Please activate JavaScript!
Please install Adobe Flash Player, click here for download
ePaper created 2017-06-21, 10:00:36 | version 1.38.0
| study temperature-related damage of the DPSC Thermal damage behaviour of human dental pulp stem cells Authors: Prof. Karsten König & Dr Anton Kasenbacher, Germany Objective Material and methods This study was designed to examine the influence of temperatures ranging from 37 to 65 °C on the cell morphology of DPSC (dental pulp stem cells) via light and electron microscopy, a synthesis of Heat Shock Proteins (HSP) with fluorescence-marked antibod- ies and vitality via the Live/Dead Fluorescence Kit. Table 1: Live/Dead Assay one hour and 24 hours after thermal treatment. Temperature Lethality % °C 37 39 42 45 46 47 48.5 50 55 56.5 58 60 65 1 h 24 h 0 0 0 0 0.5 10 18 17 24 48 100 100 100 0 0 0 0 2 17 29 27 59 54 100 100 100 30 roots 2 2017 DPSCs were cultivated at 37 °C and 5 % CO2 in sterile cell chambers (MiniCeM, JenLab GmbH). The cells were irrigated with pre-heated culture medium (Eagle’s MEM, Gibco BRL; 37 °C) with 20 % FCS, 2 mM L-Glutamin and 100 µM L-Ascorbate-2-Phosphate in order to remove cellular debris previously to the temperature trials. Filling the chamber with the cul- ture medium followed and a preheated water bath of different temperatures was introduced. Up to an incubation temperature of 46 °C, the experiments were conducted with temperatures rising every 2 °C and 0.5 °C in the sensitive temperature scale of 46 °C to 58 °C. In addition, trial series were carried out at 60 °C and 65 °C. After a total of 15 min of ther- mal treatment, the cells were cooled down in the in- cubator at a temperature of 37 °C for one hour. Some of the cells which had undergone thermal treatment were examined with the Live/Dead Fluores cence Assay (Molecular Probes) in order to assess vitality via fluorescence microscopy and Axiovert 200 (ZEISS) after incubation. A mixture of 2 µM Calcein AM and 4 µM Ethidium- homodimer-D1 was added to the cells which were slowly cooling down at 37°C in the incubator either 1 h or 24 h after thermal treatment and incubated for 10'. Vital cells exhibited a green fluorescence caused by calcein, while lethal cells showed a red core fluorescence (Ethidium-homodimer-D1 and coupled DNA). 100 cells of each type were enumerated. In order to examine the synthesis of HSP, the cells having undergone thermal treatment were processed as follows: · Opening of the chamber and removal of the cover- slip containing the cells Table 1