Effects of various growth factors on adult human articular chondrocytes / 中华骨科杂志

ABSTRACT

Objective To investigate the effects of transforming growth factor-?1(TGF-?1), fibroblast growth factor-2 (FGF-2), platelet derived growth factor-bb(PDGF-bb) and hepatocyte growth factor(HGF) on adult human articular chondrocytes(AHAC) proliferation and phenotype maintaining. Methods Isolated AHAC were cultured in DMEM/F-12 supplemented with 10% human AB serum, 50 ?g/ml ascorbic acid-2-phosphate, 0.4 mmol/L proline, 5 ?g/ml insulin and 1 mmol/L non-essential amino acids (NEAA). The cells of 2nd passage were used for proliferation kinetics studying 2.0?103 cells/well were seeded on 96-well plate, 24 h later, the cells were stimulated with various growth factors or combinations of these growth factors respectively, and the proliferation kinetics were analyzed by MTT colorimetric method. The passaged chondrocytes' phenotype were assessed by safranine O staining and immunostaining for type Ⅰ,Ⅱcollagens and aggrecan. Results All four growth factors FGF-2, TGF-?1, PDGF-bb and HGF, could promote the proliferation of AHAC, and the optimal concentrations,when used separately, were 50 ng/ml, 1 ng/ml, 1 ng/ml, 20 ng/ml respectively. While 5 ng/ml FGF-2 combined with 1 ng/ml TGF-?1 could achieve the best proliferation effect, additionally adding PDGF-bb, HGF or both could not enhance more. With the combination of FGF-2 and TGF-?1, the AHAC could expand over 2000-fold and passage over 10 times. Chondrocytes of 9th passage still excreted type Ⅱcollagen and glycosaminoglycan(GAG). Conclusion 5 ng/ml FGF-2 combined with 1 ng/ml TGF-?1 is a very appropriated circumstance for in vitro expanding of AHAC.