# The Antimicrobial Effectiveness and Cytotoxicity of the Antibiotic-Loaded Chitosan: ECM Scaffolds
## 4 Discussion  
The textbook description of wound healing presents the process as a series of overlapping stages including hemostasis, re-epithelialization, inflammation and the formation of granulation tissue and finally remodeling. However, the healing of traumatic injuries is never this simple and the process of wound healing can be delayed by factors such as excessive bleeding, bacterial and foreign object contamination. Wound dressings aim to promote hemostasis, and ideally possess antimicrobial properties and factors to promote wound healing. The development of a multifunctional wound dressing has been a goal in trauma care for several years, particularly for tactical combat casualty care (TCCC) in the armed forces. Uncontrolled hemorrhage remains the leading cause of trauma deaths in both the combat and civilian settings accounting for 60% of preventable combat death [30; 31]. Moreover, there is a growing incidence of bacterial wound infection, particularly in combat injuries [32; 33; 34], that is stimulating the development of new wound treatments. The dressing developed in the present study was built on a platform that utilizes two different extracellular matrices (ECM): ECM from decellularized mammalian tissue and ECM (chitosan) from crustaceans. These were chosen since both types of ECM have well established clinical benefits in promoting wound healing and controlling hemostasis. This hybrid ECM platform was augmented with the addition of antibiotics, aimed to boost bactericidal activity at the wound site. The use of chitosan as a wound dressing is well established with a number of groups using it as a wound dressing on its own or in combination with other polymers, proteins or proteoglycans [35; 36; 37; 38; 39; 40; 41; 42; 43; 44]. Similarly, ECM from decellularized mammalian tissue is already used in the manufacture of several wound dressing products [45; 46; 47]. While these decellularized tissue scaffolds are primarily used to promote rapid wound healing and constructive tissue remodeling, previous studies have shown that the degradation products of these materials have direct antimicrobial effects [48] and indirect antimicrobial effects through regulation of the inflammatory response [49; 50]. Chitosan has also been shown to have antimicrobial properties, although, this may be dependent on the preparation method and pH with poor solubility of chitosan and the loss of the cationic charge in neutral and alkaline environments being two of the major obstacles to its antimicrobial activity [25; 26; 51]. Since the scaffolds used in the present study were prepared in a neutral environment and used either pegsin-digested mammalian ECM or comminuted powder, it was of interest to see if these materials without antibiotic augmentation possessed any antimicrobial activity. Interestingly, under certain circumstances, the scaffolds alone did have antimicrobial activity against _Staph. Aureus_ bacteria, but not _E. Coli._ Looking at the zone of inhibition around the different scaffold formulations all the scaffolds, the scaffolds made from 100% chitosan or the 80/20 and 50/50 chitosan ECM hydrogel mix showed a small zone of inhibition around the scaffold that was only 1 mm larger than the scaffold disc at most. Only the 1% (_w/v_) scaffolds produced a clear, consistent zone of inhibition that was over 4 mm larger than the scaffold discs. Similarly, when the growth rates of _Staph. Aureus_ were compared after exposure to the scaffold supernatants, some degree of bacteriostatic effect was seen with supernatant samples from incubations up to 3 h where all the scaffold formulations were able to reduce, but not completely inhibit, bacterial growth. The inclusion of antibiotics into the scaffold formulations had an obvious effect on the antimicrobial properties of the scaffolds.