Wound healing is a complex cascade of events which diminishes the

Wound healing is a complex cascade of events which diminishes the size of the wound and reestablishes tissue integrity. cell infiltrate and increase of apoptotic fibroblasts. Analysis of the present data suggests that SFRP1 may be partly responsible for the poorer healing performance of the palatal wounds compared with dermal wounds. Blocking SFRP1 results in improvement of palatal healing outcomes. palatal wound healing and defining whether the modulation of SFRP1 affects wound-healing outcomes. MATERIALS & METHODS Animals and Wound Models Fifty 8-week-old male CD-1 mice purchased from the Charles River Laboratories (Boston MA USA) were used as models for the comparison of dermal and palatal wounds. An additional 10 age-matched mice were used for the antibody-blocking experiment. All procedures involving animals were approved by the Institutional Animal Care and Use GANT 58 Committee at Boston University Medical Center. Mice were intraperitoneally anesthetized with a ketamine (80 mg/kg) and xylazine (10 mg/kg) mixture. A palatal excisional wound (2.0 mm) was placed anterior to the soft palate or a scalp excisional wound was placed at the midline between the ears for each mouse. Mice were killed after 0 3 7 10 and 14 days. Five mice group were used at each time point. Blocking SFRP1 with Anti-SFRP1 Antibody We used anti-SFRP1 antibody (Santa Cruz Biotechnology Santa Cruz CA USA) to block the SFRP1 expression in wounded palatal tissues. The control group received IgG (Santa Cruz Biotechnology Santa Cruz CA USA). Five mice wound group were used. One dose of anti-SFRP1 antibody (30 μg) or IgG (30 μg) was injected submucosally around the wounded area on 6.5 8 Klf6 and 9.5 days (a total dose of 90 μg of anti-SFRP1 antibody or IgG). Mice were killed on the 10th day. Specimen Preparation Following the animals’ death the calvarial or palatal bone with intact surrounding tissue was dissected and fixed in cold 4% paraformaldehyde for 24 hrs. After fixation the specimens were decalcified in cold Immunocal (Decal Corporation Congers NY USA) for 7 days with the solution changed every day. Cryostat sagittal sections were prepared at a thickness of 5 microns. Quantitative Histologic Analysis The distance between the edges of the epithelium and connective tissue of the wound the area of new connective tissue (defined as the new tissue formed between the wound edges) and the percentage of new connective tissue in the defect were measured in H&E-stained sections at the widest part of each wound with the use of Image-Pro Plus Version 4 software (Media Cybernetics Silver Spring MD USA). We quantified polymorphonuclear neutrophil (PMN) and mononuclear cell infiltrates by identifying their characteristic morphology at 400× magnification. We stained several serial sections GANT 58 adjacent to those H&E-stained with Ly6G for neutrophils and Moma-2 for macrophages at each time-point in both groups. Cell counts obtained with immunostaining or H&E were similar and the differences were not statistically significant. Detection of Apoptosis Apoptotic cells were detected by TUNEL assay by means of an GANT 58 cell death detection kit (Roche Diagnostics Indianapolis IN USA) according to the manufacturer’s instructions. At high magnification (400×) TUNEL-positive fibroblasts and inflammatory cells were identified by stringent morphologic characteristics quantified and presented as percentages of apoptotic cells relative to the total of cell counts in the same field of analysis. Immunohistochemistry Immunohistochemical staining was carried out as described previously (Han and Amar 2004 At high magnification GANT 58 (400×) SFRP1-positive fibroblasts were quantified; only spindle-shaped cells were counted. Statistical Analysis Student’s test was performed for statistical analyses. RESULTS Healing Responses in Palatal and Dermal Groups In the dermal group the epithelial gap was dramatically reduced and completely covered the wounds by day 10. In contrast intact epithelial coverage was not achieved by day 10 in the palatal group (Figs. 1A 1 Connective tissue edges were bridged faster in the dermal group than the palatal group (Figs. 1B 1 Connective tissue healing was complete in dermal wounds on day 10 and the amount of new connective tissue was 1.6-fold more than that of palatal wounds (Fig. 1C). By day 10.