OBJECTIVETo investigate the effects in vitro of 17 beta-estradiol (E2) on the proliferation and metabolism of rabbit mandibular condylar cartilage cells.

METHODSChondrocytes were derived from neonatal rabbit mandibular condylar cartilage using a modified enzyme method. 17 beta-estradiol was added to the culture medium in a variety of concentrations. Cell growth and DNA, collagen, and proteoglycan synthesis were used as indicators of proliferation and differentiation of condylar chondrocytes. These were measured by cell number, 3H-proline and 35S-incorporation, respectively.

RESULTSE2 increased cell number and 3H-thymidine incorporation at 10(-8) to 10(-10) mol/L, and 10(-8) to 10(-11) mol/L in a dose-dependent manner, peaking at 10(-8) mol/L and 10(-9) mol/L, respectively. However, further increase in the concentration of estradiol caused inhibition of both cell number and 3H-thymidine incorporation, and this was significant at 10(-6) mol/L. The effect of E2 on proteoglycan synthesis was similar; the maximum stimulating effect was at 10(-8) mol/L, and inhibition was significant at 10(-6) mol/L. There was no obvious stimulatory effect of E2 on 3H-thymidine incorporation observed.

CONCLUSIONSEstradiol affects condylar chondrocyte cell growth, DNA, and proteoglycan synthesis in a biphasic manner depending on its concentration. This indicates that estrogen may be important in the proliferation and differentiation of mandibular condylar chondrocytes, and could be relevant to some aspects of certain temporomandibular joint diseases by modulating the function of the chondrocytes.

Animals ; Cartilage, Articular ; cytology ; metabolism ; Cell Differentiation ; Cell Division ; drug effects ; Cells, Cultured ; Estradiol ; pharmacology ; Mandibular Condyle ; cytology ; metabolism ; Rabbits

OBJECTIVE: To explore the effect of insulin-like growth factor (IGF-1) on the concentration of NO and PGE(2) in the supernatant of rabbit articular chondrocytes induced by IL-1, and to explore the mechanism of IGF-1 in the development of osteoarthritis (OA). METHODS: The samples were divided into 7 groups: IL-1beta 10 microg/L group, IL-1beta 10 microg/L+IGF-1 1 microg/L group, IL-1beta 10 microg/L+IGF-1 10 microg/L group, IL-1beta 10 microg/L+IGF-1 50 microg/L group, IL-1beta 10 microg/L+IGF-1 100 microg/L group, IGF-1 50 microg/L group, and a blank control group. The chondrocytes from the articular cartilage of 2 month old rabbits were cultivated and identified, and then co-cultured in the second filial generation chondrocytes on plates with or without recombinant human IGF-1 or IL-1. The concentration of NO was detected by nitrate reductase kit, and that of PGE(2) by enzyme-linked immunosorbent assay (ELISA). The results were analyzed by statistical method. RESULTS: The average value of NO and PGE(2) was (89.971+/-10.224) micromol/L and (22.028+/-8.731) micromol/L in the IL-1beta 10 microg/L group, and (12.404+/-8.809) micromol/L and (1.900+/-0.227) ng/L in the blank control group. The concentration of NO and PGE(2) in IL-1beta 10 microg/L group was significantly higher than that in the blank control group (P<0.05). At the same concentration of 10 microg/L, IGF-1 could dose-dependently decrease the increase of NO and PGE(2) concentration induced by IL-1beta in the chondrocytes supernatant in vitro, and the optimum concentration of IGF-1 was 50 microg/L. CONCLUSION IL-1 can significantly increase the concentration of NO and PGE(2), and IGF-1 can dose-dependently decrease the concentration of NO and PGE(2) in the chondrocytes supernatant in vitro. The optimum concentration of IGF-1 was 50 microg/L.
Animals ; Cartilage, Articular ; cytology ; metabolism ; Cells, Cultured ; Chondrocytes ; drug effects ; metabolism ; Dinoprostone ; metabolism ; Insulin-Like Growth Factor I ; pharmacology ; Interleukin-1 ; pharmacology ; Nitric Oxide ; metabolism ; Osteoarthritis ; metabolism ; Rabbits

OBJECTIVEThis study was to explore the cytotoxic effect and the related injury mechanism of deoxynivalenol (DON) on articular chondrocytes in human embryo.

METHODSArticular cartilage cells were isolated from knees of human embryo and cultured in DMEM/F12 medium. The cells of the 4th generation were divided into five groups and incubated with varying concentrations of DON as the followings: control group and group with DON of 0.1, 0.2, 0.4, 1.0 µg/ml. The effects of DON were observed 72 hours after incubation. Cell apoptosis was assayed by flow cytometry (FCM) with Annexin V-FITC/PI staining; MMP-13 and PGE2 were detected by ELISA kits; NO was measured by Griess assay with spectrophotometer. Inducible nitric oxide synthase (iNOS) and collagen II in cells were detected by FCM. The expression levels of iNOS, mRNA and collagen II mRNA were measured with RT-PCR.

RESULTSThe rates of cell apoptosis in DON groups were 6.78% - 19.05%, which were significantly higher than that in control (1.20%, F = 174.761, P < 0.05). The levels of NO in DON groups were 20.8 - 40.7 µmol/L, which were significantly higher than that in control (10.2 µmol/L, F = 91.966, P < 0.05). The levels of MMP-13 in DON groups were 0.25 - 0.56 µmol/L, which were significantly higher than that in control (0 µmol/L, F = 78.420, P < 0.05). The levels of PGE2 in DON groups were 3.2-20.6 µmol/L, which were significantly higher than that in control (11.6 µmol/L, F = 276.453, P < 0.05). The proportions of cells with positive iNOS in DON groups were 14.8% - 56.8% which were significantly higher than that in controls (7.1%, F = 214.614, P < 0.05). The proportions of cells with positive collagen II in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 56.7% and 52.7%, which were significantly lower than that in control (62.2%, F = 5.134, P < 0.05). The relative absorbance values of iNOS mRNA in DON groups were 1.07 - 1.33, which were significantly higher than that in control (0.62, F = 8.358, P < 0.05). The levels of collagen II mRNA in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 0.83 and 0.82, which were significantly lower than that in control (1.14, F = 7.887, P < 0.05).

CONCLUSIONDON could promote anabolism of NO in articular cartilage cells by which up-regulated the expression of PGE2 and MMP-13, which both promoted resolution of articular cartilage matrix such as collagen II. DON induced apoptosis in articular cartilage cells.

Cartilage, Articular ; cytology ; embryology ; Cells, Cultured ; Chondrocytes ; drug effects ; metabolism ; Dinoprostone ; metabolism ; Humans ; Matrix Metalloproteinase 13 ; metabolism ; Nitric Oxide ; biosynthesis ; Trichothecenes ; toxicity

2-deoxy-D-glucose (2DG) is known as a synthetic inhibitor of glucose. 2DG regulates various cellular responses including proliferation, apoptosis and differentiation by regulation of glucose metabolism in cancer cells. However, the effects of 2DG in normal cells, including chondrocytes, are not clear yet. We examined the effects of 2DG on dedifferentiation with a focus on the beta-catenin pathway in rabbit articular chondrocytes. The rabbit articular chondrocytes were treated with 5 mM 2DG for the indicated time periods or with various concentrations of 2DG for 24 h, and the expression of type II collagen, c-jun and beta-catenin was determined by Western blot, RT-PCR, immunofluorescence staining and immunohistochemical staining and reduction of sulfated proteoglycan synthesis detected by Alcain blue staining. Luciferase assay using a TCF (T cell factor)/LEF (lymphoid enhancer factor) reporter construct was used to demonstrate the transcriptional activity of beta-catenin. We found that 2DG treatment caused a decrease of type II collagen expression. 2DG induced dedifferentiation was dependent on activation of beta-catenin, as the 2DG stimulated accumulation of beta-catenin, which is characterized by translocation of beta-catenin into the nucleus determined by immunofluorescence staining and luciferase assay. Inhibition of beta-catenin degradation by inhibition of glycogen synthase kinase 3-beta with lithium chloride (LiCl) or inhibition of proteasome with z-Leu-Leu-Leu-CHO (MG132) accelerated the decrease of type II collagen expression in the chondrocytes. 2DG regulated the post-translational level of beta-catenin whereas the transcriptional level of beta-catenin was not altered. These results collectively showed that 2DG regulates dedifferentiation via beta-catenin pathway in rabbit articular chondrocytes.
Animals ; Cartilage, Articular/*cytology ; Cell Dedifferentiation/*drug effects ; Cell Nucleus/drug effects/metabolism ; Chondrocytes/*cytology/drug effects/enzymology/*metabolism ; Deoxyglucose/*pharmacology ; Endoplasmic Reticulum/drug effects/pathology ; Glycogen Synthase Kinase 3/metabolism ; Mutant Proteins/metabolism ; Protein Transport/drug effects ; Proteoglycans/metabolism ; Rabbits ; Signal Transduction/*drug effects ; beta Catenin/*metabolism

OBJECTIVETo study the influence of metabolism of articular cartilage in ovariectomized rats in the intervention of different drugs.

METHODSSixty-six virgin female Sprague-Dawley rats were randomly stratified into six groups: sham operation (SHAM) group,ovariectomized (OVX) group, estrogen replacement therapy (ERT) group, estrogen receptor modulator (ERM) group, Xianling Gubao([Chinese characters: see text], XLGB) group,and glucosamine sulfate (GS) group. One group subjected to standard sham operation and the remaining five groups were ovariectomized. The five ovariectomized groups received treatment either with the vehicle or different drugs given as an oral suspension in the vehicle. Urine samples were obtained at initial time and weeks 3, 5 and 7 after ovariectomy. ELISA method was used to measure the concentration of collagen II degradation products (CTX-II) in the urine. At study termination,histological analysis of the knee joint was used to assess the pathological changes of the articular cartilage.

RESULTSCompared with the baseline, the concentration of CTX-II at weeks 3 and 5 after ovariectomy were obviously different (P < 0.05). Compared with the OVX group, concentration of CTX-II in the ERT group and ERM group were significant different (P < 0.01). XLGB group also was obviously different with the OVX group at the two points (P < 0.05). And interestingly in the histological examination in the OVX group was different with all the other groups.

CONCLUSIONThe study indicates that estrogen deficiency accelerates destructive turnover of the collagen II in articular cartilage. The ERT, ERM, XLGB can relieve the effect. And all the drugs can delay the cartilage degradation caused by that effect.

Animals ; Cartilage, Articular ; drug effects ; metabolism ; pathology ; Collagen Type II ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Estrogen Receptor Modulators ; pharmacology ; Estrogen Replacement Therapy ; Estrogens ; pharmacology ; Female ; Humans ; Osteoarthritis ; drug therapy ; metabolism ; pathology ; Ovariectomy ; adverse effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVECulturing rabbit articular chondrocytes in vitro and seeding on polylactic acid (PLA) coated with lecithin and poly-l-lysine modulated by bFGF to find a suitable method for cartilaginous tissue engineering.

METHODSThe articular chondrocytes were isolated enzymatically from the articular cartilage of young rabbits, and cultured in vitro. Collecting the chondrocytes of the third passage and seeding on three-dimensional scaffold of polylactic acid coated with lecithin and poly-l-lysine. At the same time, basic fibroblast growth factor (bFGF) was added. Proceeding series of detections when the cell-scaffold complexes were cultured more than two weeks, such as macroscopic, invert microscope, light microscope, scanning electron microscope and immunohistochemistry of collagen II.

RESULTSThe cell-scaffold complexes modulate by bFGF could not only keep their original shapes, but also maintain the stable homogeneous three-dimensional distribution of chondrocytes without cell falling during the cultivation. At the same time, the complexes were gradually decreasing the consistency, however, increasing the Pliability with elasticity and lubrication surface. After the second week, the complexes were gradually reorganized into the mature engineered cartilage with typical cartilaginous histological structure with rich collagen II.

CONCLUSIONbFGF can facilitate the regeneration and maturation of tissue-engineered articular cartilage.

Animals ; Cartilage, Articular ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chondrocytes ; cytology ; drug effects ; metabolism ; Collagen Type II ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Lactic Acid ; Polymers ; Rabbits ; Tissue Engineering

Endoplasmic reticulum (ER) stress regulates a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells. In this study, we observed the role of 2-deoxy-D-glucose (2DG) on inflammation of chondrocytes. 2DG is well known as an inducer of ER stress, via inhibition of glycolysis and glycosylation. Treatment of 2DG in chondrocytes considerably induced ER stress in a dose- and time-dependent manner, which was demonstrated by a reduction of glucose regulated protein of 94 kDa (grp94), an ER stress-inducible protein, as determined by a Western blot analysis. In addition, induction of ER stress by 2DG led to the expression of COX-2 protein with an apparent molecular mass of 66-70kDa as compared with the normally expressed 72-74 kDa protein. The suppression of ER stress with salubrinal (Salub), a selective inhibitor of eif2-alpha dephosphorylation, successfully prevented grp94 induction and efficiently recovered 2DG-modified COX-2 molecular mass and COX-2 activity might be associated with COX-2 N-glycosylation. Also, treatment of 2DG increased phosphorylation of Src in chondrocytes. The inhibition of the Src signaling pathway with PP2 (Src tyrosine kinase inhibitor) suppressed grp94 expression and restored COX-2 expression, N-glycosylation, and PGE2 production, as determined by a Western blot analysis and PGE2 assay. Taken together, our results indicate that the ER stress induced by 2DG results in a decrease of the transcription level, the molecular mass, and the activity of COX-2 in rabbit articular chondrocytes via a Src kinase-dependent pathway.
Animals ; Cartilage, Articular/pathology ; Cells, Cultured ; Chondrocytes/drug effects/immunology/*metabolism/pathology ; Cyclooxygenase 2/genetics/*metabolism ; Deoxyglucose/*pharmacology ; Down-Regulation ; Endoplasmic Reticulum/drug effects/*metabolism/pathology ; Glycosylation/drug effects ; Inflammation ; Rabbits ; Signal Transduction/drug effects ; Stress, Physiological/drug effects/immunology ; src-Family Kinases/*metabolism

BACKGROUND/AIMS: SKI306X, a mixed extract of three herbs, Clematis mandshurica (CM), Prunella vulgaris (PV), and Trichosanthes kirilowii (TK), is chondroprotective in animal models of osteoarthritis (OA). The objectives of this study were to investigate its effect on interleukin (IL)-1beta-induced degradation of glycosaminoglycan (GAG) and the basis of its action in human OA cartilage, as well as to screen for the presence of inhibitors of matrix metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 in SKI306X and its component herbs, as well as in fractions from SKI306X. METHODS: Human OA chondrocytes and cartilage explants were obtained during total knee replacements and incubated with IL-1beta +/- oncostatin M with or without SKI306X or its component herb extracts. GAG degradation was assayed in cartilage explants using a commercial kit. Expression of genes involved in cartilage destruction was measured by real-time polymerase chain reaction using chondrocyte RNA. SKI306X was fractionated by preparative liquid chromatography to test for the presence of inhibitors of MMP-13 and ADAMTS-4. RESULTS: SKI306X and PV inhibited IL-1beta-induced GAG release from cartilage explants, and SKI306X, CM, PV, and TK inhibited IL-1beta-induced MMP gene expression. Unexpectedly, SKI306X greatly stimulated IL-1beta + oncostatin M-induced ADAMTS-4 gene expression, probably due to its TK component. Some fractions of SKI306X also inhibited ADAMTS-4 activity. CONCLUSIONS: SKI306X and its herbal components inhibit GAG degradation and catabolic gene expression in human OA chondrocytes and cartilage explants. SKI306X likely also contains one or more ADAMTS-4 inhibitor.
ADAM Proteins/antagonists & inhibitors ; Cartilage, Articular/*drug effects/*metabolism ; Cells, Cultured ; Chondrocytes/drug effects/metabolism ; Down-Regulation/drug effects ; Drugs, Chinese Herbal/*pharmacology ; Glycosaminoglycans/*metabolism ; Humans ; Interleukin-1beta/metabolism ; Matrix Metalloproteinase 13/metabolism ; Matrix Metalloproteinase Inhibitors/pharmacology ; Oncostatin M/metabolism ; Osteoarthritis, Knee/drug therapy/genetics/metabolism ; Procollagen N-Endopeptidase/antagonists & inhibitors

Withaferin A (WFA) is known as a constituent of Ayurvedic medicinal plant, Withania somnifera, and has been used for thousands of years. Although WFA has been used for the treatment of osteoarthritis (OA) and has a wide range of biochemical and pharmacologic activities, there are no findings suggesting its properties on chondrocytes or cartilage. The aim of the present study is to investigate the effects of WFA on apoptosis with focus on generation of intracellular reactive oxygen species (ROS). Here we showed that WFA significantly increased the generation of intracellular ROS in a dose-dependent manner. We also determined that WFA markedly leads to apoptosis as evidenced by accumulation of p53 by Western blot analysis. N-Acetyl-L-Cystein (NAC), an antioxidant, prevented WFA-caused expression of p53 and inhibited apoptosis of chondrocytes. We also found that WFA causes the activation of PI3K/Akt and JNKinase. Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA. Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes.
Animals ; Anti-Inflammatory Agents/administration & dosage ; Apoptosis/drug effects/physiology ; Cartilage, Articular/cytology/drug effects/*metabolism ; Cells, Cultured ; Chondrocytes/drug effects/*metabolism ; Dose-Response Relationship, Drug ; MAP Kinase Kinase 4/*metabolism ; Phosphatidylinositol 3-Kinases/*metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Rabbits ; Reactive Oxygen Species/*metabolism ; Withanolides/*administration & dosage

OBJECTIVETo identify the relationship between T-2 toxin and Kashin-Beck disease (KBD), the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro.

METHODSChondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA), soluble CD44 (sCD44), IL-1beta and TNF-alpha levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was determined by flow cytometry (FCM). CD44, hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13, 3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro.

RESULTST-2 toxin inhibited CD44, HAS-2, and aggrecan mRNA expressions, but promoted aggrecanase-2 mRNA expression. Meanwhile, CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition, ELISA results indicated that there were higher sCD44, IL-1beta and TNF-alpha levels in T-2 toxin group. Similarly, higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore, using monoclonal antibodies BC-13, 3-B-3 and 2-B-6, strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin, whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above.

CONCLUSIONT-2 toxin could inhibit aggrecan synthesis, promote aggrecanases and pro-inflammatory cytokines production, and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage, inducing aggrecan loss in the end, which may be the initiation of the cartilage degradation.

Cartilage, Articular ; drug effects ; metabolism ; Cells, Cultured ; DNA ; analysis ; Flow Cytometry ; Humans ; Hyaluronan Receptors ; analysis ; Immunohistochemistry ; Interleukin-1beta ; analysis ; Proteoglycans ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Selenium ; pharmacology ; T-2 Toxin ; toxicity ; Tumor Necrosis Factor-alpha ; analysis