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ePaper created 2017-06-21, 10:00:36 | version 1.38.0
| study temperature-related damage of the DPSC Fig. 5: REM: Thermal treatment at HSP production TEM Fig. 5 60 °C. While the exterior shape remains mostly intact, their surface does not exhibit any structuring anymore. Examinations with regard to the production of HSP via light microscope or transmission laser microscopy showed a slight, unspecific colouring of the cells after incubation of 37 °C (control, Fig. 2). An increase in HSP production (intense colouring) was noted at a temperature of 50 °C, while thermal treatment at 60 °C again resulted in slight, unspe- cific colouring of the cells. REM Scanning electron microscopy showed a typical flat, long distribution of the control cells (37 °C cells, Fig. 3). These cells exhibited many processes and microvilli-like structures. In addition, cell-to-cell connections with neighbouring cells were observed. The successive rise in temperature resulted in the first critical temperature level of 46.5 °C ± 0.5 °C. From this level onwards, significant initial changes of the cells were registered via light and electron mi- croscope, especially an initial deformation and rounding of the cells. The cell structure (microvil- li-like structures) was reduced. However, microvilli were observed at temperatures of up to 50 °C (Fig. 4). At 50 °C (chance of survival > 70 °C accord- ing to Live/Dead Assay), the cells left distinct cyto- plasm protuberances on the base of the coverslip (Fig. 4, arrow), probably caused by a rapid contrac- tion or rounding. The fibroblast-like DPSCs (Fig. 6) exhibited long, extended mitochondria (M) within the 3-D net- work 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 ribo- somes as well as the Golgi apparatus did not show any anomalies. A significantly expressed cytoskel- eton (Z) whose filaments were aligned parallely to the longitudinal axis (probably microfilaments) was observed. The cells featured a number of in- clusions. At 50 °C, cell rounding became irreversible (Fig. 7). Mitochondria (M) exhibited structural changes, especially an inflation which concurred with the destruction of the christae alignment, the paral- lelism of which got lost. There was no longer a three-dimensional network. The Golgi apparatus was significantly deformed and hardly any vesi- cles were constricted. The cytoskeleton was par- tially disintegrated and could no longer be de- tected. 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, re- sulting in a reduction of the euchromatin-areas which condensed at the heterochromatin. The nu- cleus exhibited segmented chambering (arrow). Incubation at a temperature of 60 °C, at which none of the cells survived, resulted in a different outcome. There was no apparent deformation or rounding of the cells, with the original cell shape remaining mostly intact and some small reductions. The cells appeared to have been “thermally fixed” instantly. Neither microvilli nor other surface struc- tures were visible. Cell processes in contact with the coverslip remained intact, but exhibited denatura- tion and fixation caused by rapid heating (Fig. 5). Contrarily, the external shape of DPSCs incu- bated at 60 °C (Fig. 8) remained mostly intact. However, cytoplasm was hardly detectable. Mitochondria (M) were destroyed, membranes and christae 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 het- erochromatin (*). The nuclear membrane was significantly vesiculated. 34 roots 2 2017