Chondrocyte Mineralization Research Articles

Association between serum uric acid concentration and radiographic axial spondylarthritis: a cross-sectional study of 202 patients.

Association between serum uric acid concentration and radiographic axial spondylarthritis: a cross-sectional study of 202 patients.

Iron overload promotes intervertebral disc degeneration via inducing oxidative stress and ferroptosis in endplate chondrocytes

Iron overload is a common phenomenon in the elderly population. Many clinical studies have indicated an association between iron overload and the incidence and pathological progression of intervertebral disc degeneration (IVDD). However, the role and underlying mechanism by which iron participates in the progression of IVDD has not yet been reported. In the present study, we aimed to elucidate the connection between iron overload and IVDD, and explore the underlying mechanisms of disease. Firstly, a clinical epidemiology study was conducted and revealed that iron overload is an independent risk factor for human IVDD. To elucidate the role of iron overload in IVDD, an iron overload mouse model was established, and we observed that iron overload promoted IVDD and cartilage endplate degeneration in a dose dependent manner. Endplate chondrocytes were further isolated and treated with FAC to mimic iron overload in vitro. Excess iron significantly promoted mineralization of endplate chondrocytes in addition to their degeneration via oxidative stress. Moreover, a high dose of excess iron promoted chondrocytes ferroptosis. An iron chelator (DFO), an antioxidant (NAC) and a ferroptosis inhibitor (Fer-1) demonstrated effective inhibition of endplate chondrocyte degeneration induced by iron overload, and our in vivo studies further demonstrated that DFO, NAC and Fer-1 could rescue high dose iron-induced IVDD and cartilage endplate calcification. In conclusion, our results indicate that iron overload is strongly associated with the onset and development of IVDD via oxidative stress and ferroptosis. Inhibiting oxidative stress or ferroptosis could therefore be promising therapeutic strategies for IVDD induced by iron overload.

Fibrin deposition associates with cartilage degeneration in arthritis

SummaryBackgroundCartilage damage in inflammatory arthritis is attributed to inflammatory cytokines and pannus infiltration. Activation of the coagulation system is a well known feature of arthritis, especially in rheumatoid arthritis (RA). Here we describe mechanisms by which fibrin directly mediates cartilage degeneration.MethodsFibrin deposits were stained on cartilage and synovial tissue of RA and osteoarthritis (OA) patients and in murine adjuvant-induced arthritis (AIA) in wild-type or fibrinogen deficient mice. Fibrinogen expression and procoagulant activity in chondrocytes were evaluated using qRT-PCR analysis and turbidimetry. Chondro-synovial adhesion was studied in co-cultures of human RA cartilage and synoviocytes, and in the AIA model. Calcific deposits were stained in human RA and OA cartilage and in vitro in fibrinogen-stimulated chondrocytes.FindingsFibrin deposits on cartilage correlated with the severity of cartilage damage in human RA explants and in AIA in wild-type mice, whilst fibrinogen deficient mice were protected. Fibrin upregulated Adamts5 and Mmp13 in chondrocytes. Chondro-synovial adhesion only occurred in fibrin-rich cartilage areas and correlated with cartilage damage. In vitro, autologous human synoviocytes, cultured on RA cartilage explants, adhered exclusively to fibrin-rich areas. Fibrin co-localized with calcification in human RA cartilage and triggered chondrocyte mineralization by inducing pro-calcification genes (Anx5, Pit1, Pc1) and the IL-6 cytokine. Similar fibrin-mediated mechanisms were observed in OA models, but to a lesser extent and without pseudo-membranes formation.InterpretationIn arthritis, fibrin plaques directly impair cartilage integrity via a triad of catabolism, adhesion, and calcification.FundingNone.

Nanohydroxyapatite Hydrogel Can Promote the Proliferation and Migration of Chondrocytes and Better Repair Talar Articular Cartilage.

As an important load-bearing part of the body, joints are prone to articular cartilage degradation during exercise, resulting in joint pain, swelling, and deformity, which has an adverse impact on patients' life quality and social medical security. Therefore, this study aims to test an effective biopolymer scaffold in promoting the growth of chondrocytes in talus. Hydrogel (Gel)-nanohydroxyapatite (nHA) was invented as a new type of biopolymer scaffold for osteoarthritis treatment in this research. To detect the effects of Gel-nHA on guidance, cartilage matrix secretion, mineralization, proliferation, and migration of chondrocyte, we cultured chondrocytes to study the biological properties of nHA. It was found that Gel could guide chondrocytes to permeate and migrate, so it could be used as acellular matrix scaffolds for chondrocyte regeneration. In addition, nHA could stimulate chondrocytes to secrete cartilage matrix, such as type II collagen and mucopolysaccharide (GAGs). At the same time, nHA help to induce chondrocyte mineralization and stimulate the secretion of type X collagen, so as to better maintain the integrity of bone cartilage interface. In Gel-nHA, chondrocyte viability could be better maintained, and the proliferation and migration of chondrocytes could be better promoted, so as to better repair the articular cartilage of talus. Therefore, the Gel-nHA scaffold is expected to become an effective method for repairing talus cartilage in the future.

Downregulation of miR-1-3p expression inhibits the hypertrophy and mineralization of chondrocytes in DDH

BackgroundDevelopmental dysplasia of the hip (DDH) is a highly prevalent hip disease among children. However, its pathogenesis remains unclear. MicroRNAs (miRNA) are important regulators of cartilage development. In a previous study, high-throughput miRNA sequencing of tissue samples from an animal model of DDH showed a low level of miR-1-3p in the cartilage of the acetabular roof (ARC), but its role in DDH pathogenesis was not addressed. Therefore, our aim here was to investigate the effects of miR-1-3p in the ARC.MethodsThe diagnosis of acetabular dysplasia was confirmed with X-ray examination, while imaging and HE staining were conducted to further evaluate the ARC thickness in each animal model. FISH was employed to verify miR-1-3p expression in the ARC and chondrocytes. The miR-1-3p target genes were predicted by a bioinformatics database. A dual-luciferase reporter assay was used to confirm the targeting relationship between miR-1-3p and SOX9. The gene expression of miR-1-3p, SOX9, RUNX2 and collagen type X was evaluated by qPCR analysis. The protein expression of SOX9, RUNX2 and collagen type X was detected by western blot analysis. The levels of SOX9, RUNX2, and collagen type X in the ARC were further assessed via immunohistochemistry analysis. Finally, Alizarin Red S staining was used to observe the mineralized nodules produced by the chondrocytes.ResultsWe observed low expression of miR-1-3p in the ARC of animals with DDH. SOX9 is a miR-1-3p target gene. Using miR-1-3p silencing technology in vitro, we demonstrated significantly reduced chondrocyte-generated mineralized nodules compared to those of the control. We also confirmed that with miR-1-3p silencing, SOX9 expression was upregulated, whereas the expression of genes associated with endochondral osteogenesis such as RUNX2 and collagen type X was downregulated. To confirm the involvement of miR-1-3p silencing in abnormal ossification through SOX9, we also performed a rescue experiment in which SOX9 silencing restored the low expression of RUNX2 and collagen type X produced by downregulated miR-1-3p expression. Finally, the elevated SOX9 levels and reduced RUNX2 and collagen type X levels in the ARC of rabbits with DDH were also verified using immunohistochemistry, RT-PCR, and western blots.ConclusionThe relatively low expression of miR-1-3p in the ARC may be the cause of abnormal endochondral ossification in the acetabular roof of animals with DDH.

POS0393 FIBRIN DEPOSITION IS AN ACTIVE TRIGGER OF CARTILAGE DEGENERATION IN RHEUMATOID ARTHRITIS

Background:Fibrin(ogen) maintains inflammation in various disorders but has never been linked to cartilage damage in rheumatoid arthritis (RA) or other forms of inflammatory arthritis.Objectives:To investigate the role of fibrin deposition on cartilage integrity in arthritis.Methods:Fibrin deposition on knee cartilage was analyzed by immunohistochemistry in RA patients and in murine adjuvant-induced arthritis (AIA). In chondrocytes, fibrinogen expression (Fgα, Fgβ, Fgγ) and procoagulant activity were evaluated by qRT-PCR and turbidimetry respectively. Fibrin-induced catabolic genes were assessed by qRT-PCR in chondrocytes. Fibrin-mediated chondro-synovial adhesion (CSA) with subsequent cartilage tears was studied in co-cultures of human RA cartilage with autologous synoviocytes, in the AIA model, and by MRI. The link between fibrin and calcification was examined in human RA cartilage stained for calcific deposits and in vitro in fibrinogen-stimulated chondrocytes.Results:Fibrin deposition on cartilage correlated with the severity of cartilage damage in human RA explants and in AIA wildtype (WT) mice, while fibrinogen deficient (Fg-/-) mice were protected. Accordingly, fibrin upregulated catabolic enzymes (Adamts5 and Mmp13) in chondrocytes. Secondly, CSA was present in fibrin-rich and damaged cartilage in AIA WT but not in Fg-/- mice. In line, autologous human synoviocytes, cultured on RA cartilage explants, adhered exclusively to fibrin-positive degraded areas. Gadolinium-enhanced MRI of human joints showed contrast-enhancement along cartilage surface in RA patients but not in controls. Finally, fibrin co-localized with calcification in human RA cartilage and triggered chondrocyte mineralization inducing pro-calcification genes (Anx5, Pit1, Pc1) and cytokine (IL-6). Although at a much lesser extent, we observed similar fibrin-mediated mechanisms in osteoarthritis (OA).Conclusion:Fibrin deposition directly impacts on cartilage integrity via induction of catabolism, mechanical stress, and calcification. Potentially, fibrin is a key factor of cartilage damage occurring in RA as a secondary consequence of inflammation.Disclosure of Interests:None declared

Xanthine Oxidoreductase Is Involved in Chondrocyte Mineralization and Expressed in Osteoarthritic Damaged Cartilage.

Pathologic calcification of cartilage consists of the formation of basic calcium phosphate (BCP) and/or calcium pyrophosphate dihydrate (CPPD) containing calcium crystals in mature hyaline or articular cartilage and is associated with aging, cartilage injury and likely plays a role in accelerating the pathology of osteoarthritis (OA). The pathways regulating joint calcification, in particular cartilage calcification, are not completely understood, but inflammation and the formation of reactive oxygen species (ROS) are contributory factors. The xanthine oxidase (XO) form of xanthine oxidoreductase (XOR), the key enzyme in xanthine and uric acid metabolism, is a major cellular source of superoxide. We hypothesized that XOR could be implicated in chondrocyte mineralization and cartilage calcification and degradation in OA. We showed both in murine primary chondrocyte and chondrogenic ATDC5 cells, that mineralization was inhibited by two different XOR inhibitors, febuxostat and allopurinol. In addition, XOR inhibition reduced the expression of the pro-mineralizing cytokine interleukin-6 (IL-6). We next generated XOR knock-out chondrocyte cell lines with undetectable XOR expression and XO activity. XOR knock-out chondrocyte cells showed decreased mineralization and reduced alkaline phosphatase (Alp) activity. To assess the precise form of XOR involved, primary chondrocytes of XOR mutant mice expressing either the XDH form (XDH ki) or the XO form (XO ki) were studied. We found that XO ki chondrocytes exhibited increased mineralization compared to XDH ki chondrocytes, and this was associated with enhanced Alp activity, ROS generation and IL-6 secretion. Finally, we found increased XOR expression in damaged vs. undamaged cartilage obtained from OA patients and XOR expression partially co-localized with areas showing pathologic calcification. Altogether, our results suggest that XOR, via its XO form, contribute to chondrocyte mineralization and pathological calcification in OA cartilage.

CD11b Signaling Prevents Chondrocyte Mineralization and Attenuates the Severity of Osteoarthritis.

Osteoarthritis (OA) is a progressive joint disease that is strongly associated with calcium-containing crystal formation (mineralization) by chondrocytes leading ultimately to cartilage calcification. However, this calcification process is poorly understood and treatments targeting the underlying disease mechanisms are lacking. The CD11b/CD18 integrin (Mac-1 or αMβ2), a member of the beta 2 integrin family of adhesion receptors, is critically involved in the development of several inflammatory diseases, including rheumatoid arthritis and systemic lupus erythematosus. We found that in a collagen-induced arthritis, CD11b-deficient mice exhibited increased cartilage degradation compared to WT control animals. However, the functional significance of CD11b integrin signaling in the pathophysiology of chondrocytes remains unknown. CD11b expression was found in the extracellular matrix and in chondrocytes in both healthy and damaged human and murine articular cartilage. Primary murine CD11b KO chondrocytes showed increased mineralization when induced in vitro by secondary calciprotein particles (CPP) and quantified by Alizarin Red staining. This increased propensity to mineralize was associated with an increased alkaline phosphatase (Alp) expression (measured by qRT-PCR and activity assay) and an enhanced secretion of the pro-mineralizing IL-6 cytokine compared to control wild-type cells (measured by ELISA). Accordingly, addition of an anti-IL-6 receptor antibody to CD11b KO chondrocytes reduced significantly the calcification and identified IL-6 as a pro-mineralizing factor in these cells. In the same conditions, the ratio of qRT-PCR expression of collagen X over collagen II, and that of Runx2 over Sox9 (both ratio being indexes of chondrocyte hypertrophy) were increased in CD11b-deficient cells. Conversely, the CD11b activator LA1 reduced chondrocyte mineralization, Alp expression, IL-6 production and collagen X expression. In the meniscectomy (MNX) model of murine knee osteoarthritis, deficiency of CD11b led to more severe OA (OARSI scoring of medial cartilage damage in CD11b: 5.6 ± 1.8, in WT: 1.2 ± 0.5, p < 0.05, inflammation in CD11b: 2.8 ± 0.2, in WT: 1.4 ± 0.5). In conclusion, these data demonstrate that CD11b signaling prevents chondrocyte hypertrophy and chondrocyte mineralization in vitro and has a protective role in models of OA in vivo.

Matrix vesicle biomimetics harboring Annexin A5 and alkaline phosphatase bind to the native collagen matrix produced by mineralizing vascular smooth muscle cells

BackgoundVascular smooth muscle cells (VSMCs) transdifferentiated ectopically trigger vascular calcifications, contributing to clinical cardiovascular disease in the aging population. AnxA5 and TNAP play a crucial role in (patho)physiological mineralization. MethodsWe performed affinity studies between DPPC and 9:1 DPPC:DPPS-proteoliposomes carrying AnxA5 and/or TNAP and different types of collagen matrix: type I, II, I + III and native collagenous extracellular matrix (ECM) produced from VSMCs with or without differentiation, to simulate ectopic calcification conditions. ResultsAnxA5-proteoliposomes had the highest affinity for collagens, specially for type II. TNAP-proteoliposomes bound poorly and the simultaneous presence of TNAP in the AnxA5-proteoliposomes disturbed interactions between AnxA5 and collagen. DPPC AnxA5-proteoliposomes affinities for ECM from transdifferentiating cells went up 2-fold compared to that from native VSMCs. The affinities of DPPC:DPPS-proteoliposomes were high for ECM from VSMCs with or without differentiation, underscoring a synergistic effect between AnxA5 and DPPS. Co-localization studies uncovered binding of proteoliposomes harboring AnxA5 or TNAP+AnxA5 to various regions of the ECM, not limited to type II collagen. ConclusionAnxA5-proteoliposomes showed the highest affinities for type II collagen, deposited during chondrocyte mineralization in joint cartilage. TNAP in the lipid/protein microenvironment disturbs interactions between AnxA5 and collagen. These findings support the hypothesis that TNAP is cleaved from the MVs membrane just before ECM binding, such facilitating MV anchoring to ECM via AnxA5 interaction. General significanceProteoliposomes as MV biomimetics are useful in the understanding of mechanisms that regulate the mineralization process and may be essential for the development of novel therapeutic strategies to prevent or inhibit ectopic mineralization.

The protective role of the 3-mercaptopyruvate sulfurtransferase (3-MST)-hydrogen sulfide (H2S) pathway against experimental osteoarthritis

BackgroundOsteoarthritis (OA) is characterized by the formation and deposition of calcium-containing crystals in joint tissues, but the underlying mechanisms are poorly understood. The gasotransmitter hydrogen sulfide (H2S) has been implicated in mineralization but has never been studied in OA. Here, we investigated the role of the H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) in cartilage calcification and OA development.Methods3-MST expression was analyzed in cartilage from patients with different OA degrees, and in cartilage stimulated with hydroxyapatite (HA) crystals. The modulation of 3-MST expression in vivo was studied in the meniscectomy (MNX) model of murine OA, by comparing sham-operated to MNX knee cartilage. The role of 3-MST was investigated by quantifying joint calcification and cartilage degradation in WT and 3-MST−/− meniscectomized knees. Chondrocyte mineralization in vitro was measured in WT and 3-MST−/− cells. Finally, the effect of oxidative stress on 3-MST expression and chondrocyte mineralization was investigated.Results3-MST expression in human cartilage negatively correlated with calcification and OA severity, and diminished upon HA stimulation. In accordance, cartilage from menisectomized OA knees revealed decreased 3-MST if compared to sham-operated healthy knees. Moreover, 3-MST−/− mice showed exacerbated joint calcification and OA severity if compared to WT mice. In vitro, genetic or pharmacologic inhibition of 3-MST in chondrocytes resulted in enhanced mineralization and IL-6 secretion. Finally, oxidative stress decreased 3-MST expression and increased chondrocyte mineralization, maybe via induction of pro-mineralizing genes.Conclusion3-MST-generated H2S protects against joint calcification and experimental OA. Enhancing H2S production in chondrocytes may represent a potential disease modifier to treat OA.

Latexin expression correlated with mineralization of articular cartilage during progression of post-traumatic osteoarthritis in a rat model.

As latexin has been linked with chondrocyte hypertrophic differentiation it is possible that this protein may also be involved in the mineralization of cartilage in OA. Therefore, we correlated latexin expression with the mineralization marker, alkaline phosphatase and determined the mineral deposition in the articular cartilage by analyzing the Ca/P ratio and the collagen fibrils pattern, during the progression of post-traumatic OA in a rat model. OA was induced by medial meniscectomy and post-surgery exercise for 5, 10, 20 and 45 days. Protein expression in articular cartilage was evaluated by immunofluorescence, histochemistry and Western blot. Minerals and structure of collagen fibrils in the superficial zone of cartilage were analyzed by energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM) respectively. Protein expression analysis showed time-dependent up-regulation of latexin during OA progression. In the cartilage, latexin expression correlated with the expression and activity of alkaline phosphatase. EDX of the superficial zone of cartilage showed a Ca/P ratio closer to theoretical values for basic calcium phosphate minerals. The presence of minerals was also analyzed indirectly with AFM, as the collagen fibril pattern was less evident in the mineralized tissue. Latexin is expressed in articular cartilage from the early stages of post-traumatic OA; however, minerals were detected after latexin expression was up-regulated, indicating that its activity precedes and remains during the pathological mineralization of cartilage. Thus, our results contribute to the identification of molecules involved in the mineralization of articular chondrocytes.

Apoptotic bodies from endplate chondrocytes enhance the oxidative stress-induced mineralization by regulating PPi metabolism.

This study aimed to investigate the role of apoptotic bodies (Abs) from the oxidative stressed endplate chondrocytes in regulating mineralization and potential mechanisms. Endplate chondrocytes were isolated from rats and treated with H2O2 to induce oxidative stress. The calcium deposition for matrix mineralization in the cells was examined by histological staining. The expression levels of calcification‐related genes in individual groups of cells were determined by quantitative real time‐PCR (qRT‐PCR). Subsequently, extracellular vesicles (EVs) were purified and characterized. The effect of treatment with H2O2 and/or Abs on the mineralization, extracellular PPi metabolism and related gene expression were determined. Oxidative stress significantly increased the mineralization and promoted the generation of main Abs from endplate chondrocytes. Abs were effectively endocytosed by endplate chondrocytes and co‐localized with collagen (COL)‐II in the cytoplasm, which enhanced the mineralization, alkaline phosphatase (ALP), osteocalcin (OCN), Runt‐related transcription factor 2 (RUNX2) and COL‐I expression in endplate chondrocytes. Furthermore, treatment either H2O2 or Abs significantly decreased PPi, but increased Pi production and treatment with both further enhancing the changes in endplate chondrocytes. Similarly, treatment either H2O2 or Abs significantly decreased the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and ankylosis protein (ANK) expression and ENPP1 promoter activity, but increased the tissue‐nonspecific alkaline phosphatase (TNAP) expression and TNAP promoter activity in endplate chondrocytes. Oxidative stress promoted the generation of Abs, which might enhance the oxidative stress‐mediated mineralization in endplate chondrocytes by regulating the PPi metabolism.

In vitro Chondrocyte Responses in Mg-doped Wollastonite/Hydrogel Composite Scaffolds for Osteochondral Interface Regeneration

The zone of calcified cartilage (ZCC) is the mineralized region between the hyaline cartilage and subchondral bone and is critical in cartilage repair. A new non-stoichiometric calcium silicate (10% Ca substituted by Mg; CSi-Mg10) has been demonstrated to be highly bioactive in an osteogenic environment in vivo. This study is aimed to systematically evaluate the potential to regenerate osteochondral interface with different amount of Ca-Mg silicate in hydrogel-based scaffolds, and to compare with the scaffolds containing conventional Ca-phosphate biomaterials. Hydrogel-based porous scaffolds combined with 0–6% CSi-Mg10, 6% β-tricalcium phosphate (β-TCP) or 6% nanohydroxyapatite (nHAp) were made with three-dimensional (3D) printing. An increase in CSi-Mg10 content is desirable for promoting the hypertrophy and mineralization of chondrocytes, as well as cell proliferation and matrix deposition. Osteogenic and chondrogenic induction were both up-regulated in a dose-dependent manner. In comparison with the scaffolds containing 6% β-TCP or nHAp, human deep zone chondrocytes (hDZCs) seeded on CSi-Mg10 scaffold of equivalent concentration exhibited higher mineralization. It is noteworthy that the hDZCs in the 6% CSi-Mg10 scaffolds maintained a higher expression of the calcified cartilage zone specific extracellular matrix marker and hypertrophic marker, collagen type X. Immunohistochemical and Alizarin Red staining reconfirmed these findings. The study demonstrated that hydrogel-based hybrid scaffolds containing 6% CSi-Mg10 are particularly desirable for inducing the formation of calcified cartilage.

Phospholipase D: A new mediator during high phosphate-induced vascular calcification associated with chronic kidney disease.

Vascular calcification (VC) is the pathological accumulation of calcium phosphate crystals in one of the layers of blood vessels, leading to loss of elasticity and causing severe calcification in vessels. Medial calcification is mostly seen in patients with chronic kidney disease (CKD) and diabetes. Identification of key enzymes and their actions during calcification will contribute to understand the onset of pathological calcification. Phospholipase D (PLD1, PLD2) is active at the earlier steps of mineralization in osteoblasts and chondrocytes. In this study, we aimed to determine their effects during high-phosphate treatment in mouse vascular smooth muscle cell line MOVAS, in the ex vivo model of the rat aorta, and in the in vivo model of adenine-induced CKD. We observed an early increase in PLD1 gene and protein expression along with the increase in the PLD activity in vascular muscle cell line, during calcification induced by ascorbic acid and β-glycerophosphate. Inhibition of PLD1 by the selective inhibitor VU0155069, or the pan-PLD inhibitor, halopemide, prevented calcification. The mechanism of PLD activation is likely to be protein kinase C (PKC)-independent since bisindolylmaleimide X hydrochloride, a pan-PKC inhibitor, did not affect the PLD activity. In agreement, we found an increase in Pld1 gene expression and PLD activity in aortic explant cultures treated with high phosphate, whereas PLD inhibition by halopemide decreased calcification. Finally, an increase in both Pld1 and Pld2 expression occurred simultaneously with the appearance of VC in a rat model of CKD. Thus, PLD, especially PLD1, promotes VC in the context of CKD and could be an important target for preventing onset or progression of VC.

Supplemented Chondroitin Sulfate and Hyaluronic Acid Suppress Mineralization of the Chondrogenic Cell Line, ATDC5, via Direct Inhibition of Alkaline Phosphatase.

Chondroitin sulfate (CS) is a sulfated polysaccharide produced by chondrocytes. Alkaline phosphatase (ALP) is an important enzyme involved in the mineralization of chondrocytes. In recent years, it has been reported that CS regulates the differentiation of various cells. In this study, we investigated the effect of supplemented CS on ALP activity and mineralization of the chondrogenic cell line, ATDC5. In addition, hyaluronic acid (HA), a non-sulfated and acidic polysaccharide, was used in comparison to CS. CS and HA significantly suppressed ALP activity without affecting ATDC5 cell proliferation. In addition, although the inhibition of ALP activity was observed at every time point, Alp mRNA expression level was not affected by CS. The suppressive effect of CS on ALP activity was abrogated by pre-treatment with chondroitinase ABC (CSase). CS and L-homoarginine (hArg), an inhibitor of ALP, significantly suppressed mineralization in ATDC5 cells. In conclusion, supplemented CS directly inhibits ALP to prevent the progression of chondrocytes from differentiation to mineralization.

Sodium Thiosulfate Prevents Chondrocyte Mineralization and Reduces the Severity of Murine Osteoarthritis.

ObjectivesCalcium-containing crystals participate in the pathogenesis of OA. Sodium thiosulfate (STS) has been shown to be an effective treatment in calcification disorders such as calciphylaxis and vascular calcification. This study investigated the effects and mechanisms of action of STS in a murine model of OA and in chondrocyte calcification.MethodsHydroxyapatite (HA) crystals-stimulated murine chondrocytes and macrophages were treated with STS. Mineralization and cellular production of IL-6, MCP-1 and reactive oxygen species (ROS) were assayed. STS's effects on genes involved in calcification, inflammation and cartilage matrix degradation were studied by RT-PCR. STS was administered in the menisectomy model of murine OA, and the effect on periarticular calcific deposits and cartilage degeneration was investigated by micro-CT-scan and histology.ResultsIn vitro, STS prevented in a dose-dependent manner calcium crystal deposition in chondrocytes and inhibited Annexin V gene expression. In addition, there was a reduction in crystal-induced IL-6 and MCP-1 production. STS also had an antioxidant effect, diminished HA-induced ROS generation and abrogated HA-induced catabolic responses in chondrocytes. In vivo, administration of STS reduced the histological severity of OA, by limiting the size of new periarticular calcific deposits and reducing the severity of cartilage damage.ConclusionsSTS reduces the severity of periarticular calcification and cartilage damage in an animal model of OA via its effects on chondrocyte mineralization and its attenuation of crystal-induced inflammation as well as catabolic enzymes and ROS generation. Our study suggests that STS may be a disease-modifying drug in crystal-associated OA.

The functional co-operativity of tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 during initiation of skeletal mineralization.

Phosphatases are recognized to have important functions in the initiation of skeletal mineralization. Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are indispensable for bone and cartilage mineralization but their functional relationship in the mineralization process remains unclear. In this study, we have used osteoblast and ex-vivo metatarsal cultures to obtain biochemical evidence for co-operativity and cross-talk between PHOSPHO1 and TNAP in the initiation of mineralization. Clones 14 and 24 of the MC3T3-E1 cell line were used in the initial studies. Clone 14 cells expressed high levels of PHOSPHO1 and low levels of TNAP and in the presence of β-glycerol phosphate (βGP) or phosphocholine (P-Cho) as substrates and they mineralized their matrix strongly. In contrast clone 24 cells expressed high levels of TNAP and low levels of PHOSPHO1 and mineralized their matrix poorly. Lentiviral Phospho1 overexpression in clone 24 cells resulted in higher PHOSPHO1 and TNAP protein expression and increased levels of matrix mineralization. To uncouple the roles of PHOSPHO1 and TNAP in promoting matrix mineralization we used PHOSPHO1 (MLS-0263839) and TNAP (MLS-0038949) specific inhibitors, which individually reduced mineralization levels of Phospho1 overexpressing C24 cells, whereas the simultaneous addition of both inhibitors essentially abolished matrix mineralization (85%; P<0.001). Using metatarsals from E15 mice as a physiological ex vivo model of mineralization, the response to both TNAP and PHOSPHO1 inhibitors appeared to be substrate dependent. Nevertheless, in the presence of βGP, mineralization was reduced by the TNAP inhibitor alone and almost completely eliminated by the co-incubation of both inhibitors. These data suggest critical non-redundant roles for PHOSPHO1 and TNAP during the initiation of osteoblast and chondrocyte mineralization.

Wnt5a is expressed in spondyloarthritis and exerts opposite effects on enthesis and bone in murine organ and cell cultures

Spondyloarthritis (SpA) is a chronic inflammatory joint disorder that initiates at the enthesis, where tendons attach to bone through a fibrocartilage zone. At late stages, excessive bone apposition appears within the diseased enthesis. Because Wnt5a participates to normal bone formation and appears related to inflammatory processes, we investigated the role of this Wnt growth factor in inflammation-associated ossification in SpA. The concentration of Wnt5a assessed by enzyme-linked immunosorbent assay in synovial fluids of patients with SpA (2.58 ± 0.98 ng/mL) was higher than in osteoarthritic patients (1.33 ± 0.71 ng/mL). In murine primary cultures of tendon cells, chondrocytes, and osteoblasts and in an organotypic model of mouse ankle, we showed that tumor necrosis factor α reversibly diminished Wnt5a expression and secretion, respectively. Wnt5a decreased gene expression of differentiation markers and mineralization in cultured chondrocytes and reduced alkaline phosphatase activity in Achilles tendon enthesis (-14%) and osteocalcin protein levels released by ankle explants (-36%). On the contrary, Wnt5a stimulated ossification markers' expression in cultured osteoblasts and increased the bone volume of the tibial plateau of the cultured explants (+19%). In conclusion, our results suggest that Wnt5a is expressed locally in the joints of patients with SpA. Wnt5a appears more associated with ossification than with inflammation and tends to inhibit mineralization in chondrocytes and enthesis, whereas it seems to favor the ossification process in osteoblasts and bone. Further studies are needed to decipher the opposing effects observed locally in enthesis and systemically in bone in SpA.

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways

The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/β-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active β-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced β-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the β-catenin pathway.

1,25-Dihydroxyvitamin D3 and extracellular inorganic phosphate activate mitogen-activated protein kinase pathway through fibroblast growth factor 23 contributing to hypertrophy and mineralization in osteoarthritic chondrocytes

Hypertrophy and impaired mineralization are two processes closely associated with osteoarthritis (OA). 1,25-dihydroxyvitamin D(3) (1a,25(OH)(2)D(3)) and inorganic phosphate (Pi) are two important factors that are implicated in calcium and phosphate homeostasis of bone metabolism and both can be regulated by the circulating phosphaturic factor fibroblast growth factor 23 (FGF23). The objective of this study was to investigate the role of 1a,25(OH)(2)D(3) and Pi and the molecular mechanism through which they contribute to hypertrophy and mineralization in human osteoarthritic chondrocytes. For this purpose, primary human chondrocytes were obtained from articular cartilage which was collected after total knee replacement surgery in OA patients. FGF23, fibroblast growth factor receptor 1c (FGFR1c), vitamin D(3) receptor (VDR), and phosphate inorganic transporter-1 and -2 (PiT-1 and PiT-2) expression levels were evaluated and found to be significantly higher in OA chondrocytes compared with normal. In addition, we observed that the binding of FGF23 to FGFR1c was stronger in OA chondrocytes compared with normal. Chromatin immunoprecipitation (ChIP) assay revealed, for the first time, the presence of two vitamin D response elements (VDREs) in the FGF23 promoter. Treatment of normal chondrocytes with 1a,25(OH)(2)D(3) or Pi resulted in significant up-regulation of VDR, FGF23, PiT-1, PiT-2 mRNA and protein levels, extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation and induction of hypertrophy markers collagen type X (COL10A1), osteopontin (OPN), osteocalcin (OC), catabolic markers metalloproteinase-13 (MMP-13) and the apoptotic marker caspase-9. Furthermore, VDR silencing in OA chondrocytes negatively regulated FGF23, COL10A1, OPN, OC, MMP-13 and caspase-9 expressions and ERK1/2 phosphorylation. Finally, combined VDR silencing and PiT-1, PiT-2 inhibition in OA chondrocytes resulted in additive down-regulation of FGF23 expression, ERK1/2 activation and COL10A1, OPN, OC, MMP-13 and caspase-9 expression levels. We propose that 1a,25(OH)(2)D(3) and Pi act synergistically through FGF23 signaling and ERK1/2 phosphorylation contributing to late hypertrophic events and impaired mineralization in osteoarthritic chondrocytes.