Fibroblast growth factor-2 maintains the differentiation potential of nucleus pulposus cells in vitro: implications for cell-based transplantation therapy.

摘要

STUDY DESIGN: To investigate effects of FGF-2 on nucleus pulposus cell growth and differentiation. OBJECTIVES: To elucidate the phenotypic changes that occur during expansion of nucleus pulposus cells in monolayer culture, and to investigate the effects of fibroblast growth factor (FGF)-2 on cell growth and differentiation. SUMMARY OF BACKGROUND DATA: Nucleus pulposus cells would have a limited application for autologous cell transplantation if phenotypic dedifferentiation takes place during culture expansion. FGF-2 has been shown to retain the differentiation potential of monolayer expanded chondrocytic cells. However, its effect on nucleus pulposus cells is not known. METHODS: Bovine nucleus pulposus cells were serially passaged in the presence or absence of FGF-2 (1 and 10 ng/mL). After passage numbers 1 and 7, cells were immobilized in alginate beads and treated with transforming growth factor (TGF)-beta1 for 1 week to assess their differentiation. RESULTS: During culture expansion in monolayer, nucleus pulposus cells maintained the expression of aggrecan messenger ribonucleic acid (mRNA). However, mRNA levels of collagen type I, collagen type II, Sox-9, and versican decreased with increasing passage number for both control (untreated) cells and FGF-2 treated cells. When grown in alginate with TFG-beta1, passage 7 cells that received FGF-2 during culture expansion restored the mRNA expression of type II collagen, Sox-9, COMP, chondroadherin, and fibromodulin. Moreover, FGF-2 treatment resulted in increased sulfated proteoglycan synthesis and lower aggrecan turnover compared to untreated controls under identical culture conditions. FGF-2 treated cells continued to express HIF-1alpha protein till passage 7, while MMP-2 expression was evident in cells treated with TGF-beta1. In addition, cells pretreated with FGF-2 showed higher induction of phospho ERK1/2 after treatment with TGF-beta1. Also, FGF-2 maintained smad 2/smad 3 mediated signaling in cells after TGF-beta treatment. FGF-2 action resulted in reduced actin stress fiber formation and migratory cell morphology, with no effect on cell proliferation. CONCLUSIONS: The presence of FGF-2 during culture expansion of nucleus pulposus cells in monolayer can sustain a differentiated cell phenotype by maintaining responsiveness to TGF-beta1. Our results suggest that FGF-2 should be tested for its ability to maintain the reactivity of the nucleus pulposus cells to other morphogenic factors that may be used for cell-based transplantation therapy.