## 4 Discussion
Support for this strategy came from few studies that reported use of peracetic acid-ethanol solution for sterilizing bone allografts [37; 38]. Even though peracetic acid itself was sufficient for achieving sterilization, additional steps--application of negative pressure (200 mbar); addition of ethanol (approximately 24%) and gentle agitation were required to ensure predictable sterilization throughout the thickness of the allografts. We hypothesized that ethanol and peracetic acid being similar sized molecules and with ethanol possessing better wetting capabilities, PA-treatment followed by ethanol wash may result in effective desorption of PA residues. We confirmed the effectiveness of ethanol wash in additional experiments on solid, non-porous, poly-ether-ether ketone (PEEK) surfaces and MG-63 cells (data not shown). While observing cytotoxicity in PA-ePCL scaffolds, we recognized that effect of PA cannot be generalized and may depend on substrate chemical composition and architecture. The evidence came from a recently published study, which reported no cytotoxicity following PA treatment of decellularized cadaveric tendons. [39] While differences in terms of available surface area and post-PA alkali neutralization could have accounted for different observations, it provided an impetus to look into an alternate scaffold to compare. The decision to choose Alvetex was because it was commercially available, made from polystyrene, generated by high internal phase emulsion and has porosity \(>\)90% with regular (\(\sim\)40 um) interconnected pores [40]. We reasoned direct comparison of commercially available Alvetex and in-house generated ePCL would allow identify if the effect of PA would be affected by scaffold composition, geometry and architecture. While Alvetex was superior to ePCL in terms of cell proliferation both short and long term, the more interesting finding was the improved performance of Alvetex scaffolds that were treated with PA indicating some favorable surface changes induced on polystyrene surfaces. The mechanisms underlying these changes are currently being investigated. Contact angle measurements done on Alvetex scaffolds following different treatments revealed no significant differences between conventional ethanol treatment (manufacturer recommendation) and PA treatment (data not shown). These observations suggest that parameters other than scaffold hydrophilicity are responsible for the favorable cell response. Among the limitations of the current study are use of two time points to assess cell proliferation. In the context of tissue engineering, a longer time point would prove beneficial. This would be possible with the use of bioreactors. We used scanning electron micrographs to provide a visual representation of cell-scaffold interactions at microscopic level. However, use of more sophisticated confocal microscopy and molecular markers of adhesion and spreading (f-Actin, vinculin, integrins etc.) would provide more insights into cell behavior. Overall, the data from the current study has demonstrated that peracetic acid can be a viable sterilant for polymeric tissue engineering scaffold especially after verifying if the cytotoxic adsorbed residuals are removed. PA poses an interesting challenge because its adsorption profile was different in ePCL and polystyrene. Possible explanations could be differences in polymer chemistry, fabrication technique (including solvents used), scaffold architecture (porosity and available surface area) as well as additional post fabrication steps. Furthermore, by mechanisms that are not clear, PA treatment also positively influences cell proliferation in Alvetex. Importantly, we have developed a reliable strategy to reverse the effects of PA treatment using ethanol. One of the important issues to consider in developing polymeric scaffolds for clinical application is the effect of sterilization on degradation kinetics. Since most polymers undergo hydrolysis (enzymatic or non-enzymatic) upon implantation, when implanted, this needs to be investigated further.