INTRODUCTION
Wound healing occurs through multiple stages of coagulation, inflammation, proliferation, and remodeling, which are accompanied by re-epithelialization, contraction, the formation of granulation tissue, and collagen synthesis [
1-
4]. Currently, various dressings and treatment methods utilizing diverse substances have been developed in an attempt to treat excisional wounds of the skin.
Human placenta-derived natural substances, such as hormones and glycosaminoglycan, are used to treat various diseases and conditions. Many biochemicals have been developed as synthetic drugs. Of interest, studies have reported that DNA fragments extracted from human placentas can play positive roles in various diseases, with particular benefits for tissue regeneration. Subsequent studies identified polydeoxyribonucleotide (PDRN), which regulates cytokines and growth factors, and novel extraction methods have been devised to extract PDRN from natural substances. PDRN contains deoxyribonucleotide polymers and is a DNA fragment of 50–2,000 bases. It initiates intracellular signaling through A2 purinergic receptors, which are a type of G-protein coupled receptors. Numerous studies have shown that PDRN also serves as an energy source, thus influencing cellular growth and differentiation. The activation of A2 receptors is known to be associated with tissue regeneration and anti-inflammatory effects, and is recognized to promote wound healing by stimulating tissue repair. Although PDRN can be extracted from human placentas, PDRN medications have recently been extracted from the semen of trout (
Oncorhynchus mykiss) and salmon (
Oncorhynchus keta ) [
5,
6]. Such medications are obtained through high-temperature extraction and are characterized by a purity >95% without any additional pharmacologically-active proteins or peptides (Registration Dossier, Italian Ministry of Health) [
7]. In terms of the chemical structure, PDRN is composed of low-molecular weight DNA and is a linear polymer comprising 50–2,000 base pairs. PDRN is degraded by active membrane enzymes to serve as a source of purine and pyrimidine deoxynucleosides and deoxyribonucleotides, which increase cellular proliferation and activity in different tissues [
8-
12].
A PDRN injection can be used in the clinical setting to treat wounds, including diabetic foot ulcers. This treatment is already being prepared from the semen of
O. mykiss , and is imported from Italy (Placentex Integro, Mastelli, Sanremo, Italy). In South Korea, PDRN has been extracted and purified from the semen of chum salmon (
O. keta), which belongs to the same species as
O. mykiss and returns to South Korea for spawning; the equivalence of PDRN from difference sources has been confirmed [
9,
13]. Further, PDRN for injection (Rejuvenex, Pharma-Research, Seongnam, Korea) and a PDRN cream (Rejuvenex Cream, Pharma-Research) have been developed from the semen of
O. keta .
In order to provide basic data for the development of new O. keta-derived PDRN products for wound healing, the present study was designed to evaluate the wound healing effects of O. keta-derived PDRN for injection (Rejuvenex) and PDRN cream (Rejuvenex Cream) in comparison with those of O. mykiss-derived PDRN injection (Placentex). For this purpose, full-thickness skin defects were created in white mice, and the effects of different PDRN-based treatments were evaluated.
DISCUSSION
Various dressings are being used to treat excisional wounds of the skin caused by trauma or burns. Recently, dressings that utilize growth factors, including PDGF (becaplermin), epidermal growth factor, and basic fibroblast growth factor, have been developed and used. Multiple studies have reported therapeutic roles for adenosine and A2A receptor agonists in promoting wound healing [
14-
16]. Adenosine A2A receptors, belonging to the P1 subtype of purinergic receptors, are expressed in most cell types involved in wound healing, including macrophages, fibroblasts, and endothelial cells. In these cells, activation of A2A reduces inflammation, promotes the proliferation of endothelial cells and fibroblasts, increases collagen synthesis and the proportion of granulation tissue, and stimulates the secretion of VEGF, ultimately promoting wound healing [
10,
12,
17,
18].
PDRN, an A2A receptor agonist, is a polymer in which purine and pyrimidine nucleotides have bonded together through phosphodiester bonds, and is reported to mostly influence nucleotides. Such nucleotides and nucleosides promote the growth of various cell types while also stimulating the synthesis of nucleic acids and wound healing, by stimulating salvage pathways, which produce nucleic acids at a low energy expenditure, and by activating A2A purinergic receptors [
18,
19].
Studies investigating the safety of PDRN, particularly its acute and chronic toxicity, have confirmed through gross and histological examinations that PDRN does not result in any mortality or have any toxic effect on the liver, lungs, brain, skeletal muscles, or heart [
11,
20]. The
O. mykiss-derived PDRN injection used in the present study has been clinically approved and is used in Italy as a local and non-oral drug [
6]. Several studies have reported that the injection of PDRN promotes the proliferation of human fibroblasts and osteoblasts in in vitro models [
8,
9,
12]. In animal experiments, intraperitoneal injections of PDRN were observed to have beneficial effects in a rat model of ischemic skin flaps [
21]. Moreover, intraperitoneal injections of PDRN were also used in diabetic mice to stimulate angiogenesis and promote wound healing [
10]. In addition, in mice with deep dermal second-degree burns, intraperitoneal PDRN injections were reported to improve wound healing [
11]. Clinically, wound healing was promoted when PDRN was injected into the muscle and tissues surrounding wounds at skin transplantation donor sites [
22]. In patients with pressure sores, injecting PDRN into the muscle and tissues around the sore was reported to promote healing and to decrease the need for additional treatment as well as the length of the hospital stay [
20].
The present study compared the treatment effects of O. mykiss-derived PDRN injection, O. keta-derived PDRN injection, O. keta-derived PDRN cream, and 0.9% normal saline soaked dressing on a full-thickness skin defect wound on the backs of ICR mice. The changes in the wounds were observed through gross examination and biopsy after 4, 7, and 10 days. In particular, tissue staining (hematoxylin and eosin staining, Masson trichrome staining, and elastin staining), immunostaining (p63 and CD31 stain), and ELISA (VEGF and TGF-β tests) were conducted on the biopsy samples, and the results were analyzed.
According to the gross examinations and an evaluation of the changes in wound sizes, there was no delayed response in wound healing, including skin necrosis, inflammation, or other adverse reactions, observed in any of the groups. However, considering that Tegaderm was fixed on the wound immediately after it creation in order to reduce variations in wound contraction resulting from the dressing, it was interpreted that the formation of the epidermis would tend to be delayed compared to wounds without the Tegaderm [
23,
24].
Nonetheless, in terms of the changes in wound sizes, marked decreases in size were observed in the O. keta-derived PDRN injection and O. mykiss-derived PDRN injection groups when compared to the normal saline soaked dressing group. The wound healing effects of the O. keta-derived PDRN injection did not differ significantly from those of the O. mykiss-derived PDRN injection. The O. keta-derived PDRN cream group also showed more pronounced would healing effects in comparison to the normal saline soaked dressing group, even though the effects were not as great as those of the PDRN injections. If the content of PDRN could be increased in the PDRN cream, it is likely that the effects would be similar to those observed following PDRN injection.
When the biopsy findings were evaluated, the O. keta-derived PDRN injection and O. mykiss-derived PDRN injection groups were found to have more active wound healing and collagen formation than the normal saline soaked dressing group at all time points, as evidenced by re-epithelialization, granulation tissue proliferation, inflammatory responses, and neovascularization; in other words, the O. keta-derived PDRN injection was effective in promoting wound contraction and healing.
The elastic fiber formation observed by elastin staining was similar in all groups. Based on the above findings, it is speculated that the O. keta-derived PDRN injection maintain the normal wound healing process while also promoting wound healing.
ELISA was conducted to compare the TGF and VEGF levels, markers of inflammation and neovascularization, respectively. The levels of both markers in the O. keta-derived PDRN injection and O. mykiss-derived PDRN injection groups increased at all time points compared to that at baseline (days 4, 7, and 10); this finding implies that the O. keta-derived PDRN injection has similar wound healing-promoting effects as the O. mykiss-derived PDRN injection. Moreover, although the effects of the PDRN cream were not as great as those of injections, the cream was still more effective than normal saline soaked dressing.
When the wound healing effects were analyzed 4, 7, and 10 days after the creation of full-thickness skin defect wounds on the backs of ICR mice, the O. keta-derived PDRN injection and PDRN cream were both found to be more effective than normal saline soaked dressing. Moreover, since the wound healing effects of the O. keta-derived PDRN injection did not reveal any significant difference compared to those of the O. mykiss-derived injection, which is currently approved for clinical use, the O. keta-derived PDRN injection is thought to be effective in promoting wound healing for full-thickness skin defects.
However, a limitation of the present study is that it was conducted in animals; in order to apply the findings in the clinical setting, multiple variables regarding wounds, including the size and local condition around the wounds and the systemic condition of the patients, should be considered. Thus, future studies should investigate the mechanisms of action of O. keta-derived PDRN and its safety and efficacy should be evaluated in large randomized clinical trials.