# The Antimicrobial Effectiveness and Cytotoxicity of the Antibiotic-Loaded Chitosan: ECM Scaffolds
## 3 Results
### Growth Kinetics Assays
Supernatants from minocycline-loaded chitosan:ECM scaffolds showed clear differences in the effectiveness of the scaffold formulations (Figure 4E). The supernatant samples taken at 1 h and 2 h from all the scaffold formulations completely inhibited bacterial growth and there were no differences between the different scaffold formulations. However, the supernatant samples taken at 3 h for the 80/20 and 50/50 chitosan:ECM formulations did not inhibit bacterial growth, and both showed exponential growth. Bacterial growth was also seen in the 1% (_w/v_) ECM supernatant group but this was a continuous growth that did not reach the exponential phase suggesting this scaffold formulation still had a bacteriostatic effect even though its bactericidal effects had been lost. The supernatants from the 10% (_w/v_) ECM group completely inhibited bacterial growth at 3 h and no growth was seen at any other timepoint all the way to 24 h indicating that this scaffold formulation continued to have a prolonged bactericidal effect and a slower release of the antibiotic than the other scaffold formulations. None of the other scaffold formulations were able to inhibit bacterial growth after 3 h, while there were differences in the growth rates between scaffolds all the growth curves followed the typical bacterial growth curve. _Staph. Aureus_ exposed to supernatants from rifampicin-loaded chitosan:ECM scaffolds showed growth that was completely inhibited by supernatant samples from all the scaffolds taken at 1 and 2 h (Figure 4D). Supernatant samples taken from the 80/20 chitosan:ECM and 10% (_w/v_) ECM groups at 3 h were still able to completely inhibit bacterial growth. The 100% chitosan, 50/50 chitosan:ECM and 1% (_w/v_) ECM groups all showed bacterial growth that reached the exponential phase. However, the lag times for each scaffold were extended to 13.26, 9.25 and 11.77 h for each scaffold, respectively, compared to a normal lag time of between 0.56 and 1.03 h for the equivalent scaffolds without antibiotic. Interestingly, while the supernatant samples from 100% chitosan at 3 h resulted in exponential bacterial growth, samples from the same scaffold at 4 h and 8 h did not reach the exponential phase. Supernatants from the 1% (_w/v_) and 10% (_w/v_) ECM groups at 4, 8 and 24 h completely inhibited bacterial growth. These results suggest that in these scaffolds there may be a prolonged release of the antibiotic, or the release of other antimicrobial molecules from the scaffold as the scaffold begins to degrade. Supernatants from the 80/20 chitosan:ECM group inhibited growth at 4 h but did not inhibit growth at 8 or 24 h although the lag time before reaching exponential growth was extended to 3.60 and 4.20 h at 8 and 24 h, respectively. Similarly, the 50/50 chitosan:ECM scaffolds did not inhibit bacterial growth at 4, 8 or 24 h, but the lag time to reach exponential phase was extended to 10.60, 3.70 and 4.56 h at each timepoint, respectively. This extension of the lag phase is probably indicative of the fact that a population of the _Staph. Aureus_ clinical isolate was resistant to rifampicin, as demonstrated on the bacterial growth plates and this population of bacteria was able to proliferate while the rifampicin-sensitive population was killed. Growth of _Staph. Aureus_ was completely inhibited by supernatants from minocycline-loaded chitosan:ECM scaffolds taken at 1 and 2 h (Figure 4F). Supernatant samples taken at 3 h from all the scaffolds except the 80/20 chitosan:ECM group were able to completely inhibit growth. The 80/20 chitosan:ECM group did show bacterial growth, but this failed to reach the exponential phase. Exponential bacterial growth was subsequently seen with supernatant samples from the 80/20 chitosan:ECM group at all the remaining time points except 24 h where the growth rate did not become exponential.