Male Sprague-Dawley rats, 240-260 g, were obtained from the laboratory animal center of Seoul National University (Seoul, Korea). The rats were kept in a colony room with an ambient temperature of 22℃ and a 12 h light-dark cycle (7 : 00 AM onset). Food and water were provided ad libitum. The Animal Care and Use Committee at Seoul National University approved all of the methods used in this study.

The rhEGF was obtained from the biotechnology department of Daewoong Pharmaceutical Company (Korea). The drug formulation was prepared by the manufacturer at a dose of 10 µg rhEGF/g of ointment. The ointment base was formulated with 80% vaseline and 20% liquid paraffin. The rhEGF dose (10 µg/g) was selected according to the therapeutic guidelines of the manufacturer and a previous report [5].

The rats were anesthetized using an intraperitoneal injection of 4% chloral hydrate (1 ml/100 g body weight), and the dorsal hair of the rat was shaved with clippers, and disinfected with 70% ethanol and betadine. Two full-thickness wounds of 2 cm in diameter were marked using a template and the tissue was excised to the level of the panniculus carnosus using dissecting scissors and forceps. The rhEGF ointment (n = 7) or the ointment base (n = 7) was administered topically to the animals every 12 h for 14 days beginning on the day of the incision. It was reported that the interaction between rhEGF and its receptor has to be maintained for 10-12 h in order to achieve an effective cellular response in terms of better-organized granulation tissue, a greater DNA and protein content, and a higher rate of cell proliferation [5]. The animals were housed individually throughout the experimental period.

The wound-healing curve and half healing time (HT₅₀) were determined by drawing the wound margin daily with tracing film. The labeled film was scanned, and the wound area was calculated using image analysis software (Metamorph, USA). The rate of wound healing is expressed as the percentage area remaining. The Residual Wound Area (%) = [R_(2~12)/R₍₁₎] × 100, where R₍₁₎ and R_(2~12) denote the remaining area at postoperative days 1 and 2~12, respectively. The wound-healing curve was obtained using the Boltzman equation, and the HT₅₀ value was calculated.

This study was performed in a separated group [rhEGF treatment group (n = 6) and control (ointment base treatment) group (n = 6)]. However, all experimental procedures were same as for the previous wound closure measurements. On the postoperative 7th day, a 0.5 cm × 2.5 cm skin tissue sample was harvested from the central regions of the wound, fixed in 10% neutral formalin, and embedded in paraffin. Five mm thick sections were cut and mounted on poly-lysin coated slides. The sections were incubated at 43℃ for 4 h, deparaffinized in xylene, and hydrated in a graded series of ethanol. The proliferative cells in the wound area were identified in four tissue sections per skin sample using a monoclonal antibody for the proliferating cell nuclear antigen (PCNA). The deparaffinized section was incubated with 0.3% hydrogen peroxide in PBS, and preblocked with 1% normal rabbit serum and 0.3% triton X-100 in PBS. The sections were incubated overnight with the monoclonal mouse anti-PCNA antibody (1 : 100; Dako, USA) at 4℃, and the sections were incubated with biotinylated rabbit anti-mouse IgG (1 : 200; Vector, USA) for 1 h at room temperature. After 3 washes, the tissue sections were processed using the avidin biotin (ABC) method (Vector, USA), as described elsewhere [11]. Finally, the PCNA immunoreactive neurons were visualized using a 3,3'-diamino-benzidine reaction with 0.2% nickel chloride intensification, which produced black labeled neuronal nuclei. The myofibroblasts were also evaluated by measuring the alpha-smooth muscle actin (α-SMA) immunoreactivity in four tissue sections per sample. The sections were stained with monoclonal mouse anti α-SMA (1 : 100; Oncogene, USA) overnight at 4℃, which was followed by incubation with Cy3-conjugated anti-mouse IgG (1 : 100; Jackson, USA) for 1 h at room temperature. Each slide was observed by optical microscopy with the image being displayed on a monitor screen via a CCD camera (Micromax Kodak1317; Princeton, USA) connected to a computer-assisted image analysis system (Metamorph; Universal Imaging, USA). The number of PCNA-positive epidermal cells and immunoreactive areas of the α-SMA positive myofibroblasts at the epidermal basement membrane zone were measured in three different areas (300 µm² dimension) at both ends as well as in the middle, with the mean value being recorded.

The skin from the post 7day wound was homogenized in a lysis buffer [50 mM Tris at pH 8.0, 150 mM NaCl, 0.02% sodium azide, 1% sodium dodecyl sulfate (SDS), 100 µg/ml phenylmethylsulfonylfluoride (PMSF), 1 µg/ml of aprotinin, 1% igagel 630 (Sigma-Aldrich, USA), and 0.5% deoxycholate] and centrifuged at 14,000 × g for 30 min at 4℃. The protein concentration was determined using a Bradford analysis kit (Bio-Rad, USA). The total protein was prepared by boiling the samples for 10 minutes in a SDS sample buffer [50 mM Tris (pH 6.8), 100 mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol]. Equal amounts of the protein were separated on 12% SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia, UK). The membrane was blocked for 1 h at room temperature with a blocking buffer (5% of non-fat dried milk and 0.1% of BSA in PBS buffer containing 0.1% Tween 20). The membrane was then incubated for 2 h at room temperature with 2.5 µg/ml of the anti-prolyl 4-hydroxylase antibody (Chemicon, USA). After washing with PBS, the membrane was reincubated with a horseradish peroxidase labeled secondary antibody and visualized using a Westzol enhanced chemiluminescence (ECL) detection kit (Intron, Korea). The bands were detected with LAS-3000 (Fujifilm, Japan). The prolyl 4-hydroxylase bands were scanned and quantified using the Image J program (v 1.29, NIH, USA).

All values are expressed as the mean ± SE. Statistical significance in the wound healing time was assessed using the Bonferroni-test. Unpaired t-tests were used to determine the probability values between the EGF treated group and the control group. A value of p < 0.05 was considered significant.