International Dentistry - Vol. 12, No. 2 - 34

KÖNIG / KASENBACHER

Figure 4: REM: Thermally treated DPSC showed external signs of cellular damage at 46.5 °C: The
cell usually changes its elongated shape and starts to round. At 50 °C, an increased rounding can
be observed. The cell seems to contract so fast that a part of the cytoplasm processes tears off
(arrows). The surface structure of the cells is effected as appearance and number of microvilli change.

TEM
The fibroblast-like DPSC cells (Fig. 6) exhibited long, extended
mitochondria (M) within the 3-D network of the cell at 37 °C
(control). The nucleus (K) appeared to be undivided and to have
a normal nuclear envelope (arrows). ER/RER, free ribosomes as
well as the Golgi apparatus did not show any anomalies. A
significantly expressed cytoskeleton (Z) whose filaments were
aligned parallel to the longitudinal axis (probably microfilaments)
was observed. The cells featured a number of inclusions.
At 50 °C, cell rounding became irreversible (Fig. 7).
Mitochondria (M) exhibited structural changes, especially an
inflation which concurred with the destruction of the cristae
alignment, the parallelism of which got lost. There was no
longer a three-dimensional network. The Golgi apparatus
was significantly deformed and hardly any vesicles were
constricted. The cytoskeleton was partially disintegrated and
could no longer be detected. The cell membrane appeared
to have increases vacuolisation. The nucleus (K) appeared
to be damaged irreversibly. The nuclear envelope was
inflated and partially disintegrated (*). The nuclear plasma
condensed at the chromatin, resulting in a reduction of the
euchromatin-areas which condensed at the heterochromatin.
The nucleus exhibited segmented chambering (arrow).
Contrarily, the external shape of DPSC cells incubated at
60 °C (Fig. 8) remained mostly intact. How-ever, cytoplasm
was hardly detectable. Mitochondria (M) were destroyed,

membranes and cristae were partially wound up (arrows).
Golgi apparatus and cytoskeleton were not detected. The
euchromatin areas were reduced at the nucleus (K) and
condensed at the heterochromatin (*). The nuclear
membrane was significantly vesiculated.

Discussion
The first indications to a temperature-related damage of the
DPSC were seen in the Live/Dead Assay.
Calcein is able to penetrate the membrane and is only
converted to a fluorescent colouring agent inside of an intact
cell. If the cell membrane becomes permeable as a result of
damages, calcein will not remain inside the cell. As a
consequence, Ethidium-homodimer-1 will enter the cell in
exchange. This substance is not permeable for intact
membranes and will fluoresce red when combined with
DNA.
Interestingly significant thermally-induced dam-ages were
only observed at temperatures ranging from 46.5 °C ± 0.5
°C. Starting at this temperature, cell membranes are
destroyed apparently. Temperatures from 56.5 °C ± 0.5 °C
form another threshold at which the 50 % lethality limit was
reached.
If the vitality test was conducted 24 h after thermal
treatment, almost twice as much lethal cells as observed 1 h
after incubation were seen at temperatures from 46.5 °C to

34 INTERNATIONAL DENTISTRY - AUSTRALASIAN EDITION VOL. 12, NO. 2

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