The combined effects of transforming growth factor-β and basic fibroblast growth factor on the human degenerated nucleus pulposus cells in monolayer culture

Abstract

This study investigated the changes in the growth and phenotype of human degenerated intervertebral disc cells depending on the frequency of subculture and the growth factors effects in vitro monolayer culture system. The twice passaged nucleus pulposus (NP) cells were replated six well plates at a density 1×104 cells/cm2. The replated NP cells were divided into four subgroups: no treatment (control group), the treatment of 10 ng/mL transforming growth factor (TGF)-β1 (TGF group), treatment of 5 ng/mL basic fibroblast growth factor (bFGF, FGF group), and treatment of TGF-β plus bFGF (TGF/FGF group). Analyses of proliferation, morphology, histological and reverse transcriptase — polymerase chain reaction (RT-PCR) were made at different time point. The NP cells gradually showed a fibroblast-like spindle shape from their irregular, polygonal shape while undergoing the third time of subculture. The cell proliferation was the highest at the second subculture time. On RT-PCR, the type II collagen expression was gradually decreased and almost unexpressed at the third subculture, but the type I collagen expression was gradually increased in the NP cells. The expression of proteoglycan was gradually decreased in NP cells and it was almost unexpressed at the third time of subculture. FGF can stimulate NP cell proliferation and TGF-β showed increase collagen type II and aggrecan synthesis, and the combination of TGF and FGF appeared both effects of proliferation and extracellular matrix (ECM) synthesis on the NP metabolism. To obtain a great number of cells using a small frequency of subculture and to increase the ECM synthesis, new culture methods based on growth factors or mechanical stimulation must be considered.