## 4 Discussion  
In addition to being non-toxic, tissue-engineering scaffolds need to be biologically active; integrate with the tissues, undergo remodeling and be eventually replaced by the host tissue. In order for these events to occur, it is imperative that the scaffolds act as a favorable substrate that support appropriate cell activity, including attachment, migration and proliferation through the thickness of the scaffold. PCL is aliphatic polyester, readily soluble in a variety of solvents used in manufacturing. Further, being a synthetic polymer, the degradation rates and mechanical properties can be readily controlled. The fact that a number of drug-delivery devices fabricated with PCL already have FDA approval makes this polymer really attractive for tissue engineering purposes [33]. Irrespective of its biomaterial, fabrication technique or its intended clinical application any implanted biomaterial must be sterile prior to implantation. Even though a variety of physical (autoclaving, dry heat and irradiation) or chemical (ethylene oxide gas or peracetic acid liquid) methods exist, our lab has reported conventional sterilization techniques (including EtO) induced PCL scaffold degradation, while peracetic acid treatment (1000 ppm for 15 min at room temperature) effectively sterilized the scaffolds without adversely affecting its properties. Since PA is an equilibrium mixture of acetic acid, hydrogen peroxide and water, presence of toxic residuals within the biomaterial can contribute to cytotoxicity and hence remains a concern. Given the efficacy of PA in sterilizing polymeric scaffolds at room temperature, the focus of this study was  
Figure 8: Differential effects of PAA on various substrates. Data is presented as a ratio of optical densities from day 7 to day 1 to account for significant differences in initial (day 1) cell attachment on different surfaces. Since each scaffold serves as its internal control, this approach allows comparison of cell proliferation across scaffolds. While ES+PA+ EtOH supported early cell survival and limited cell proliferation, it was significantly lower than Ctrl ES. PA+ Alvetex showed maximal cell proliferation, which decreased following ethanol wash.  
to address the issue of cytotoxicity after PA treatment. Our cytotoxicity results in ePCL is consistent with the findings of [PERSON] et al. [34], who reported significant cytotoxicity associated with low temperature hydrogen peroxide gas plasma treatment of PCL scaffolds. The authors suggested that the leaching of residual hydrogen peroxide from within or upon the porous scaffolds could explain observed cytotoxic effects. Other studies have reported adsorption of known chemical and gas sterilants (hydrogen peroxide and EtO) onto polymers with some polymer absorbing considerable amounts of H\({}_{2}\)O\({}_{2}\) in less than an hour. [32; 35] Studies exist in the literature regarding use of STS and catalase to remove residual concentrations of PA. [36]. [PERSON] et al. demonstrated that desorption kinetics from polymers exposed to hydrogen peroxide showed a strong dependence on the material composition and temperature [29]. These studies formed the basis for the physico-chemical methods we employed to desorb PA residues from electrospun scaffolds in this study. Since conventional processing of scaffolds prior to experiments is to treat them with ethanol followed by 3 washes with DI/ PBS 10 min per wash and the solutions being changed for every wash, we started off with this strategy. Extensive washing (irrespective of volume or the number of times solutions were changed) or total incubation time (up to 2 days) were any more effective than washing for 30 min. Looking at the minimal effect of washing on removing PA residuals, we turned our attention to using published physical and chemical methods of neutralizing PA residuals using STS, catalase and aeration. With limited success, we chose to investigate the effect of ethanol wash on PA-ePCL scaffolds.