The Role of Topical Estrogen, Phenytoin, and Silver Sulfadiazine in Time to Wound Healing in Rats

Many recent studies have focused on the potential role of topical agents in the wound healing process. To compare the time to healing of full-thickness wounds treated with topical estrogen, phenytoin, or silver sulfadiazine (SSD), an in vivo study was conducted using 32 male Wistar rats.Animals were housed individually in standard cages in similar environmental conditions, and a single, circular (4 mm in diameter), full-thickness skin wound was created on the dorsum of each rat. Animals were randomly divided into 4 groups of 8 rats each and treated with topical phenytoin, SSD, estrogen cream, or no treatment/control. Each wound was measured and examined daily until healing, defined as complete reepithelialization and closure of the wound. Group mean healing times were calculated, and Tukey’s multiple comparison test was used to compare these data. Average times to healing were 11 days in estrogen group, 10 days in phenytoin group, 7.62 days in SSD group, and 11.87 days in control group. Wound healing was significantly faster in the SSD compared to control (P <.01) and the estrogen group (P <.01). No other differences were statistically significant. Further studies, especially randomized clinical trials on human beings with larger sample sizes, are recommended to elucidate if these topical agents affect wound outcomes.

Because the skin protects against dehydration, bleeding, and invasion of microorganisms, maintaining its integrity is important.¹ A wound, defined as the destruction of the epidermis and dermis, compromises the skin and prolonged healing time increases the risk of complications. Therefore, clinicians and researchers aim to decrease the healing time of wounds. 

Phenytoin was introduced as an antiepileptic drug in 1937; owing to the related complication of gingival hypertrophy, 2 in vivo studies,^2,3 a systematic review,⁴ and a comparison study⁵ investigated the probable effects of phenytoin on the wound healing process. These studies concluded phenytoin improves angiogenesis and increases pain tolerance during the healing process. In a comparative study by Hokkam et al,⁵ 54 patients with chronic venous ulcers received topical phenytoin once daily and 50 patients (the control group) received a dressing with normal saline. After 8 weeks, complete healing was observed in 64.8% of the patients in the study group and 52% of the patients in the control group (P = .04). The authors concluded topical phenytoin can be administered to improve chronic venous ulcer healing. In an experimental study,⁶ 24 Wistar rats with tibia fractures were divided into 2 groups; the study group was provided phenytoin 20 mg/kg, injected directly to the fracture site every 72 hours, while the control group had normal saline injected at the same intervals. After 28 days, histomorphometric assessment demonstrated the total periosteal callus was mineralized significantly more in the phenytoin group than the control group (P = .011), showing phenytoin had a positive effect in fracture healing. In a single-blind, placebo-controlled study (N = 100), Pereira et al⁷ excised 2 melanocytic nevi from each patient. One (1) of the injuries was provided a dressing with topical phenytoin 0.5% cream and the other a dressing with just cream (not specifically described in the study). Wounds treated with phenytoin demonstrated shorter healing time, more intense epithelialization, a smaller scar, and better cosmetic results. In a prospective, randomized controlled trial, Subbanna et al⁸ compared 28 patients with Stage 2 pressure ulcers, 14 randomly dressed with phenytoin and 14 with normal saline, for 15 days. Pressure ulcer healing scores were slightly higher and ulcer size slightly reduced with topical phenytoin treatment, but neither sets of data were significantly different statistically. However, measuring the serum levels of phenytoin in this study showed unremarkable systemic absorption and safety.

Silver sulfadiazine, a topical sulfonamide with bactericidal and bacteriostatic properties, has been traditionally used for burns. This drug has been reported effective in a systematic review⁹ on both Staphylococcus aureus and Pseudomonas and improves the wound healing process by inducing epithelialization and granulation tissue formation.¹⁰ Olawoye et al¹¹ included 144 patients with burn injuries of various thickness in their descriptive study and applied a layer of silver sulfadiazine over the entire wound. The mean duration from time of injury to wound healing was 21.5 days with a median of 17 days. The authors concluded open dressing with topical silver sulfadiazine is safe and efficient and should be regarded as a viable option for burn wound patients. 

In vivo study¹² has shown estrogen can affect neovascularization, proliferation of myofibroblasts, and secretion of growth factors. A significant difference in wound healing between men and women has been noted, as well as between women before and after menopause,^13-16 indicating sex hormones have an effect on the wound healing process. In vivo studies¹³ of estrogen and its derivatives have shown it can accelerate the wound healing process¹⁷ while androgens decelerate it. In an in vivo investigation by Gal et al,¹⁷ 16 rats were ovariectomized and 8 additional rats were sham operated. Two (2) parallel full-thickness skin incisions and 2 round full-thickness skin excisions were made on the dorsum of each rat. Eight (8) ovariectomized rats received estradiol benzoate for 6 days after the operation, while the other rats received a placebo. Histological examination of ovariectomized, estrogen-treated rats demonstrated a significant augmentation of neovascularization associated with enhancement of collagen deposition in their wounds. Systemic or topical administration of 17-beta-estradiol has been proposed to accelerate the wound healing process in postmenopausal women as well.^13-16 In these studies, different derivatives of estrogen were used, including conjugated estrogen, a topical derivate of estrogen. 

The aim of the present study was to compare the effects of topical phenytoin, silver sulfadiazine, and conjugated estrogen (the derivative available to the authors) on the healing of skin wounds in male rats.

All experiments in this study were reviewed and approved by Arak University Animal Experiment Committee and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Arak University of Medical Sciences, Arak, Iran. In the present experimental study, 32 10-week-old male Wistar rats weighing 275 g to 300 g were housed individually in standard cages under similar environmental conditions (temperature 18˚ C to 23˚ C, humidity 50% to 55%, 12-hour light/dark cycle using fluorescent light, and free access to food and water). Animals first were anesthetized in a glass vessel containing cotton wool soaked in chloroform; then their dorsal hair was shaved using a standard electric shaver, and the shaved area was rinsed with normal saline. Next, under sterile conditions, a single, circular (4 mm in diameter), full-thickness skin wound was made on the dorsum of each rat using a sterile disposable biopsy punch (Kai Industries Co. Ltd, Gifu, Japan). The depth of the induced wound was controlled by excising the epithelial tissue to the extent that the dorsal muscular fascia was exposed. Then, the animals were randomly divided into 4 groups (8 rats with 8 wounds in each group), and each sample was given a specific code and a checklist sheet. Wound surfaces were rinsed with normal saline and dried gently every day. In group 1, wounds were covered with 2-mm thick phenytoin 1% ointment; in group 2, silver sulfadiazine 1% ointment; in group 3, conjugated estrogen 0.625% ointment; and in group 4 (control) simple ointment (Eucerin cream, Beiersdorf AG, Wilton, CT) was applied daily, all without cover dressing. The base vehicles of these ointments (base vehicles were not the same but did not differ regarding wound adhesion) had no wound healing properties. Each wound was examined for possible infection every day, and its unepithelialized area was measured using a mm ruler. Results were recorded until complete healing, defined as complete reepithelialization and closure of wound surface. After completion of the experiment, rats were sacrificed by decapitation with a guillotine. The healing times in the 4 groups were compared using one-way variance analysis, and Tukey’s multiple comparison test was used to compare mean healing times. A P value <.05 was considered significant.

The mean time to healing was 10 ± 1.7 days in the phenytoin group, 7.62 ± 0.72 days in the silver sulfadiazine group, 11 ± 1.81 days in the conjugated estrogen group, and 11.87 ± 2.01 days in the control group (see Table). In other words, the shortest healing time occurred in the silver sulfadiazine group and the longest in the control group. The difference in time to healing was statistically significant only between the silver sulfadiazine and conjugated estrogen cream groups (P ≤.01). 

The results of this study showed silver sulfadiazine is effective in wound healing and reduces the healing time in comparison with the control group and conjugated estrogen. In this study, the effects were approximately similar in phenytoin, estrogen, and control groups and these treatments had no advantage over one another. To the authors’ knowledge, this comparison has not been made in the literature; most studies investigated the effects of only 1 of these agents on the process of wound healing.

Phenytoin. The mechanism of action of topical phenytoin in wound healing remains a subject for debate. According to several clinical trials,^18-20 phenytoin promotes proliferation of myofibroblasts and fibroblasts, the production of the extracellular matrix and its proteins, and the activity of growth factors. Eventually, collagen synthesis is enhanced, resulting in increased wound strength. Phenytoin also decreases collagenase activity, edema, wound exudate, and bacterial load. In a study conducted by Hasamnis et al,² excisional wounds in 20 male Wistar rats were divided randomly to the control group (A) that received no drug and the treatment group (B) that received 1% phenytoin cream on their wounds for 16 days. This study showed the average number of days required for complete epithelialization of wounds in group B (20.60 ± 1.51) was significantly less than the period required for complete epithelialization in group A (23.00 ± 2.26) (P = .0120). The experimental study by Mathew et al⁶ demonstrated phenytoin had a positive effect in fracture healing. Beigom Taheri et al²¹ divided 60 rats into 4 groups: group 1 served as the control and the wounds in groups 2, 3, and 4 were treated with 1% phenytoin cream, diode laser, and both phenytoin cream and diode laser, respectively. Histopathologic features of reepithelialization of wounds in these groups were compared; reepithelialization was complete in all animals of the 4 groups and a keratin layer was generated, but in the phenytoin group the healing process was slower. The result for the phenytoin-treated group was not better than in the control group, which was similar to current study results.

Estrogen. The discovery of the crucial role of estrogen in skin physiology resulted in the hypothesis that it may have an important role in wound healing.^14-16 Several studies have demonstrated estrogen might be involved in wound healing in terms of altering the inflammatory response, enhancing reepithelialization, inducing granulation formation, modifying proteolysis, and balancing collagen biosynthesis and degradation.¹⁶ However, conflicting results were obtained in the research. In the review of histological process of wound healing in animals by Gal et al,¹⁷ significant augmentation of neovascularization associated with enhancement of collagen deposition was reported. In the clinical trial conducted by Ghazizadeh Hashemi et al,¹⁵ patients with traumatic wounds were divided to 2 groups of 15. The case group was treated with topical conjugated estrogen cream, while the control received Eucerin cream as a placebo. Wound area, healing rate, and required time for wound healing were compared, and no significant differences were found between the 2 groups for these factors. These findings are comparable with the current results. A clinical trial conducted by Asilian et al²² found topical estrogen resulted in slower wound healing in comparison with phenytoin and silver sulfadiazine, also similar to the results of the current study.

Silver sulfadiazine. Silver sulfadiazine is best known for its antibacterial properties. The application of silver sulfadiazine was associated with favorable results in surgical wounds, burns, and wound healing in systematic reviews⁹ and descriptive studies,¹⁰ albeit with conflicting results. A recent systematic review by Wasiak and Cleland²³ reported silver sulfadiazine may prolong healing time and increase pain, although the authors noted the existing evidence was limited by small sample sizes and the heterogeneity of the patient population. In a large systematic review, Miller et al¹⁰ concluded data are lacking to either confirm or disprove the routine use of silver sulfadiazine for patients with partial-thickness burns or stasis dermatitis ulcers.

In the current study, the average time to healing in the silver sulfadiazine group was shorter than in the phenytoin group, although the difference was not statistically significant. These 2 substances appeared to have almost similar effects, a finding comparable to results of the in vivo study by Shamseddini et al.²⁴ Furthermore, the authors of the current study found a significant decrease in the time to healing in the silver sulfadiazine group in comparison with the estrogen and control groups. Although the results of some studies^9,10,25-27 support these findings, other in vivo and review studies found increased rates of healing were obtained with other agents such as argan oil,²⁸ silver nanoparticles,²⁹Lithospermum officinale,³⁰ and honey³¹ in comparison with silver sulfadiazine.

The current study was limited by the rather small sample size and the inherent limitations of in vivo research.

A study of wounds in rats that compared 3 topical products to each other and to a control group showed wound healing was significantly faster in the silver sulfadiazine compared to the control and estrogen groups. No other differences were statistically significant. However, conflicting data in the literature about these topical agents, as well as the limitations of animal research, infer that additional studies, especially randomized clinical trials on human beings involving larger samples, are recommended to elucidate the best therapeutic options for patients with wounds.