Correct functioning of chondrocytes is crucial for long bone growth and fracture repair. These cells are highly anabolic but survive and function in an avascular environment, implying specific ...metabolic requirements that are, however, poorly characterized. Here, we show that chondrocyte identity and function are closely linked with glutamine metabolism in a feedforward process. The master chondrogenic transcription factor SOX9 stimulates glutamine metabolism by increasing glutamine consumption and levels of glutaminase 1 (GLS1), a rate-controlling enzyme in this pathway. Consecutively, GLS1 action is critical for chondrocyte properties and function via a tripartite mechanism. First, glutamine controls chondrogenic gene expression epigenetically through glutamate dehydrogenase-dependent acetyl-CoA synthesis, necessary for histone acetylation. Second, transaminase-mediated aspartate synthesis supports chondrocyte proliferation and matrix synthesis. Third, glutamine-derived glutathione synthesis avoids harmful reactive oxygen species accumulation and allows chondrocyte survival in the avascular growth plate. Collectively, our study identifies glutamine as a metabolic regulator of cartilage fitness during bone development.
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•The chondrogenic transcription factor SOX9 induces glutamine metabolism•Glutamine-derived acetyl-CoA epigenetically regulates chondrogenic gene expression•Transaminase-driven glutamine-derived aspartate controls chondrocyte biosynthesis•Glutamine-dependent glutathione synthesis maintains chondrocyte redox homeostasis
Growth plate chondrocytes survive and function in an avascular environment, but their nutritional requirements are poorly characterized. Stegen et al. now demonstrate that the key chondrogenic transcription factor SOX9 induces glutamine metabolism, and this metabolic adaptation is in turn necessary to support chondrocyte gene expression, biosynthesis, and redox homeostasis.
Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation ...remains incompletely understood. In this study, using a Tbx18:Cre (Tbx18Cre/+) gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 (Smad4f/f) in the limbs of mice. We found that the Smad4-deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including Col2a1 and Acan, in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy–related markers Col10a1 and Panx3 were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 (Runx2), a critical mediator of chondrocyte hypertrophy, was also down-regulated in Smad4-deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the Runx2 promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating Runx2 expression during skeletal development. The regulatory mechanism involving Smad4-mediated Runx2 activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia.
Degradation of extracellular matrix (ECM) underlies loss of cartilage tissue in osteoarthritis, a common disease for which no effective disease-modifying therapy currently exists. Here we describe ...BNTA, a small molecule with ECM modulatory properties. BNTA promotes generation of ECM components in cultured chondrocytes isolated from individuals with osteoarthritis. In human osteoarthritic cartilage explants, BNTA treatment stimulates expression of ECM components while suppressing inflammatory mediators. Intra-articular injection of BNTA delays the disease progression in a trauma-induced rat model of osteoarthritis. Furthermore, we identify superoxide dismutase 3 (SOD3) as a mediator of BNTA activity. BNTA induces SOD3 expression and superoxide anion elimination in osteoarthritic chondrocyte culture, and ectopic SOD3 expression recapitulates the effect of BNTA on ECM biosynthesis. These observations identify SOD3 as a relevant drug target, and BNTA as a potential therapeutic agent in osteoarthritis.
Electrospun poly(lactic acid) (PLLA) nanofibers (NF) were modified with cationized gelatin (CG) to improve their compatibility with chondrocytes and to show in vitro and in vivo the potential ...applications of CG-grafted PLLA nanofibrous membranes (CG-PLLA NFM) as a cartilage tissue engineering scaffold. PLLA NF were first treated with oxygen plasma to introduce –COOH groups on the surface, followed by covalent grafting of CG molecules onto the fiber surface, using water-soluble carbodiimide as the coupling agent. The effects of CG grafting and properties of NFM were characterized by scanning electron microscopy (SEM), transmission electron microscopy, thermogravimetric analysis, atomic force microscope, X-ray photoelectron spectra and Fourier transform infrared spectroscopy. In vitro studies indicated that CG-PLLA NFM could enhance viability, proliferation and differentiation of rabbit articular chondrocytes compared with pristine PLLA NFM. SEM observations of the cell–scaffold construct confirmed the tight attachment of chondrocytes to CG-PLLA NF and in-growth of cells into the interior of the membrane with proper maintenance of cell morphology. Improved cell differentiation in CG-PLLA NFM was confirmed by enhanced glycoaminoglycan and collagen secretion, histological analysis and reverse transcription-polymerase chain reaction studies, which showed that the cells were able to maintain the expression of characteristic markers (collagen II, aggregan and SOX 9) of chondrocytes. Subcutaneous implantation of the cell–scaffold constructs with autologous chondrocytes also confirmed the formation of ectopic cartilage tissues after 28
days by histological examination and immunostaining.
Abstract Hydrogels prepared from poly(ethylene glycol) (PEG) are widely applied in tissue engineering, especially those derived from a combination of functional multi-arm star PEG and linear ...crosslinker, with an expectation to form a structurally ideal network. However, the poor mechanical strength still renders their further applications. Here we examined the relationship between the dynamics of the pre-gel solution and the mechanical property of the resultant hydrogel in a system consisting of 4-arm star PEG functionalized with vinyl sulfone and short dithiol crosslinker. A method to prepare mechanically strong hydrogel for cartilage tissue engineering is proposed. It is found that when gelation takes place at the overlap concentration, at which a slow relaxation mode just appears in dynamic light scattering (DLS), the resultant hydrogel has a local maximum compressive strength ∼20 MPa, while still keeps ultralow mass concentration and Young's modulus. Chondrocyte-laden hydrogel constructed under this condition was transplanted into the subcutaneous pocket and an osteochondral defect model in SCID mice. The in vivo results show that chondrocytes can proliferate and maintain their phenotypes in the hydrogel, with the production of abundant extracellular matrix (ECM) components, formation of typical chondrocyte lacunae structure and increase in Young's modulus over 12 weeks, as indicated by histological, immunohistochemistry, gene expression analyses and mechanical test. Moreover, newly formed hyaline cartilage was observed to be integrated with the host articular cartilage tissue in the defects injected with chondrocytes/hydrogel constructs. The results suggest that this hydrogel is a promising candidate scaffold for cartilage tissue engineering.
Osteoarthritis (OA) is a chronic disease pathologically characterized by articular cartilage degeneration and damage. Currently, studies have found that circular RNA (circRNA) is involved in ...intracellular RNA regulating network and is closely related to the occurrence and development of diseases, therefore it may become a new biological marker and therapeutic target. After stimulating chondrocytes with interleukin-1 beta (IL-1β), hsa_circ_0005105 expression was significantly upregulated, while miR-26a expression was significantly inhibited. Hsa_circ_0005105 did not influence miR-26a expression but inhibited its transcriptional activity so as to upregulate the expression of its target NAMPT. Studies further indicated that hsa_circ_0005105 can inhibit the expression of type II collagen and aggrecan, promote the expression of MMP-13 and ADAMTS-4, and the generation of PGE2, IL-6, and IL-8, but the linear sequence of hsa_circ_0005105 cannot. MiR-26a has the opposite effect, and hsa_circ_0005105 can antagonize the function of miR-26a. When NAMPT expression was downregulated, the above function of hsa_circ_0005105 was significantly weakened. Therefore, hsa_circ_0005105 can promote extracellular matrix (ECM) degradation by regulating the expression of miR-26a target NAMPT. These findings will provide new targets for treatment and prevention of OA and other orthopedic diseases.
Mitochondrial dysfunction leads to osteoarthritis (OA) and disc degeneration. Hypoxia inducible factor-1α (HIF-1α) mediated mitophagy has a protective role in several diseases. However, the ...underlying mechanism of HIF-1α mediated mitophagy in OA remains largely unknown. This current study was performed to determine the effect of HIF-1α mediated mitophagy on OA. Therefore, X-ray and tissue staining including HE staining, safranin O-fast green (S-O) and Alcian Blue were used to assess imageology and histomorphology differences of mouse knee joint. Transcriptional analysis was used to find the possible targets in osteoarthritis. Western blot analysis, RT-qPCR and immunofluorescence staining were used to detect the changes in gene and protein levels in the vitro experiment. The expression of HIF-1α was increased in human and mouse OA cartilage. HIF-1α knockdown by siRNA further impair the hypoxia-induced mitochondrial dysfunction; In contrast, HIF-1α mediated protective role was reinforced by prolylhydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG). In addition, HIF-1α stabilization could alleviate apoptosis and senescence via mitophagy in chondrocytes under hypoxia condition, which could also ameliorate surgery-induced cartilage degradation in mice OA model. In conclusion, HIF-1α mediated mitophagy could alleviate OA, which may serve as a promising strategy for OA treatment.
Background/Aims: Chronic inflammation contributes to cartilage degeneration during the progression of osteoarthritis (OA). Adipose tissue-derived mesenchymal stem cells (AD-MSC) show great potential ...to treat inflammatory and degradative processes in OA and have demonstrated paracrine effects in chondrocytes. In the present work, we have isolated and characterized the extracellular vesicles from human AD-MSC to investigate their role in the chondroprotective actions of these cells. Methods: AD-MSC were isolated by collagenase treatment from adipose tissue from healthy individuals subjected to abdominal lipectomy surgery. Microvesicles and exosomes were obtained from conditioned medium by filtration and differential centrifugation. Chondrocytes from OA patients were used in primary culture and stimulated with 10 ng/ml interleukin(IL)-1β in the presence or absence of AD-MSC microvesicles, exosomes or conditioned medium. Protein expression was investigated by ELISA and immunofluorescence, transcription factor-DNA binding by ELISA, gene expression by real-time PCR, prostaglandin E2 (PGE2) by radioimmunoassay, and matrix metalloproteinase (MMP) activity and nitric oxide (NO) production by fluorometry. Results: In OA chondrocytes stimulated with IL-1β, microvesicles and exosomes reduced the production of inflammatory mediators tumor necrosis factor-α, IL-6, PGE2 and NO. The downregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 would lead to the decreased PGE2 production while the effect on NO could depend on the reduction of inducible nitric oxide synthase expression. Treatment of OA chondrocytes with extracellular vesicles also decreased the release of MMP activity and MMP-13 expression whereas the production of the anti-inflammatory cytokine IL-10 and the expression of collagen II were significantly enhanced. The reduction of inflammatory and catabolic mediators could be the consequence of a lower activation of nuclear factor-κB and activator protein-1. The upregulation of annexin A1 specially in MV may contribute to the anti-inflammatory and chondroprotective effects of AD-MSC. Conclusions: Our data support the interest of AD-MSC extracellular vesicles to develop new therapeutic approaches in joint conditions.
The aim of this work is to investigate the capability of PRP as an adjuvant therapy to autologous chondrocyte implantation (ACI) in combination with multi-axial load with respect to cartilage ...regeneration. Articular cartilage shows poor repair capacity and therapies for cartilage defects are still lacking. Well-established operative treatments include ACI, and growing evidence shows the beneficial effects of PRP. Platelets contain numerous growth factors, among them transforming growth factor beta (TGF-β). Dynamic mechanical loading is known to be essential for tissue formation, improving extracellular matrix (ECM) production. For our ACI model monolayer expanded human chondrocytes were seeded into polyurethane scaffolds and embedded in fibrin (hChondro), in PRP-Gel (PRP), or in fibrin with platelet lysate (PL), which was added to the media once a week with a concentration of 50 vol%. The groups were either exposed to static conditions or multi-axial forces in a ball-joint bioreactor for 1 h per day over 2 weeks, mimicking ACI under physiological load. The culture medium was collected and analyzed for glycosaminoglycan (GAG), nitrite and transforming growth factor beta 1 (TGF-β1) content. The cell-scaffold constructs were collected for DNA and GAG quantification; the expression of chondrogenic genes, TGF-β and related receptors, as well as inflammatory genes, were analyzed using qPCR. Loading conditions showed superior chondrogenic differentiation (upregulation of COL2A1, ACAN, COMP and PRG4 expression) than static conditions. PRP and PL groups combined with mechanical loading showed upregulation of COL2A1, ACAN and COMP. The highest amount of total TGF-β1 was quantified in the PL group. Latent TGF-β1 was activated in all loaded groups, while the highest amount was found in the PL group. Load increased TGFBR1/TGFBR2 mRNA ratio, with further increases in response to supplements. In general, loading increased nitrite release into the media. However, over time, the media nitrite content was lower in the PL group compared to the control group. Based on these experiments, we conclude that chondrogenic differentiation is strongest when simulated ACI is performed in combination with dynamic mechanical loading and PRP-gel or PL supplementation. An inflammatory reaction was reduced by PRP and PL, which could be one of the major therapeutic effects. Loading presumably can enhance the action of TGF-β1, which was predominantly activated in loaded PL groups. The combination of load and PRP represents an effective and promising synergy concerning chondrocyte-based cartilage repair.
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Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage degradation but also synovial membrane inflammation, osteophyte formation and subchondral ...bone sclerosis. Medical care is mainly based on alleviating pain symptoms, but to date, no effective drug can stop the disease progression. Cartilage is a tissue composed of only one cell type, chondrocytes, wrapped in a collagen rich extracellular matrix they synthesize. Chondrocytes can adopt different phenotypes in vivo and in vitro, defined by the collagen type they produce. Isolated from their matrix, chondrocytes present the particularity to dedifferentiate, producing fibroblastic type I and III collagens. With OA onset, chondrocytes undergo multiple changes, in terms of proliferation, viability, but also secretory profile. The acquisition of a hypertrophic phenotype (producing aberrant type X collagen and catabolic MMP-13 protease) by chondrocytes is well documented and contributes to OA development. However, it is increasingly believed that chondrocytes rather acquire a variety of degenerated phenotypes at the onset of OA, including a “dedifferentiated-like” phenotype that might also contribute to OA progression. In this review, we will (i) present molecular knowledge underlying dedifferentiation process, (ii) emphasize connections between dedifferentiation and OA and (iii) consider OA therapeutic strategies aiming at the maintenance of chondrogenic phenotype.
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