Abstract: The aim of the present study is to investigate the markers of cell cycle arrest and senescence, i.e. betagalactosidase and telomerase activity, after long term cultivation (up to 16 passages) of periodontal ligament cells. Periodontal ligament stem cells were isolated from routinely extracted third molars via enzymatic digestion (3 mg/mL collagenase type I and 4 mg/mL dispase) and cultured continuously up to passage 16. Cell count and population doubling were evaluated at each passage. The enzymatic activity of telomerase and betagalactosidase were assessed, as these are wellknown markers for cell senescence and aging. The following kits were used for these purpose, following the manufacturer`s instructions: Human TERT / Telomerase Reverse Transcriptase ELISA Kit (ELISAGenie, Dublin, Ireland) and ELISA Kit for Galactosidase Beta (GLb) (Cloud Clone Corp, Katy, TX, USA). Total protein amount was previously identified in all samples using Nanodrop 1000 (Thermo Scientific). The results demonstrate 2 peaks with significantly increased cellular proliferation rate at passage 6 and passage 12. Slight differences in the proliferative ability were identified between cells from 1 st and 16 th passages without any statistical significance. Significant decrease in telomerase activity was observed starting after the first passage. Betagalactosidase activity was found to be uninterrupted following longterm in vitro cultivation. Our study indicates that PDL stem cells do not enter cell proliferation arrest phase after longterm in vitro cultivation (16 passages – about 40 doublings), as the PDL stem cells did not show significant decrease in the proliferation ability. The telomerase activity is known to be quite typical for neoplastic cells. Therefore, our data suggest lack of tumorigenic potential in human PDL stem cell culture as we revealed suppression of telomerase activity following continuous cultivation.
Funding Source: Bulgarian National Science Fund KП-06-H73/12 15.12.2023
