Scar Prevention and Enhanced Wound Healing Induced by Polydeoxyribonucleotide in a Rat Incisional Wound-Healing Model

Abstract

High-mobility group box protein-1 (HMGB-1) plays a central role in the inflammatory network, and uncontrolled chronic inflammation can lead to excessive scarring. The aim of this study was to evaluate the anti-inflammatory effects of polydeoxyribonucleotide (PDRN) on scar formation. Sprague-Dawley rats (n = 30) underwent dorsal excision of the skin, followed by skin repair. PDRN (8 mg/kg) was administered via intraperitoneal injection for three (PDRN-3 group, n = 8) or seven (PDRN-7 group, n = 8) days, and HMGB-1 was administered via intradermal injection in addition to PDRN treatment for three days (PDRN-3+HMGB-1 group; n = 6). The scar-reducing effects of PDRN were evaluated in the internal scar area and by inflammatory cell counts using histology and immunohistochemistry. Western blot, immunohistochemistry and immunofluorescence assays were performed to observe changes in type I and type III collagen and the expression of HMGB-1 and CD45. Treatment with PDRN significantly reduced the scar area, inflammatory cell infiltration and the number of CD45-positive cells. In addition, the increased expression of HMGB-1 observed in the sham group was significantly reduced after treatment with PDRN. Rats administered HMGB-1 in addition to PDRN exhibited scar areas with inflammatory cell infiltration similar to the sham group, and the collagen synthesis effects of PDRN were reversed. In summary, PDRN exerts anti-inflammatory and collagen synthesis effects via HMGB-1 suppression, preventing scar formation. Thus, we believe that the anti-inflammatory and collagen synthesis effects of PDRN resulted in faster wound healing and decreased scar formation.

Keywords: cicatrix, inflammation; polydeoxyribonucleotide; rats; wounds and injuries.

Figure 1

H&E- and M-T-stained tissues from the sham, Polydeoxyribonucleotide (PDRN)-3, and PDRN-7 groups on Days 7 and 14 (magnification, 40×). (A) All groups exhibited complete re-epithelialization and the formation of granulation tissue on Day 7. However, the PDRN-3 group showed the narrowest granulation tissue area among all groups. (B) Hematoxylin and eosin (H&E)- and Masson’s trichrome (M-T)-stained tissues from the sham, PDRN-3, and PDRN-7 groups on Day 14 (magnification, 40×). The sham group continued to show a wide granulation tissue area with inflammation. However, the PDRN-3 and PDRN-7 groups showed more collagen deposition within narrower scar areas, as demonstrated by M-T staining. (C) Quantitative analyses of the scar areas in each treatment group. The scar areas were significantly narrower in the PDRN-3 and PDRN-7 groups than in the sham group on Days 7 and 14. However, no significant difference in scar size was observed between the PDRN-3 and PDRN-7 groups on Day 7 or 14 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

The sham group still exhibited granulation tissue and fewer collagen fibers with inflammatory cell infiltration on Day 14. However, the PDRN-treated groups demonstrated reduced inflammatory cell infiltration and collagen fibers (arrows) within the scar area.

Figure 3

(A) Immunofluorescence analysis of CD45-expressing leukocytes on Day 7 (magnification, 400×). The sham group exhibited abundant cellular infiltration, and the majority of the cells were CD45-positive (green) leukocytes. The PDRN-3 and PDRN-7 groups exhibited diminished cellular infiltration and CD45-positive leukocytes. (B) Inflammatory cell infiltration in the scar area, as demonstrated by H&E staining (magnification, 400×). On Days 7 and 14, the sham group exhibited significant inflammatory cell infiltration into the scar area compared with the PDRN-3 and PDRN-7 groups. (C) Comparison of inflammatory cell counts. The infiltration of inflammatory cells in the scar area was lower in the PDRN-treated groups than in the sham group on Days 7 and 14 (* p < 0.05, *** p < 0.001).

Figure 3

(A) Immunofluorescence analysis of CD45-expressing leukocytes on Day 7 (magnification, 400×). The sham group exhibited abundant cellular infiltration, and the majority of the cells were CD45-positive (green) leukocytes. The PDRN-3 and PDRN-7 groups exhibited diminished cellular infiltration and CD45-positive leukocytes. (B) Inflammatory cell infiltration in the scar area, as demonstrated by H&E staining (magnification, 400×). On Days 7 and 14, the sham group exhibited significant inflammatory cell infiltration into the scar area compared with the PDRN-3 and PDRN-7 groups. (C) Comparison of inflammatory cell counts. The infiltration of inflammatory cells in the scar area was lower in the PDRN-treated groups than in the sham group on Days 7 and 14 (* p < 0.05, *** p < 0.001).

Figure 4

(A) Immunohistochemistry of high mobility group box-1 (HMGB-1) (magnification, 400×). All groups exhibited extracellular expression of HMGB-1, though the PDRN-3 and PDRN-7 groups displayed weaker expression within the narrow scar areas on Day 7. On Day 14, the sham group continued to show high extracellular expression of HMGB-1 in the wide scar areas, whereas extracellular expression of HMGB-1 was absent in the PDRN-3 and PDRN-7 groups. (B) Semi-quantitative analysis of HMGB-1 expression levels. The sham group showed significantly higher levels of HMGB-1 expression than the PDRN-3 and PDRN-7 groups on Days 7 and 14 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

(A) H&E- and M-T-stained tissues from the sham, PDRN 3 and PDRN-3 + HMGB-1 groups on Day 7 (magnification, 40×). The PDRN-3 + HMGB-1 group exhibited wider scars compared with the PDRN 3 group. (B) H&E- and M-T-stained tissues from the sham, PDRN 3 and PDRN-3 + HMGB-1 groups on Day 14 (magnification, 40×). Although the rats in the PDRN-3 + HMGB-1 group were treated with PDRN, this group exhibited wide scars similar to the sham group on Day 14. The scar-narrowing effect of PDRN was reversed by administration of HMGB-1. (C) The scar areas in the PDRN-3 + HMGB-1 group were significantly wider than those in the PDRN-3 group. Additional administration of HMGB-1 reversed the scar-narrowing effect of PDRN (* p < 0.05, ** p < 0.01). (D) Inflammatory cell infiltration (arrow) in the PDRN-3 + HMGB-1 group, as demonstrated by H&E staining (magnification, 400×). Many inflammatory cells had infiltrated the scar area on Days 7 and 14. (E) The total inflammatory cell count was significantly higher in the PDRN-3 + HMGB-1 group than in the PDRN-3 group on Day 14 (* p < 0.05, *** p < 0.001). This result indicated that the anti-inflammatory effect of PDRN was reversed by HMGB-1 administration.

Figure 6

(A) Western blots for type I and type III collagen on Day 7. The expression of type I and III collagen was significantly higher in the PDRN-3 group compared with the sham and PDRN-3 + HMGB-1 group (* p < 0.05, ** p < 0.01, *** p < 0.001). (B) Western blots for type I and type III collagen on Day 14. The PDRN-3 group also demonstrated higher collagen synthesis compared with the other groups. PDRN accelerated wound healing by promoting early collagen synthesis. This effect of PDRN was reversed by HMGB-1 administration (* p < 0.05, ** p < 0.01, *** p < 0.001).