## 3 Results
### Live and Dead Cell Assay  
The Live and Dead Cell assay was conducted to evaluate the viability of MC3T3-E1 cells following exposure to different treatment groups, including different concentrations of EEP at both fermentation periods, 2.5% NaOCl and control solutions. This assay utilises dual-colour fluorescence staining, where viable cells are stained with green fluorescence and dead cells are stained with red fluorescence. The positive control group (Figure 4a), treated with 10% DMSO, displayed an abundance of cells with red fluorescence, indicating a higher number of dead cells with increased intercellular space and less cell density. Conversely, the untreated cells in the negative control group (Figure 4b) showed increased cell density and an abundance of cells with green fluorescence, indicating viable cells.  
Figure 3: Visual analysis of Alamar Blue assay of MC3T3-E1 cells exposed to different concentrations of EEP at 3 and 6 months of fermentation. Blue colour indicates non-viable cells, while pink colour indicates viable cells.  
Figure 2: The mean percentage of reduction for MC3T3-E1 cells exposed to control solutions, NaOCl and different concentrations of EEP at 3 and 6 months of fermentation. * Indicates \(p\) < 0.05 versus NaOCl; # indicates \(p\) > 0.05 versus negative control.  
Figure 4: Live and Dead Cell assay of MC3T3-E1 cells exposed to control, 2.5% NaOCl and EEP at 100% and 50%, observed under fluorescence microscopy (\(\times\)100): (**a**) positive control (10% DMSO); (**b**) negative control; (**c**) 2.5% NaOCl; (**d**) 3 M 100% EEP; (**e**) 6 M 100% EEP; (**f**) 3 M 50% EEP; (**g**) 6 M 50% EEP. 3 M: 3 months of fermentation; 6 M: 6 months of fermentation. Green: viable cells; Red: dead cells.  
The qualitative assessment from the Live and Dead Cell assay illustrated the cytotoxicity of EEP in a concentration-dependent manner for both fermentation periods. For the cells exposed to 100% and 50% EEP at both fermentation periods (Figure 4d-g), a notable number of intensely bright red fluorescent cells were observed, indicating increased cell death. When MC3T3-E1 cells were treated with 25% and 12.5% EEP at both fermentation periods (Figure 5a-d), there was a mixture of green and red fluorescence, with red fluorescence predominating, suggesting a combination of viable and dead cells. At an EEP concentration of 6.25% (Figure 5e,f), there was a noticeable increase in the density of cells with green fluorescence, and red fluorescent cells were barely seen, indicating a higher proportion of viable cells. Further reduction in the concentration of EEP to below 3.13% (Figure 6a-f) resulted in a significant increase in the density of cells with green fluorescence, similar to the negative control (Figure 4b), indicating comparable viable cell numbers. This trend was observed for both fermentation periods. On the other hand, cells exposed to 2.5% NaOCl (Figure 4c) exhibited red fluorescence with a fragmented pattern, indicating the presence of dead cells with fragmented nuclei.  
Figure 5: Live and Dead Cell assay of MC3T3-E1 cells exposed to EEP at 25%, 12.5% and 6.25%, observed under fluorescence microscopy (\(\times\)100): (**a**) 3 M 25% EEP; (**b**) 6 M 25% EEP; (**c**) 3 M 12.5% EEP; (**d**) 6 M 12.5% EEP; (**e**) 3 M 6.25% EEP; (**f**) 6 M 6.25% EEP; 3 M: 3 months of fermentation; 6 M: 6 months of fermentation. Green: viable cells; Red: dead cells; Yellow: cells undergoing initial stage of necrosis.