Experimental Study On Promotion Of Chondrogenesis In Vitro By Poly-L-lysine

Objective To investigate the effect and possible mechanism of poly-L-lysine(PL) on promoting chondrogenesis in vitro.Methods High density cultures of rat limb bud cell wereexposed to culture media containing PL(0-15y. g/ml) and culturedfor four days, Chondrogenesis was assayed based on cartilagenodule number , Alcian blue bound (OD 600) and expression of typeII collagen. By adding PL to the cultures at different periods,The best culture time point of exposing the cultures to PL wasinfered. The possible mechanism of the promotion ofchondrogenesis in vitro by PL were analysed by examining neuralcadherin(N-cadherin) expression in cells treated by PL throughimmunohistochemistry and adding polyclonal antibody ofN-cadherin to the culture medium to test whether it could affectchondrogenic differentiation.Results Compared with untreated control, PL-treatedcultures contained a significantly elevated number of cartilage nodules in a dose-dependent manner, Which was as follows: 59 + 13 (Ou g/ml) 82+14 (lug/ml); 97 + 19 (3ug/ml); 108 + 24 (5u g/ml); By lOug/ml of PL , chondrogenesis was stimulated to such an extent that nodules which confounded with each other were connected to be flat; Cultures treated with 15 u g/ml of PL produced less detectable nodules. So the best concentration of PL may be 1011 g/ml. Adding PL to culture medium at different time, The number of cartilage nodules were 108 + 24 (0-12h); 105 + 20 (12-24h); 86 + 15 (24-36h); 73 + 15 (36-48h); 62 + 12 (48-60h); 57+15 (60-72h) , which suggested that chondrogenesis stimulated by PL mainly happen on first 24h. The immunohistochemistry showed that expression of type II collagen and N-cadherin were increasing in PL-treated cultures(5 y. g/ml). When polyclonal antibody of N-cadherin was present, for the first 24 hours of culture , the number of cartilage nodules were 37 + 8 (untreatedX 23 + 6 (AbX 59 + 8 (PL), 31 + 6 ( PL+Ab) respectively, Therefore polyclonal antibody of N-cadherin could inhibit the chondrogenic differentiation and depress the effect of PL.Conclusions PL promote chondrogenesis in vitro, which may be achieved by enhancing the expression of N-cadherin.