We purchased Normal Human Primary Dermal Fibroblasts-Neonatal (HDF-n), isolated from human neonatal foreskin, from GIBCO® (Invitrogen, Carlsbad, CA, USA). We injected human fibroblasts into the bottom of cell-culture dishes and added Dulbecco's Modified Eagle's Medium (DMEM; Welgene, Gyeongsan, Korea) containing penicillin (100 IU/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum (GIBCO®). Then, we cultivated the dishes in an incubator with a 5% CO₂ atmosphere at 37℃.

HDF-n cells (5×10³ cells) were seeded into a microtiter plate with 96 wells and cultivated for 24 hours under standard cell culture conditions. We removed the media and rinsed the plates with Dulbecco's phosphate-buffered saline (DPBS). We then induced starvation and maintained it for 24 hours. Next, we added new media with PDRN (except for media supplements) into the plate and cultivated the cells for 24 hours. After this, we again removed media with PDRN and rinsed the plate with DPBS. To assess cell viability, we diluted the CCK-8 reaction solution with media with no added supplements at a ratio of 1:10, added 100 µl of the diluted solution into each well, incubated the cells for one hour, and measured absorbance at 450 nm. To assess cell proliferation, we measured cell viability at 24, 48, and 72 hours.

We seeded HDF-n cells in Ibidi™ chambers in 60 mm dishes at a density of 1.0×10⁴ cells/chamber and cultivated them for 24 hours under standard cell culture conditions. Then, we removed the chambers, rinsed them with DPBS, each plate added 25, 50, 100, and 200 µg/ml of PDRN with the media, and cultivated them under standard cell culture conditions. We observed cell migration with an optical microscope at each time point (0, 24, 48, and 72 hours).

Every animal experiment conducted for this study was approved by Chung-Ang University's Institutional Animal Care and Use Committee (approval number: 2014-00007). We purchased seven-week-old BKS.Cg-+Lepr^db/+Lepr^db and ^m+/+Lepr^db female mice strains from The Jackson Laboratory in Korea. Diabetic mutations are the result of spontaneous point mutations in the leptin receptor gene (Lepr). This mouse becomes obese after 3 to 4 weeks. The rise in plasma insulin begins at 10 to 14 days based on the information provided by the vendor, and blood glucose begins at 4 to 8 weeks. All mice were housed in personally air-conditioned animal rooms with standard environmental controls of 24℃±2℃ (temperature), 50%±10% (humidity), and a 12-hour day/night cycle. Animals were allowed free access to food and underwent a one-week stabilization period before testing.

Mice were anesthetized with Zoletil (30 mg/kg) and Rompun (10 mg/kg). The back of each mouse was divided into two areas according to the centerline along the back. Each area was inflicted with a full-thickness skin wound 4 mm in diameter using a biopsy punch (Kai Industries Co., Ltd., Tokyo, Japan). The size of a wound area was measured right after the wound was inflicted by a 4-mm biopsy punch, in consideration of skin contraction. The size of the wounds inflicted by a 4-mm biopsy punch was 12.56 mm², which is within one standard deviation of the average size of the wounds of all mice (12.83±0.83 mm²). As a result, each mouse had two wounds, one serving as the control and the other as the test. The PBS and PDRN group also had their wounds injected with 50 µl into the wound edges of each of the wound sites on each animal in groups of two, using a 0.5 ml syringe and a 28.5 G needle. Each wound was dressed, and PDRN were applied once a day. We observed mice for general symptoms once a day until the test was complete. Observations were made six hours after PDRN intradermal injection. To minimize contamination of the wounds and prevent the PDRN from migrating and dropping out, we used Tegaderm™ films (3M, St. Paul, MN, USA). When 90% of more of the wound was regenerated, the test was considered complete, and the mice were humanely euthanized.

We observed the surface change in the wounds of every mouse using a DSLR (D3200; Nikon, Tokyo, Japan) and Folliscope (LeedM, Seoul, Korea) at days 0, 3, 6, 9, and 12. We captured three-dimensional (3D) images of the wounds with a PRIMOS^LITE optical 3D skin measuring device (LMI Technologies GmbH, Teltow, Germany) and analyzed them with PRIMOS software version 5.7. We measured the size and depth of the wounds to assess the time required for wound healing.

After the mice were euthanized, we extracted skin tissue from the wounds and fixed them with 10% neutral-buffered formalin fixative. We embedded the tissues in molten paraffin wax and the embedded tissues were sectioned into 5-µm slices. We stained the slices with hematoxylin and eosin (H&E) to observe infiltration of inflammatory cells, location of the muscular layer, numbers of foreign body reactions and inflammatory cells, re-epithelialization, proliferation of fibroblasts, and formation of blood vessels. We used Masson's trichrome and van Gieson's staining protocols to determine whether collagen and elastic fibers were created at the wound site.

VEGF and CD31 immunostaining was performed using tissue samples collected from day 12 after the surgical procedure in order to investigate angiogenesis. Using immunohistochemical staining for VEGF and CD31, the slides from each group were assessed for intensity of expressed diaminobenzidine (DAB) chromogen. CD31 represents a highly specific marker for vascular and endothelial cells.

Paraffin-embedded tissues were sectioned (5 µm), and the slides were subjected to antigen retrieval, with endogenous peroxidase activity quenched using hydrogen peroxide. After blocking non-specific proteins with normal serum in PBS (0.1% Tween-20), slides were incubated with rabbit polyclonal antibodies against CD31 (ab28364; Abcam, Cambridge, UK) or VEGF (SC-152; Santa Cruz Biotechnology, Santa Cruz, CA) (both at a 1:50 dilution). After washing with PBS, slides were incubated with the appropriate biotinylated secondary antibody, followed by conjugated horseradish peroxidase-labeled streptavidin (Thermo Fisher, Waltham, MA, USA), and then washed with PBS. The slides were then incubated with DAB as the chromogen, followed by counterstaining with diluted Harris' H&E and mounting with coverslips. After staining, three high-power fields (×400) were randomly selected in each slide, and the average proportion of positive cells in each field was counted using the true color multi-functional cell image analysis management system (Image-Pro plus 7.0; Media Cybernetics Inc., Silver Spring, MD, USA). To rule out any non-specific staining, PBS was used instead of the primary antibody as a negative control.

Data are expressed as the mean plus or minus the standard error of the mean (mean±SEM), and statistical tables in this document contain figures as well as the mean of each group for each statistical period. Comparisons between the control group and the test group were carried out using PASW Statistics ver. 18.0 (IBM Corp., Armonk, NY, USA). The Shapiro–Wilk test was used to test whether normal distributions were present in the data. Probability errors were measured by the Kruskal–Wallis one-way analysis of variance (ANOVA) on ranks. In all cases, a probability error <0.05 was selected as the criterion for statistical significance. When comparisons with the control group were made, the Mann–Whitney U test and Student's t-test using independent samples were used. Probability errors are expressed as follows: p<0.05, p<0.01, p<0.001.

We measured the cytotoxicity of the PDRN using CCK-8 reaction solution, which can determine the viability of cells, in human dermal fibroblast neonatal (HDF-n) cells. The percentage of viable cells was expressed as a proportion of control cell viability, which was set to 100%. The control group was comprised of untreated HDF-n cells, while the other groups consisted of HDF-n cells treated with PDRN at a concentration of 12.5, 25, 50, 100, or 200 µg/ml. To assay cell proliferation in the cells treated with PDRN, we first determined the concentration of PDRN at which no cytotoxicity was observed and then measured cell proliferation in cells treated with that concentration after 48 and 72 hours. When treated with PDRN at concentrations of 12.5, 25, 50, 100, and 200 µg/ml, the number of HDF-n cells increased by 98.4%, 105.4%, 112.6%, 125.2%, and 130.1%, respectively, 48 hours after treatment and by 100.5%, 103.7%, 107.8%, 117.8%, and 120.4% 72 hours after treatment (Fig. 1). We observed migration of HDF-n cells using an optical microscope at each time point (0, 24, 48, and 72 hours). Compared with control cells, the space between sides of the scratched wound became narrower after 48 hours in cells treated with PDRN, but this difference was not statistically significant. Together, these results show that PDRN has no cytotoxic effect and enhances cell proliferation.

We conducted a visual inspection to assess the progress of wound closure. There was no significant difference between the PBS-injected group and the PDRN-injected group in the ^m+/+Lepr^db mice (hetero type) however +Lepr^db/+Lepr^db mice (db/db) showed significant increase in granulation tissue and capillary blood vessels in the PDRN injected group compared to PBS injected group (Fig. 2). At the same time as the visual inspection, we also measured and analyzed the size and depth of wounds and the associated decreases (Fig. 3). As for the ^m+/+Lepr^db mice, those injected with PBS and those with PDRN showed no significant differences in terms of the size of wound areas and wound depth. However, for +Lepr^db/+Lepr^db mice, those injected with PDRN showed a significant decrease in wound depth compared with those injected with PBS at day 6 (40.33%±3.61%, p<0.05) and day 9 (22.75%±1.95%, p<0.05).

H&E revealed that the wounds were recovered in order of PDRN injected group, PBS injected group in the ^m+/+Lepr^db mice and PDRN injected group, PBS injected group in the +Lepr^db/+Lepr^db mice in the order of date (Fig. 4). In the ^m+/+Lepr^db mice group, the difference between PBS injected group and PDRN injected group was not significant in visual examination, but tissue evaluation showed a clear difference between PBS injected group and PDRN injected group. The proliferation of fibroblasts was promoted at the wound site, the epidermis with re-epithelialization thickened, and the wound quickly closed. Day 12 after wound, hair follicles were formed normally, and skin thickness was restored normally in the ^m+/+Lepr^db mice. Thus, in the +Lepr^db/+Lepr^db mice, the proliferation of fibroblasts and keratinocytes in the PDRN-injected group compared to the PBS-injected group was confirmed to be rapidly restored.

In the hetero-type ^m+/+Lepr^db mice, those injected with PBS and those with PDRN showed no significant histological differences (Fig. 5). However, for diabetic +Lepr^db/+Lepr^db mice, those injected with PDRN showed epithelialization of the epidermis around the wound. In addition, since collagen fibers (the main component of the extracellular matrix) grew relatively quickly and new capillary networks were formed and expanded, tissues at the wound site rapidly achieved a normal morphology.

When the intensity of DAB in the VEGF antibody was analyzed, the hetero-type ^m+/+Lepr^db mice, those PBS injected group was 38.52±0.60%, PDRN injected group was 67.21%±4.21%, and diabatic +Lepr^db/+Lepr^db mice, those PBS injected group was 24.38%±1.55%, PDRN injected group was 32.57%±1.40%. Both hetero-type and homozygous types mice showed a significant increase (p<0.01) in PDRN injected group compared to PBS injected group. Also, when the intensity of DAB in the CD31 antibody was analyzed, the hetero-type ^m+/+Lepr^db mice, those PBS injected group was 47.36%±0.68%, PDRN injected group was 58.051%±2.68%, and diabatic +Lepr^db/+Lepr^db mice, those PBS injected group was 8.98%±1.60%, PDRN injected group was 13.96%±0.64%. Both hetero-type and homozygous types mice showed a significant increase (p<0.01) in PDRN injected group compared to PBS injected group (Fig. 6).