# Micro-Structured Patches for Dermal Regeneration Obtained via Electrophoretic Replica Deposition
## 3 Results
### In Vivo Tests  
No postoperative adverse effects, such as infection, pyogenesis, and body fluid effusions, occurred in any animal: diet, drinking, and defecation were normal throughout the experiment. On day 0, a full thickness skin wound of \(30\times 30\) mm\({}^{2}\) was made in every group: Figure 3a shows the typical implant sites at day 0, immediately after full thickness skin wound (FTSW) preparation. Figure 3b-d show the appearance of the representative wound sites in the different groups at day 14: chitosan patches, regardless of their structure, promoted wound healing from day 1 to 14. On day 14, hairs were grown on the surrounding area of the un-epithelialized wound in all groups. To take photographs of the un-epithelialized wound area, the surrounding hairs were re-shaved to clearly show the wound margins. On day 14, the largest un-epithelialized wound was found in the CP1000 group, followed by the CPR and CP500 groups. A reduction in wound size has been observed for all the samples (Figure 3b-d): a significant difference has been observed between CPR samples (WR 89%) vs. non-treatment control condition (81%) and between CP500 (WR 87%) vs. non-treatment control condition (81%). No differences have been observed between CP1000 and non-treatment control condition of 81% in either sample. Histological staining of wound areas at day 14 is reported in Figure 4. On day 14, necrotic fibrinoid debris (FD), inflammatory infiltration, fibroblast, and capillary (CP, Figure 5) hyperproliferation were  
Figure 3: Wound sizes at (**a**) day 0 and (**b–d**) after 14 days of implantation. (**b**) CPR wound rate (WR) after 14 days, WR = 89%; (**c**) CP500 WR = 87%; (**d**) CP1000 WR = 81%; (**b–d**) specimens are compared with non-treatment condition (control), showing WR = 81%. Figure 2: Uniaxial tensile tests on flat and micro-patterned patches in wet conditions: (**a**) representative preconditiong cycle for CPR specimens, (**b**) tensile tests up to break, and (**c**) Young modulus (E). still observed in the CP1000 group, indicating a still persistent inflammation process for this group (Figure 4, CP1000). Wounds in CPR and CP500 groups demonstrated epithelialization (EP) without capillary hyperplasia. Compared with normal skin, more layers of keratinocytes were seen in CPR and CP500 groups, indicating the proliferating stage of keratinocytes in these two groups. A thin epidermal layer with several skin appendages, including hair follicles (HF) and sebaceous glands (SB), were shown in the CPR group as compared to CP500 and CP1000 groups (Figure 4). Moreover H&E and [PERSON]'s trichrome staining showed an important inflammatory infiltrate present only on CP1000, where a significant amorphous fibrotic mass is also present. Moreover, the extracellular matrix appears still not organized, while CPR showed granular and cornified layers and cutaneous annex formation (hair follicles, sebaceous glands), comparable with controls. CP500 also shows the epithelial layers and the presence of cutaneous annexes (Figure 4). Figure 4: Hematoxylin and eosin and [PERSON]’s trichrome staining in different groups on day 14. FD, fibrinous debris; EP, epithelialization; HF, hair follicle; SB, sebaceous gland. (Scale bar = 50 \(\upmu\)m.). Figure 5: Capillary formation in deep dermal layers of different implantation groups on day 14. (**a**) CPR; (**b**) CP500; (**c**) CP1000; (**d**) control. Capillary hyperplasia (arrows) was markedly observed in CP1000 group and in control group. Scale bar: 30 \(\upmu\)m.  
In addition, coarse bundles of collagen were observed in the dermis for CPR and CP500 groups, as stained with [PERSON]'s technique, whereas reticular fibers and thin collagen bundles were mostly observed for the CP1000 group, as shown by silver staining (SI3).