# Micro-Structured Patches for Dermal Regeneration Obtained via Electrophoretic Replica Deposition
## Materials and Methods
### Methods
#### 2.2.2 In Vivo Characterization  
A full thickness skin wound (FTSW) animal model was tested in rats: all protocols were performed according to the Ethics Approval Committee of Ahvaz Jundishapur University of Medical Sciences (1394/657). The back areas of the rats were shaved and swabed with 70% ethanol solution, under general anesthesia by ketamine/xylazine. A sterile square template (30 \(\times\) 30 mm\({}^{2}\)) was placed on the skin, and its boundaries were traced using a sterile fine felt-tipped pen. Then, an FTSW of the same size was made by excising the skin within the confines of the square down to the level of the subcutaneous panniculus carnosus. The wounds were then subjected to four different treatments (\(n\) = 5 for each group): (1) implantation of patches with flat morphology (CPR); (2) implantation of patches with 500 \(\upmu\)m pore size (CP500); (3) implantation of patches with 1 mm pore size (CP1000); (4) untreated wounds (control). Patches were disinfected/sterilized by immersion in a 70% ethanol solution for 20 min. Patch skin grafts were then sutured over the wound and the wound site covered with a sterilized gauze and a biocompatible adhesive, LEUKOLFAST(r) ELASTOMUL(r) HAFT glue, was used. After the surgery, each animal was maintained in a cage: postsurgical care included analgesic and antibiotic injections. The appearance of any post-surgery complication was checked daily after surgery. After 14 days, rats in each group were euthanized by excessive dose of ketamine/xylazine and the reconstituted skin was evaluated.  
Digital photographs of wound sites were acquired postoperatively on day 14 by a 10-megapixel digital camera (Panasonic DMC-FP3, Osaka, Japan) and analyzed with image software (Image J, [[http://rsb.info.nih.gov/ij/](http://rsb.info.nih.gov/ij/)]([http://rsb.info.nih.gov/ij/](http://rsb.info.nih.gov/ij/))) by two blinded observers. In order to determine the differences in healing rate among the 4 groups, the wound closure percentage, \(Wr\) (%), was evaluated according to Equation (1):  
\[Wr(\%)=100*\left(1-\frac{A_{14}}{A_{0}}\right) \tag{1}\]  
where \(A_{0}\) is the wound area on postoperative day and \(A_{14}\) is the wound area after 14 days.  
The reconstituted skin of all groups was excised to the level of panniculus carnosus layer, fixed in 10% formaldehyde, dehydrated, and then paraffin-embedded. Serial 10 \(\upmu\)m paraffin sections were cut with a rotating microtome and stained with hematoxylin and eosin (H&E, kit from Asiapajouhesh, Tehran, Iran) and [PERSON]'s trichrome staining (Asiapajouhesh, Tehran, Iran), according to routine histological protocols.  
The full thickness of the reconstituted skin was excised and fixed with 2.5% formaldehyde solution for 1 h to detect the expression level of keratin 10 (CK10) by immunohistochemistry. After dehydration by graded ethanol series (from 30% to 100%), the sample was embedded in a paraffin block to prepare tissue sections. Immunohistochemistry biochemistry was performed according to the manufacturer's instructions. Briefly, a monoclonal anti-keratin 10 antibody (mouse IgG) (Abcam Inc., Cambridge, MA, USA) was incubated for 2 h and a secondary goat anti-mouse antibody (IgG) (1/1000) was subsequently incubated for 10 min. After extensive washes, the sample was incubated with 3,3'- diaminobenzzidine until a brownish color appeared. The sample was counterstainedined with hematoxylin for 10 min.