Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro

Abstract

Study design: Human cells from the anulus were grown in tissue culture in an experimental design to study the anti-apoptotic effect of two selected cytokines.

Objectives: To determine whether two selected cytokines, insulin-like growth factor-1 and platelet-derived growth factor, were effective in decreasing apoptosis in human cells from the anulus grown in culture for 10 days.

Summary of the background data: Previous studies have shown that there is a small cell population in the aging human intervertebral disc. Earlier work from the authors' laboratory suggested that apoptosis (programmed cell death) may be a major contributing factor to the decrease in cell number. A wide variety of inhibitors of apoptosis have now been identified; the present report presents findings on the actions of insulin-like growth factor-1 and platelet-derived growth factor in retarding or preventing apoptosis.

Methods: Using previously published culture methods, cells from the anulus of 25 subjects (mean age, 41.7 years) were grown in monolayer culture for 10 days and tested under the following conditions: 1) control growth in the presence of 20% fetal bovine serum; 2) positive control conditions promoting the development of apoptosis in the absence of serum; or 3) in dose-response regimes where insulin-like growth factor-1 or platelet-derived growth factor were added in the presence of only 1% fetal bovine serum (necessary for basal cell maintenance). Specimens were derived from 18 lumbar, 9 cervical, and 1 thoracic sites; the average Thompson score was III. Cells were grown on chambered slides and evaluated in situ using the TdT in situ apoptosis detection reaction to identify apoptotic cells. An average of 300 cells were counted in replicate cultures at each dose to determine the incidence of apoptosis; results were analyzed with standard statistical techniques. Cultured cells also were examined with transmission electron microscopy.

Results: Serum withdrawal to a 1% level was used as a positive apoptosis control in vitro and resulted in a significantly greater percentage of apoptosis compared with the 20% serum negative control (1.02% +/- 0.34 (28) versus 0.14% +/- 0. 04 (27; mean +/- SEM (n)), P < 0.0001). Exposure to 50 ng/mL insulin-like growth factor-1 significantly reduced the percentage of apoptosis (vs.- 1% serum) to 0.49% +/- 0.26 (P = 0.005); 500 ng/mL was also significantly effective (% apoptosis = 0.09% +/- 0.04 (P = 0.0001). Platelet-derived growth factor at a dose of 100 ng/mL also significantly reduced apoptosis (0.18 +/- 0.11, P = 0.0001).

Conclusions: Data demonstrate a significant reduction in the percentage of apoptotic disc cells after exposure to 50-500 ng/mL insulin-like growth factor-1 or exposure to 100 ng/mL platelet-derived growth factor. These findings expand the understanding of the cell biology of the disc cell and show that selected cytokines can retard or prevent programmed cell death in vitro. The administration of these cytokines may have future therapeutic potential in the treatment of disc degeneration.