Study Of Chondroitin Sulfate、PGE2on Porcine Chondrocytes Proliferation And Differentiation And The Effects Of Intracellular Calcium Response

Osteoarthritis (OA) is a kind of common and until now untreatable joint disease.OA accompanies by releasing a large amount of inflammatory mediator prostaglandinE2(PGE2) and its pathogenesis is not understood adequately. The damage of cartilagewhich is the major component of joints is the main pathological features ofosteoarthritis. Cartilage is composed of chondrocytes and cartilage matrix. So it is verynecessary to understand the influence on chondrocytes aroused by themicroenvironment change of cartilage matrix. In this study, the influence of differentconcentration of inflammatory mediators PGE2and cartilage matrix chondroitin sulfate(CS) on different layers of normal pig knee chondrocytes were investigated. Our resultscould do help in revealing the pathogenesis of osteoarthritis and provide certainscientific basis for the prevention and treatment of osteoarthritis in clinic.The main contents and conclusions of this paper were as follows：(1) The primary superficial-middle and deep layers of chondrocytes were separatedfrom the normal femoral head of porcine by the0.25%trypsin,0.3%type II collagenasegradually digestion method. Trypan blue exclusion test results showed that the viabilityof collected chondrocytes was up to90%approximately. Cellular adherence wascompleted after48h. Cells achieved fusion to80~90%by9-10d anddisplayed typical cobblestone-like morphology. Cells of different layers were in shapeof a flat triangle or a polygon. While the volume of deep layer of chondrocytes waslarger than superficial-middle layer of chondrocytes. The results suggested that thechondrocytes we obtained were of good viability and large quantity which could beused for next experiments.(2) The effects of different concentration of CS and PGE2alone or combined withon superficial-middle and deep layers of chondrocytes proliferation and differentiationwere investigated. Results showed that2mg/ml of CS、2mg/ml of CS combined with2.5×10-8mg/ml of PGE2、2mg/ml of CS combined with10-6mg/ml of PGE2in2mLof culture medium could significantly promote the both layers of normal chondrocytesproliferation as well as deep cartilage glycosaminoglycan (GAG) synthesis, andinhibited the deep layer of chondrocytes alkaline phosphatase (AKP) activity. The effecton promoting proliferation of deep layer of chondrocytes by2mg/ml of CS combinedwith10-6mg/ml of PGE2was significantly greater than2mg/ml of CS. Different concentrations of PGE2had no effect on normal chondrocytes proliferation anddifferentiation. Results suggested that CS could inhibit differentiation of chondrocytesand maintain the chondrocyte phenotype, and played a role in the protection and repairof articular cartilage.(3) The effects of different concentration of CS and PGE2alone or combined withon superficial-middle and deep layers of chondrocytes intracellular calcium responsewere investigated. There were no significant differences in the cellular calcium responsebetween different layers of cartilage cells without CS or PGE2treatment. Butsuperficial-middle layer of chondrocytes calcium response was significantly higher thanthe deep layer of chondrocytes after CS or/and PGE2treatment. The treatment of lowconcentration of PGE2could promote the cellular calcium response percentage ofsuperficial-middle chondrocytes while it was reduced by the treatment of highconcentration of PGE2. The treatment of high concentrations of CS and PGE2significant promote calcium response frequency of superficial-middle layer ofchondrocytes. Results suggested that superficial-middle layer of chondrocytes weremore sensitive to CS and PGE2than deep layer of chondrocytes. The induction ofexogenous signaling of cartilage might mainly be mediated by the superficial-middlelayer of chondrocytes. The reduction of intracellular calcium responsive percentage andincreasment the frequency of response induced by the treatment of high concentrationsof PGE2might be related to the onset of osteoarthritis and intensify. The treatment ofhigh concentrations of CS promoted chondrocytes calcium response frequency.