In vitro clonogenic capacity of periodontal ligament fibroblasts cultured with Emdogain

The aim of the present study was to evaluate the efficiency of Emdogain (EMD) in preserving the size of the periodontal ligament progenitor pool (clonogenic capacity) and in promoting their proliferation. Periodontal ligament fibroblasts (PDLF) were obtained from explants of young permanent healthy tooth. After initial outgrowth (10 days to 2 weeks following explantation), the culture medium of experimental flasks was replaced with medium supplemented with 100 mu g ml(-1) EMD, whereas the other served as controls and were fed with regular medium. Following 5 weeks, the cells were washed (3x), harvested (trypsin + EDTA), and evaluated for their viability. Viable cells from each group were inoculated into six 96-well plates at a concentration of one Viable cell per two wells and were allowed to grow for 5 weeks. The percentage of cells with clonogenic capacity was determined as the number of colonies formed/number of cells seeded X 100 in the experimental and control groups. Three degrees of dish area coverage were utilized: up to 25%, between 25% and 75% and higher than 75%. This experiment was repeated four times from four different donors. A total of 2328 cells were evaluated, half of which, were cultured with EMD. The mean percentage of cells (from all donors) who exhibited any clonogenic capacity in the presence of EMD was comparable with that of cells cultured in the absence of EMD: 26.6 +/- 14.3% when compared with 34.6 +/- 20.6% respectively (P = 0.186). Similarly, the percentage of clones that proliferated to cover up to 25% of the well area was comparable in the two groups 7.5 +/- 8.6 for EMD-treated clones and 7.1 +/- 7.8 for untreated clones (P = 0.674). The percentage of clones that proliferated to cover 25% up to 75% of the well area was greater EMD-treated clones as compared with the untreated cells: 8.1 +/- 6.7% vs 3.8 +/- 3%. However this difference was not statistically significant (P = 0.277). In contrast, the percentage of clones that covered more than 75% of the well area was significantly lower in the EMD-treated clones when compared with the untreated clones (10.9 +/- 11.1 vs 23.8 +/- 14.7; P = 0.022). In conclusion, EMD decreased the percentage of PDLF with capabilities of arising colonies with 75-100% confluency probably by increasing their differentiation.