Topical N-Acetylcysteine Improves Wound Healing Comparable to Dexpanthenol: An Experimental Study

Thirty albino Wistar rats weighing 250–300 g were used in this study. The animals were kept under 12-hour light/dark cycle at 21 ± 2°C with a humidity of 45 to 55%. The rats were fed standard rat chow and allowed ad libitum access to water. This study was approved by the Ethics Committee for Animal Use at Dicle University.

A commercially available mixture containing a 5% dexpanthenol water-in-oil emulsion was used (Bepanthol cream, 5% dexpanthenol, Bayer, Germany).

After weighing each component for the mixture, a 3% NAC cream was prepared, and then the necessary number of NAC (Asist 200-mg capsule, Husnu Arsan, Istanbul, Turkey) was crushed into tiny pieces and added to water, producing a paste. The targeted amount of this mixture was added into cold cream and mixed until a thorough mixture was achieved.

The anesthesia of the rats was achieved with administration of ketamine hydrochloride (25 mg/kg) through the muscles. Once the back hair was shaved, the target field was marked using a pen, and the back was opened via a 2-cm linear full-depth incision on the skin. Three groups were formed with 10 rats each. The control group undertook no treatment. The second group received dexpanthenol cream, and the third group was administered with 3% NAC cream. Daily cleaning of the wounds was performed with saline. Following the cleaning process, the whole wound area was covered with adequate amount of cream. On the 21st day, the rats were anesthetized using intra muscular ketamine hydrochloride (25 mg/kg), the wound and surrounding tissues were removed, and the defects on the skin were closed with nonabsorbable suture.

The wound areas in all 3 groups were measured on the first day and on the third, fifth, seventh, 10th, 14th, and 21st days after the wound. The measurements were performed using transparencies and a permanent marker. Graph paper was used to record and measure the wound areas, and the Walker formula was utilized for assessing the ratio of wound healing.⁷ 

The samples obtained from the skin were put into 10% neutral formalin, processed via the classical histologic preparation technique, and then embedded in paraffin. The blocks were used to obtain 5-μm serial sections, and the general appearance of the tissues was evaluated by staining the sections with hematoxylin-eosin. The slides were examined using a light microscope (Axiophot, Zeiss, Germany). The scoring of morphologic findings, including inflammation, fibrosis, and angiogenesis was as follows: score 0, “none”; score 1, “few”; score 2, “moderate”; and score 3, “many.” The assessment of epithelialization and granulation was determined as either present or absent. The histologic analyses and the scoring process were conducted in a blinded fashion.

Data analyses were performed using SPSS for Windows Version 13.0 (SPSS, Chicago, Illinois). Descriptive statistics were presented as mean ± SD. ANOVA was used to evaluate the group differences on the continuous variables specified at baseline, and the wound closure rate method was employed as the grouping variable. Group differences on categoric variables were assessed using the chi-square test. Repeated-measures ANOVA were performed to evaluate the group differences across time in overall percent wound closure ratio (preliminary wound closure ratio and maintenance). In repeated-measures analyses, the sphericity was controlled by Greenhouse-Geisser correction, as appropriate. Both main effect and simple effect post hoc contrasts were managed with the Bonferroni adjustment. A P value of <0.05 was considered statistically significant.

The epithelialization and granulation rates between the groups were revealed similar in microscopic evaluations (P > 0.05). Less inflammation was revealed in the control group (P = 0.027). Although there was more fibrosis in control group than in the other groups, it was similar in the NAC and dexpanthenol groups (P = 0.002). Angiogenesis rate was more remarkable in the NAC group than in the others (P = 0.04; Table 1; Fig. 1).

Wound closure rates of dexpanthenol and NAC groups were similar and were significantly higher than the control group's rates (P < 0.001; Table 2; Fig. 2).

In multiple comparison analysis, dexpanthenol and NAC groups had similar results in terms of wound healing rates (P < 0.05), which were both higher than in the control group (P > 0.05).

As the largest and the most essential part of the body, skin plays a vital role in maintaining body homeostasis. In terms of skin health, the wound healing process is a principal activity, since it is the key element for the restoration and regeneration of tissues and functions that have been impaired or injured as a result of physical, chemical, bacterial, or viral attacks.⁸  The wound-healing process is comprised of 4 stages: hemostasis, inflammation, proliferation, and tissue remodeling.^2,3  An ideal wound healing process is a multi-element activity involving the collaboration of the inflammatory cells, biochemical mediators, extracellular matrix molecules, the microenvironment of the cell, and remodeling. This process can be facilitated by a number of natural or artificial agents.⁹  However, the wound healing process can be interrupted by a number of factors, which may impair 1 or more stages of the process and thus lead to inappropriate or failed tissue repair. Failed processes of wound healing lead to large amounts of health care expenses, which are estimated to exceed $3 billion per year.¹⁰  Because of this high cost, finding new methods to facilitate wound healing process has been studied by many researchers and scholars.⁴ 

When administered in tissues, dexpanthenol is transformed to pantothenic acid, which is crucial for the epithelia to sustain their normal function. Following the topical administration of dexpanthenol, both skin regeneration and wound healing process are facilitated. This is the main reason why dexpanthenol is commonly used in skin care and also in the treatment of a number of skin diseases.⁵  As Biro et al and Proksch et al reported, the topical administration of dexpanthenol provides effective results in the treatment of and protection against skin irritation and inflammation.^11,12  Weimann et al documented that the need for pantothenic acid in wound healing processes increases since the turnover of coenzyme A in the skin wound is elevated. For this reason, the level of local pantothenic acid could be raised to enhance most of the cellular activities associated with wound healing.¹³  Wiederholt et al reported that the gene expression in proliferating human dermal fibroblasts is altered by calcium pantothenate.¹⁴  In a study by Ebner et al, dexpanthenol is reported to activate fibroblast proliferation and speed up re-epithelialization in wound healing.⁵  It is shown that dexpanthenol is effective for promoting epidermal renewal and speeding up the wound healing process in in vivo models of skin damages. It also revealed that dexpanthenol promotes epithelialization and granulation.⁵  Another study argues that the fibroblast proliferation during the wound healing process is influenced by dexpanthenol through its anti-inflammatory and positive effects.¹⁵  Similar to these studies, in our study, the dexpanthenol group had higher values in wound healing ratios, epithelialization ratios, and the mean fibrosis scores than the control group. Based on these data, we hypothesize that the improvement in wound healing ratios is a result of increased fibroblast proliferation as well as accelerated epithelialization. In addition, we observed significantly less inflammation in the dexpanthenol group than in the NAC group and the control group, which we believe was due to the anti-inflammatory effects of dexpanthenol.

N-acetylcysteine (NAC) supplies bioavailable cysteine for glutathione replenishment and prevents oxidative damage and inflammation. In the wound healing process, reactive oxygen species (ROS) are generated by a number of inflammatory cells, including neutrophils, macrophages, endothelial cells, and fibroblasts.¹⁶  With the generation of ROS in cutaneous wounds, the wound healing process is constrained since they damage the cellular membranes, lipids, proteins, and DNA. GSH is an antioxidant that prevents the damage to important cellular components caused by ROS.¹⁷  Angiogenesis plays a key role in the proliferation of wound healing.¹  Aktunç et al reported that NAC facilitates the wound healing process by stimulating angiogenesis and also by scavenging free oxygen radicals.⁶  Yunsook Lim et al demonstrated that the early inflammatory reactions during cutaneous wound healing in protein-malnourished mice are increased by dietary administration of NAC.¹⁸  Rosato et al reported that the healing of ulcers in patients with bullous morphea is facilitated by the administration of NAC.¹⁹  Murat et al described NAC as a potent antioxidant since it prevents the necrosis of zone of stasis.²⁰  Demir et al reported that the healing of the wounds in irradiated rats was improved by intraperitoneal or oral administration of NAC.²¹  Min-Ling Tsai et al reported that the topical administration of 3% NAC was significantly effective on the cell proliferation, migration, scratch wound healing, and collagenous expression of MMP–1 via the PKC/Stat3 signaling pathway.⁹  In addition, topical NAC 8% eye drops have also been reported to improve the initial treatment of experimental alkali corneal burns.²²  In our study, better wound healing ratios and epithelialization ratios were obtained in the NAC group than in the control group, and the mean fibrosis score was significantly higher in the NAC group than in the control group.

Although the dexpanthenol and NAC groups had better wound healing ratios and epithelialization ratios than the control group, there were no statistically significant differences between the dexpanthenol and NAC groups.

In light of these findings, we believe that NAC and dexpanthenol have positive effects on fibroblast proliferation and fibroplasia and thus improve wound healing.

This study is limited by the small number of animals used. More experimental and clinical studies should be performed in order to evaluate the clinical benefits of topical NAC administration on wound healing.

The efficacy of NAC in wound healing is comparable to dexpanthenol, which is widely used in the treatment of wounds, and both substances can be used to improve wound healing. However, some clinical trials are needed to assess the use of NAC as a standard treatment in wound healing.