Articular chondrocytes reside in lacunae distributed in cartilage responsible for the remodelling of the tissue with limited ability of damage repairing. The in vitro expanded chondrocytes enhanced ...by factors/agents to obtain large numbers of cells with strengthened phenotype are essential for successful repair of cartilage lesions by clinical cell implantation therapies. Because the salvianolic acid B (Sal B), a major hydrophilic therapeutic agent isolated from Salvia miltiorrhiza, has been widely used to treat diseases and able to stimulate activity of cells, this study examines the effects of Sal B on passaged chondrocytes. Chondrocytes were treated with various concentrations of Sal B in monolayer culture, their morphological properties and changes, and mitochondrial membrane potential were analysed using microscopic analyses, including cellular biochemical staining and confocal laser scanning microscopy. The proteins were quantified by BCA and Western blotting, and the transcription of genes was detected by qRT‐PCR. The passaged chondrocytes treated with Sal B showed strengthened cellular synthesis and stabilized mitochondrial membrane potential with upregulated expression of the marker genes for chondrocyte phenotype, Col2‐α1, Acan and Sox9, the key Wnt signalling molecule β‐catenin and paracrine cytokine Cytl‐1. The treatments using CYTL‐1 protein significantly increased expression of Col2‐α1 and Acan with no effect on Sox9, indicating the paracrine cytokine acts on chondrocytes independent of SOX9. Sal B has ultimately promoted cell growth and enhanced chondrocyte phenotype. The chondrocytes treated with pharmaceutical agent and cytokine in the formulated medium for generating large number of differentiated chondrocytes would facilitate the cell‐based therapies for cartilage repair.
Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines ...may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes.
Primary chondrocytes were treated with IL-1β to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy.
Pretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1β-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1β-induced down-regulation of collagen type II, cartilage specific proteoglycans, β1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1β signaling.
The results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1β-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects.
Autologous chondrocyte implantation (ACI) is a cell therapy for the treatment of focal cartilage defects. The ACI product that is currently approved for use in the European Union (EU) consists of ...spheroids of autologous matrix-associated chondrocytes. These spheroids are spherical aggregates of ex vivo expanded human autologous chondrocytes and their self-synthesized extracellular matrix. The aim is to provide an overview of the preclinical and nonclinical studies that have been performed to ensure reproducible quality, safety, and efficacy of the cell therapy, and to evaluate the clinical data on ACI with spheroids. A systematic review was performed to include all English publications on self-aggregated spheroids of chondrocytes cultured in autologous serum without other supplements. A total of 20 publications were included, 7 pre- and nonclinical and 13 clinical research publications. The pre- and nonclinical research publications describe the development from concept to in vivo efficacy and quality- and safety-related aspects such as biodistribution, tumorigenicity, genetic stability, and potency. The evaluation of clinical research shows short- to mid-term safety and efficacy for the ACI with spheroid-based treatment of cartilage defects in both randomized clinical trials with selected patients, as well as in routine treatment providing real-world data in more complex patients.
Purpose
Traumatic lesions of articular cartilage represent a crucial risk factor for osteoarthritis. Even if several strategies exist to treat such damages, the optimal solution has not yet been ...found. A new strategy represents the scaffold-free spheroid-based autologous chondrocyte transplantation. In this method, spheroids of chondrocytes are synthesized after chondrocyte isolation and expansion, followed by the implantation in a second intervention.
Methods
Fine Jamshidi-needle biopsies from five patients (one from each patient, Ø 2 mm) treated with a spheroid-based autologous chondrocyte implantation (ACI) after traumatic lesions of the articular cartilage of the knee were analysed histologically and immunohistologically for collagen II, collagen X and aggrecan expression. The indication for a second look arthroscopy was given by arthrofibrosis or meniscus-lesions, respectively. The time between ACI and second-look arthroscopy ranged between 6 and 16 months.
Results
In all patients, the histological examinations revealed an avascular cartilage tissue with a homogenic extracellular matrix. The subchondral bone neither showed bleeding, necrosis nor hypertrophy. A homogenous alcian blue staining indicated high amounts of mucopolysaccharides and glycosaminoglycans. Collagen II staining was highly positive, whereas collagen X staining was negative in every patient, ruling out hypertrophic chondrocyte differentiation. In addition, intense aggrecan staining indicated a strong expression of this extracellular matrix component.
Conclusion
The present case series represents the first histological and immunohistological analyses of spheroid-based ACI in humans. Spheroid-based ACI revealed excellent histological results regarding the regeneration of hyaline articular cartilage. These results indicate that spheroid based ACI is a promising strategy for treating traumatic lesions of the articular cartilage of the knee.
Berberine, a plant alkaloid used in Chinese medicine, has broad cell‐protective functions in a variety of cell lines. Chondrocyte apoptosis contributes to the pathogenesis of cartilage degeneration ...in osteoarthritis (OA). However, little is known about the effect and underlying mechanism of berberine on OA chondrocytes. Here, we assessed the effects of berberine on cartilage degeneration in interleukin‐1β (IL‐1β)‐stimulated rat chondrocytes and in a rat model of OA. The results of an MTT assay and western blotting analysis showed that berberine attenuated the inhibitory effect of IL‐1β on the cell viability and proliferating cell nuclear antigen expression in rat chondrocytes. Furthermore, berberine activated Akt, which triggered p70S6K/S6 pathway and up‐regulated the levels of aggrecan and Col II expression in IL‐1β‐stimulated rat chondrocytes. In addition, berberine increased the level of proteoglycans in cartilage matrix and the thickness of articular cartilage, with the elevated levels of Col II, p‐Akt and p‐S6 expression in a rat OA model, as demonstrated by histopathological and immunohistochemistry techniques. The data thus strongly suggest that berberine may ameliorate cartilage degeneration from OA by promoting cell survival and matrix production of chondrocytes, which was partly attributed to the activation of Akt in IL‐1β‐stimulated articular chondrocytes and in a rat OA model. The resultant chondroprotective effects indicate that berberine merits consideration as a therapeutic agent in OA.
Oxidative stress has been implicated in the pathogenesis of osteoarthritis and has become an important therapeutic target. Investigations of various antioxidant supplements, reactive oxidative ...species (ROS) pathway mediators, and free radical scavengers for treating osteoarthritis have demonstrated common disadvantages including poor bioavailability and stability, as well as rapid joint clearance or release profiles from delivery vehicles. Moreover, these therapies do not target cartilage, which irreversibly degenerates in the presence of oxidative stress. The goal of this study was to engineer a nanoparticle system capable of sustained retention in the joint space, localization to cartilage, and mitigation of oxidative stress. Towards this goal, ROS scavenging manganese dioxide nanoparticles with physicochemical properties (less than 20 nm and cationic) that facilitate their uptake into cartilage were developed and characterized. These particles penetrated through the depth of cartilage explants and were found both in the extracellular matrix as well as intracellularly within the resident chondrocytes. Furthermore, the particles demonstrated chondroprotection of cytokine-challenged cartilage explants by reducing the loss of glycosaminoglycans and release of nitric oxide. Quantitative PCR analysis revealed that the particles mitigated impacts of oxidative stress related genes in cytokine-challenged chondrocytes. When injected intra-articularly into rats, the particles persisted in the joint space over one week, with 75% of the initial signal remaining in the joint. Biodistribution and histological analysis revealed accumulation of particles at the chondral surfaces and colocalization of the particles with the lacunae of chondrocytes. The results suggest that the manganese dioxide nanoparticles could be a promising approach for the chondroprotection of osteoarthritic cartilage.
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Osteoarthritis (OA) is a degenerative and chronic joint disease characterized by clinical symptoms and distortion of joint tissues. It primarily damages joint cartilage, causing pain, swelling, and ...stiffness around the joint. It is the major cause of disability and pain. The prevalence of OA is expected to increase gradually with the aging population and increasing prevalence of obesity. Many potential therapeutic advances have been made in recent years due to the improved understanding of the underlying mechanisms, diagnosis, and management of OA. Embryonic stem cells and induced pluripotent stem cells differentiate into chondrocytes or mesenchymal stem cells (MSCs) and can be used as a source of injectable treatments in the OA joint cavity. MSCs are known to be the most studied cell therapy products in cell-based OA therapy owing to their ability to differentiate into chondrocytes and their immunomodulatory properties. They have the potential to improve cartilage recovery and ultimately restore healthy joints. However, despite currently available therapies and advances in research, unfulfilled medical needs persist for OA treatment. In this review, we focused on the contents of non-cellular and cellular therapies for OA, and briefly summarized the results of clinical trials for cell-based OA therapy to lay a solid application basis for clinical research.
Abnormal expression of KDM6A and SOX9 is a key factor in the pathogenesis of osteoarthritis (OA). Cellular treatments of OA with articular cartilage chondrocytes (ACCs) and bone marrow mesenchymal ...stem cells (BMSCs) are promising, but their underlying mechanisms remain to be explored. The pellet size, weight and sulfated glycosaminoglycan/DNA content of ACCs were measured to evaluate the effect of BMSCs on the chondrogenic differentiation of SCCs. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was used to analyze the proliferation of ACCs cultured along or cocultured with BMSCs. Quantitative polymerase chain reaction (qPCR) was performed to evaluate the messenger RNA expression of KDM6A, SOX9, type2 collagen, and Aggrecan in ACCs and OA rats. Western blot and immunohistochemistry were performed to analyze the expression of KDM6A and SOX9 proteins. Bisulfite sequencing PCR was performed to assess the DNA methylation level of the SOX9 promoter. Flow cytometry was used to evaluate the apoptotic status of ACCs. The chondrogenic differentiation of ACCs was significantly enhanced by coculturing with BMSCs, especially under a hypoxic condition. The expression of KDM6A, SOX9, type2 collagen, and Aggrecan was remarkably elevated in ACCs cocultured with BMSCs. Also, the DNA methylation of SOX9 promoter was decreased in ACCs cocultured with BMSCs, along with notably reduced apoptosis. Moreover, ACCs cocultured with BMSCs could repair cartilage lesions and prevent the abnormal expression of KDM6A, SOX9, type2 collagen, and Aggrecan in OA rats. In this study, we cocultured ACCs with BMSCs and used them to treat OA rats. Our findings presented a mechanistic basis for explaining the therapeutic effect of BMSCs on OA treatment.
Our data suggested that hypoxia improved the differentiation and proliferation of bone marrow mesenchymal stem cells (BMSCs) by promoting KDM6A expression and activating the SOX9 signaling pathway. This is a novel mechanism underlying the differentiation of BMSCs that highlights a promising future for osteoarthritis (OA) treatment.
Mechanical loading of joints plays a critical role in maintaining the health and function of articular cartilage. The mechanism(s) of chondrocyte mechanotransduction are not fully understood, but ...could provide important insights into new physical or pharmacologic therapies for joint diseases. Transient receptor potential vanilloid 4 (TRPV4), a Ca ²⁺-permeable osmomechano-TRP channel, is highly expressed in articular chondrocytes, and loss of TRPV4 function is associated with joint arthropathy and osteoarthritis. The goal of this study was to examine the hypothesis that TRPV4 transduces dynamic compressive loading in articular chondrocytes. We first confirmed the presence of physically induced, TRPV4-dependent intracellular Ca ²⁺ signaling in agarose-embedded chondrocytes, and then used this model system to study the role of TRPV4 in regulating the response of chondrocytes to dynamic compression. Inhibition of TRPV4 during dynamic loading prevented acute, mechanically mediated regulation of proanabolic and anticatabolic genes, and furthermore, blocked the loading-induced enhancement of matrix accumulation and mechanical properties. Furthermore, chemical activation of TRPV4 by the agonist GSK1016790A in the absence of mechanical loading similarly enhanced anabolic and suppressed catabolic gene expression, and potently increased matrix biosynthesis and construct mechanical properties. These findings support the hypothesis that TRPV4-mediated Ca ²⁺ signaling plays a central role in the transduction of mechanical signals to support cartilage extracellular matrix maintenance and joint health. Moreover, these insights raise the possibility of therapeutically targeting TRPV4-mediated mechanotransduction for the treatment of diseases such as osteoarthritis, as well as to enhance matrix formation and functional properties of tissue-engineered cartilage as an alternative to bioreactor-based mechanical stimulation.
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