# The Antimicrobial Effectiveness and Cytotoxicity of the Antibiotic-Loaded Chitosan: ECM Scaffolds
## 4 Discussion
The two antibiotics, minocycline and rifampicin, were chosen because these were antibiotics already approved to the use in topical wound dressings and the dosage was matched to that used in antibiotic sensitivity discs to allow easy comparison to a representative control. Consequently, this proof of principle experiment did not attempt to optimize the antibiotic concentration and it is possible that some of the results of the antimicrobial and cytotoxicity tests would have been different had an optimal dose been used. The clinical isolates of bacteria that were used in this study also had different sensitivities to the antibiotic. Both _E. Coli_ and _Staph. Aureus_ showed resistance to the rifampicin antibiotic, but in the case of _Staph. Aureus_, it was only a specific subpopulation of bacteria that were resistant. The presence of these resistant strains explains the growth rate results seen with the scaffold supernatants. In these studies, the antibiotic was effective at killing the rifampicin-sensitive bacteria but allowed the resistant strain to survive and eventually proliferate at an exponential rate resulting in the long lag times of 10-13 h that were seen. Other groups have shown that chitosan scaffolds can be loaded with other antibiotics with similar results to the present study. [PERSON] and [PERSON] prepared chitosan-polyethylene oxide scaffolds that were loaded with cefazolin and cefazolin-loaded silica nanoparticles [52]. They showed that the antibiotic was released from the scaffolds easily and that they were 100% effective against _Staph. Aureus_ and _E. Coli_ in vitro. [PERSON] et al. used a chitosan scaffold as a delivery vehicle for silver sulfradiazine which showed similar release profiles to the scaffolds in the present study by had antimicrobial effectiveness up to 1 week and was successful at inhibiting _Pseudomonas Aeruginosa_ and _Staph. Aureus_ infection in wounds in vivo [53]. [PERSON] et al. used chitosan sponges to deliver a combination of vancomycin and tobramycin, testing the effectiveness using bioluminescent strains _Staph. Aureus_ and _P. Aeruginosa_ in a large animal model. While the wound healing properties of the scaffolds were not investigated, they showed that the antibiotic-loaded scaffolds effectively reduced, but did not completely eliminate, the bacterial infection, while unloaded scaffolds allowed the bacteria to grow and proliferate [54]. Interestingly, the incorporation of mammalian ECM, whether in hydrogel form or particulates, appeared to alter the effectiveness and release rate of the antibiotics. 1% (_w/v_) and 10% (_w/v_) ECM scaffolds had a sustained release of minocycline and rifampicin that was effective at inhibiting bacterial growth for up to 24 h, while the 100% chitosan, 80/20, and 50/50 chitosan:ECM scaffolds released the antibiotic faster and lost their effectiveness typically after around 3 h. Moreover, when looking at the effect of minocycline-loaded scaffolds on _E. Coli_, while the antibiotic sensitivity discs inhibited bacterial growth of up to 24 h, all the scaffold formulations lost their original effectiveness after approximately 2-3 h suggesting that the effectiveness of the antibiotic was being altered by the material properties of the scaffolds. Antibiotic release by the scaffolds was expected to be by passive diffusion and so prolonged release of the antibiotic promoted by the presence of ECM is not thought to be the result of direct protein interaction between the ECM and the antibiotics. However, this interaction was not investigated. The more likely explanation is that the presence of ECM increases the ability of the scaffolds to retain water since the hydrated ECM is approximately 90% water, resulting in slower diffusion of the antibiotic out of the scaffold material. Modulating the release profile of the antibiotics could be modified further in a number of ways. Chemical cross-linking of the scaffold materials would prevent degradation of the material potentially prolonging antibiotic release.