In vitro evaluation of casein phosphopeptide-amorphous calcium phosphate as a potential tooth transport medium:: viability and apoptosis in L929 fibroblasts

Casein phosphopeptides (CPP) are derived from casein, which accounts for 80% of the total protein in bovine milk .The purpose of this in vitro study was to evaluate the potential use of a CPP-amorphous calcium phosphate (CPP-ACP) preparation as a transport medium for avulsed teeth. L929 fibroblastic cell line was plated in 24-well culture plates. Following incubation, the cells were treated with 10(-3), 10(-4), 10(-6), 10(-8), 10(-12) dilutions of a water-based CPP-ACP paste (Tooth Mousse, GC Corp., Tokyo, Japan). Untreated cells served as controls. The L929 cells were counted at the 1st, 3rd and 7th days. Propidium iodide/acridine orange staining was used to assess apoptosis of treated cells and of the positive control. For each concentration (dilution), statistical analysis of cell survival within time was performed using two-way analysis of variance (anova, P = 0.05). One way anova and Tukey tests were applied to compare the effect of different concentrations on cell survival at each evaluation day (P = 0.05). Except for the 10(-3)and 10(-4) dilutions, all groups demonstrated an increase in cell numbers at days 1 and 3, followed by a decrease at day 7. Irrespective of the increase or decrease in cell viability, time-dependent changes for each dilution group were significantly different. Cells in the 10(-3)and 10(-4) dilution groups demonstrated a rapid apoptotic response. A relatively few number of apoptotic cells were observed in the 10(-6) and 10(-8) dilution groups, while no sign of apoptosis was evident in the 10(-12) dilution group and control. These results suggest that when highly diluted, the tested CPP-ACP preparation may help preserve L929 cell viability in the short term without inducing apoptosis.