metabolismo , Humanos , Inflamación/genética , Inflamación/metabolismo , MicroARNs/genética , Señales de Clasificación de Proteína , ARN Circular/genética , Sinoviocitos/metabolismo , Vimentina/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
To evaluate the performance of a composite scaffold of Wharton's jelly (WJ) and chondroitin sulfate (CS) and the effect of the composite scaffold loaded with human umbilical cord mesenchymal stem cells (hUCMSCs) in repairing articular cartilage defects, two experiments were carried out. The in vitro experiments involved identification of the hUCMSCs, construction of the biomimetic composite scaffolds by the physical and chemical crosslinking of WJ and CS, and testing of the biomechanical properties of both the composite scaffold and the WJ scaffold. In the in vivo experiments, composite scaffolds loaded with hUCMSCs and WJ scaffolds loaded with hUCMSCs were applied to repair articular cartilage defects in the rat knee. Moreover, their repair effects were evaluated by the unaided eye, histological observations, and the immunogenicity of scaffolds and hUCMSCs. We found that in vitro, the Young's modulus of the composite scaffold (WJ-CS) was higher than that of the WJ scaffold. In vivo, the composite scaffold loaded with hUCMSCs repaired rat cartilage defects better than did the WJ scaffold loaded with hUCMSCs. Both the scaffold and hUCMSCs showed low immunogenicity. These results demonstrate that the in vitro construction of a human-derived WJ-CS composite scaffold enhances the biomechanical properties of WJ and that the repair of knee cartilage defects in rats is better with the composite scaffold than with the single WJ scaffold if the scaffold is loaded with hUCMSCs.
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Cartílago Articular/metabolismo , Sulfatos de Condroitina/química , Miembro Posterior/fisiología , Células Madre Mesenquimatosas/citología , Andamios del Tejido/química , Cordón Umbilical/metabolismo , Gelatina de Wharton/química , Animales , Fenómenos Biomecánicos , Cartílago , Diferenciación Celular , Condrocitos/citología , Inmunohistoquímica , Técnicas In Vitro , Interleucina-6/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Ingeniería de TejidosRESUMEN
INTRODUCTION: Mechanical stimulation is important for maintaining cartilage function. We used a loading device to exert rolling-sliding mechanical stimulation on cartilage preserved in vitro to investigate cartilage viability and the involved mechanisms. METHODS: Osteochondral grafts from pig knees were randomly classified into loading and control groups. The loading group cartilage was subjected to cycles of mechanical stimulation with specified frequency/time/pressure combinations every 3 days; Then the DMEM was refreshed, and the cartilage was preserved in vitro. The control group cartilage was preserved in DMEM throughout the process and was changed every 3 days. On days 14 and 28, the chondrocyte survival rate, histology, and Young's modulus of the cartilage were measured. Western blots were performed after 2 h of loading to evaluate the protein expression. RESULTS: The loading group showed a significantly higher chondrocyte survival rate, proteoglycan and type II collagen content, and Young's modulus than did the control group on day 14, but no statistically significant differences were found on day 28. After two hours of the loading, the phosphorylation levels of MEK and ERK1/2 increased, and the expression of caspase-3, cleaved caspase-3 and bax decreased. CONCLUSION: These results suggest that periodic rolling-sliding mechanical stimulation can increase cartilage vitality in 2 weeks; a possible mechanism is that mechanical stimulation activates the MEK/ERK signalling pathway, thus inhibiting apoptotic protein expression. This loading preservation scheme could be used by cartilage tissue banks to improve cartilage preservation in vitro and enhance the quality of cartilage repair.
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The viability of cartilage explants preserved in vitro decreases with time, which limits its use for transplantation. The effect of mechanical stimulation to cartilage explants in vitro is unknown. In this study, we observed the effects of mechanical stimulation on chondrocyte viability and the mechanical properties of cartilage explants preserved in vitro using a rolling-sliding loading device designed by us, and the optimal stimulation protocol was established. A cylindrical osteochondral mass drilled on the femoral condyle of a healthy pig was divided into two groups (loading group and control group), and changes in the chondrocyte survival rate, matrix composition and cartilage biomechanical properties was observed at different time points. Additionally, the mRNA expression of the apoptosis-related proteins caspase-3/Bax/Bcl-2, the cytoskeletal proteins actin/vimentin, and the matrix-related protein MMP13 were detected. The loading group exhibited delayed collagen and aggrecan degeneration and improved chondrocyte viability for three days. Protein and mRNA detection showed that apoptotic factors such as caspase-3 and Bax decreased rapidly in cartilage tissue after loading. The cytoskeletal proteins actin and vimentin showed no significant changes in mRNA expression in the control group, but was significantly higher in the loading group. MMP-13 mRNA expression was significantly higher in both the control group and loading group. Overall, this study suggests that suitable mechanical stimulation decreases the loss of chondrocyte viability and the mechanical properties of cartilage explants in vitro and improves cartilage preservation.
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Cartílago Articular/citología , Condrocitos/citología , Técnicas de Cultivo de Tejidos/instrumentación , Animales , Apoptosis , Fenómenos Biomecánicos , Supervivencia Celular , Células Cultivadas , Diseño de Equipo , Presión , Estrés Mecánico , PorcinosRESUMEN
As an ideal antioxidant and anti-apoptotic substance, hydrogen (H2) has protective effects on many isolated organs, such as the heart, lung and kidney. In this study, we explore whether H2 improves the preservation effect of osteochondral allograft by adding it to Dulbecco's Modified Eagle Medium (DMEM) solution during the tissue culture stage. The osteochondral allograft apparatus was used to harvest 60 pieces of cylindrical allografts (l = 10 mm, d = 6 mm) cartilage in the lateral loading area of the femoral condyle from the pig knee joint in the aseptic condition, and the grafts were randomly divided into 4 groups: Control group (DMEM solution without hydrogen); H-1 group (DMEM solution with hydrogen concentration of 0.2 mmol/L); H-2 group (DMEM solution with hydrogen concentration of 0.4 mmol/L); and H-3 group (DMEM solution with hydrogen concentration of 0.8 mmol/L). The chondrocyte viability, histological changes (hematoxylin and eosin staining, Safranine O staining, and collagen type II immunohistochemistry staining) and biomechanical properties (Young's modulus) of the osteochondral allograft were investigated after 28 days' storage. The chondrocyte viability and proteoglycan and collagen type II contents in the H-3 and H-2 groups were higher than that in the Control and H-1 groups, and the H-3 group had the highest values. However, significant differences were not observed between the four groups based on Young's modulus. Hydrogen as an additive to the DMEM solution improved the preservation effect of osteochondral allograft. The preservation effect of hydrogen occurred in a concentration-dependent manner.
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Cartílago Articular/cirugía , Condrocitos/trasplante , Hidrógeno/análisis , Articulación de la Rodilla/cirugía , Soluciones Preservantes de Órganos/química , Conservación de Tejido/métodos , Animales , Supervivencia Celular/efectos de los fármacos , Condrocitos/fisiología , Medios de Cultivo/química , Hidrógeno/química , Porcinos , Trasplante HomólogoRESUMEN
To compare the effects of Tsmu solution with vitrification on chondrocyte viability and examine histological and biomechanical properties of osteochondral allografts (OCAs) after storage, OCAs from femoral condyles of New Zealand rabbits were harvested, stored for 35 days in Tsmu solution or by in vitro vitrification, and subjected to in vivo and in vitro assays. Stored OCAs were transplanted into knee femoral condyle cartilage defects in recipient rabbits. Chondrocyte viability and histological changes of cartilage grafts were assessed in vitro. Gross assessment, chondrocyte viability, histological assessment, OCA biomechanics, and immunological markers were evaluated in vivo 6 months after transplantation. Fresh OCAs served as in vitro and in vivo controls. Chondrocyte viability and scores for cartilage surface and histological quantitative assessment were superior for Tsmu solution compared with vitrification, but inferior compared with fresh OCAs in vitro and in vivo. With the exception of interleukin 6 content, biomechanical features of samples stored in Tsmu solution were superior to vitrification, and inferior to fresh OCAs in vivo. Thus, Tsmu solution provided suitable storage that improved chondrocyte viability, intact OCA cartilage matrix architecture, and transplantation outcomes.
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Aloinjertos/efectos de los fármacos , Huesos/efectos de los fármacos , Condrocitos/citología , Crioprotectores/farmacología , Conservación de Tejido , Animales , Fenómenos Biomecánicos/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Conejos , Soluciones , Trasplante HomólogoRESUMEN
The purpose of this study is to evaluate the reliability of cartilage digestion and fluorescein diacetate-ethidium bromide (FDA-EB) fluorescence staining for the detection of chondrocyte viability in osteochondral grafts. Sixteen fresh osteochondral grafts were harvested from pig knee condyles, and the articular cartilage tissue was preserved. Each cartilage graft was cut into two 70-µm thick pieces and randomly allocated to Group A or Group B. The cell viability of Group A was detected using FDA-EB fluorescence staining of the digested cartilage, and the viability of Group B was detected with FDA-EB fluorescence staining of cartilage sections. Comparisons of chondrocyte viability and correlation analyses of the two groups were performed using the paired sample t test and Pearson correlation test, respectively. No significant difference was found in the chondrocyte viability between Groups A and B (p > 0.05), and a strong correlation was observed (r = 0.70, p < 0.05). Therefore, cartilage digestion with FDA-EB fluorescence staining is a reliable method for detecting chondrocyte viability in osteochondral grafts.