RESUMO
BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by chronic inflammation and progressive cartilage degradation, ultimately leading to joint dysfunction and disability. Oleocanthal (OC), a bioactive phenolic compound derived from extra virgin olive oil, has garnered significant attention due to its potent anti-inflammatory properties, which are comparable to those of non-steroidal anti-inflammatory drugs (NSAIDs). This study pioneers the investigation into the effects of OC on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway in OA, aiming to validate its efficacy as a functional food-based therapeutic intervention. METHODS: To simulate cartilage tissue in vitro, human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes. An inflammatory OA-like environment was induced in these chondrocytes using lipopolysaccharide (LPS) to mimic the pathological conditions of OA. The therapeutic effects of OC were evaluated by treating these inflamed chondrocytes with various concentrations of OC. The study focused on assessing key inflammatory markers, catabolic enzymes, and mitochondrial function to elucidate the protective mechanisms of OC. Mitochondrial function, specifically mitochondrial membrane potential (ΔΨm), was assessed using Rhodamine 123 staining, a fluorescent dye that selectively accumulates in active mitochondria. The integrity of ΔΨm serves as an indicator of mitochondrial and bioenergetic function. Additionally, Western blotting was employed to analyze protein expression levels, while real-time polymerase chain reaction (RT-PCR) was used to quantify gene expression of inflammatory cytokines and catabolic enzymes. Flow cytometry was utilized to measure cell viability and apoptosis, providing a comprehensive evaluation of OC's therapeutic effects on chondrocytes. RESULTS: The results demonstrated that OC significantly downregulated PAR-2 expression in a dose-dependent manner, leading to a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1ß, and MCP-1. Furthermore, OC attenuated the expression of catabolic markers such as SOX4 and ADAMTS5, which are critically involved in cartilage matrix degradation. Importantly, OC was found to preserve mitochondrial membrane potential (ΔΨm) in chondrocytes subjected to inflammatory stress, as evidenced by Rhodamine 123 staining, indicating a protective effect on cellular bioenergetics. Additionally, OC modulated the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL)/Receptor Activator of Nuclear Factor Kappa-Β (RANK) pathway, suggesting a broader therapeutic action against the multifactorial pathogenesis of OA. CONCLUSIONS: This study is the first to elucidate the modulatory effects of OC on the PAR-2 mediated inflammatory pathway in OA, revealing its potential as a multifaceted therapeutic agent that not only mitigates inflammation but also protects cartilage integrity. The preservation of mitochondrial function and modulation of the RANKL/RANK pathway further underscores OC's comprehensive therapeutic potential in counteracting the complex pathogenesis of OA. These findings position OC as a promising candidate for integration into nutritional interventions aimed at managing OA. However, further research is warranted to fully explore OC's therapeutic potential across different stages of OA and its long-term effects in musculoskeletal disorders.
Assuntos
Anti-Inflamatórios , Condrócitos , Monoterpenos Ciclopentânicos , Células-Tronco Mesenquimais , Osteoartrite , Receptor PAR-2 , Humanos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Osteoartrite/metabolismo , Osteoartrite/tratamento farmacológico , Receptor PAR-2/metabolismo , Anti-Inflamatórios/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Monoterpenos Ciclopentânicos/farmacologia , Células Cultivadas , Alimento Funcional , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Lipopolissacarídeos/farmacologia , Aldeídos , FenóisRESUMO
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.
Assuntos
Ferroptose , Melatonina , Mitocôndrias , NADPH Oxidase 4 , Osteoartrite , Melatonina/farmacologia , Ferroptose/efeitos dos fármacos , Osteoartrite/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/patologia , NADPH Oxidase 4/metabolismo , Animais , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Estresse Oxidativo/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Humanos , CamundongosRESUMO
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.
Assuntos
Cartilagem Articular , Condrócitos , Regeneração , Condrócitos/metabolismo , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Animais , Humanos , Cartilagem Articular/metabolismo , Ratos , Osteoartrite/metabolismo , Osteoartrite/terapia , Hidrogéis/química , Movimento Celular/efeitos dos fármacosRESUMO
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.
Assuntos
Morte Celular , Condrócitos , Progressão da Doença , Osteoartrite , Condrócitos/patologia , Humanos , Osteoartrite/patologia , Osteoartrite/terapia , Morte Celular/fisiologia , Apoptose/fisiologia , Cartilagem Articular/patologia , Autofagia/fisiologia , Animais , Piroptose/fisiologia , Ferroptose/fisiologiaRESUMO
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.
Assuntos
Proteínas ADAM , Condrócitos , Progressão da Doença , Proteínas de Membrana , RNA Longo não Codificante , Fator de Transcrição STAT3 , Transdução de Sinais , Regulação para Cima , Animais , Humanos , Masculino , Camundongos , Proteínas ADAM/metabolismo , Proteínas ADAM/genética , Adenosina/análogos & derivados , Apoptose , Artrite Experimental/metabolismo , Artrite Experimental/genética , Artrite Experimental/patologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Linhagem Celular , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Interleucina-1beta/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Camundongos Endogâmicos C57BL , Osteoartrite/metabolismo , Osteoartrite/genética , Osteoartrite/patologia , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genéticaRESUMO
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.
Assuntos
Condrócitos , Fator 2 Relacionado a NF-E2 , NF-kappa B , Osteoartrite , Quercetina , Transdução de Sinais , Animais , Humanos , Masculino , Camundongos , Proteína ADAMTS5/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Ciclo-Oxigenase 2/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Osteoartrite/patologia , Quercetina/farmacologia , Quercetina/análogos & derivados , Quercetina/química , Quercetina/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
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].
Assuntos
Diferenciação Celular , Condrócitos , Condrogênese , Células-Tronco Mesenquimais , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Alicerces Teciduais , Fator de Crescimento Transformador beta3 , Fator de Crescimento Transformador beta3/farmacologia , Fator de Crescimento Transformador beta3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Alicerces Teciduais/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Animais , Humanos , Engenharia Tecidual/métodos , Células Cultivadas , Concentração de Íons de Hidrogênio , Ácido Poliglicólico/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Nanopartículas/químicaAssuntos
Condrócitos , Células Epiteliais , Células-Tronco Mesenquimais , Engenharia Tecidual , Animais , Condrócitos/transplante , Engenharia Tecidual/métodos , Células Epiteliais/transplante , Suínos , Sobrevivência de Enxerto , Traqueia/transplante , Transplante de Células-Tronco Mesenquimais/métodosRESUMO
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.
Assuntos
Agrecanas , Cartilagem Articular , Condrócitos , Proteoglicanas , Transdução de Sinais , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa , Vitamina D , Humanos , Condrócitos/metabolismo , Condrócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Agrecanas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vitamina D/farmacologia , Proteoglicanas/metabolismo , Proteoglicanas/farmacologia , Cartilagem Articular/metabolismo , Cartilagem Articular/efeitos dos fármacos , Células Cultivadas , Sobrevivência Celular/efeitos dos fármacos , Osteoartrite/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptores de Calcitriol/metabolismoRESUMO
EphrinB2, a member of the Ephrin family, has been linked to several orthopaedic conditions. Nevertheless, the correlation between ephrinB2 and post-traumatic arthritis (PTOA) remains unclear. Human PTOA cartilage from human and mouse knee joints was systematically analysed to investigate the relationship between EphrinB2 and PTOA using SO-FG and toluidine blue staining, micro-CT, histomorphometry, immunohistochemistry, immunofluorescence, lentiviral articular injection and in situ end labeling (TUNEL) assays. EphrinB2 expression was significantly downregulated in PTOA chondrocytes. Blocking EphrinB2 increased the breakdown of cartilage matrix in mice with PTOA via reducing the process of chondrocyte autophagy. The presence of severe cartilage damage was evident, as indicated by a considerable decrease in both cartilage thickness and area, accompanied by an increase in chondrocyte death. Altogether, EphrinB2 is required for the maintenance of cartilage homeostasis in post-traumatic arthritis, and EphrinB2 ablation is associated with accelerated chondrocyte matrix degeneration, finally causing damage to the articular cartilage.
Assuntos
Autofagia , Cartilagem Articular , Condrócitos , Efrina-B2 , Homeostase , Condrócitos/metabolismo , Condrócitos/patologia , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Efrina-B2/metabolismo , Efrina-B2/genética , Humanos , Camundongos , Masculino , Camundongos Endogâmicos C57BL , FemininoRESUMO
BACKGROUND: The uncultured adipose-derived stromal vascular fraction (SVF), consisting of adipose-derived stromal cells (ADSCs), M2 macrophages (M2Φ) and others, has shown therapeutic potential against osteoarthritis (OA), however, the mechanisms underlying its therapeutic effects remain unclear. Therefore, this study investigated the effects of the SVF on OA in a human-immunodeficient rat xenotransplantation model. METHODS: OA model was induced in the knees of female immunodeficient rats by destabilization of the medial meniscus. Immediately after the surgery, human SVF (1 × 105), ADSCs (1 × 104), or phosphate buffered saline as a control group were transplanted into the knees. At 4 and 8 weeks postoperatively, OA progression and synovitis were analyzed by macroscopic and histological analyses, and the expression of collagen II, SOX9, MMP-13, ADAMTS-5, F4/80, CD86 (M1), CD163 (M2), and human nuclear antigen (hNA) were evaluated immunohistochemically. In vitro, flow cytometry was performed to collect CD163-positive cells as M2Φ from the SVF. Chondrocyte pellets (1 × 105) were co-cultured with SVF (1 × 105), M2Φ (1 × 104), and ADSCs (1 × 104) or alone as a control group, and the pellet size was compared. TGF-ß, IL-10 and MMP-13 concentrations in the medium were evaluated using enzyme-linked immunosorbent assay. RESULTS: In comparison with the control and ADSC groups, the SVF group showed significantly slower OA progression and less synovitis with higher expression of collagen II and SOX9, lower expression of MMP-13 and ADAMTS-5, and lower F4/80 and M1/M2 ratio in the synovium. Only the SVF group showed partial expression of hNA-, CD163-, and F4/80-positive cells in the rat synovium. In vitro, the SVF, M2Φ, ADSC and control groups, in that order, showed larger pellet sizes, higher TGF-ß and IL-10, and lower MMP-13 concentrations. CONCLUSIONS: The M2Φ in the transplanted SVF directly affected recipient tissue, enhancing the secretion of growth factors and chondrocyte-protecting cytokines, and partially improving chondrocytes and joint homeostasis. These findings indicate that the SVF is as an effective option for regenerative therapy for OA, with mechanisms different from those of ADSCs.
Assuntos
Tecido Adiposo , Macrófagos , Osteoartrite , Animais , Humanos , Ratos , Feminino , Osteoartrite/terapia , Osteoartrite/patologia , Macrófagos/metabolismo , Tecido Adiposo/citologia , Condrócitos/metabolismo , Condrócitos/citologia , Fração Vascular Estromal/metabolismo , Modelos Animais de DoençasRESUMO
Endochondral ossification is the process by which cartilage is mineralized into bone, and is essential for the development of long bones. Osteocalcin (OCN), a protein abundant in bone matrix, also exhibits high expression in chondrocytes, especially hypertrophic chondrocytes, while its role in endochondral ossification remains unclear. Utilizing a new CRISPR/Cas9-mediated bglap-bglap2 deficiency (OCNem) mouse model generated in our laboratory, we provide the first evidence of OCN's regulatory function in chondrocyte differentiation and endochondral ossification. The OCNem mice exhibited significant delays in primary and secondary ossification centers compared to wild-type mice, along with increased cartilage length in growth plates and hypertrophic zones during neonatal and adolescent stages. These anomalies indicated that OCN deficiency disturbed endochondral ossification during embryonic and postnatal periods. Mechanism wise, OCN deficiency was found to increase chondrocyte differentiation and postpone vascularization process. Furthermore, bone marrow mesenchymal stromal cells (BMSCs) from OCNem mice demonstrated an increased capacity for chondrogenic differentiation. Transcriptional network analysis implicated that BMP and TGF-ß signaling pathways were highly affected in OCNem BMSCs, which is closely associated with cartilage development and maintenance. This elucidation of OCN's function in chondrocyte differentiation and endochondral ossification contributes to a more comprehensive understanding of its impact on skeletal development and homeostasis.
Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular , Condrócitos , Condrogênese , Osteocalcina , Osteogênese , Animais , Condrócitos/metabolismo , Condrócitos/citologia , Osteogênese/genética , Camundongos , Osteocalcina/metabolismo , Osteocalcina/genética , Diferenciação Celular/genética , Condrogênese/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Cartilagem/metabolismo , Transdução de Sinais , Camundongos KnockoutRESUMO
Osteoarthritis of the knee (OAK), a progressive degenerative disease affecting quality of life, is characterized by cartilage degeneration, synovial inflammation, and osteophyte formation causing pain and disability. Platelet-rich plasma (PRP) is an autologous blood product effective in reducing OAK-associated pain. PRP compositions depend on their purification. In clinical practice, PRP is typically administered immediately after purification, while cryopreserved PRP is used in research. Platelets are activated by freezing followed by release of their humoral factors. Therefore, PRP without any manipulation after purification (utPRP) and freeze-thawed PRP (fPRP) may differ in their properties. We purified leukocyte-poor PRP (LPPRP) and autologous protein solution (APS) to compare the properties of utPRPs and fPRPs and their effects on OAK target cells. We found significant differences in platelet activation and humoral factor content between utPRPs and fPRPs in both LPPRP and APS. Freeze-thawing affected the anti-inflammatory properties of LPPRP and APS in chondrocytes and synovial cells differed. Both utPRPs and fPRPs inhibited polarization toward M1 macrophages while promoting polarization toward M2 macrophages. Freeze-thawing specifically affected humoral factor production in macrophages, suggesting that evaluating the efficacy of PRPs requires considering PRP purification methods, properties, and conditions. Understanding these variations may enhance therapeutic application of PRPs in OAK.
Assuntos
Congelamento , Plasma Rico em Plaquetas , Plasma Rico em Plaquetas/metabolismo , Plasma Rico em Plaquetas/química , Humanos , Osteoartrite do Joelho/terapia , Condrócitos/metabolismo , Macrófagos/metabolismo , Ativação Plaquetária , Masculino , Criopreservação/métodosRESUMO
BACKGROUND: Hesperetin, a flavonoid derived from citrus fruits, exhibits potent antioxidant and anti-inflammatory activities and has been implicated in cartilage protection. However, its effectiveness against T-2 toxin-induced knee cartilage damage remains unclear. METHODS: In this study, high-throughput sequencing analysis was employed to identify the key signaling pathways involved in T-2 toxin-induced articular cartilage damage in rats. Animal models were divided into the following groups: control, low-dose T-2 toxin, high-dose T-2 toxin, T-2 toxin + hesperetin, hesperetin, and vehicle. Pathological staining and immunohistochemistry were used to assess pathological changes, as well as the expression levels of the cartilage matrix-related proteins MMP13 and collagen II, along with the activation of the p38 MAPK signaling pathway. Additionally, primary rat chondrocytes were cultured to establish an in vitro model for investigating the underlying mechanism. RESULTS: High-throughput sequencing analysis revealed the involvement of the MAPK signaling pathway in T-2 toxin-induced articular cartilage damage in rats. Hesperetin intervention in T-2 toxin-exposed rats attenuated pathological cartilage damage. Immunohistochemistry results demonstrated a significant reduction in collagen II protein expression in the high-dose T-2 toxin group (p < 0.01), accompanied by a significant increase in MMP13 protein expression (p < 0.01). In both the articular cartilage and the epiphyseal plate, the T-2 toxin + hesperetin group exhibited significantly higher collagen II protein expression than the high-dose T-2 toxin group (p < 0.05), along with significantly lower MMP13 protein expression (p < 0.05). Hesperetin inhibited the over-activation of the p38/MEF2C signaling axis induced by T-2 toxin in primary rat chondrocytes. Compared to the T-2 toxin group, the T-2 toxin + hesperetin group showed significantly reduced phosphorylation levels of p38 and protein expression levels of MEF2C (p < 0.001 or p < 0.05). Moreover, the T-2 toxin + hesperetin group exhibited a significant decrease in MMP13 protein expression (p < 0.05) and a significant increase in collagen II protein expression (p < 0.01) compared to the T-2 toxin group. CONCLUSIONS: T-2 toxin activates the p38 MAPK signaling pathway, causing knee cartilage damage in rats. Treatment with hesperetin inhibits the p38/MEF2C signaling axis, regulates collagen II and MMP13 protein expression, and reduces cartilage injury significantly.
Assuntos
Cartilagem Articular , Condrócitos , Hesperidina , Toxina T-2 , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Masculino , Ratos , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Modelos Animais de Doenças , Hesperidina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Toxina T-2/toxicidadeRESUMO
OBJECTIVE: Osteoporosis is a common bone disease. miR-26b regulates OA-induced osteogenesis and induces osteoporosis. miR-26b is elevated in bone marrow stromal cells (BMSCs) during bone formation; however, we haven't fully revealed whether it is directly involved in this process, which was the aim of this study. METHODS: An oophorectomized rat model of osteoporosis was used. BMSCs were detected by electron microscopy of exosomes, and mir-26b levels were detected by RT-PCR. The correlation between mir-26b and sirt2 was detected by bioinformatics and luciferase activity analysis. Bone microstructure and cartilage moisture content were also measured. The proliferation ability of mir-26b and sirt2 on chondrocytes was detected by cell viability test and flow cytometry. RESULTS: Western blotting further proved that the surface markers of isolated granular exosomes were positive for CD63 and CD81. Further analysis showed that exosomes' diameters ranged from 50 to 150 nm. Mir-26b is elevated in BMSC, and its mimics can promote proliferation. Luciferase showed that mir-26b targets sirt2 and the effect of elevated mir-26b on chondrocytes was completely reversed by silencing sirt2. The proliferation ability of C28/I2 chondrocytes in Mir MICs group was lower than other two groups, while that in Mir inhibition group had stronger proliferation ability than in the Mir NC group. mir-26b was highly expressed in BMSC, indicating that mir-26b comes from secretion of BMSC. CONCLUSION: Mir-26 is highly expressed in OP. mir-26b can therefore target sirt2 to promote proliferation and inhibit apoptosis of OP chondrocytes. It may offer a possibility of a treatment of OP in the future.
Assuntos
Proliferação de Células , Condrócitos , Células-Tronco Mesenquimais , MicroRNAs , Osteoporose , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Condrócitos/metabolismo , Condrócitos/patologia , Células-Tronco Mesenquimais/metabolismo , Osteoporose/patologia , Osteoporose/genética , Osteoporose/metabolismo , Ratos , Exossomos/metabolismo , Ratos Sprague-Dawley , Feminino , Sirtuína 2/metabolismo , Sirtuína 2/genéticaRESUMO
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.
Assuntos
Cartilagem Articular , Condrócitos , Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Cartilagem Articular/metabolismo , Humanos , Condrócitos/metabolismo , Condrócitos/citologia , Condrogênese/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Proliferação de Células , Transplante de Células-Tronco Mesenquimais/métodos , AnimaisRESUMO
BACKGROUND: Small extracellular vesicles (sEV) derived from synovial fibroblasts (SF) represent a novel molecular mechanism regulating cartilage erosion in osteoarthritis (OA). However, a comprehensive evaluation using disease relevant cells has not been undertaken. The aim of this study was to isolate and characterise sEV from OA SF and to look at their ability to regulate OA chondrocyte effector responses relevant to disease. Profiling of micro (mi) RNA signatures in sEV and parental OA SF cells was performed. METHODS: SF and chondrocytes were isolated from OA synovial membrane and cartilage respectively (n = 9). sEV were isolated from OA SF (± IL-1ß) conditioned media by ultracentrifugation and characterised using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Particle size was confirmed by nanoparticle tracking analysis (NTA). sEV regulation of OA chondrocyte and cartilage effector response was evaluated using qPCR, ELISA and sulphated glycosaminoglycan assay (sGAG). RNA-sequencing was used to establish miRNA signatures in isolated sEV from OA SF. RESULTS: OA SF derived sEV were readily taken up by OA chondrocytes, with increased expression of the catabolic gene MMP 13 (p < 0.01) and decreased expression of the anabolic genes aggrecan and COL2A1 (p < 0.01) observed. Treatment with sEV derived from IL-1ß stimulated OA SF significantly decreased expression of aggrecan and COL2A1 (p < 0.001) and increased SOX 9 gene expression (p < 0.05). OA chondrocytes cultured with sEV from either non-stimulated or IL-1ß treated OA SF, resulted in a significant increase in the secretion of IL-6, IL-8 and MMP-3 (p < 0.01). Cartilage explants cultured with sEV from SF (± IL-1ß) had a significant increase in the release of sGAG (p < 0.01). miRNA signatures differed between parental SF cells and isolated sEV. The recently identified osteoclastogenic regulator miR182, along with miR4472-2, miR1302-3, miR6720, miR6087 and miR4532 were enriched in sEV compared to parental cells, p < 0.01. Signatures were similar in sEVs derived from non-stimulated or IL-1ß stimulated SF. CONCLUSIONS: OA SF sEV regulate chondrocyte inflammatory and remodelling responses. OA SF sEV have unique signatures compared to parental cells which do not alter with IL-1ß stimulation. This study provides insight into a novel regulatory mechanism within the OA joint which could inform future targeted therapy.
Assuntos
Condrócitos , Vesículas Extracelulares , Fibroblastos , MicroRNAs , Osteoartrite , Membrana Sinovial , Humanos , Condrócitos/metabolismo , Condrócitos/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Osteoartrite/metabolismo , Osteoartrite/genética , Osteoartrite/patologia , Células Cultivadas , Idoso , Masculino , Feminino , Pessoa de Meia-IdadeRESUMO
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% ± 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 ± 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.
Assuntos
Condrócitos , Medidas de Resultados Relatados pelo Paciente , Transplante Autólogo , Humanos , Condrócitos/transplante , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Adulto Jovem , Cartilagem Articular/cirurgia , Cartilagem Articular/lesões , Traumatismos do Joelho/cirurgia , Seguimentos , Imageamento por Ressonância Magnética , Adolescente , Resultado do Tratamento , Articulação do Joelho/cirurgiaRESUMO
Chronic ankle pain significantly impairs daily activities and athletic performance with osteochondral lesions of the talus (OLT) in Hepple stages IV and V, which are often causative factors. This study aimed to assess the efficacy and safety of autologous osteochondral transplantation (AOT) for the treatment of these conditions. This retrospective study was conducted from May 2020 to May 2023 at Cangzhou Traditional Chinese and Western Medicine Combined Hospital, including patients with a diagnosis of Hepple stage IV or V OLT confirmed by magnetic resonance imaging (MRI) and arthroscopy. Surgical interventions involved arthroscopic debridement, followed by AOT or limited arthrotomy based on the location and size of the lesion. Preoperative and postoperative evaluations used the Visual Analog Scale, American Orthopedic Foot and Ankle Society Ankle-Hindfoot Scale, MRI-Based Cartilage Repair Tissue Scoring, and the International Knee Documentation Committee Knee Evaluation Form. Statistical analysis was conducted using paired-sample t tests to compare the preoperative and postoperative data. Twenty patients were included, revealing significant postoperative improvements in Visual Analog Scale, American Orthopedic Foot and Ankle Society, and MRI-based cartilage repair tissue scores (Pâ <â .05). The radiographic findings suggested effective cartilage regeneration. No adverse effects were observed in the donor knee sites, as confirmed by the stable pre- and postoperative International Knee Documentation Committee Knee Evaluation Form scores. Recovery of physical abilities was achieved on average within 7.3 weeks for daily activities and 13.4 weeks for sports activities. AOT effectively treats Hepple stage IV-V OLT, improves ankle function, promotes cartilage regrowth, and allows quick resumption of daily and athletic activities without compromising donor-site integrity.
Assuntos
Transplante Ósseo , Condrócitos , Ílio , Transplante Autólogo , Humanos , Estudos Retrospectivos , Feminino , Masculino , Adulto , Transplante Ósseo/métodos , Transplante Autólogo/métodos , Ílio/transplante , Condrócitos/transplante , Periósteo/transplante , Tálus/cirurgia , Pessoa de Meia-Idade , Cartilagem Articular/cirurgia , Artroplastia Subcondral/métodos , Artroscopia/métodos , Imageamento por Ressonância Magnética , Desbridamento/métodos , Resultado do Tratamento , Adulto Jovem , Articulação do Tornozelo/cirurgia , Articulação do Tornozelo/diagnóstico por imagemRESUMO
Introduction: Osteoarthritis is a degenerative condition of the cartilage, often common among the population and occurs frequently with aging. Many factors are decisive for the development of its pathogenesis such as age, obesity, trauma, mechanical load, and modification of synovial biology. The main features of osteoarthritis are chondrocytes and cartilage matrix loss, which lead to pain, loss of function of the whole joint, and disability, representing a relevant health problem. Recently, a new therapeutic approach based on cell therapy has been studying the regenerative ability of mesenchymal stem cells for osteoarthritic chondrocytes. Aim: This in vitro study clarifies the regenerative effects of multipotent adipose-derived stem cells and the pluripotent amniotic epithelial stem cells on arthrosis chondrocytes by performing co-culture experiments. Methods: We studied the regenerative potential of secretome (soluble factors and extracellular vesicles), mesenchymal stem cells, and the adipose stromal vascular fraction. The regenerative effects were evaluated by gene and protein expression analysis of articular cartilage-specific genes and proteins like col2a1, acan, and sox9. Results: Mesenchymal stem cells, secretome, and adipose stromal vascular fractions influenced the cartilage genes and protein expression. Conclusions: The results indicate that the treatment with mesenchymal stem cells could be the best biological approach for cartilage regenerative medicine.