# The Antimicrobial Effectiveness and Cytotoxicity of the Antibiotic-Loaded Chitosan: ECM Scaffolds
## 4 Discussion
However, cross-linking would reduce the constructive remodeling properties of the ECM scaffolds [55; 56]. An alternative approach would be to encapsulate the antibiotic in some form of microsphere. Chitosan microspheres have been developed to provide a variety of controlled-release molecules including antimicrobial chemicals [57; 58; 59; 60]. These microspheres could easily be incorporated into the current scaffold design to provide prolonged antibiotic release, or even incorporation of a secondary antibiotic or wound healing factor to further promote wound healing. The antimicrobial effectiveness of the scaffolds is of little consequence if the scaffold formulations prove to be cytotoxic. Two tests for cytotoxicity were performed flowing ISO standards to test the direct and indirect cytotoxic properties of the scaffolds. Chitosan and mammalian ECM were not expected to be cytotoxic, although some studies have shown that these materials, or their structural composition, can affect cell growth, proliferation and cell infiltration. Under certain conditions and preparation methods, chitosan has been shown to support poor cell growth with cells remaining rounded and clumping together [61; 62]. While mammalian ECM contains components that promote cell attachment, dense ECM scaffold materials such as multilaminated scaffolds, can slow cellular growth and inhibit cellular infiltration [56; 63; 64]. The neutral red uptake assay is designed to test cytotoxicity independently of the ability of the scaffolds to support cell growth as the scaffold materials are applied to the cells, rather than vice versa as is more commonly done. Interestingly, the 1% (_w/v_) and 10% (_w/v_) ECM formulations showed reduced cell viability with or without antibiotic loading, with the 10% (_w/v_) ECM scaffold meeting the criteria for being classified cytotoxic. Since ECM is not known to be cytotoxic, this was an unexpected result and one that requires further analysis. Endotoxin testing of the chitosan component of the scaffolds showed that the starting solution (100% chitosan) contained approximately 5 EU/mL (approximately 0.5 mg/mL). Since endotoxin activity depends on a variety of factors such as polysaccharide chain length, solubility and source, the possibility that endotoxin is the cause of the cytotoxicity cannot be ruled out. Studies of the effects of endotoxin generally use much higher endotoxin levels than those found in the present study, typically using levels in the microgram rather than nanogram range. [PERSON] et al. showed that 100 ug/mL were capable of altering the morphology and could reduce the proliferation rate of hybridoma cells by 62% [65]. [PERSON] et al. have compared the LAL endotoxin assay to a cell-based assay using microvascular endothelial cells. While they did not specifically look at viability of the cells, they did show that microvascular endothelial cellswere particularly sensitive to endotoxin, resulting in significant changes in e-selectin expression [66]. The neutral red uptake assay does have a high sensitivity that may lead to false positive results and the experiment must be carefully controlled using known standards for cytotoxicity. It is a possibility that since this scaffold formulation was shown to swell and lose integrity when hydrated, cell attachment to the scaffold or sloughing of the cell monolayer during scaffold removal may have occurred, reducing the cell density and therefore reducing neutral red uptake relative to the other materials. Specific cell attachment assays would help to understand this result and will be performed in the future. While the direct cytotoxicity assays showed that only the two scaffold formulations containing particulate ECM reduced neutral red uptake, the indirect cytotoxicity assays showed that neutral red uptake was reduced by all the supernatant samples of all the scaffold formulations.