# The Antimicrobial Effectiveness and Cytotoxicity of the Antibiotic-Loaded Chitosan: ECM Scaffolds
## 3 Results
### Growth Kinetics Assays  
Growth kinetic assays were performed with _E. Coli_ and _Staph. Aureus_ exposed to the same supernatant samples from the chitosan:ECM scaffold formulations to investigate the duration of antimicrobial activity further. The growth rate of the bacteria was calculated to determine the effectiveness of the antibiotic-loaded scaffolds (Figure 4). Scaffold formulations without antibiotics were tested to determine if the chitosan:ECM formulations had any inherent antimicrobial activity. When _E. Coli_ were exposed to the supernatants from the scaffolds no antimicrobial effects were seen, all samples tested resulted in the characteristic bacterial growth curve with a lag time to reach exponential growth of between 1 and 1.67 h. No significant differences were seen between the different scaffold materials or between the different supernatant samples (Figure 4A). Interestingly, when the same supernatant samples were applied to _Staph. Aureus_ bacteria some antimicrobial activity was seen (Figure 4B). In the 1-h supernatant samples the 100% chitosan, 80/20 chitosan:ECM and 1% (_w/v_) ECM scaffolds all reduced the growth rate of the bacteria. Bacterial growth was not completely inhibited, but the growth rate was reduced and never reached the exponential phase over the 18 h of the test. These results suggest that these formulations may have a bacteriostatic effect inhibiting growth but not killing the bacteria. The 50/50 chitosan:ECM and 10% (_w/v_) ECM scaffolds at this timepoint did not inhibit bacterial growth; however, the 2-h supernatant samples from all the scaffold formulations reduced bacterial growth and prevented exponential growth phase. At three hours the 50/50 chitosan:ECM and 1% and 10% (_w/v_) ECM scaffolds continued to show reduced bacterial growth, however the 1% chitosan and 80/20 chitosan:ECM supernatants no longer inhibited bacterial growth. Moreover, the 100% chitosan and 80/20 chitosan:ECM supernatant samples between 1 and 4 h showed a gradual increase in bacterial growth suggesting that any antimicrobial molecules release by the scaffolds had completed eluted by 4 h. All the scaffold formulations had lost their antimicrobial effect by 4 h with no differences in bacterial growth being seen between the different scaffolds or between different supernatant samples between hours 4 and 24. Supernatants from scaffolds containing antibiotic showed similar result to the agar diffusion assay, but the growth curves allowed for a more detailed analysis of the bacterial growth. When _E. Coli_ were exposed to supernatants from rifampicin-loaded chitosan:ECM scaffolds bacterial growth was seen in all supernatant samples taken between 2- and 24-h indicative of the resistance _E. Coli_ had to the rifampicin antibiotic (Figure 4C). However, supernatant samples taken after 1 h did show inhibition of growth. While the 1 h supernatant samples from 100% chitosan and 10% (_w/v_) ECM scaffolds showed bacterial growth, in the 10% (_w/v_) ECM group this growth rate never reach the exponential phase while in the 100% chitosan group the exponential phase of growth was delayed with a lag time of 6.63 h compared to lag time of 1.30 h for the same scaffold without antibiotic. No bacterial growth was seen in the 80/20 or 50/50 chitosan:ECM groups or in the 1% (_w/v_) ECM group. Figure 4: Graphs showing the calculated growth rate of _E. Coli_ (**A**,**C**,**E**) and _Staph. Aureus_ (**B**,**D**,**F**) bacteria exposed to different scaffold formulation supernatants. (**A**,**B**) growth rate of _E. Coli_ (**A**) and _Staph. Aureus_ bacteria when exposed to supernatant samples from scaffold formulations without antibiotics; (**C**,**D**) growth rate of _E. Coli_ (**C**) and _Staph. Aureus_ (**D**) when exposed to supernatant samples of rifampicin-loaded scaffold formulations; (**E**,**F**) growth rate of _E. Coli_ (**E**) and _Staph. Aureus_ (**F**) when exposed to supernatant samples of minocycline-loaded scaffold formulations.