Tissue-engineered skin represents a useful strategy for the treatment of deep skin injuries and might contribute to the understanding of skin regeneration. less scarring and wound contraction. Oddly enough, only DPC-containing constructs showed embryonic hair bud-like structures with cells of human source, presence of precursor epithelial cells, and manifestation of a hair differentiation marker. Although initial, these findings have exhibited the importance of the presence of DPCs for proper skin repair. = 6 per group) after being anesthetized with ketamine/xylazine (110 mg/10 mg per kilogram body excess weight). No shaving in the surgical area was necessary because nude mice were used. The air-liquid interphase skin constructs were grafted, locating the graft borders between the hypodermis and muscle-fascia without any type of surgical suture. The grafts were only coated by an auto-adhesive polyurethane transparent bandage (Tegaderm, 3M, St. Paul, MN, http://www.3m.com) that allows gas exchange and avoids fluid loss. A control group was grafted with ADM alone. In all cases, three impartial grafting assays were performed. In each, six animals per group for each time point were grafted with the different types of constructs. The mice whose grafts were lost or experienced relocated from their initial place P005672 HCl were discarded for statistical analysis (one in ADM control group at the 14-day point; two each in the HFSC-DF and HFSC-DPC groups at the 70-day point). At the indicated time points (14, 30, and 70 days), the grafts were photographed, and the mice were sacrificed. The grafted skin constructs, including the rodent skin border, were dissected and fixed in formaldehyde and paraffin-embedded for histological and immunohistochemical analysis. The skin contraction ratio (SCR) of the hurt and grafted area was P005672 HCl calculated as follows: SCR = 1 ? (RLA/OLA), with RLA indicating the remaining lesion area 70 days after grafting and OLA, the initial lesion area. Histological and Immunohistochemical Analysis Paraffin-embedded histological photo slides from in vitro and in vivo assays were stained with hematoxylin and eosin for tissue architecture analysis. The number of epidermal layers was evaluated in 10 fields of 400 magnification, and epidermal invaginations were evaluated in six 100 fields for each construct. Immunohistochemical assays were performed with anti-p63 antibody (mouse monoclonal antibody IgG2a, Santa Cruz Biotechnology Inc., Dallas, TX, http://www.scbt.com) for epidermal stem cell detection, anti-murine AWS CD34 antibody (mouse monoclonal antibody IgG2a, Abcam, Cambridge, U.K., http://www.abcam.com) for neovessel detection in the grafting experiments, anti-human leukocyte antigen type I (HLA I) ABC (mouse monoclonal antibody IgG2a, Abcam) for detection of human cells in the grafted mice, and anti-k6hf (polyclonal guinea pig antibody, Progen Biotechnik GmbH, Heidelberg, Philippines, http://www.progen.de) for detection of hair committed cells in mouse skin injured areas grafted with the constructs containing DPCs and HFSCs. Main antibodies were developed using Universal LSAB kit (Dako, Glostrup, Denmark, http://www.dako.com) according to the manufacturers recommendations. Positive epidermal cells for p63 in the in P005672 HCl vitro and in vivo constructs were evaluated in ten 1,000 and five 400 fields, respectively, and were normalized to the total number of epidermal cells in each field. Neovessels in grafted constructs were evaluated by CD34 immunostaining in five 400 fields and normalized to the dermis surface (number of blood vessels per 0.01 mm2) in each field. All the results shown in bar graphs represent the mean P005672 HCl values SD from three impartial experiments. Statistical Analysis One-way analysis of variance to evaluate statistical.