The Effects of Nitrofurazone on Wound Healing in Thoracoabdominal Full-thickness Skin Defects

Introduction. The use of topical antibiotics on wound healing has been a matter of debate for many years because of the effectiveness. Objective. The aim of this study is to investigate the potential effects of topical nitrofurazone, an antibacterial agent, on the healing of full-thickness skin defects created in a laboratory setting. Materials and Methods. A total of 42 adult male Sprague Dawley rats were divided into 2 groups: group A (control group; n = 21) and group B (nitrofurazone group; n = 21). Circular full-thickness skin defects about 1 cm x 1 cm in size were formed in the left thoracoabdominal regions of all rats. Local physiological saline was applied to the wound once daily in the control group, and a thin layer of nitrofurazone cream was applied to the wound topically once daily in the nitrofurazone group. The defect sizes of all rats were photographed at baseline and days 3, 7, and 10 of the experiment, and wound size reduction was measured macroscopically on the computer to calculate the healing rates. A total of 7 rats from each group were euthanized on days 3, 7, and 10, and their defected regions were resected. The removed specimens were evaluated histopathologically and scored for inflammatory cells, collagen accumulation, granulation tissue formation, reepithelization, and features of the skin defect (eg, layers of the skin affected, size, whether it involves any abscess-necrosis). Statistical significance was set at P < .05. Results. The healing rate had higher values in group B at days 7 and 10 of the experiment (P < .001). A comparison of the group scores showed that there were statistically significant differences in favor of group B. No statistically significant difference was found between the 2 groups with respect to granulation tissue formation. Conclusions. Topically applied nitrofurazone produced positive effects accelerating the wound healing process. 

Wound healing is a complex, active, and dynamic process that aims to reestablish tissue integrity and reverse loss of function. It is not yet fully understood how wound healing is regulated at the molecular level.^1,2 Ideal wound healing should occur within an acceptable timeframe and be cosmetically acceptable to the patient. Various factors affecting certain stages of the wound healing process lead to positive or negative developments in the morphological and functional integrity of the tissue.^1-3 These factors include age, sex, race, ethnicity, nutrition, circulation, tissue oxygenation, infections, and some hormones such as glucocorticoids, insulin, and thyroid hormones.^1,2 If a wound infection occurs in any region of the body, healing can be delayed.¹ In acute and chronic wounds (eg, burns, pressure injuries, granulated wounds), with high positive culture rates prior to treatment, topically applied antibiotics aim to reduce the bacterial load, which eliminates the unfavorable effect of bacterial colonization on wound healing, and reduce the need for systemic antibiotic use.^{4, 5} Moreover, topical antibiotics are easily applied, typically have limited systemic absorption and toxicity, produce low systemic concentration but high wound concentration, and prevent the development of resistance to antibiotics.⁴ However, the effect of these drugs on the wound healing rate and epithelization should be investigated.⁵

In the literature, there is documentation of the use of various topical antibiotics, such as bacitracin, polymyxin B, neomycin, silver sulfadiazine, nystatin, gentamicin, acetic acid, silver nitrate, and nitrofurazone.^3,6,7 Several animal studies have focused on wound healing with topically applied nitrofurazone.^6,8,9 In an experimental study on adult Japanese quails (Coturnix japonica), nitrofurazone was found to accelerate wound healing macroscopically and had no negative effect on recovery.⁶ Geronemus et al⁸ studied partial-thickness wounds in white domestic pigs and assessed epithelization; the authors reported that nitrofurazone significantly delayed wound healing by 24%.⁸ Saydam et al⁹ found nitrofurazone, which was topically applied on full-thickness tissue wounds formed in the back regions of rats, significantly delayed wound healing when used alone but had no negative effect on wound healing when combined with rifampicin.⁹

The present experimental study investigated the effects of topical application of nitrofurazone on acute surgical wound healing in full-thickness skin defects formed at the thoracoabdominal regions of rats. 

Population
This experimental study was conducted on 42 adult male Sprague Dawley rats from the same colony, with each rat weighing between 250 g and 350 g. The rats were obtained from the Experimental Animals Laboratory of the Karadeniz Technical University in Trabzon, Turkey. The purpose of using rats was due to their availability, being safe to use, and resistance to infections and surgical procedures when compared with other experimental animals, as well as having a high ratio of repeating the experiment and having substantial data in the literature on the characteristics of rats’ skin and the cicatricial process.^10,11 Male rats were used to ensure there would be no interference in hormonal variation due to females’ estrous cycle, which could impede in the tissue repair process.¹⁰

The study was approved by the local ethics committee at the Karadeniz Technical University Faculty of Medicine, Animal Care and Use Committee. The rats were handled in accordance with the 8^th edition of the Guide for the Care and Use of Laboratory Animals.

Design
The rats were randomly divided into 2 groups: group A (control group; n = 21) and group B (nitrofurazone group; n = 21). In both groups, circular full-thickness skin defects of about 1 cm x 1 cm in size were formed in the left thoracoabdominal regions of the rats. On days 3, 7, and 10, 7 rats from each group were euthanized, and their defected regions were resected (Figure 1). 

Group A. In the control group, with the exception of application of local saline to the wounds once daily to prevent the wound from drying, no treatment was given to the rats.

Group B. In the nitrofurazone group, a thin layer of cream containing nitrofurazone (Furacin Pomade, 0.2%; Zentiva) was applied to the wounds on the rats once daily. 

Based on previous experiment methodology,¹¹ the rats were kept under special pathogen-free conditions to prevent infections and were placed separately in a light-controlled room with a 12-hour light/12-hour dark cycle. The temperature was kept at 22°C ± 0.5°C, and the relative humidity remained between 65% and 70%. Care was taken to avoid unnecessary stress during the study. The rats were given standard laboratory rodent chow and water. The animals had neither been used in another study nor been given any drugs previously. Their feeding was discontinued 12 hours before the experiment began, but they were allowed to drink water.

Technical and surgical procedures
All of the rats were anesthetized by administering ketamine hydrochloride (Ketalar; Eczacıbaşı) 50 mg/kg and xylazine hydrochloride (Rompun; Bayer) 3 mg/kg intraperitoneally. The anesthetic deepness was assessed with extremity pulling response, and additional doses were administered when necessary. The rats were placed in the supine position during the procedures, which allowed spontaneous breathing under sterile conditions. A heating pad was used to keep their body temperature at 37.0°C in order to prevent the effects of hypothermia and maintain the stability of hemodynamic parameters. The left thoracoabdominal regions of the rats were shaved without harming the skin, and the area was cleaned with 10% povidone-iodine solution (Baticon 10% Solution; Adeka Pharmaceuticals). A circular full-thickness wound tissue measuring 1 cm x 1 cm was formed by using a no. 15 surgical blade to remove the skin and subcutaneous tissue without damaging the underlying aponeurosis. Firm pressure was immediately applied to the defect areas to control bleeding.

No complications occurred in the rats, and none were lost during the surgical procedure. Wound care for all 42 rats was performed daily.

Macroscopic assessment
The defect sizes of all the rats were photographed at baseline and days 3, 7, and 10 of the experiment. The reductions in wound size were measured macroscopically on the computer using the metric system. The Formula¹² was used to calculate the healing rates based on the present data.

Histopathological examination
When the aforementioned times mentioned in the study protocol expired, the rats were euthanized with high doses of the anesthetic drug administered intraperitoneally, and their defect areas were resected as full thickness, retaining at least 1 cm of unharmed tissue around the defect. The specimens were promptly fixed in 10% formalin and processed for paraffin embedding. Then, tissue sections of 5 µm in thickness were obtained with a microtome. Light microscopy (CX41 Upright Microscope; Olympus) was used for histopathological analysis of the hematoxylin and eosin-stained and Masson’s trichrome-stained sections. The histopathological assessment was carried out by the same pathologist, who did not know which tissue specimen belonged to which group; the assessment was conducted through random selection. The histopathological examination was performed according to the scoring of the wound healing assessment (Table 1); inflammatory cells, collagen accumulation, granulation tissue formation, reepithelization, and features of skin defect (eg, what layers of the skin were affected by it, defect size, any abscess-necrosis) were assessed in the specimens. 

In this scale, the parameters were scored from 0 to 3 and were recorded separately for the rats in each group. For inflammatory cells, the following points occurred: 0 points for no inflammation; 1 point for a dispersed, small amount of mixed inflammation; 2 points for moderate mixed inflammation concentrating around vessels; and 3 points for intensive mixed inflammation concentrating around vessels and forming clusters. For collagen accumulation, scores included: 0 points for none; 1 point for patch-like collagenization in the form of short strips; 2 points for strip-like, thin collagenization; and 3 points for strip-like, coarse, thick collagenization. For granulation tissue formation, scores included: 0 points for none; 1 point for involving less than 10 new vessel formations in 1 high power field (HPF); 2 points for involving 11 to 20 new vessel formations in 1 HPF; and 3 points for much and mature granulation tissue involving more than 20 new vessel formations in 1 HPF. For reepithelization, scores included: 0 points for none; 1 point for partial reepithelization in less than one-third of the tissue; 2 points for thin reepithelization in one-third to two-thirds of the tissue; and 3 points for mature reepithelization in the entire tissue. For features of skin defect, scores included: 0 points for none, 1 point for an ulcer limited to the epidermis and smaller than 0 cm to 0.4 cm in diameter microscopically, 2 points for an ulcer limited to the epidermis and papillary dermis of 0.4 cm to 0.6 cm in diameter microscopically, and 3 points for an ulcer in the epidermis and extending to reticular dermis, wider than 0.7 cm microscopically or involving necrosis-abscess formation (Table 1).

Statistical analyses
All statistical data analyses were performed using the SPSS Statistics Version 15.0 for Windows (SPSS Inc.). The histopathological scores of the results obtained for the groups at days 3, 7, and 10 were individually compared within group A and group B, as well as between the 2 groups, using the nonparametric Friedman and Wilcoxon signed-rank tests. The results of the healing rates obtained for group A and group B were compared using the independent samples t test. Statistical significance was set at P < .05.

The healing rate values at day 3 were 28.4% ± 2.8% for group A and 25.9% ± 4.9% for group B. At day 3, the statistical comparison of these parameters did not produce any significant difference (P = .276). The healing rate values at day 7 were 49.4% ± 1.6% in group A and 57.0 ± 2.8% in group B. The healing rate values at day 10 were 59.4% ± 3.1% in group A and 78.1% ± 2.4% in group B. The values obtained at days 7 and 10 for each group were compared statistically and a fairly significant difference was found in favor of group B (P < .001 for both).

The resected specimens were histologically evaluated and scored for inflammatory cells, collagen accumulation, granulation tissue formation, reepithelization, and features of skin defect in line with the scoring system (Table 1). All scores obtained for the above parameters in both groups and the average values of the scores are reported in Table 2. All of the histopathological results were statistically analyzed for significance. 

The number of inflammatory cells was found to remain relatively constant throughout the experiment in group A (P = 1.000), whereas it decreased day by day in group B. In group B, the most significant statistical result was obtained between days 3 and 10 (P =.024). The scores relating to collagen accumulation tended to remain constant for the first 7 days in group A, showing an increase afterwards between days 7 and 10 (P = .038). Collagen accumulation increased day by day in group B; statistically significant results occurred between days 3 and 7 and between days 3 and 10 (P = .038 and P = .024, respectively). 

The scores relating to granulation tissue formation went up in group A from day 7 and remained constant on the average through day 10; statistically significant differences were found between days 3 and 7 and between days 3 and 10 (P = .016 for both). The granulation tissue also increased in group B until day 7, but scores averaging close to 0 were observed at day 10; statistically significant differences were found between days 3 and 7 (P = .024), between days 3 and 10 (P = .020), and between days 7 and 10 (P = .011). 

Reepithelization remained constant, with low scores throughout the experiment in group A (P >.05). Similar low scores were obtained on the average in group B until day 7, but there was a considerable increase in the scores between days 7 and 10 (P =.011). 

For the features of the skin defect, high scores were obtained in group A from days 3 to 10 (P >.05), while the high scores seen at day 3 gradually decreased in group B averaging to 0 at day 10. Statistically significant differences were found in group B in terms of skin defect between days 3 and 10 and between days 7 and 10 (P =.014 and P =.026, respectively) (Figure 2, Figure 3).

A comparison of the scores between groups A and B with respect to inflammatory cells, collagen accumulation, reepithelization, and features of skin defect showed there were statistically significant differences in favor of group B. No statistically significant difference was found between the 2 groups with respect to granulation tissue formation. All results obtained are shown in Table 2 and Figure 4 and Figure 5.

This experimental study on the use of nitrofurazone in full-thickness skin defects underlines 6 points. First, nitrofurazone caused a marked decrease in inflammatory cell concentration in the defected region. Collagen accumulation significantly increased in the defected region in the group in which nitrofurazone was used. Nitrofurazone caused extensive granulation tissue formation within the first week, but the granulation tissue disappeared almost completely in the specimens at the end of the experiment. Nitrofurazone caused significant increases in reepithelization in the defected region. With the use of nitrofurazone, the skin defect area formed at the beginning showed minimal superficial tissue loss at the end of the experiment and disappeared almost completely. The healing rate values were distinctly higher in the rats in the nitrofurazone group compared with the control group.  

Skin integrity impairing with defects that occur after an operation or biopsy — due to a trauma or burn or those that occur in patients who have been bedridden for a long time (eg, pressure injuries) — are the defects frequently encountered in many surgical clinics.⁷ As a barrier against external factors, the skin is the most important organ in terms of general functioning.^4,8 After a wound occurs, the recovery involves a process starting with hemostasis and inflammation and continuing with cell proliferation and remodeling/scar formation.^1,4 Inflammation is an important phase in wound healing, and the number of inflammatory cells and particularly that of neutrophils increase in the defected area during the early days of the recovery process.^1,7 However, the number of these inflammatory cells should decrease during the following periods, as a strong inflammatory response increases tissue damage, delays epithelialization and collagen synthesis, and plays an important role in prolonging healing by slowing it down.¹³ In the present study, the strong inflammatory response that occurred at day 3 in the defected regions of the control group retained the same severity until day 10. The reason for the strong inflammatory response to stay at the same severity throughout the experiment could have been due to the major effects of the inflammation phase, which is the first stage of the normal wound healing process. 

In the group in which nitrofurazone was used, however, a gradual decrease occurred in the number of inflammatory cells during the experiment, and the scores dropped to the lowest level at day 10 when the experiment was terminated. Occurrence of severe inflammation in the defected region at an early stage is an expected situation, but a decrease occurs in this inflammation during the healing process.^1,13 Nitrofurazone facilitated and accelerated this physiological decrease in inflammation, which enabled acceleration in wound healing and defect closure. It is believed the nitrofurazone had a bactericidal effect due to its antimicrobial activity against many microorganisms (primarily streptococci) that exist in the normal skin flora of the rats, but could become pathogenic due to the defect, and this process reduced the inflammatory response.^5,6 

The main function of the fibroblasts concentrated in the tissue at the proliferation phase of wound healing is the collagen synthesis that begins at day 2 of injury and shows the highest activity at day 7.¹⁴ This new collagen tissue formation in the defected area enables newly developing cells to bind to each other with stronger bonds, facilitates closure of wound edges, contributes to contraction, and makes the new tissue stronger and firmer.^1,15 Nitrofurazone increased the collagen formation in the tissue throughout the experiment, which had a positive impact on the wound healing process. However, collagen normally decreases at the remodeling stage due to its increased collagenase activity, and its shape turns into collagen networks with small fibrils.¹ Since the present study ended after day 10, the effects of nitrofurazone that would be used for a longer time on collagen production remained unknown. 

Generally, the amount of granulation in the tissue increases from day 5 in the wound healing process, but when healing is completed and reepithelization becomes predominant, the granulation tissue is observed to disappear.^1,13 In this study, nitrofurazone caused formation of a dense granulation tissue in the defected region within 7 days; interestingly, the granulation tissue disappeared almost completely in the specimens at day 10 when the experiment ended. Known as the last suitable point for detecting wound healing, reepithelization was seen in low amounts in group B in the first week and showed a considerable increase between days 7 and 10. These 2 histopathological parameters manifested in group A with dense granulation tissue and partially increased reepithelization This result shows there was no healing in these periods in the wound that was left on its own, but wound healing accelerated with the use of nitrofurazone. In addition, the absence of granulation tissue and highest level of reepithelization in the nitrofurazone group at day 10 meant wound healing was almost complete. The absence of granulation tissue and highest level of reepithelization indicated that the once daily application of nitrofurazone provided the maximum benefit at day 10 of the present study. 

Widespread skin defects often occur at the beginning of the wound formation process due to medical issues, including vascular problems, feeding difficulties, and infections.^3,16 During wound healing, certain factors, such as infection control and angiogenesis, enable the aforementioned skin defects to disappear. In the present study, a skin defect area was seen in the control group from the beginning of the study, but the skin defect areas gradually decreased in group B; minimal defects were found in the tissue at day 10 for group B. No superficial tissue loss should be present in the tissue, and extensive fibrosis should be seen at the stage of completion of wound healing. The current findings may indicate that nitrofurazone could be used to prevent the formation of superficial tissue loss from being infected by local or external microorganisms; the present study found nitrofurazone accelerated the wound healing process.

The similar healing rate values observed in groups A and B during the first 3 days of this study suggest nitrofurazone does not have any effect on wound healing during the first few days. However, the healing rate was noticeably higher at days 7 and 10 in the nitrofurazone group than in the control group. At day 10, the healing rate in group B reached 80%. With an approximated 20% difference between the nitrofurazone group and control group, the results in the present study could be interpreted as a macroscopic indication that nitrofurazone potentially accelerates wound healing.

In the wound healing process, neutrophils, macrophages and endothelial cells, and fibroblasts generate a number of reactive oxygen products.¹⁶ Similarly, free oxygen radicals cause formation of reactive oxygen products and lipid peroxidation.¹⁷ As a result, the wound healing process is negatively affected due to decreased protein synthesis, elevated leukocyte infiltration, impaired extracellular matrix formation, and prolonged inflammatory response.^17,18 The major antioxidant enzymes that fight with this situation are glutathione peroxidase and superoxide dismutase. Glutathione peroxidase not only prevents initiation of lipid peroxidation but also enables metabolization of hydroperoxides that emerge as a result of lipid peroxidation. A decrease in the activity of this enzyme leads to hydrogen peroxide accumulation and cell damage. Superoxide dismutase exists in all aerobic cells. This enzyme is present in cytosol and mitochondria and protects cells from the harmful effects of superoxide radicals by deactivating them. In a study conducted by Hong et al,¹⁹ depending on the dose, nitrofurazone was found to increase these antioxidant enzymes in protozoan Euplotes vannus cells. Further animal studies showing in vivo that similar effects of nitrofurazone can occur in the cells of wounds would increase the value of the positive results obtained in the present study. The authors believe these results will lead to future human studies.

Nitrofurazone is a derivative of nitrofuran and has a broad spectrum, including Gram-positive and Gram-negative bacteria (eg, Staphylococcus aureus, Streptococcus, Escherichia coli, Clostridium perfringens, Aerobacter aerogenes, Proteus spp). Nitrofurazone has a bactericidal effect on most of these microorganisms that cause surface infections.⁹ The mechanism of action of nitrofurazone is the inactivation of ribosomal proteins and other macromolecules, with consequent inhibition of proteins, DNA, RNA, and cell wall synthesis, blocking the aerobic metabolism of bacterial cells and inhibiting the carbohydrate metabolism enzymes of bacteria.¹⁰ It can be used as an adjuvant in the healing process of cutaneous wounds, since along with the antimicrobial activity, it interferes in the formation of granulation tissue.¹⁰ The microorganisms colonized on wounded tissue are killed and digested by the phagolysosomes of leukocytes.²⁰ In the meantime, reactive superoxide and hydroxyl radicals are formed as a result of oxidative reactions.^18,20 Although these actions take place in a time as short as milliseconds, they are toxic if uncontrolled.²¹ Owing to its antibacterial property, topically applied nitrofurazone reduces the number of bacteria in the wound site and protects the tissue from toxic effects that would occur through the aforementioned mechanism.^5,6

In a study on white domestic pigs, nitrofurazone was found to delay wound healing.⁸ However, that study⁸ was conducted about 40 years ago and only evaluated the reepithelialization rates. The result of the study⁸ can be contributed to the fact that the study was not a large-scale histopathological study in which the entire phases of the wound healing process were evaluated by investigating various parameters involved in it. In a study included in the literature, Ozkaya et al⁵ formed partial-thickness wounds on the backs of rats and applied topical nitrofurazone, rifampicin, nitrofurazone-rifampicin, and bacitracin-neomycin. The authors found nitrofurazone did not delay wound healing, but the wound surface was larger during the healing process as compared with the other agents.⁵ No histopathological examination was performed in that study⁵; the wound healing times and sizes were explored through observation and macroscopic examination. The results of the current study, in conjunction with the histopathological data collected herein, support the positive effects of nitrofurazone obtained in the study by Ozkaya et al.⁵

The wound healing processes of rats and humans are quite different, especially in terms of the time in which wounds take to heal. Unlike humans, wound healing in rats occurs primarily through contracture. Based on the processes, including timing and the predominant stage of healing, the result obtained here can only be valid for the present study. As such, clinical studies on human subjects should be conducted. Additional limitations of the present study include the small number of rats in the groups, study period being limited to 10 days, and completion of the study with results based on histopathological examination outcomes. With further studies in which more rats are used and nitrofurazone is applied for longer times, more objective results can be obtained and nitrofurazone’s appropriate doses, the time it takes for it to show its full effect in wound healing, and its side effects will be better understood. Additionally, microbiology-based experimental studies revealing nitrofurazone’s effects at certain times on the number and variety of microorganisms in a wound site and biochemistry-based studies investigating its effect on the mediators, cytokines, and growth factors that play a role in wound healing will provide more comprehensive information on this subject.

Topically applied nitrofurazone showed a positive effect and accelerated the wound healing process. In light of this result, the authors hope nitrofurazone, which has lost its popularity in many countries in recent years, will be used again as an important pharmacological agent in the treatment of wounded tissues, which is a great public health problem in terms of workload and cost. 

Authors: Sami Karapolat, MD¹; Banu Karapolat, MD²; Alaaddin Buran, MD¹; Burcu Kemal Okatan, MD³; AtilaTurkyilmaz, MD¹; Turan Set, MD⁴; and CelalTekinbas, MD¹

Affiliations: ¹Department of Thoracic Surgery, Karadeniz Technical University Medical School, Trabzon, Turkey; ²Department of General Surgery, Kanuni Training and Research Hospital, Trabzon, Turkey; ³Department of Pathology, Kanuni Training and Research Hospital; and ⁴Department of Family Medicine, Karadeniz Technical University Medical School

Correspondence: Sami Karapolat, MD, Department of Thoracic Surgery, Karadeniz Technical University Medical School, Trabzon, Turkey; samikarapolat@yahoo.com

Disclosure: The authors disclose no financial or other conflicts of interest.