# The Antimicrobial Effectiveness and Cytotoxicity of the Antibiotic-Loaded Chitosan: ECM Scaffolds
## 3 Results
### Indirect Cytotoxicity Testing  
The potential cytotoxic effects of the scaffold formulations were further elucidated using indirect cytotoxicity testing. Using culture media supernatants taken from scaffold samples after 1, 2, 3, 4, 8 and 24 h would determine whether components eluted from the scaffolds had a cytotoxic effect. These supernatant samples represented the total eluted molecules after each time point. The neutral red uptake results for the scaffolds alone and for each antibiotic-loaded group are shown in Figure 6.  
Figure 5: Graph showing the neutral red uptake by human microvascular endothelial cells exposed directly to the different scaffold formulations with and without antibiotics. Red line indicates the average neutral red uptake of cells exposed to ISO standard cytotoxic control, while the blue line shows the level of neutral red uptake in cells grown without scaffold exposure. Figure 6: Graph showing the neutral red uptake by human microvascular endothelial cells exposed to supernatant samples from scaffold formulations with and without antibiotics. Supernatant samples were collected at 1, 2, 3, 4, 8 and 24 h. (**A**) Neutral red uptake of cells exposed to supernatant samples from scaffold formulations without antibiotic; (**B**) neutral red uptake of cells exposed to supernatant samples from scaffold formulations loaded with minocycline; (**C**) neutral red uptake of cells exposed to supernatant samples from scaffold formulations loaded with rifampicin. Blue line shows neutral red uptake of cells cultured in culture media alone (no scaffold control). Supernatant samples from all the scaffold formulations, regardless of the presence or absence of antibiotic, showed reduced neutral red uptake compared to the no scaffold control at all the time points tested, only the 80/20 chitosan:ECM supernatant at 4 h and the rifampicin-loaded 100% chitosan scaffold supernatant at 2 h showed similar uptake to the positive control. These results suggest that molecules eluted from these scaffolds did have the ability to inhibit cells growth. Comparison with the negative control would subsequently identify those samples that had a measurable cytotoxic effect on the cells. Supernatant samples from the scaffolds without antibiotic (Figure 6A) showed that neutral red uptake from cells exposed to the eluted products from the 100% chitosan scaffolds was no different to the negative control at any time point, indicative of a cytotoxic effect. Cytotoxic effects were also seen with the other scaffold formulations, but the effect varied depending of the time point sampled. For example, the eluted molecules in the supernatant samples from 1% and 10% (_w/v_) ECM scaffolds did not become cytotoxic to the microvascular endothelial cells until 4 h, while the 80/20 and 50/50 chitosan:ECM scaffolds were more variable with some time points showing a difference to the negative control and others showing no difference. The results suggest that while cell growth was inhibited, the cytotoxic effects may be borderline and affected by the variability of the neutral red uptake by the negative control cells. Supernatant samples from the scaffolds loaded with minocycline (Figure 6B) again showed that neutral red uptake from cells exposed to the eluted products from the 100% chitosan scaffolds was no different to the negative control at any time point except at 24 h. In addition, eluted molecules from the minocycline-loaded 1% (_w/v_) ECM again showed no cytotoxic effects until hour 4. The minocycline-loaded 50/50 chitosan:ECM scaffolds and the 10% (_w/v_) ECM scaffolds showed reduced cell growth but no cytotoxicity at hour 1, whereas the supernatants had a cytotoxic effect at all other time points. In contrast, the 80/20 chitosan:ECM scaffolds were more variable and alternated between cytotoxic at hours 1, 4, 8 and 24 and not cytotoxic at hours 2 and 3, although these still had reduced neutral red uptake compared to the positive samples.