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Azerbaycan Saytlari

 »  Home  »  Endodontic Articles 1  »  In vitro cytotoxicity of a composite resin and compomer
In vitro cytotoxicity of a composite resin and compomer
Results.



In pilot studies, examination under the light microscope ( 40) indicated that when dentine sections were placed directly on the surface of the cell-culture medium they sank to the base of the well within about 5 min, disturbing some of the adherent cells. Thus it was necessary to melt a layer of soft wax around three sides of the material attached to the dentine, to ensure that it was in contact with the culture medium but did not sink down to the base of the well.

MTT assay.
The MTT assay was used to determine the effect of each restorative material on cell viability over a three-day time period. The restorative materials had little effect on day 1 (Fig. 1). However, after 2 days of incubation, Spectrum® resulted in a significant reduction in cell number relative to the dentine control ( P < 0.01), whereas Dyract® AP had no significant effect. By the third day both materials had caused significant reductions in viable cells, with Spectrum® ( P < 0.01) being a more potent inhibitor than Dyract® AP ( P < 0.05).

LDH assay.
The relationship between material cytotoxicity and cell viability was further investigated using the release of LDH from cultured cells (Fig. 2). This is a cytosolic enzyme that is only released from lysed cells. There was no release of LDH from the treated cells on the first day. Although some LDH release could be detected after 2 days’ incubation, this was not statistically different from the control samples. However, after 3 days of incubation Spectrum® resulted in substantial cell lysis (47.4% of total LDH), whereas Dyract® AP (18.7% of total LDH) was less potent.

Cell viability of ECV-304 cells following exposure to restorative materials.
Figure 1. Cell viability of ECV-304 cells following exposure to restorative materials. Viability was assessed using the MTT assay. *, denotes significant difference from the control (dentine), P < 0.05 (Dyract), P < 0.01 (Spectrum).

Cell lysis ECV-304 cells following exposure to restorative materials measured by LDH release
Figure 2. Cell lysis ECV-304 cells following exposure to restorative materials measured by LDH release. *, denotes significant difference from the control (dentine) P < 0.01 (Spectrum).

Time-course of the death of HL- 60 cells exposed to Spectrum composite resin
Figure 3. Time-course of the death of HL- 60 cells exposed to Spectrum® composite resin. Cells were incubated with fully cured (m,d) and partially cured (n,s) samples. Apoptosis (n,m) and necrosis (s,d) were determined microscopically (40).
Time-course of the death of HL-60 cells exposed to Dyract AP compomer
Figure 4. Time-course of the death of HL-60 cells exposed to Dyract AP® compomer. Cells were incubated with fully cured (m,d) and partially cured (n,s) samples. Apoptosis (n,m) and necrosis (s,d) were determined microscopically (40).

Morphological staining of cells.
Fully (40 s) and partially (4 s) cured samples of Dyract® AP and Spectrum® were incubated with HL-60 cells over a period of 2–12 h. Unlike the previous studies, where the materials were separated from the cells by dentine, each material sample was in direct contact with the cells in this case. This resulted in an increase in cell death over the 12 h (Figs 3, 4) for both the partially and fully cured sample materials. When fully cured material was used, the most common form of cell death was by apoptosis (Spectrum® 55%, Dyract® AP 40%) with little necrosis being evident (Spectrum® 14%, Dyract® AP 6%) at the end of the 12 h incubation. However, with partially cured material, there was a marked reduction in the level of apoptotic cell death (Spectrum® 26%, Dyract® AP 20%) with necrosis becoming more prevalent (Spectrum® 70%, Dyract® AP 53%).

Caspase-3 fluorogenic assay.
The process of apoptotic cell death is often associated with activation of the peptidase, caspase-3. No significant activity was observed in the control (untreated) cells. The fully cured (40 s) samples resulted in the greatest caspase-3 activation, relative to the positive, etoposidetreated controls (Fig. 5), with Spectrum® (547 pmol AMC min–1 mg–1 protein) having a greater effect than Dyract® AP (490 pmol AMC min–1 mg–1 protein). The extent of caspase-3 activation increased with the time of light-curing, with partially cured Dyract® AP (caspase-3 activities of 227 and 401 pmol AMC min–1 mg–1 protein after 1 and 4 s light-curing, respectively) being more potent than Spectrum® (110 and 352 pmol AMC min–1 mg–1 protein, respectively).

Caspase activity in HL-60 cells after exposure to restorative materials
Figure 5. Caspase activity in HL-60 cells after exposure to restorative materials. *, Denotes significant difference from the control, **P < 0.0001, *P < 0.05 compared to the untreated cells and curing light alone.