RESUMEN
Recent evidence indicates that the damaged regions in osteoarthritis are accompanied by the accumulation of iron ions. Ferroptosis, as an iron-dependent form of cell death, holds significant implications in osteoarthritis. Melatonin, a natural product with strong scavenging abilities against reactive oxygen species and lipid peroxidation, plays a crucial role in the treatment of osteoarthritis. This study aims to demonstrate the existence of ferroptosis in osteoarthritis and explore the specific mechanism of melatonin in suppressing ferroptosis and alleviating osteoarthritis. Our findings reveal that melatonin reverses inflammation-induced oxidative stress and lipid peroxidation while promoting the expression of extracellular matrix components in chondrocytes, safeguarding the cells. Our research has revealed that NADPH oxidase 4 (NOX4) serves as a crucial molecule in the ferroptosis process of osteoarthritis. Specifically, NOX4 is located on mitochondria in chondrocytes, which can induce disorders in mitochondrial energy metabolism and dysfunction, thereby intensifying oxidative stress and lipid peroxidation. LC-MS analysis further uncovered that GRP78 is a downstream binding protein of NOX4. NOX4 induces ferroptosis by weakening GRP78's protective effect on GPX4 and reducing its expression. Melatonin can inhibit the upregulation of NOX4 on mitochondria and mitigate mitochondrial dysfunction, effectively suppressing ferroptosis and alleviating osteoarthritis. This suggests that melatonin therapy represents a promising new approach for the treatment of osteoarthritis.
Asunto(s)
Ferroptosis , Melatonina , Mitocondrias , NADPH Oxidasa 4 , Osteoartritis , Melatonina/farmacología , Ferroptosis/efectos de los fármacos , Osteoartritis/metabolismo , Osteoartritis/tratamiento farmacológico , Osteoartritis/patología , NADPH Oxidasa 4/metabolismo , Animales , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Condrocitos/patología , Estrés Oxidativo/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Humanos , RatonesRESUMEN
The rapid advancement of cell therapies underscores the importance of understanding fundamental cellular attributes. Among these, cell fitness-how transplanted cells adapt to new microenvironments and maintain functional stability in vivo-is crucial. This study identifies a chemical compound, FPH2, that enhances the fitness of human chondrocytes and the repair of articular cartilage, which is typically nonregenerative. Through drug screening, FPH2 was shown to broadly improve cell performance, especially in maintaining chondrocyte phenotype and enhancing migration. Single-cell transcriptomics indicated that FPH2 induced a super-fit cell state. The mechanism primarily involves the inhibition of carnitine palmitoyl transferase I and the optimization of metabolic homeostasis. In animal models, FPH2-treated human chondrocytes substantially improved cartilage regeneration, demonstrating well-integrated tissue interfaces in rats. In addition, an acellular FPH2-loaded hydrogel proved effective in preventing the onset of osteoarthritis. This research provides a viable and safe method to enhance chondrocyte fitness, offering insights into the self-regulatory mechanisms of cell fitness.
Asunto(s)
Cartílago Articular , Condrocitos , Regeneración , Condrocitos/metabolismo , Condrocitos/citología , Condrocitos/efectos de los fármacos , Animales , Humanos , Cartílago Articular/metabolismo , Ratas , Osteoartritis/metabolismo , Osteoartritis/terapia , Hidrogeles/química , Movimiento Celular/efectos de los fármacosRESUMEN
Low back pain (LBP) is largely attributed to intervertebral disc degeneration (IVDD), of which the endplate changes are an important component. However, the alterations in cell fate and properties within the endplates during degeneration remain unknown. Here, we firstly performed the single-cell RNA-sequencing analysis (scRNA-seq) of the cells focusing on degenerative human endplates. By unsupervised clustering of the 8,534 single-cell based on the gene expression, we identified nine distinct cell types. We employed Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, and the single-cell regulatory network inference and clustering (SCENIC) to determine the enriched pathways and transcriptional activities across seven chondrocyte subpopulations. Furthermore, two cell fates of chondrocyte differentiation were found by trajectory analysis, one was enriched in inflammation-related genes, and the other was related to extracellular matrix (ECM). Additionally, the intercellular interactions of macrophages (MA) and chondrocytes, T cells/natural killer cells (T/NK) and chondrocytes were examined by ligand-receptor pairs analysis, showing the important regulative function of FN1 from MA and CD74 from T/NK during endplate degeneration. Overall, our findings provide novel perspectives on the endplate degeneration at the single-cell level and a whole-transcriptome size.
Asunto(s)
Diferenciación Celular , Condrocitos , Degeneración del Disco Intervertebral , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Humanos , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/patología , Análisis de la Célula Individual/métodos , Condrocitos/metabolismo , Condrocitos/patología , Diferenciación Celular/genética , Perfilación de la Expresión Génica , Femenino , Masculino , Redes Reguladoras de Genes , Persona de Mediana Edad , Macrófagos/metabolismo , Adulto , Disco Intervertebral/patología , Disco Intervertebral/metabolismoRESUMEN
BACKGROUND: Articular cartilage degeneration can result from injury, age, or arthritis, causing significant joint pain and disability without surgical intervention. Currently, the only FDA cell-based therapy for articular cartilage injury is Autologous Chondrocyte Implantation (ACI); however, this procedure is costly, time-intensive, and requires multiple treatments. Mesenchymal stromal cells (MSCs) are an attractive alternative autologous therapy due to their availability and ability to robustly differentiate into chondrocytes for transplantation with good safety profiles. However, treatment outcomes are variable due to donor-to-donor variability as well as intrapopulation heterogeneity and unstandardized MSC manufacturing protocols. Process improvements that reduce cell heterogeneity while increasing donor cell numbers with improved chondrogenic potential during expansion culture are needed to realize the full potential of MSC therapy. METHODS: In this study, we investigated the potential of MSC metabolic modulation during expansion to enhance their chondrogenic commitment by varying the nutrient composition, including glucose, pyruvate, glutamine, and ascorbic acid in culture media. We tested the effect of metabolic modulation in short-term (one passage) and long-term (up to seven passages). We measured metabolic state, cell size, population doubling time, and senescence and employed novel tools including micro-magnetic resonance relaxometry (µMRR) relaxation time (T2) to characterize the effects of AA on improved MSC expansion and chondrogenic potential. RESULTS: Our data show that the addition of 1 mM L-ascorbic acid-2-phosphate (AA) to cultures for one passage during MSC expansion prior to initiation of differentiation improves chondrogenic differentiation. We further demonstrate that AA treatment reduced the proportion of senescent cells and cell heterogeneity also allowing for long-term expansion that led to a > 300-fold increase in yield of MSCs with enhanced chondrogenic potential compared to untreated cells. AA-treated MSCs with improved chondrogenic potential showed a robust shift in metabolic profile to OXPHOS and higher µMRR T2 values, identifying critical quality attributes that could be implemented in MSC manufacturing for articular cartilage repair. CONCLUSIONS: Our results suggest an improved MSC manufacturing process that can enhance chondrogenic potential by targeting MSC metabolism and integrating process analytic tools during expansion.
Asunto(s)
Cartílago Articular , Condrocitos , Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Cartílago Articular/metabolismo , Humanos , Condrocitos/metabolismo , Condrocitos/citología , Condrogénesis/efectos de los fármacos , Diferenciación Celular , Células Cultivadas , Proliferación Celular , Trasplante de Células Madre Mesenquimatosas/métodos , AnimalesRESUMEN
Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.
Asunto(s)
Diferenciación Celular , Condrogénesis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Factor de Transcripción SOX9 , Ubiquitina Tiolesterasa , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Diferenciación Celular/genética , Condrogénesis/genética , Animales , Humanos , Cartílago/metabolismo , Condrocitos/metabolismo , Condrocitos/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Sistemas CRISPR-Cas , RatonesRESUMEN
The periosteum contains skeletal stem/progenitor cells that contribute to bone fracture healing. However, the in vivo identity of periosteal skeletal stem cells (P-SSCs) remains unclear, and membrane protein markers of P-SSCs that facilitate tissue engineering are needed. Here, we identified integral membrane protein 2A (Itm2a) enriched in SSCs using single-cell transcriptomics. Itm2a+ P-SSCs displayed clonal multipotency and self-renewal and sat at the apex of their differentiation hierarchy. Lineage-tracing experiments showed that Itm2a selectively labeled the periosteum and that Itm2a+ cells were preferentially located in the outer fibrous layer of the periosteum. The Itm2a+ cells rarely expressed CD34 or Osx, but expressed periosteal markers such as Ctsk, CD51, PDGFRA, Sca1, and Gli1. Itm2a+ P-SSCs contributed to osteoblasts, chondrocytes, and marrow stromal cells upon injury. Genetic lineage tracing using dual recombinases showed that Itm2a and Prrx1 lineage cells generated spatially separated subsets of chondrocytes and osteoblasts during fracture healing. Bone morphogenetic protein 2 (Bmp2) deficiency or ablation of Itm2a+ P-SSCs resulted in defects in fracture healing. ITM2A+ P-SSCs were also present in the human periosteum. Thus, our study identified a membrane protein marker that labels P-SSCs, providing an attractive target for drug and cellular therapy for skeletal disorders.
Asunto(s)
Curación de Fractura , Proteínas de la Membrana , Periostio , Animales , Periostio/metabolismo , Periostio/citología , Ratones , Curación de Fractura/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Células Madre/metabolismo , Células Madre/citología , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Fracturas Óseas/patología , Fracturas Óseas/metabolismo , Fracturas Óseas/terapia , Fracturas Óseas/genética , Osteoblastos/metabolismo , Osteoblastos/citología , Diferenciación Celular , Condrocitos/metabolismo , Condrocitos/citología , Masculino , Linaje de la CélulaRESUMEN
Articular cartilage phenotypic homeostasis is crucial for life-long joint function, but the underlying cellular and molecular mechanisms governing chondrocyte stability remain poorly understood. Here, we show that the protein tyrosine phosphatase SHP2 is differentially expressed in articular cartilage (AC) and growth plate cartilage (GPC) and that it negatively regulates cell proliferation and cartilage phenotypic program. Postnatal SHP2 deletion in Prg4+ AC chondrocytes increased articular cellularity and thickness, whereas SHP2 deletion in Acan+ pan-chondrocytes caused excessive GPC chondrocyte proliferation and led to joint malformation post-puberty. These observations were verified in mice and in cultured chondrocytes following treatment with the SHP2 PROTAC inhibitor SHP2D26. Further mechanistic studies indicated that SHP2 negatively regulates SOX9 stability and transcriptional activity by influencing SOX9 phosphorylation and promoting its proteasome degradation. In contrast to published work, SHP2 ablation in chondrocytes did not impact IL-1-evoked inflammation responses, and SHP2's negative regulation of SOX9 could be curtailed by genetic or chemical SHP2 inhibition, suggesting that manipulating SHP2 signaling has translational potential for diseases of cartilage dyshomeostasis.
Asunto(s)
Cartílago Articular , Condrocitos , Osteoartritis , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Factor de Transcripción SOX9 , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/genética , Animales , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Condrocitos/metabolismo , Condrocitos/patología , Ratones , Cartílago Articular/metabolismo , Cartílago Articular/patología , Osteoartritis/metabolismo , Osteoartritis/patología , Proliferación Celular , Células Cultivadas , Ratones Endogámicos C57BL , Ratones Noqueados , MasculinoRESUMEN
Intervertebral disc degeneration (IDD) is a major cause of discogenic pain, and is attributed to the dysfunction of nucleus pulposus, annulus fibrosus, and cartilaginous endplate (CEP). Osteopontin (OPN), a glycoprotein, is highly expressed in the CEP. However, little is known on how OPN regulates CEP homeostasis and degeneration, contributing to the pathogenesis of IDD. Here, we investigate the roles of OPN in CEP degeneration in a mouse IDD model induced by lumbar spine instability and its impact on the degeneration of endplate chondrocytes (EPCs) under pathological conditions. OPN is mainly expressed in the CEP and decreases with degeneration in mice and human patients with severe IDD. Conditional Spp1 knockout in EPCs of adult mice enhances age-related CEP degeneration and accelerates CEP remodeling during IDD. Mechanistically, OPN deficiency increases CCL2 and CCL5 production in EPCs to recruit macrophages and enhances the activation of NLRP3 inflammasome and NF-κB signaling by facilitating assembly of IRAK1-TRAF6 complex, deteriorating CEP degeneration in a spatiotemporal pattern. More importantly, pharmacological inhibition of the NF-κB/NLRP3 axis attenuates CEP degeneration in OPN-deficient IDD mice. Overall, this study highlights the importance of OPN in maintaining CEP and disc homeostasis, and proposes a promising therapeutic strategy for IDD by targeting the NF-κB/NLRP3 axis.
Asunto(s)
Inflamasomas , Degeneración del Disco Intervertebral , Macrófagos , Ratones Noqueados , FN-kappa B , Proteína con Dominio Pirina 3 de la Familia NLR , Osteopontina , Transducción de Señal , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Adulto Joven , Cartílago/patología , Cartílago/metabolismo , Condrocitos/metabolismo , Condrocitos/patología , Inflamasomas/metabolismo , Degeneración del Disco Intervertebral/patología , Degeneración del Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/genética , Macrófagos/metabolismo , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Osteopontina/metabolismo , Osteopontina/deficiencia , Osteopontina/genéticaRESUMEN
The extracellular matrix of cartilage primarily constitutes of collagen and aggrecan. Cartilage degradation starts with aggrecan loss in osteoarthritis (OA). Vitamin D (VD) plays an essential role in several inflammation-related diseases and can protect the collagen in cartilage during OA. The present study focused on the role of VD in aggrecan turnover of human articular chondrocytes treated with tumor necrosis factor α (TNF-α) and the possible mechanism. Treatment with different doses of VD and different periods of intervention with TNF-α and TGF-ß1 receptor (TGFßR1) inhibitor SB525334 were investigated. The viability of human chondrocytes and extracellular secretion of TGF-ß1 were measured. The expression of intracellular TGFßR1 and VD receptor was examined. Transcriptional and translational levels of aggrecan and the related metabolic factors were analyzed. The results showed that TNF-α markedly reduced the viability, TGFßR1 expressions and aggrecan levels of human chondrocytes, and increased disintegrin and metalloproteinase with thrombospondin motifs. The alterations were partially inhibited by VD treatment. Furthermore, the effects of VD were blocked by the TGFßR1 inhibitor SB525334 in TNF-α-treated cells. VD may prevent proteoglycan loss due to TNF-α via TGF-ß1 signaling in human chondrocytes.
Asunto(s)
Agrecanos , Cartílago Articular , Condrocitos , Proteoglicanos , Transducción de Señal , Factor de Crecimiento Transformador beta1 , Factor de Necrosis Tumoral alfa , Vitamina D , Humanos , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Agrecanos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Vitamina D/farmacología , Proteoglicanos/metabolismo , Proteoglicanos/farmacología , Cartílago Articular/metabolismo , Cartílago Articular/efectos de los fármacos , Células Cultivadas , Supervivencia Celular/efectos de los fármacos , Osteoartritis/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Receptores de Calcitriol/metabolismoRESUMEN
Articular cartilage receives nutrients and oxygen from the synovial fluid to maintain homeostasis. However, compared to tissues with abundant blood flow, articular cartilage is exposed to a hypoxic environment (i.e., physioxia) and has an enhanced hypoxic stress response. Hypoxia-inducible factors (HIFs) play a pivotal role in this physioxic environment. In normoxic conditions, HIFs are downregulated, whereas in physioxic conditions, they are upregulated. The HIF-α family comprises three members: HIF-1α, HIF-2α, and HIF-3α. Each member has a distinct function in articular cartilage. In osteoarthritis, which is primarily caused by degeneration of articular cartilage, HIF-1α is upregulated in chondrocytes and is believed to protect articular cartilage by acting anabolically on it. Conversely, in contrast to HIF-1α, HIF-2α exerts a catabolic influence on articular cartilage. It may therefore be possible to develop a new treatment for OA by controlling the expression of HIF-1α and HIF-2α with drugs or by altering the oxygen environment in the joints.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cartílago Articular , Condrocitos , Homeostasis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Osteoartritis , Humanos , Cartílago Articular/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Osteoartritis/metabolismo , Condrocitos/metabolismo , Oxígeno/metabolismo , Hipoxia/metabolismo , Hipoxia/fisiopatologíaRESUMEN
BACKGROUND: Matrix-induced autologous chondrocyte implantation (MACI), the third-generation of the technique, is an established procedure for the treatment of focal cartilage defects in the knee. However, the literature lacks long-term results of MACI with good statistical power. PURPOSE: To determine long-term survival and patient-reported outcomes (PROs) in a representative cohort and to identify patient- and surgery-related parameters that may influence long-term clinical outcomes. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A total of 103 patients were clinically evaluated at the current follow-up of 8.1 years (range, 5-11.9 years). PRO measures (PROMs) included the Knee injury and Osteoarthritis Outcome Score (KOOS), EQ-5D, visual analog scale for pain, and Tegner Activity Scale. Magnetic resonance imaging results were evaluated by using the AMADEUS (area measurement and depth and underlying structures) and MOCART (magnetic resonance observation of cartilage repair tissue) 2.0 knee score classification systems. Potential factors influencing PROs were first identified univariately and investigated in a multivariate regression model. RESULTS: The defects had a mean size of 4.8 cm2 (range, 1.2-12 cm2) and were predominantly femorotibial (66%). The mean Kaplan-Meier survival rate of revision for any reason was 97.2% 6 1.6% at 10 years. In comparison to preoperative values, all PROMs were significantly improved at the current follow-up (P < .05). The MOCART 2.0 score peaked at 12 months (mean, 80.2 6 15.3 months) and showed no significant change at 96 months (mean, 76.1 ± 19.5 months; P = .142). The linear multivariate regression model identified an association of body mass index (BMI), MOCART 2.0 score, and number of previous knee surgeries with KOOS (R2 = 0.41; f2 = 0.69). Further analysis of the individual determinants revealed an optimal BMI range of 20 to 29 for favorable PROs at 96 months. Significant correlations of MOCART subscores with the overall KOOS were found for graft surface and structure, bony reaction, and subchondral detectable changes. Only 30% of patients with 2 previous surgeries and 20% of patients with 3 previous surgeries achieved a Patient Acceptable Symptom State (χ2 = 10.93; P = .012). CONCLUSION: The present study shows consistently good long-term clinical outcomes after MACI with a low revision rate and high patient satisfaction. BMI and number of previous knee surgeries may influence clinical outcomes and should be considered in patient selection and education. There is a correlation between graft structure, subchondral bone changes on magnetic resonance imaging, and long-term PROMs.
Asunto(s)
Condrocitos , Medición de Resultados Informados por el Paciente , Trasplante Autólogo , Humanos , Condrocitos/trasplante , Femenino , Masculino , Adulto , Persona de Mediana Edad , Adulto Joven , Cartílago Articular/cirugía , Cartílago Articular/lesiones , Traumatismos de la Rodilla/cirugía , Estudios de Seguimiento , Imagen por Resonancia Magnética , Adolescente , Resultado del Tratamiento , Articulación de la Rodilla/cirugíaRESUMEN
BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative disease. We explored the role and regulatory mechanisms of lncRNA-FAS-AS1 in OA progression. METHODS: We exposed human immortalized chondrocytes to IL-1ß for 24 h to induce an OA cell model. The target molecule levels were assessed using western blot and quantitative real-time PCR (RT-qPCR). Cell viability and apoptosis were measured using CCK-8 and flow cytometry. The m6A modification of FAS-AS1 was determined using MeRIP. We examined the binding relationships between FAS-AS1, Fragile X mental retardation 1 (FMR1), and A disintegrin and metalloproteinase 8 (ADAM8) using RIP and RNA pull-down. The OA animal model was established by separating the medial collateral ligament and medial meniscus. Safranin-O staining and Mankin's scale were employed to evaluate pathological changes within the cartilage. RESULTS: FAS-AS1, METTL14, and ADAM8 were upregulated, and the JAK/STAT3 signaling pathway was activated in OA mice and IL-1ß-induced chondrocytes. FAS-AS1 knockdown inhibited extracellular matrix degradation in IL-1ß-induced chondrocytes; however, ADAM8 overexpression reversed this effect. FAS-AS1 maintained the stability of ADAM8 mRNA by recruiting FMR1. METTL14 knockdown repressed FAS-AS1 expression in an m6A-dependent manner. FAS-AS1 overexpression reversed the inhibitory effects of METTL14 knockdown on JAK/STAT3 signaling and cartilage damage in the OA model both in vitro and in vivo. CONCLUSION: METTL14-mediated FAS-AS1 promotes OA progression through the FMR1/ADAM8/JAK/STAT3 axis.
Asunto(s)
Proteínas ADAM , Condrocitos , Progresión de la Enfermedad , Proteínas de la Membrana , ARN Largo no Codificante , Factor de Transcripción STAT3 , Transducción de Señal , Regulación hacia Arriba , Animales , Humanos , Masculino , Ratones , Proteínas ADAM/metabolismo , Proteínas ADAM/genética , Adenosina/análogos & derivados , Apoptosis , Artritis Experimental/metabolismo , Artritis Experimental/genética , Artritis Experimental/patología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Línea Celular , Condrocitos/metabolismo , Condrocitos/patología , Modelos Animales de Enfermedad , Interleucina-1beta/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Ratones Endogámicos C57BL , Osteoartritis/metabolismo , Osteoartritis/genética , Osteoartritis/patología , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/patología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
Osteoarthritis (OA) is a progressive joint disease characterized by extracellular matrix (ECM) degradation and inflammation, which is involved with pathological microenvironmental alterations induced by damaged chondrocytes. However, current therapies are not effective in alleviating the progression of OA. Isoquercetin is a natural flavonoid glycoside compound that has various pharmacological effects including anticancer, anti-diabetes and blood lipid regulation. Previous evidence suggests that isoquercetin has anti-inflammatory properties in various diseases, but its effect on OA has not been investigated yet. In this study, through western bolt, qRT-PCR and ELISA, it was found that isoquercetin could reduce the increase of ADAMTS5, MMP13, COX-2, iNOS and IL-6 induced by IL-1ß, suggesting that isoquercetin could inhibit the inflammation and ECM degradation of chondrocytes. Through nuclear-plasma separation technique, western blot and immunocytochemistry, it can be found that Nrf2 and NF-κB pathways are activated in this process, and isoquercetin may rely on this process to play its protective role. In vivo, the results of X-ray and SO staining show that intra-articular injection of isoquercetin reduces the degradation of cartilage in the mouse OA model. In conclusion, the present work suggests that isoquercetin may benefit chondrocytes by regulating the Nrf2/NF-κB signaling axis, which supports isoquercetin as a potential drug for the treatment of OA.
Asunto(s)
Condrocitos , Factor 2 Relacionado con NF-E2 , FN-kappa B , Osteoartritis , Quercetina , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Proteína ADAMTS5/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Ciclooxigenasa 2/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Osteoartritis/patología , Quercetina/farmacología , Quercetina/análogos & derivados , Quercetina/química , Quercetina/uso terapéutico , Transducción de Señal/efectos de los fármacosRESUMEN
Aim: RADA16-PLGA composite scaffolds constructed with simultaneous loading of BMSCs and TGF-ß3 and explored their ability for chondrogenic differentiation in vitro.Methods: The performance of the composite scaffolds is assessed by rheometer assay, electron microscopic structural observation and ELISA release assay. The biosafety of the composite scaffolds is assessed by cytocompatibility assay and cell migration ability. The chondrogenic differentiation ability of composite scaffolds is evaluated by Alisin blue staining, PCR and immunofluorescence staining.Results: The composite scaffold has a good ECM-like structure, the ability to control the release of TGF-ß3 and good biocompatibility. More importantly, the composite scaffolds can induce the differentiation of BMSCs to chondrocytes.Conclusion: Composite scaffolds are expected to enhance the endogenous NP repair process.
[Box: see text].
Asunto(s)
Diferenciación Celular , Condrocitos , Condrogénesis , Células Madre Mesenquimatosas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Andamios del Tejido , Factor de Crecimiento Transformador beta3 , Factor de Crecimiento Transformador beta3/farmacología , Factor de Crecimiento Transformador beta3/metabolismo , Diferenciación Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Andamios del Tejido/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Animales , Humanos , Ingeniería de Tejidos/métodos , Células Cultivadas , Concentración de Iones de Hidrógeno , Ácido Poliglicólico/química , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Nanopartículas/químicaRESUMEN
Osteoarthritis is a degenerative joint disease with joint pain as the main symptom, caused by fibrosis and loss of articular cartilage. Due to the complexity and heterogeneity of osteoarthritis, there is a lack of effective individualized disease-modifying osteoarthritis drugs in clinical practice. Chondrocyte senescence is reported to participate in occurrence and progression of osteoarthritis. Here we show that small molecule 10-hydroxy-2-decenoic acid suppresses cartilage degeneration and relieves pain in the chondrocytes, cartilage explants from osteoarthritis patients, surgery-induced medial meniscus destabilization or naturally aged male mice. We further confirm that 10-hydroxy-2-decenoic acid exerts a protective effect by targeting the glycosylation site in the Asp_Arg_Hydrox domain of aspartyl ß-hydroxylase. Mechanistically, 10-hydroxy-2-decenoic acid alleviate cellular senescence through the ERK/p53/p21 and GSK3ß/p16 pathways in the chondrocytes. Our study uncovers that 10-hydroxy-2-decenoic acid modulate cartilage metabolism by targeting aspartyl ß-hydroxylase to inhibit chondrocyte senescence in osteoarthritis. 10-hydroxy-2-decenoic acid may be a promising therapeutic drug against osteoarthritis.
Asunto(s)
Cartílago Articular , Senescencia Celular , Condrocitos , Ácidos Grasos Monoinsaturados , Osteoartritis , Animales , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/patología , Masculino , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/tratamiento farmacológico , Osteoartritis/prevención & control , Ratones , Senescencia Celular/efectos de los fármacos , Humanos , Ácidos Grasos Monoinsaturados/farmacología , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Cartílago Articular/patología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , FemeninoRESUMEN
Osteoarthritis (OA) is the most common form of arthritic disease, and phenotypic modification of chondrocytes is an important mechanism that contributes to the loss of cartilage homeostasis. This study identified that Fascin actin-bundling protein 1 (FSCN1) plays a pivotal role in regulating chondrocytes phenotype and maintaining cartilage homeostasis. Proteome-wide screening revealed markedly upregulated FSCN1 protein expression in human OA cartilage. FSCN1 accumulation was confirmed in the superficial layer of OA cartilage from humans and mice, primarily in dedifferentiated-like chondrocytes, associated with enhanced actin stress fiber formation and upregulated type I and III collagens. FSCN1-inducible knockout mice exhibited delayed cartilage degeneration following experimental OA surgery. Mechanistically, FSCN1 promoted actin polymerization and disrupted the inhibition of Decorin on TGF-ß1, leading to excessive TGF-ß1 production and ALK1/Smad1/5 signaling activation, thus, accelerated chondrocyte dedifferentiation. Intra-articular injection of FSCN1-overexpressing adeno-associated virus exacerbated OA progression in mice, which was mitigated by an ALK1 inhibitor. Moreover, FSCN1 inhibitor NP-G2-044 effectively reduced extracellular matrix degradation in OA mice, cultured human OA chondrocytes, and cartilage explants by suppressing ALK1/Smad1/5 signaling. These findings suggest that targeting FSCN1 represents a promising therapeutic approach for OA.
Asunto(s)
Proteínas Portadoras , Condrocitos , Proteínas de Microfilamentos , Osteoartritis , Animales , Humanos , Masculino , Ratones , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Cartílago Articular/metabolismo , Cartílago Articular/patología , Condrocitos/metabolismo , Condrocitos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Osteoartritis/patología , Osteoartritis/metabolismo , Osteoartritis/genética , Fenotipo , Receptores Odorantes , Transducción de SeñalRESUMEN
Osteoarthritis (OA) is a chronic joint disease characterized by the degeneration, destruction, and excessive ossification of articular cartilage. The prevalence of OA is rising annually, concomitant with the aging global population and increasing rates of obesity. This condition imposes a substantial and escalating burden on individual health, healthcare systems, and broader social and economic frameworks. The etiology of OA is multifaceted and not fully understood. Current research suggests that the death of chondrocytes, encompassing mechanisms such as cellular apoptosis, pyroptosis, autophagy, ferroptosis and cuproptosis, contributes to both the initiation and progression of the disease. These cell death pathways not only diminish the population of chondrocytes but also exacerbate joint damage through the induction of inflammation and other deleterious processes. This paper delineates the morphological characteristics associated with various modes of cell death and summarizes current research results on the molecular mechanisms of different cell death patterns in OA. The objective is to review the advancements in understanding chondrocyte cell death in OA, thereby offering novel insights for potential clinical interventions.
Asunto(s)
Muerte Celular , Condrocitos , Progresión de la Enfermedad , Osteoartritis , Condrocitos/patología , Humanos , Osteoartritis/patología , Osteoartritis/terapia , Muerte Celular/fisiología , Apoptosis/fisiología , Cartílago Articular/patología , Autofagia/fisiología , Animales , Piroptosis/fisiología , Ferroptosis/fisiologíaRESUMEN
Inflammation models are widely used in the in vitro investigation of new therapeutic approaches for osteoarthritis. TNFα (tumor necrosis factor alpha) plays an important role in the inflammatory process. Current inflammation models lack uniformity and make comparisons difficult. Therefore, this study aimed to systematically investigate whether the effects of TNFα are concentration-dependent and whether chondrocyte expansion has an effect on the inflammatory model. Bovine chondrocytes were enzymatically isolated, expanded to passages 1-3, and transferred into a 3D pellet culture. Chondrocyte pellets were stimulated with recombinant bovine TNFα at different concentrations for 48 h to induce inflammation. Gene expression of anabolic (collagen 2, aggrecan, cartilage oligomeric protein (COMP)), catabolic (matrix metalloproteinases (MMP3, MMP13)), dedifferentiation (collagen 1) markers, inflammation markers (interleukin-6 (IL-6), nuclear factor kappa B (NFkB), cyclooxygenase-2 (COX), prostaglandin-E-synthase-2 (PTGES2)), and the apoptosis marker caspase 3 was determined. At the protein level, concentrations of IL-6, nitric oxide (NO), and sulfated glycosaminoglycans (GAG) were evaluated. Statistical analysis was performed using the independent t-test, and significance was defined as p < 0.05. In general, TNFα caused a decrease in anabolic markers and an increase in the expression of catabolic and inflammatory markers. There was a concentration-dependent threshold of 10 ng/mL to induce significant inflammatory effects. Most of the markers analyzed showed TNFα concentration-dependent effects (COMP, PRG4, AGN, Col1, MMP3, and NFkB). There was a statistical influence of selected gene expression markers from different passages on the TNFα chondrocyte inflammation model, including Col2, MMP13, IL-6, NFkB, COX2, and PTGES2. Considering the expression of collagen 2 and MMP3, passage 3 chondrocytes showed a higher sensitivity to TNFα stimulation compared to passages 1 and 2. On the other hand, MMP13, IL-6, NFkB, and caspase 3 gene expression were lower in P3 chondrocytes compared to the other passages. On the protein level, inflammatory effects showed a similar pattern, with cytokine effects starting at 10 ng/mL and differences between the passages. TNFα had a detrimental effect on cartilage, with a clear threshold observed at 10 ng/mL. Although TNFα effects showed concentration-dependent patterns, this was not consistent for all markers. The selected passage showed a clear influence, especially on inflammation markers. Further experiments were warranted to explore the effects of TNFα concentration and passage in long-term stimulation.
Asunto(s)
Condrocitos , Inflamación , Factor de Necrosis Tumoral alfa , Animales , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Bovinos , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Inflamación/metabolismo , Inflamación/patología , Células Cultivadas , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Interleucina-6/metabolismo , Interleucina-6/genética , BiomarcadoresRESUMEN
OBJECTIVE: The aim of this study was to investigate whether ASA VI controls osteoarthritis (OA) by regulating mitochondrial function. METHODS: Primary chondrocytes were isolated and cultured from rat knee joints. The chondrocytes were treated with ASA VI and interleukin-1ß (IL-1ß) to simulate the inflammatory environment of OA. Cell viability, apoptosis, inflammatory cytokine levels, and extracellular matrix (ECM) component levels were assessed. Mitochondrial function, including ATP levels, mitochondrial membrane potential, reactive oxygen species (ROS) levels, and mitochondrial DNA content, was evaluated. The expression of Sirtuin 3 (Sirt3), a key regulator of mitochondrial homeostasis, was examined. Additionally, a rat OA model was established by destabilizing the medial meniscus, and the effects of ASA VI on cartilage degeneration were assessed. RESULTS: ASA VI treatment improved cell viability, reduced apoptosis, and decreased IL-6 and TNF-α levels in IL-1ß-induced chondrocytes. ASA VI also upregulated Collagen II and Aggrecan expression, while downregulating ADAMTS5 and MMP-13 expression. Furthermore, ASA VI mitigated IL-1ß-induced mitochondrial dysfunction by increasing ATP levels, restoring mitochondrial membrane potential, reducing ROS production, and preserving mitochondrial DNA content. These effects were accompanied by the activation of Sirt3. In the rat OA model, ASA VI treatment increased Sirt3 expression and alleviated cartilage degeneration. CONCLUSION: ASA VI exerts chondroprotective and anti-inflammatory effects on IL-1ß-induced chondrocytes by improving mitochondrial function through Sirt3 activation. ASA VI also attenuates cartilage degeneration in a rat OA model. These findings suggest that ASA VI may be a potential therapeutic agent for the treatment of osteoarthritis by targeting mitochondrial dysfunction.
Asunto(s)
Condrocitos , Homeostasis , Mitocondrias , Osteoartritis , Saponinas , Sirtuina 3 , Animales , Masculino , Ratones , Ratas , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Apoptosis/efectos de los fármacos , Cartílago Articular/patología , Cartílago Articular/metabolismo , Cartílago Articular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Interleucina-1beta/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Osteoartritis/patología , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 3/metabolismo , Sirtuina 3/genética , Saponinas/farmacologíaRESUMEN
Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel's cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel's cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish.