# Micro-Structured Patches for Dermal Regeneration Obtained via Electrophoretic Replica Deposition
## 4 Discussion
In contrast, thinner scaffolds, especially those with a thickness of less than 0.5 mm, promote earlier angiogenesis, ensuring skin-graft viability with a mild FBR, ordered fibroblast infiltration, and better collagen remodeling [12; 34]. It is worth mentioning that the application of nanofibrous mats with a thickness of 500 \(\upmu\)m can accelerate wound healing by around one week in rats [35]. Finally, the EPrD technique allowed us to obtain structures showing mechanical properties suitable to support protective functions and maintain the elastic properties of the skin during wound healing, with elastic behavior and a strain at break of up to 40%. The Young's modulus of the samples was comparable to that found by [PERSON] et al. [34] and in the range of elastic modulus in normal human skin (E = 0.2-20 MPa), indicating the acceptable mechanical strength and elasticity of these patches and making them suitable for in vivo grafting [36]. Altogether, these results support the use of the obtained chitosan  
Figure 7: Scheme of the electrophoretic replica deposition process (EPrD). patches as dermal templates for the treatment of skin defects using a one-stage grafting procedure in a full thickness wound skin model in rats. Previous studies demonstrated that CS has an anti-inflammatory and antimicrobial nature [14; 37] and can accelerate wound healing [38]. Some authors found that chitosan promotes the contraction and epithelialization of wounds by means of facilitating the migration and proliferation of epithelial cells and myofiboblasts [38]. In this work, the efficacy of chitosan patches in large wound sites has been assessed in vivo. Ethanol disinfection was used prior to implantation. Disinfection techniques, particularly the use ethanol, are widely applied in the development of novel biomaterials prior to in vitro testing [39]. Ethanol solutions (60-90% in water) are known to possess bactericidal properties [39]. Some authors define the ethanol soaking of scaffolds as a sterilization technique [40], even though there are concerns about the efficacy of ethanol disinfection for implanted biomaterials [41]. However, ethanol solutions are used by some authors prior to in vivo implantation when other sterilization techniques can alter the physical or chemical structure of the scaffolds or their degradation profiles [12; 41; 42]. In our study, no postoperative adverse effects such as infection, pyogenesis, and body fluid effusion occurred in any animals throughout the experimental period. More importantly, it is worth noting that the FTWS of 900 mm\({}^{2}\) reported here is higher than in previous studies on chitosan showing re-epithelialization at 14 days, with an FTWS ranging from 20 mm\({}^{2}\) to 150 mm\({}^{2}\)[43; 44; 45; 46]. In all cases, the wound sites were significantly lower than the case reported here. Histological sections found a thin epidermal layer similar to normal skin. More skin appendages, including hair follicles (HF) and sebaceous glands (SB), were formed in the CPR group as compared to CP500 and CP1000 groups for 14 days. Immunohistochemistry demonstrated that keratin 10 (CK10) was uniformly expressed in the epithelium of the CP1000 group, whereas it was mostly expressed in the basal and suprabasal layers of CPR and CP500 groups, like normal skin. The wound contraction observed 14 days after implantation seemed to be very high for all types of patches, especially CPR and CP500, considering that the initial scar dimensions were higher than those presented in many other studies [44; 45; 47]. Random structure (CPR) and hierarchical structure with lower dimensions (CP500) seem to better support the migration and proliferation of fibroblasts from peripheral zones in proximity to the wound as compared to chitosan porous patches with larger channels (CP1000). Both the pore size and the structure of the scaffold are key points influencing cell interaction and migration.