The experimental protocol used in the present study, including the use of animals, was approved by the Institutional Animal Care and Use Committee, Yonsei University Wonju College of Medicine (Wonju, Korea; approval no. YWC-160504-1). Male hairless mice (SHK1; n=32) aged 8 weeks and weighing 25–30 g were obtained from Orient Bio, Inc. (Seongnam, Korea). Mice were randomly divided into four groups: Control (n=8); low (low-PDRN; <50 kDa; n=8); middle (classic PDRN; 50–1,500 kDa; n=8); and high (high-PDRN; >1,500 kDa; n=8) molecular weight PDRNs. All PDRN products were obtained from Pharma Research Products Co., Ltd. (Pangyo, Korea). All animals were provided with standard laboratory food and water ad libitum. The mice were raised at a constant temperature (22±3°C) and relative humidity (50±10%) in a 12 h light/dark cycle. Wound production was performed under general anesthetic isoflurane inhalation. Under aseptic conditions, a 4 mm biopsy punch (Kai Europe GmbH, Solingen, Germany) was used to produce wounds in the skin of the mice. Following the procedure, mice were treated with a daily intraperitoneal (i.p.) administration of PDRNs at a dose of 8 mg/kg (day 0 to 6). The control group received i.p. injection of vehicle (0.9% NaCl) at the same volume and time as the treatment group.

A stable human fibroblast cell line (CCD-986SK; Korean Cell Line Bank, Seoul, Korea) was cultured in 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin and streptomycin in high glucose Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) in a 37°C humidified atmosphere of 5% CO₂.

The human fibroblast cells were seeded at 1×10⁶ cells/well in a 6-well plate and grown to full confluence. Cells were scratched with a 200 µl pipette tip across the center of the wells. In order to distinguish cell migration from proliferation, all wound-healing assays were performed with or without anti-tumor drug mitomycin C (cat. no. M4287; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) treatment, at a final concentration of 5 µg/ml. Images were captured using a light microscope (×100), 24 h after treatment at 37°C with the drug (time 0 was the initial time point). The healing area (%) was measured using Image J software version 1.8 (National Institutes of Health, Bethesda, MD, USA).

Cells were incubated in 1% FBS and 1% antibiotics in high glucose DMEM for 48 h, treated at 37°C with PDRNs (50 µg/ml) or EGF (30 ng/ml) for 24 h, and subsequently washed with PBS and lysed with radioimmunoprecipitation assay buffer (Intron Biotechnology, Inc., Seongnam, Korea) containing protease and phosphatase inhibitors (Roche Diagnostics, Basel, Switzerland). Extracts were isolated by centrifugation at 4°C and 12,000 × g for 15 min. Protein concentration was measured using the Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Protein (10 µg/lane) were loaded and electrophoresis was conducted on 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% bovine serum albumin (Bioshop Canada, Inc., Burlington, ON, Canada) in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. The blocked membranes were incubated with primary antibodies overnight at 4°C with agitation, followed by incubation with a secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:3,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature. The blots were visualized using the Chemiluminescence Western Blot Detection System (BioSpectrum^® 600 Imaging System; UVP, LLC, Upland, CA, USA). Primary antibodies against phosphorylated (p)-c-Jun N-terminal kinase (JNK; 1:1,000, cat. no. 9251S), total (t)-JNK (1:1,000; cat. no. 9252S), p-focal adhesion kinase (FAK; 1:1,000; cat. no. 8556S), t-FAK (1:1,000; cat. no. 13009S), p-extracellular signal-regulated kinase (ERK)1/2 (1:3,000; cat. no. 9101S) and t-ERK1/2 (1:3,000; cat. no. 9102S) were acquired from Cell Signaling Technology, Inc. (Danvers, MA, USA), and an antibody against α-tubulin (1:1,000; cat. no. sc-8035) was obtained from Santa Cruz Biotechnology, Inc.

For immunohistochemistry, mice were sacrificed at days 3 and 7 and tissues were harvested from normal and wounded areas. All tissues were fixed in 4% paraformaldehyde for at least 48 h at room temperature. Following fixation, tissues were dehydrated in graded ethanol from 70, 80, 90, 95 to 100%, cleared in xylene and embedded in paraffin. Sections (4 µm) were mounted on glass slides at 45°C, dewaxed using xylene, rehydrated with graded ethanol from 100, 95, 90, 80 and 70% to distilled water and stained at room temperature with hematoxylin and eosin (H&E) or Masson's Trichrome. As part of the histological evaluation, all slides were examined by a pathologist without knowledge of treatments under a light microscope with magnification, ×20-100. The area of collagen portion (%) was measured using Image J software.

All experiments were repeated at least three times. Data analysis was performed with the Prism software (version 5; GraphPad Software, Inc., La Jolla, CA, USA). All data are presented as the mean ± standard error of the mean. Statistical comparisons between two groups were determined using a two-tailed unpaired Student's t-test. Multiple comparisons were determined using one-way analysis of variance followed by Tukey's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

Multiple previous studies have demonstrated that PDRNs promote wound healing in various animal models (6,10,18–20). To evaluate the effect of PDRNs on wound healing, 8-week old hairless (SKH1) mice were given different sizes of PDRNs daily for a week. Based on previous results of in vivo pharmacological tests (21), 8 mg/kg doses of PDRNs were administered (6,19). To evaluate wound closure, images were taken of PDRN-treated mouse skin at different healing stages (days 3 and 7). Administration of classic PDRN demonstrated no significant differences in wound closure or wound area compared with low- and high-PDRNs (Fig. 1).

Although differences in PDRN molecular weight did not affect the apparent surface wound closure, it did affect the quality of wound repairing. To analyze wound healing quality following treatment with PDRNs, the tissue repair process was monitored by H&E staining. Among all the treatments, classic PDRN-treated mice demonstrated the best wound closure quality (Fig. 2). Although the wound closure was similar among the different groups, treatment with classic PDRN resulted in less lipid accumulation and a normal wound healing process. This result suggests that among the DNA mixtures, classic PDRN improved the quality of wound regeneration in mice the most.

The normal wound healing process primarily progresses based on the balance between collagen synthesis and collagen catabolism by matrix metalloproteinases (MMPs), and the adult wound bed predominantly contains type I collagen deposition during ECM remodeling (22). To determine whether classic PDRN affected collagen fiber accumulation, Masson's Trichrome staining was examined to analyze collagen composition. On day 3, the collagen portion was significantly increased in classic PDRN-treated mouse skin compared with skin treated with other sizes of PDRN (Fig. 3A and B). At day 7, the majority of the PDRN-treated groups demonstrated similar collagen composition (Fig. 3C). However, compared with the other groups, the classic PDRN-treated group demonstrated increased collagen production and a decreased wound area; whereas, the other groups exhibited blood clots and were still in the early stages of wound healing. This data strongly supports the hypothesis that classic PDRN facilitates accumulation of collagen in earlier wound healing stages compared with other DNA mixtures.

Although PDRN has been implicated in the process of repair and regeneration, the underlying mechanisms remain largely elusive. Wound healing involves cell migration and proliferation during wound closure. The mitogen-activated protein kinase (MAPK) pathway serves an important role in the regulation of cell migration and wound healing (23). Signaling by JNK is required for wounded epithelial cells to close the wound (24). Furthermore, extracellular signals stimulate cellular processes, including migration and proliferation, which are stimulated by FAK (25). To determine the mechanism underlying PDRN-mediated wound healing and determine whether it is mediated through the activation of FAK and JNK, FAK and JNK activation with vehicle and the different PDRN treatments (50 µg/ml) were examined (Fig. 4A). Classic PDRN induced the phosphorylation of JNK 24 h after treatment. FAK and ERK phosphorylation increased in the high and classic PDRN treatments compared with the vehicle treatment. However, the total forms additionally increased; FAK and ERK were not significantly activated by high and classic PDRN (Fig. 4B). These results suggest that classic PDRN activates JNK, which may be important for the classic PDRN-mediated wound healing process. To determine whether classic PDRN-induced wound closure was mediated by proliferation or migration, an in vitro scratch assay was conducted and subsequently incubated with EGF (a known promoter of wound healing), PDRNs or vehicle with mitomycin C, a known inhibitor of proliferation, for 24 h. Images were captured from the initial point of cell migration at 0 h and at a scratch after 18 h (Fig. 4C). In the presence of vehicle, the wound area gradually closed and the rate of wound closure was significantly increased with EGF or classic PDRN treatment, suggesting that classic PDRN accelerated wound closure in human fibroblasts (Fig. 4D). EGF increased cell proliferation significantly compared with vehicle without mitomycin C; whereas, treatment with PDRNs did not affect proliferation (data not shown). This result suggests that classic PDRN may promote wound healing by increasing cell migration to attempt closure of the wound bed in human fibroblasts.