Antioxidant sol-gel improves cutaneous wound healing in streptozotocin-induced diabetic rats

Abstract

We examined the effects of vitamin C in Pluronic F127 on diabetic wound healing. Full-thickness excision skin wounds were made in normal and diabetic Wistar rats to evaluate the effect of saline, saline plus vitamin C (antioxidant sol), Pluronic F127, or Pluronic F127 plus vitamin C (antioxidant sol-gel). The rate of wound contraction, the levels of epidermal and dermal maturation, collagen synthesis, and apoptosis production in the wound tissue were determined. In vitro data showed that after 6 hours of air exposure, the order of the scavenging abilities for HOCl, H(2)O(2), and O(2) (-) was antioxidant sol-gel > antioxidant saline > Pluronic F127 = saline. After 7 and 14 days of wound injury, the antioxidant sol-gel improved wound healing significantly by accelerated epidermal and dermal maturation, an increase in collagen content, and a decrease in apoptosis formation. However, the wounds of all treatments healed mostly at 3 weeks. Vitamin C in Pluronic F127 hastened cutaneous wound healing by its antioxidant and antiapoptotic mechanisms through a good drug delivery system. This study showed that Pluronic F127 plus vitamin C could potentially be employed as a novel wound-healing enhancer.

Figure 1

Scavenging abilities of saline (S), Pluronic F127 (F), antioxidant saline (SC), and antioxidant sol-gel (FC) for HOCl (a), H₂O₂ (b), and O₂ ⁻ (c) after 0 and 6 hours of preparation. Fresh-prepared (0 hour) antioxidant saline and antioxidant sol-gel significantly decreased HOCl, H₂O₂, and O₂ ⁻ counts when compared to saline. After 6 hours of air exposure, the antioxidant ability is significantly reserved in FC group when compared to SC group. Data are expressed as mean ± SEM. *P < 0.05, ^# P < 0.01 when compared to saline group. S: saline control; F: Pluronic F127; SC: saline plus vitamin C; FC: Pluronic F127 plus vitamin C.

Figure 2

The effect of saline, Pluronic F127, antioxidant saline, and antioxidant Pluronic F127 on wound closure in the normal rats (a) and diabetic rats (b). Full-thickness skin wounds of 1.0 × 1.0 cm were measured from the time of wounding until closures. The skin defect was compared to the initial wound size to determine wound closure rate by tracing the wound. (a) Closure of full-thickness skin wounds of normal rats showed no significant difference in the healing rate between four groups of treatment. (b) Wound closure of diabetic skin showed that the antioxidant sol-gel-treated wounds closed faster than the saline-, sol- and antioxidant saline-treated wounds on days 7 and 14. Data is expressed as mean ± SEM for three separate experiments, each in quadruplicate. *P < 0.05 when compared to saline control. (c) Representative pictures of skin wounds in group saline (a), Pluronic F127 (b), antioxidant saline (c), and antioxidant sol-gel (d) at day 7.

Figure 3

Representative data of HOCl, H₂O₂, and O₂ ⁻ contents are measured in the normal and diabetic rat skins. The level of HOCl (a), H₂O₂ (b), and O₂ ⁻ (c) counts is higher in the skin of diabetic rats than the normal rats. A summary data (d) shows that increased HOCl, H₂O₂, and O₂ ⁻ counts are found in diabetic rats when compared to the normal rats. Data are expressed as mean ± SEM in 5 rats each. *P < 0.05 when compared to normal rats.

Figure 4

H & E stain in the saline control (a), Pluronic F127 (b), saline plus vitamin C (c), and Pluronic F127 plus vitamin C (d). Epidermal maturation was scored histologically from “no migration” (0) to “completed migration with keratinization” (4). The mean score of epidermal maturation is displayed in (e). Significant epidermal maturation indicated by migration of keratinization was shown here in the groups of SC and FC when compared with the group of Saline 14 days after wounding (e). Data are expressed as mean ± SEM. *Four groups are significantly different when compared with Kruskal-Wallis test and posttest comparing all pairs of columns. S: saline control; F: Pluronic F127; SC: saline plus vitamin C; FC: Pluronic F127 plus vitamin C. Original magnifications taken at ×200.

Figure 5

Effect of vitamin C on dermal maturation in diabetic rat wounds. Sections stained with H & E are displayed in the saline control (a), Pluronic F127 sol (b), saline plus vitamin C (c), and Pluronic F127 plus vitamin C (d). Histological evaluation of the 14-day wounds by H & E stain demonstrated enhanced healing characteristics including wound of proliferation, remodeling, and maturation in the antioxidant saline (c) or antioxidant sol-treated wound (d). Saline (a) or Pluronic 127 treatment (b) did not show any marked healing responses in the diabetic wounds. This advancement correlates with the fibroblast infiltration into the wounded area which was scored based on their maturity from reactive to normal. The mean score of dermal maturation is displayed in (e). The degree of dermal maturation is demonstrated in an order of FC > SC > F > S 14 days after wounding (e). Data are expressed as mean ± SEM. *Four groups are significantly different but not significant between saline and F127; vitamin C in saline and vitamin C in PF127. S: saline control; F: Pluronic F127; SC: saline plus vitamin C; FC: Pluronic F127 plus vitamin C. Original magnifications taken at ×100.

Figure 6

Effect of antioxidant sol-gel on collagen expression and collagen content at 14th day. Masson's trichrome staining of collagen in the saline control (a), Pluronic F127 sol (b), saline plus vitamin C (c), and Pluronic F127 plus vitamin C (d). The mean score of blue stain is displayed in (e). Four groups are significantly different when compared with Kruskal-Wallis test and posttest comparing all pairs of columns. Significant blue stain was demonstrated in an order of FC > SC > F > S. Increased hydroxyproline content was consistently increased in an order of FC > SC > F > S 14 days after wounding (f). Data are expressed as mean ± SEM. *P < 0.05 when compared to saline group. S: saline control; F: Pluronic F127; SC: saline plus vitamin C; FC: Pluronic F127 plus vitamin C. Original magnifications taken at ×200.

Figure 7

Immunohistochemical evidence of keratinization by loricrin at 14th day. Wounds treated with saline control (a), Pluronic F127 sol (b), saline plus vitamin C (c), and Pluronic F127 plus vitamin C (d) were demonstrated. Original magnifications taken at ×100 and ×200 as inset. Immunohistochemical staining by antiloricrin (epidermal differentiation marker) antibodies showed that loricrin was highly expressed in the upper granular cell layer, especially in the regenerated epidermis of Pluronic F127 plus vitamin C or saline plus vitamin C groups.

Figure 8

Effect of antioxidant sol-gel on apoptosis production of the healing skin in diabetic rats at 7th day. Apoptosis analyzed by TUNEL stain (brown color in the nucleus) was shown in the saline control (a), Pluronic F127 sol (b), saline plus vitamin C (c), and Pluronic F127 plus vitamin C (d). The data were expressed as the number of apoptosis (high power field) in each section (400x) is displayed in (e). The percentage of apoptosis appearance in the wounds is demonstrated in an order of FC > SC > F > S 14 days after wounding (e). Data are expressed as mean ± SEM. *P < 0.05 when compared to saline group. S: saline control; F: Pluronic F127; SC: saline plus vitamin C; FC: Pluronic F127 plus vitamin C. Original magnifications taken at ×200.