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.
Wound healing represents a well-orchestrated reparative response that occurs after all surgical procedures or traumatic injury. Wound healing is a complex multifactorial process, involving inflammation, migration of different cell types, fibroplasia, collagen deposition, and wound contraction. During the inflammation phase, inflammatory cells significantly increased in the wound site [
Diabetes mellitus is one of the major contributors to chronic wound-healing problems, because minor skin wounds can lead to chronic, nonhealing ulcers and ultimately result in infection, gangrene, or even amputation. In critical ill diabetic patients, the antioxidant vitamin C in plasma was reported lower than nondiabetic critical ill patients [
Vitamin C is an important water-soluble antioxidant, which may successfully scavenge ROS, protect against lipid damage, protein oxidation, and DNA oxidation [
Pluronic F127 is one member of a family of triblock copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), generically called poloxamers. At low temperatures, poly(propylene oxide) blocks have only weak hydrophobic properties. With increasing temperature, poly(ethylene oxide) blocks are dehrydrated and promote the aggregation to micelles and become gel form [
Female Wistar rats (
Pluronic F127 and vitamin C were purchased from Sigma-Aldrich Chemical Co. (USA). The Pluronic F127 (13% w/w) was dissolved in saline at 4°C by stirring into homogeneous sol-gel. Vitamin C powder was dissolved in the Pluronic F127 sol-gel (1 mg vitamin C/mL Pluronic F127 solution) as antioxidant sol-gel. The vitamin C powder was dissolved in saline (1 mg/mL) as vitamin C solution. We compared the
To measure the production of ROS in the samples, chemiluminescence (CL) method was adopted using lucigenin (0.25 mM) as an amplifier for measuring superoxide (
Diabetes was induced by a single 65 mg/kg intraperitoneal injection of streptozotocin (STZ; Sigma, Inc., St. Louis, MO, USA), a toxin specific for insulin-producing cells, in normal saline. Blood glucose levels were measured using an acute glucometer. The diabetic state was confirmed 3 weeks after STZ injections by blood glucose levels above 300 mg/dL. Under brief anesthesia with intraperitoneal Nembutal (65 mg/kg), the dorsal skin of the animals was shaved and cleaned with povidone-iodine solution, and a full-thickness skin wound (approximately 1 × 1 cm2) was created after marking the area with a wooden ink stamp before cutting the outlined skin. We applied 0.2 mL of antioxidant sol-gel, antioxidant saline, Pluronic F127, or saline on wounds twice per day for 21 days. Wound size was recorded with photographs, after anesthesia each time at 0, 7, 14, and 21 days after wounding. The wound size was then calculated with a free program called Image J. Animals were euthanized at each time point and the wound samples and adjacent normal skin were harvested and fixed in 10% paraformaldehyde for histological or snap-frozen in liquid nitrogen and stored at −80°C for further analysis. For detecting skin ROS, the intact skin (0.5 g) of diabetic rats as well as normal rats was biopsied, snap-frozen in liquid nitrogen, and homogenized by mortar and pestle, followed by adding 1 mL normal saline.
The percentage of wound closure was calculated as follows by using the initial and final area drawn on glass slides during the experiments:
Wound tissues stored at −80°C were dried to a constant weight and hydrolyzed in 6 M HCl for 16 h at 120°C. Samples were dried on a hot plate and then washed three times with distilled water. The acid-free samples were reconstituted in 2.0 mL of acetate-citrate buffer (1.2% sodium acetate trihydrate, 5% citric acid, 12% sodium acetate, and 3.4% sodium hydroxide, pH 4–9). Five hundred microliters of 0.05 M chloramine-T was added to 1 mL of each sample, after which the samples were incubated for 15 min at room temperature, followed by the addition of 0.5 mL 15% perchloric acid and 15% 4-dimethyl aminobenzaldehyde in 1-propanol. After incubation at 60°C for 15 min, each sample was transferred to a microliter plate and the absorbance read at 550 nm. Hydroxyproline concentrations were calculated from the linear standard curve and presented as
Wound bed biopsies were collected at days 7, 14, and 21 after wounding. Tissue samples were fixed in 10% buffered formalin, processed, and embedded in paraffin. Sections were stained with hematoxylin and eosin (H & E). Microscopic assessment of these slides was coded by a technician, and read-blinded to the sample identification. The sections were scored on a scale of 0–4 for epidermal healing (0 = no migration, 1 = partial migration, 2 = complete migration with partial keratinization, 3 = complete keratinization, and 4 = hypertrophic epidermis) and dermal healing (0 = no healing, 1 = inflammatory infiltrate, 2 = granulation tissue present-fibroplasias and angiogenesis, 3 = collagen deposition replacing granulation tissue > 50%, and 4 = hypertrophic fibrotic response) [
To investigate this further, differentiation of the neo-epidermis was studied by immunohistology using loricrin as late differentiation marker. Structural proteins, including involucrin and loricrin, are produced as skin matures imparting biomechanical strength to the epidermis [
After tissue sections were dewaxed and rehydrated conventionally, sections were incubated with 3% H2O2 for 30 minutes. The slides were washed with PBS (pH 7.4) twice. The sections were blocked with 5% BSA in TBS for 20 minutes. After the redundant liquid had been discarded, the sections were incubated with loricrin antibody (Abcam, Cambridge, UK) at 4°C overnight. After slides had been washed with PBS, the slides were incubated with rabbit secondary antibody for 1 hour, followed by incubation with streptavidin-HRP for 20 minutes. The antibody binding sites were visualized by incubation with DAB-H2O2 solution.
Sections were dewaxed and rehydrated conventionally, placed in Weigert’s hematoxylin stain for 1 h, rinsed under lukewarm water for 5 min, immersed in Masson solution for 15 min, and rinsed in deionized water before placing in phosphomolybdic acid for 10 min. Subsequently, sections were immersed in 2% aniline blue for 15 min, rinsed in 1% acetic acid, 95% ethanol, and absolute ethanol in turn, immersed in xylene for 10 min, and mounted with resin. Collagen fibers were stained blue, cytoplasm and erythrocyte were stained red, and nuclei were stained bluish brown.
Apoptosis assay was performed using the TACS.XL DAB In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD, USA). Briefly, sections were blocked by incubation in 3% H2O2 in methanol for 5 minutes at 25°C. Then the sections were labeled with TdT labeling reaction mix at 37°C for 1 h. Nuclei exhibiting DNA fragmentation were visualized by incubation in 3′,3-diaminobenzidine (DAB) for 15 min.
All values are expressed as mean ± SEM. For comparisons of parametric data, one-way analysis of variance and then the Student’s unpaired
We showed that the CL counts of H2O2, HOCl, and
Scavenging abilities of saline (S), Pluronic F127 (F), antioxidant saline (SC), and antioxidant sol-gel (FC) for HOCl (a), H2O2 (b), and
Rats receiving STZ have significant elevation of blood glucose level (>300 mg/dL) after 3 weeks, which was sustained throughout the duration of the study. The wound healing of various treatments was evaluated in a full-thickness wound model. The wounds decreased in size gradually with time, closed at 2 weeks in normal rats and at 3 weeks in diabetic rats. We did not note any statistical difference in the wound closure of the normal rats with four kinds of treatment (Figure
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. *
As shown in Figure
Representative data of HOCl, H2O2, and
With the help of hematoxylin & eosin stain (Figure
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.
Normally, dermal recovery is assessed for three stages: proliferation, remodeling, and maturation. Histopathological examination with hematoxylin & eosin staining showed that the antioxidants sol-gel-treated wounds exhibited advancement in all these three stages. The histologic expression showed that dermal maturation was ranked in an order of antioxidant sol-gel > antioxidant saline > Pluronic F127 > saline (Figure
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.
Collagen deposition and cellular proliferation can be measured in the histological cross-sections of wounds with Masson’s trichrome staining. Significant increase in blue collagen stain was found in the antioxidant sol-gel-treated group (Figure
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. *
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.
Hydroxyproline is a major component of the protein collagen. Therefore, hydroxyproline content was used as an indicator to determine collagen content. As shown in Figure
The apoptosis formation analyzed by TUNEL stain showed that a marked increase of apoptosis in the wound tissue of diabetic rats treated with topical saline 7 days, after injury. The topical application of antioxidant sol-gel or antioxidant saline significantly decreased the apoptosis production in the diabetic wounds (Figure
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. *
The present study showed that the effect of
Pluronics (also called Poloxamers) have been particularly interesting because this polymer shows a critical solution temperature (reverse sol-gel transition temperature) below the human physiological temperature and, thus, exists to a gel state in the body at 37°C. Yamaoka et al. [
Wound healing is a complex multifactorial process that results in the contraction and closure of the wound and restoration of a functional barrier. One of the leading causes of impaired wound healing is diabetes mellitus. In diabetic rats, a minor skin wound often leads to chronic, nonhealing ulcers and ultimately results in gangrene, even amputation. ROS and oxidative stress arise from inflammatory cells, which are strongly implicated in the pathogenesis of several diseases including chronic ulcers [
Several studies from rat dermal wound have shown that the treatment of antioxidants to depress ROS is beneficial for wound healing. An improvement in the quality of wound healing has been attempted by slow delivery of antioxidants-like curcumin from collagen, which also acts as a supportive matrix for the regenerative tissue [
ROS can affect proliferative and cell survival signaling to alter apoptotic pathways in the skin diseases. Excess production of ROS in the skin can foster the development of dermatological diseases. One approach to prevent or treat these ROS-mediated disorders is based on the administration of various antioxidants in an effort to restore homeostasis. Many antioxidants have shown substantive efficacy in cell culture systems and in animal models of oxidant injury. On the other hand, increased apoptosis formation delayed wound healing [
We demonstrate that vitamin C incorporated into Pluronic F127 exerts continuous effects of antioxidant and anti-apoptotic activities, which enhance epidermal and dermal maturation and collagen synthesis in the diabetic skins. The antioxidant sol-gel can decrease potentially harmful factors such as ROS production and apoptosis cell death present in chronic wound of the diabetic rats. These characteristics suggest a beneficial role for this preparation in helping rebalance the chronic wound environment and therefore promote healing.
The authors report no conflict of interests.
This work was supported by Grants from the National Science Council of Taiwan (NSC 98-2320-B-002-043-MY3, 96-2221-E-002-256-MY3, and 98-2320-B002-016-MY3). The authors also want to thank Dr. Gunnar Johansson for critical reading of this paper and National Taiwan University Hospital the Third Core Lab for providing lab facilities.