Ann Dermatol.  2019 Aug;31(4):403-413. 10.5021/ad.2019.31.4.403.

Polydeoxyribonucleotides Improve Diabetic Wound Healing in Mouse Animal Model for Experimental Validation

• Kwon TR
• Han SW
• Kim JH
• Lee BC
• Kim JM
• Hong JY
• Kim BJ

• KMID: 2451477
• DOI: http://doi.org/10.5021/ad.2019.31.4.403

Abstract

BACKGROUND
Wound healing mechanisms is believed to have effects similar to wound healing disorders in diabetic patients, including abnormal inflammatory cells, angiogenesis disorders, and reduced collagen synthesis. Therefore, reestablishment of structural and promoted angiogenesis could be beneficial to promote wound healing process.
OBJECTIVE
Therefore, we investigated whether the polydeoxyribonucleotide (PDRN) that was self-production in Korea, could be useful as an intradermal injection for promoting wound healing. Also, we validate for wound healing effect of PDRN using healing-impaired (db/db) mice.
METHODS
In this study, we confirmed the effects of PDRN by creating wound models in in vitro and in vivo model. Using an in vitro wound healing assay, we observed that PDRN stimulated closure of wounded monolayers of human fibroblast cells. PDRN (8.25 mg/ml) or phosphate-buffered saline (0.9% NaCl) was injected once daily into the dermis adjacent to the wound for 12 days after skin injury.
RESULTS
Time course observations revealed that mice treated with PDRN showed accelerated wound closure and epidermal and dermal regeneration, enhanced angiogenesis. The wound area and depth decreased at 3, 6, 9, and 12 days after skin injury. Histological evaluation showed an increase of vascular endothelial growth factor, CD31, and collagen fibers in the PDRN group compared with the control group, indicating that PDRN was effective in the treatment of delayed wound healing caused by diabetes.
CONCLUSION
This study suggests that our PDRN has a wound healing effect in transgenic animal models with cells and diabetes through angiogenesis.

Keyword

CD31; Diabetes mouse model; Polyribonucleotide; Vascular endothelial growth factor A; Wound healing

MeSH Terms

Animals
Animals*
Animals, Genetically Modified
Collagen
Dermis
Fibroblasts
Humans
In Vitro Techniques
Injections, Intradermal
Korea
Mice*
Models, Animal*
Polydeoxyribonucleotides*
Regeneration
Skin
Vascular Endothelial Growth Factor A
Wound Healing*
Wounds and Injuries*
Collagen
Polydeoxyribonucleotides
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 Effects of polydeoxyribonucleotide (PDRN) on cytotoxicity and proliferation in human dermal fibroblast neonatal (HDF-n) cells. (A) The cytotoxicity of PDRN was measured using CCK-8 reaction solution in on HDF-n cells. The percentage of viable cells was expressed as a proportion of control cell viability, which was set to 100%. (B) The cell proliferation of HDF-n cells at 48 and 72 hours. The number of HDF-n cells treated with PDRN. (C) The cell migration of HDF-n. We observed these results with optical microscopes (CKX41; OLYMPUS, Tokyo, Japan) at each time point (0, 24, 48, and 72 hours). Significant differences from the control group are *p<0.05, **p<0.01.

• 

  Fig. 2 Polydeoxyribonucleotide (PDRN) stimulates wound healing at diabetic mouse model. (A) Folliscope images (×15) (LeedM, Seoul, Korea). (B) Digital camera images (D3200; Nikon, Tokyo, Japan). It was confirmed that the area of the wounds was reduced visually with time after skin damage. PBS: phosphate-buffered saline.

• 

  Fig. 3 Polydeoxyribonucleotide (PDRN) decreases wound area and wound depth in diabetic mice. (A) Color-coded surface topography image captured by a PRIMOSLITE optical 3D skin measuring device, (B) graph showing changes in wound area over time after injury. In +Leprdb/+Leprdb mice, the reduction of the wound area in the PDRN-injected group occurred more rapidly than in the phosphate-buffered saline (PBS)-injected group. In both m+/+Leprdb mice and +Leprdb/+Leprdb mice, the wound area decreased faster than in controls, but this difference was not significant. (C) Graph showing changes in wound depth over time after injury. The PDRN-injected +Leprdb/+Leprdb mice recovered at a rate similar to that of PDRN-injected m+/+Leprdb mice, but the recovery of the wound depth was delayed in the PBS-injected +Leprdb/+Leprdb mice. The wound depth of the PBS-injected group was significantly different from that of the PDRN-injected group on days 6 and 9 (*p<0.05) in +Leprdb/+Leprdb mice.

• 

  Fig. 4 Polydeoxyribonucleotide (PDRN) promotes wound healing with rapid re-epithelization. Hematoxylin-eosin (H&E) staining was performed on paraffin block sections of the skin tissue 3, 6, 12 days after initial wounding. H&E staining revealed that the wounds were recovered in order of PDRN injected group, phosphate-buffered saline (PBS) injected group in the m+/+Leprdb mice and PDRN injected group, PBS injected group in the +Leprdb/+Leprdb mice in the order of date. In the m+/+Leprdb 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. Scale bars=1 mm.

• 

  Fig. 5 Polydeoxyribonucleotide (PDRN) promotes skin repair in diabetic mice. (A) Masson's trichrome stain. Red: keratin & muscle fiber, blue: collagen & bone, pink: cytoplasm (×40). (B) van Gieson's stain. Blue: nuclei, bright red: collagen, yellow: cytoplasm, muscle, fibrin, and red blood cells. For the hetero-type m+/+Leprdb mice, those injected with phosphate-buffered saline (PBS) and those with PDRN showed no significant differences histologically. However, for diabetic BKS.Cg-+Leprdb/+Leprdb mice, those injected with PDRN showed faster epithelialization of the epidermis around the wound and faster formation of collagen fiber grows compared with those injected with PBS (×100).

• 

  Fig. 6 Polydeoxyribonucleotide (PDRN) increases expression of vascular endothelial growth factor (VEGF) and CD31 in the regenerating wounds of diabetic mice. Expression levels of VEGF and CD31 in the tissues of animals sacrificed at day 12 after skin injury were confirmed by immunohistochemical staining. (A) VEGF and (B) CD31. (C) Graph of VEGF intensity. (D) Graph of CD31 intensity. In both m+/+Leprdb mice and +Leprdb/+Leprdb mice, the PDRN treatment group showed significantly higher expression of VEGF and CD31 than the PBS treatment group (analysis by Image-Pro plus 7.0), one-way ANOVA (Shapiro–Wilk p>0.05), independent t-test assay (**p<0.01).

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