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Osteoarthritis (OA) is the most prevalent form of arthritis and a major cause of pain and disability. The pathology of OA involves the whole joint in an inflammatory and degenerative process, especially in articular cartilage. OA may be divided into distinguishable phenotypes including one associated with the metabolic syndrome (MetS) of which dyslipidemia and hyperglycemia have been individually linked to OA. Since their combined role in OA pathogenesis remains to be elucidated, we investigated the chondrocyte response to these metabolic stresses, and determined whether a n-3 polyunsaturated fatty acid (PUFA), i.e., eicosapentaenoic acid (EPA), may preserve chondrocyte functions. Rat chondrocytes were cultured with palmitic acid (PA) and/or EPA in normal or high glucose conditions. The expression of genes encoding proteins found in cartilage matrix (type 2 collagen and aggrecan) or involved in degenerative (metalloproteinases, MMPs) or in inflammatory (cyclooxygenase-2, COX-2 and microsomal prostaglandin E synthase, mPGES) processes was analyzed by qPCR. Prostaglandin E 2 (PGE 2 ) release was also evaluated by an enzyme-linked immunosorbent assay. Our data indicated that PA dose-dependently up-regulated the mRNA expression of MMP-3 and -13 . PA also induced the expression of COX-2 and mPGES and promoted the synthesis of PGE 2 . Glucose at high concentrations further increased the chondrocyte response to PA. Interestingly, EPA suppressed the inflammatory effects of PA and glucose, and strongly reduced MMP-13 expression. Among the free fatty acid receptors (FFARs), FFAR4 partly mediated the EPA effects and the activation of FFAR1 markedly reduced the inflammatory effects of PA in high glucose conditions. Our findings demonstrate that dyslipidemia associated with hyperglycemia may contribute to OA pathogenesis and explains why an excess of saturated fatty acids and a low level in n-3 PUFAs may disrupt cartilage homeostasis.

The low efficiency in transfecting rat- and human-derived chondrocytes have been hampering developments in the field of cartilage biology. Transforming growth factor (TGF)-β1 has shown positive effects on chondrocytes, but its applications remain limited due to its short half-life, low stability and poor penetration into cartilage. Naturally derived liposomes have been shown to be promising delivery nanosystems due to their similarities with biological membranes. Here, we used agro-based rapeseed liposomes, which contains a high level of mono- and poly-unsaturated fatty acids, to efficiently deliver encapsulated TGF-β1 to rat chondrocytes. Results showed that TGF-β1 encapsulated in nano-sized rapeseed liposomes were safe for chondrocytes and did not induce any alterations of their phenotype. Furthermore, the controlled release of TGF-β1 from liposomes produced an improved response in chondrocytes, even at low doses. Altogether, these outcomes demonstrate that agro-based nanoliposomes are promising drug carriers.

Investigations in cartilage biology have been hampered by the limited capacity of chondrocytes, especially in rats and humans, to be efficiently transfected. Liposomes are a promising delivery system due to their lipid bilayer structure similar to a biological membrane. Here we used natural rapeseed lecithin, which contains a high level of mono- and poly-unsaturated fatty acids, to evaluate the cytocompatibility of these phospholipids as future potential carriers of biomolecules in joint regenerative medicine. Results show that appropriate concentrations of nanoliposome rapeseed lecithin under 500 µg/mL were safe for chondrocytes and did not induce any alterations of their phenotype. Altogether, these results sustain that they could represent a novel natural carrier to deliver active substances into cartilage cells.

Objectives: Chondrocytes hypertrophy is a physiological process observed in endochondral ossification during development until adolescence in human. It can also be observed during pathophysiological conditions such as osteoarthritis. Hypertrophic chondrocytes synthesise collagen X and express matrix metalloproteinase 13 and alkaline phosphatase. The cellular models available to study this process are either not convenient, they might lead to a rapid dedifferentiation of chondrocytes, or they are far from the physiological conditions. The objective of this study was to design an user-friendly 2D-primary cell culture of young articular chondrocytes of rat able to follow the terminal differentiation process., Experimental Design: After confluence, chondrocytes were cultured according to 4 differentiation protocols. Protocol 1 contained DMEM/F12 supplemented with 10% foetal bovine serum (FBS) and 2 μg/ml insulin. Protocol 2 contained alpha-MEM supplemented with 5% FBS and 2 μg/ml insulin. Protocol 3 contained 2% FBS and 2 μg/ml insulin. Protocol 4 contained DMEM/F12 supplemented with 2% FBS in absence or in presence of 2 μg/ml insulin and 37.5 μg/ml ascorbate. The cell morphology was observed by phase-contrast microscopy and the expression of markers specific of mature and hypertrophic chondrocytes were assessed by RT-qPCR., Results: The effect of a decrease in nutrient quality of the culture medium after confluence was tested using protocols 1, 2 and 3. Protocol 1 did not allow the maintenance of chondrocyte phenotype more than one week, because cells became fibroblastic. A decrease in Sox9 mRNA expression, in collagen II/collagen I and in aggrecan/versican mRNA ratios was also found with protocol 1. Protocol 3 was the best when compared with protocols 1 and 2. It allowed chondrocytes to adopt a hypertrophic morphology. Cells also expressed the collagen X specific hypertrophic marker, and presented an increase in collagen II/I and aggrecan/versican ratios after 15 days of culture post-confluence. The effect of the insulin/ascorbate supplementation was studied using protocol 4. The insulin/ascorbate supplementation allowed an earlier chondrocytes conversion to terminal differentiation with a prolonged effect till 3 weeks post-confluence, compared to control without insulin/ascorbate. Finally, the profile of chondrocyte differentiation was checked during 5 successive sub-cultures. Only the first passage could be used to study hypertrophy., Conclusion: A convenient protocol to study chondrocyte hypertrophy is proposed. Protocol 4 offers the possibility to study this differentiation phenotype which is crucial for the development of articular diseases such as osteoarthritis. Our model could also be used in tissue engineering for cartilage repair strategies in which hypertrophic differentiation of chondrocyte should be avoided.

Introduction: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage., Methods: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients., Results: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages., Conclusions: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.

Transforming growth factor (TGF)-β1 stimulates extracellular PP(i) (ePP(i)) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was up-regulated by TGF-β1 activation of ERK1/2 and Ca(2+)-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePP(i) metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa(2+)) or cytosolic Ca(2+) (cCa(2+)) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePP(i) levels (radiometric assay), and cCa(2+) input (fluorescent probe). Voltage-operated Ca(2+)-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa(2+) and ePP(i) levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa(2+) dose-dependent manner. TGF-β1 effects were suppressed by cCa(2+) chelation or L- and T-VOC blockade while being mostly reproduced by ionomycin. In the same experimental conditions, the activation of Ras, the phosphorylation of ERK1/2 and PKCα, and the stimulation of Ank promoter activity were affected similarly. Activation of SP1 (specific protein 1) and ELK-1 (Ets-like protein-1) transcription factors supported the regulatory role of Ca(2+). SP1 or ELK-1 overexpression or blockade experiments demonstrated a major contribution of ELK-1, which acted synergistically with SP1 to activate Ank promoter in response to TGF-β1. TGF-β1 promotes input of eCa(2+) through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePP(i) production in chondrocyte.

The differentiated phenotype of chondrocyte is lost in pathological situations and after interleukin (IL)-1beta challenge. Wnt proteins and the inorganic pyrophosphate (PP(i)) transporter Ank regulate the differentiation process in many cell types. We investigated the possible contribution of Ank and/or PP(i) to the maintenance of the differentiated chondrocyte phenotype with special care to Wnt signaling. Primary articular chondrocytes lost their phenotype upon IL-1beta challenge, with cessation of type II collagen and Sox-9 expression. Ank expression and PP(i) transport were strongly reduced by IL-1beta, whereas Wnt-5a was the only Wnt protein increased. Transient overexpression of Ank counteracted most of IL-1beta effects on Type II collagen, Sox-9, and Wnt-5a expression. When resting chondrocytes were transfected with a siRNA against Ank, this reproduced the phenotype induced by IL-1beta. In both cases, no markers for hypertrophic chondrocytes were detected. The conditioned supernatant from chondrocytes knocked-down for Ank contained Wnt-5a, which activated Tcf/Lef reporter plasmids and promoted translocation of beta-catenin into the nucleus without activating the c-Jun N-terminal kinase (JNK) pathway. Supplementation with PP(i) compensated for most effects of Ank deficiency on Type II collagen, Sox-9, and Wnt-5 expression, both in IL-1beta and Ank knock-down conditions. Phenotype changes induced by IL-1beta were also supported by activation of the JNK pathway, but this latter was not sensitive to PP(i) supplementation. Altogether our data demonstrate that the transport of PP(i) by ANK contributed to the maintenance of the differentiated phenotype of chondrocyte by controlling the canonical Wnt pathway in a Wnt-5a-dependent manner.

In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.