International Dentistry - Vol. 12, No. 2 - 30
SCIENTIFIC
Thermal damage behaviour of human
dental pulp stem cells
Karsten König1 and Anton Kasenbacher2
Objective
This study was designed to examine the influence of temperatures ranging from 37 to 65
°C on the cell morphology of DPSC stem cells via light and electron microscopy, a
synthesis of Heat Shock Proteins (HSP) with fluorescence-marked antibodies and vitality
using the Live/Dead Fluorescence Kit.
Material and methods
1
Prof. Dr Karsten König
Saarland University Campus
A5.1, Room 2.35 66123
Saarbrücken, Germany
Tel.: +49 681 3023451
Fax: +49 681 3023090
k.koenig@blt.uni-saarland.de
2
Dr Anton Kasenbacher
Private Practice, Obere
Hammerstr. 5 83278 Traunstein,
Germany
Tel.: +49 861 4692
Fax: +49 861 12853
a.k@ts-net.de
DPCS were cultivated at 37 °C and 5 % CO2 in sterile cell chambers (MiniCeM,
JenLab GmbH, Jena, Germany). The cells were irrigated with pre-heated culture medium
(Eagle's MEM, Gibco BRL, Paisley, Scotland, 37 °C) with 20 % FCS, 2 mM L-Glutamin
and 100 µM L-Ascorbate-2-Phosphate in order to re-move cellular debris previously to
the temperature trials. Filling the chamber with the culture medium followed and a
preheated water bath of different temperatures was introduced. Up to an incubation
temperature of 46 °C, the experiments were con-ducted 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 thermal treatment,
the cells were cooled down in the incubator at a temperature of 37 °C for 1 hour.
Some of the cells which had undergone thermal treatment were examined with the
Live/Dead Fluorescence Assay (Molecular Probes, Eugene, OR, USA) in order to assess
vitality via fluorescence microscopy and Axiovert 200 (ZEISS, Jena) 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 (Ethidiumhomodimer-D1 and coupled DNA). 100 cells of each type were enumerated.
In order to examine the synthesis of HSP proteins, the cells having undergone thermal
treatment were processed as follows:
- Opening of the chamber and removal of the cover-slip containing the cells
- Suction of the nutritive medium, two rinses with PBS (isotonic: 67 mM phosphate
buffer pH 7.2-7.4, 0.5 % NaCl)
- 12' fixation in 2 % paraformaldehyde in 0.1 M cacodylate buffer pH 7.2; Rinse: 3
x PBS, 2 x TBS (Tris buffered saline, 50 mM Tris-HCl buffer, 1.25 % NaCl)
- Parting of the coverslip with Pap-Pen pen (oil pen), possibly correct with paraffin
- Incubate one half of the coverslip overnight at 4 °C with 1:500 diluted antibody AK
HSP25, Rabbit (Bio-mol), diluting solution: fish gelatin 1 %, Triton x 100 1 % in TBS)
- Cover the other half of the coverslip exclusively in diluting solution (without AK)
- Wash in TBS for 3 x 10'
- 15` Alkaline-Phosphatase verification with 3 mM Levamisol in Chedium (induces bluebrown colouring according to Seidel).
30 INTERNATIONAL DENTISTRY - AUSTRALASIAN EDITION VOL. 12, NO. 2
Table of Contents for the Digital Edition of International Dentistry - Vol. 12, No. 2
Contents
International Dentistry - Vol. 12, No. 2 - Cover1
International Dentistry - Vol. 12, No. 2 - Cover2
International Dentistry - Vol. 12, No. 2 - Contents
International Dentistry - Vol. 12, No. 2 - 2
International Dentistry - Vol. 12, No. 2 - 3
International Dentistry - Vol. 12, No. 2 - 4
International Dentistry - Vol. 12, No. 2 - 5
International Dentistry - Vol. 12, No. 2 - 6
International Dentistry - Vol. 12, No. 2 - 7
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International Dentistry - Vol. 12, No. 2 - Cover3
International Dentistry - Vol. 12, No. 2 - Cover4
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