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Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage.

ABSTRACT: Intracellular calcium ([Ca2+]i) signaling is an essential universal secondary messenger in articular chondrocytes. However, little is known about its spatiotemporal features in the context of osteoarthritis (OA). Herein, by examining the cartilage samples collected from patients undergoing knee arthroscopic surgery, we investigated the spatiotemporal features of spontaneous [Ca2+]i signaling in in situ chondrocytes at different OA stages. Our data showed zonal dependent spontaneous [Ca2+]i signaling in healthy cartilage samples under 4?mM calcium environment. This signal was significantly attenuated in healthy cartilage samples but increased in early-degenerated cartilage when cultured in 0?mM calcium environment. No significant difference was found in [Ca2+]i intensity oscillation in chondrocytes located in middle zones among ICRS 1-3 samples under both 4 and 0?mM calcium environments. However, the correlation was found in deep zone chondrocytes incubated in 4?mM calcium environment. In addition, increased protein abundance of Cav3.3 T-type voltage dependent calcium channel and Nfatc2 activity were observed in early-degenerated cartilage samples. The present study exhibited OA severity dependent spatiotemporal features of spontaneous [Ca2+]i oscillations of in situ chondrocytes, which might reflect the zonal specific role of chondrocytes during OA progression and provide new insight in articular cartilage degradation during OA progression.

SUBMITTER: Gong X

PROVIDER: S-EPMC5719003 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage.

Gong Xiaoyuan X Xie Wenbin W Wang Bin B Gu Lingchuan L Wang Fuyou F Ren Xiang X Chen Cheng C Yang Liu L

Scientific reports 20171206 1

Intracellular calcium ([Ca2+]i) signaling is an essential universal secondary messenger in articular chondrocytes. However, little is known about its spatiotemporal features in the context of osteoarthritis (OA). Herein, by examining the cartilage samples collected from patients undergoing knee arthroscopic surgery, we investigated the spatiotemporal features of spontaneous [Ca2+]i signaling in in situ chondrocytes at different OA stages. Our data show ...[more]

PMID: 29213100

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Project description:Basic calcium phosphate (BCP)-based calcification of cartilage is a common finding during osteoarthritis (OA) and is directly linked to the severity of the disease and hypertrophic differentiation of chondrocytes. Chondrocalcinosis (CC) is associated with calcium pyrophosphate dihydrate (CPPD) deposition disease in the joint inducing OA-like symptoms. There is only little knowledge about the effect of CPPD crystals on chondrocytes and the signaling pathways involved in their generation. The aim of this study was to investigate the chondrocyte phenotype in CC cartilage and the effect of CPPD crystals on chondrocytes. Cartilage samples of patients with CC, patients with severe OA, and healthy donors were included in this study. The presence of CC was evaluated using standard X-ray pictures, as well as von Kossa staining of cartilage sections. OA severity was evaluated using the Chambers Score on cartilage sections, as well as the radiological Kellgren-Lawrence Score. Patients with radiologically detectable CC presented calcification mainly on the cartilage surface, whereas OA patients showed calcification mainly in the pericellular matrix of hypertrophic chondrocytes. OA cartilage exhibited increased levels of collagen X and matrix metalloproteinase 13 (MMP13) compared with CC and healthy cartilage. This observation was confirmed by qRT-PCR using cartilage samples. No relevant influence of CPPD crystals on hypertrophic marker genes was observed in vitro, whereas BCP crystals significantly induced hypertrophic differentiation of chondrocytes. Interestingly, we observed an increased expression of p16 and p21 in cartilage samples of CC patients compared with OA patients and healthy controls, indicating cellular senescence. To investigate whether CPPD crystals were sufficient to induce senescence, we incubated chondrocytes with BCP and CPPD crystals and quantified senescence using β-gal staining. No significant difference was observed for the staining, but an increase of p16 expression was observed after 10 days of culture. Primary chondrocytes from CC patients produced CPPD crystals in culture. This phenotype was stabilized by mitomycin C-induced senescence. Healthy and OA chondrocytes did not exhibit this phenotype. BCP and CPPD crystals seem to be associated with two different chondrocyte phenotypes. Whereas BCP deposition is associated with chondrocyte hypertrophy, CPPD deposition is associated with cellular senescence.

Project description:Intracellular calcium ([Ca2+]i) oscillation is a fundamental signaling response of cartilage cells under mechanical loading or osmotic stress. Chondrocytes are usually considered as nonexcitable cells with no spontaneous [Ca2+]i signaling. This study proved that chondrocytes can exhibit robust spontaneous [Ca2+]i signaling without explicit external stimuli. The intensity of [Ca2+]i peaks from individual chondrocytes maintain a consistent spatiotemporal pattern, acting as a unique "fingerprint" for each cell. Statistical analysis revealed lognormal distributions of the temporal parameters of [Ca2+]i peaks, as well as strong linear correlations between their means and sds. Based on these statistical findings, we hypothesized that the spontaneous [Ca2+]i peaks may result from an autocatalytic process and that [Ca2+]i oscillation is controlled by a threshold-regulating mechanism. To test these 2 mechanisms, we established a multistage biophysical model by assuming the spontaneous [Ca2+]i signaling of chondrocytes as a combination of deterministic and stochastic processes. The theoretical model successfully explained the lognormal distribution of the temporal parameters and the fingerprint feature of [Ca2+]i peaks. In addition, by using antagonists for 10 pathways, we revealed that the initiation of spontaneous [Ca2+]i peaks in chondrocytes requires the presence of extracellular Ca2+, and that the PLC-inositol 1,4,5-trisphosphate pathway, which controls the release of calcium from the endoplasmic reticulum, can affect the initiation of spontaneous [Ca2+]i peaks in chondrocytes. The purinoceptors and transient receptor potential vanilloid 4 channels on the plasma membrane also play key roles in the spontaneous [Ca2+]i signaling of chondrocytes. In contrast, blocking the T-type or L-type voltage-gated calcium channel promoted the spontaneous calcium signaling. This study represents a systematic effort to understand the features and initiation mechanisms of spontaneous [Ca2+]i signaling in chondrocytes, which are critical for chondrocyte mechanobiology.-Zhou, Y., Lv, M., Li, T., Zhang, T., Duncan, R., Wang, L., Lu, X. L. Spontaneous calcium signaling of cartilage cells: from spatiotemporal features to biophysical modeling.

Project description:Shenmai injection (SMI) has been widely used as a therapy to treat a number of diseases. However, its anti-osteoarthritic properties have not yet been fully investigated. In the present study, the protective effect of SMI on knee articular cartilage of anterior cruciate ligament transected rabbits and interleukin-1β (IL-1β)-stimulated human chondrocytes was investigated. For the in vivo study, knee osteoarthritis (KOA) was induced in female New Zealand white rabbits by anterior cruciate ligament transection (ACLT) in the knee of right hind limb. Rabbits either underwent sham surgery or ACLT surgery. Out of the rabbits receiving ACLT surgery, half of the rabbits received one 0.3 ml Shenmai intra-articular injection in the knee per week for four weeks, following ACLT surgery. The other rabbits received the same volume of normal saline solution. The cartilage was subsequently collected for histological evaluation. For the in vitro study, cultured human chondrocytes were treated with 10 ng/ml IL-1β in the presence or absence of 5 and 2% (v/v) SMI for 24 h. Nitric oxide (NO) and prostaglandin E2 (PGE2) levels in cell culture supernatant were assessed using a Griess reaction and ELISA respectively. The mRNA expression of cyclooxgenase-2 (COX-2), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP)-1, MMP-13 and tissue inhibitors of metalloproteinase-1 (TIMP-1) in chondrocytes were detected by reverse transcription-quantitative polymerase chain reaction. The results of the current study revealed that treatment with SMI ameliorated cartilage degradation in the ACLT rabbit model, and decreased levels of NO and PGE2. Furthermore, treatment with SMI decreased levels of COX-2, iNOS, MMP-1 and MMP-13 mRNA expression and increased TIMP-1 mRNA expression in IL-1β-stimulated human chondrocytes. These results indicate that SMI suppresses inflammation and ameliorated cartilage degradation, making it a potential and promising therapeutic option to treat KOA.

Project description: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.

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Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage.

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Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage.

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