Comparing the effects of nebivolol and dexpanthenol on wound healing: an experimental study

Abstract

Wound healing is a dynamic, interactive process that is initiated in response to injury. A number of investigations and clinical studies have been performed to determine new approaches for the improvement of wound healing. The aim of this study was to compare the effects of dexpanthenol, a molecule that is widely used for improving wound healing, and nebivolol, a molecule that increases nitric oxide release, on wound healing. A total of 30 rats were divided into three equal groups (n = 10). A linear 2 cm incision was made in the rats' skin. No treatment was administered in the first (control) group. Dexpanthenol cream was administered to the rats in the second group and 5% nebivolol cream was administered to the rats in the third group. The wound areas of all of the rats were measured on certain days. On the 21^st day, all wounds were excised and histologically evaluated. The wound healing rates of the dexpanthenol and nebivolol groups were higher than those of the control group (P < 0·05). However, the wound healing rates of the dexpanthenol and nebivolol groups were not significantly different. Nebivolol and dexpanthenol have comparable effects on wound healing.

Introduction

Wound healing is a dynamic, interactive process that is initiated in response to injury 1. There are many factors that may negatively affect the process of wound healing. A number of investigations and clinical studies have been performed to determine new approaches to improve wound healing 2.

Dexpanthenol is the stable alcoholic analogue of pantothenic acid, which is a component of coenzyme A. The topical form of dexpanthenol is widely used because it easily penetrates the skin at high local concentrations. The most prominent effects of formulations containing dexpanthenol include the stimulation of epithelialisation, granulation and the mitigation of itching 3.

Nitric oxide has been shown to play a major role in wound healing 4. It activates angiogenesis, acts as a chemoattractant to fibroblasts and promotes fibroplasia 5. Some molecules have been shown to improve wound healing by improving the release of nitric oxide 6. Nebivolol is a compound that has been reported to improve nitric oxide release 7. Recently, nebivolol was reported to improve wound healing in diabetic rats 8.

The aim of this study was to compare the effects of dexpanthenol, a molecule that is widely used to improve wound healing, and nebivolol, a molecule that increases nitric oxide release, on wound healing.

Materials and methods

Animals

Albino Wistar rats (n = 30) weighing 250–300 g were used in this study. They were housed in polycarbonate cages in a temperature (21 ± 1°C) and humidity controlled (45–55%) room that was maintained on a 12/12 reversed light cycle. The rats were fed a standard rat chow and allowed to drink water ad libitum. This study was approved by the Ethics Committee for Animal Use of Dicle University.

Dexpanthenol cream

A commercial preparation containing a 5% dexpanthenol water‐in‐oil emulsion was used (Bepanthol^® cream, 5% dexpanthenol, Bayer, Germany).

Nebivolol cream

A nebivolol cream base was formulated with cold cream. The ingredients used for the preparation of 5% nebivolol cream were accurately weighed, and the required number of nebivolol tablets (Vasoxen^® tablet, 5 mg nebivolol HCl, Berlin Chemie AG, Germany) were pulverised. Then, purified water was added to the nebivolol powder to form a paste, which was then incorporated into the cold cream at the intended final weight and mixed well.

Surgical procedure and application

Rats were anaesthetised with intramuscular ketamine hydrochloride (25 mg/kg). Their back hair was shaved, the application field was outlined with a marking pen and a 2 cm linear full‐thickness incision was made in the skin of the back. A total of 30 rats were equally divided into three groups (n = 10). No treatment was administered in the first (control) group. Dexpanthenol cream was administered to rats in the second group, and 5% nebivolol cream was administered to rats in the third group. All wounds were cleaned daily with saline. After cleaning, the creams were applied to the wounds in sufficient amounts to cover the entire wound area. On the 21^st day, rats were anaesthetised by intramuscular ketamine hydrochloride (25 mg/kg), the wound and adherent tissues were removed and the skin defects were closed with non‐absorbable suture.

Evaluation and measurement of wound healing

The measurements of the wound areas for all three groups were taken on the initial day and on the 3^rd, 5^th, 7^th, 10^th, 14^th and 21^st days following the wound. The wounds were measured using transparencies and a permanent marker. The wound areas were recorded and measured on graph paper, and the percentage of wound healing was calculated by the Walker Formula 9.

Wound healing rate (%) = 100 × (1 − wound area/initial wound area)

Histology

All skin samples were fixed at 10% neutral formalin, processed using the classical histological preparation technique and embedded in paraffin. Serial sections (5 µm) were taken from these blocks and stained with haematoxylin–eosin for the evaluation of general tissue appearance. All slides were observed under a light microscope (Axiophot, Zeiss, Germany). Morphological findings, including inflammation, fibrosis and angiogenesis were scored as follows: none, score 0; few, score 1; moderate, score 2; many, score 3. Epithelialisation and granulation were determined as either present or absent. The histological examination and scoring system were performed in a blinded fashion.

Statistical analysis

Statistical analyses were performed using SPSS for Windows 13.0 (SPSS Inc., Chicago, IL). Data were expressed as mean ± SD (standard deviation) values. A chi‐square test was used for qualitative data, whereas groups were compared using the non‐parametric Kruskal‐Wallis test. A Mann–Whitney U test was used for binary comparisons, whereas repeated measures of analysis of variance (ANOVA) test were used for the reduction of the wound area. A P‐value <0·05 was considered significant.

Results

Multiple comparison analysis showed that the wound healing rates were higher in the dexpanthenol and nebivolol groups than in the control group (P < 0·05), and that the difference between the wound healing rates of the dexpanthenol and nebivolol groups was not statistically significant (P > 0·05) (Figure 1).

Figure 1.

The wound healing rates of the groups.

Microscopic analysis showed that the dexpanthenol group developed less inflammation than the other two groups (P = 0·027), and that the control group developed less fibrosis than the other two groups (P = 0·002) (Figure 2).

Figure 2.

The microscopic images of the (A) control group, (B) dexpanthenol group, (C) nebivolol group. (Haematoxylin–eosin ×25).

Although the dexpanthenol and nebivolol groups had higher epithelialisation rates than the control group, these differences were not statistically significant (P > 0·05). Similarly, the mean angiogenesis and granulation scores of the dexpanthenol and nebivolol groups were higher than those of the control group, but these differences were not statistically significant (P > 0·05) (Table 1).

Table 1.

The microscopic histopathological findings of the rats according to the groups

		Control n (%)	Dexpanthenol n (%)	Nebivolol n (%)	P *
Epithelisation	Present	3 (30)	5 (50)	6 (60)	NS
	Absent	7 (70)	5 (50)	4 (40)
Inflammation	0	3 (30)	2 (20)	0 (0)	0·027
	1	1 (10)	6 (60)	2 (20)
	2	6 (60)	2 (20)	8 (80)
	3	0 (0)	0 (0)	0 (0)
Fibrosis	0	0 (0)	0 (0)	0 (0)	0·002
	1	5 (50)	0 (0)	0 (0)
	2	5 (50)	10 (100)	10 (100)
	3	0 (0)	0 (0)	0 (0)
Angiogenesis	0	0 (0)	0 (0)	0 (0)	NS
	1	7 (70)	5 (50)	1 (10)
	2	3 (30)	4 (40)	7 (70)
	3	0 (0)	1 (10)	2 (20)
Granulation	Present	5 (50)	7 (70)	8 (80)	NS
	Absent	5 (50)	3 (30)	2 (20)

NS = not significant.

^* Chi‐square test.

Discussion

Skin is the largest organ of the body, and it plays a critical role in maintaining body homeostasis. Wound healing is a dynamic, interactive process that is initiated in response to injury and restores the function and integrity of damaged tissues 1. There are four phases of skin wound healing, which include haemostasis, inflammation, proliferation and tissue remodelling 10. Optimal wound healing requires the collaboration of the inflammatory cells, biochemical mediators, extracellular matrix molecules, the microenvironment of the cell and remodelling 11, 12, 13. There are many factors that may negatively affect the process of wound healing in patients, therefore causing improper or impaired tissue repair. The quality of skin wound healing can be improved by the application of various natural or synthetic materials, several of which are currently in use 14. It is estimated that morbidity because of non‐healing wounds cost an extra $3 billion annually for health services 15. Therefore, a number of investigations and clinical studies have been performed to determine new approaches to improve wound healing 2.

Pantothenic acid, a vitamin of the B complex and the inactive form of coenzyme A, is essential to normal epithelial function. Dexpanthenol, the stable alcoholic analogue of pantothenic acid, is used for topical application because it has good skin penetration and high local concentrations when administered in water‐in‐oil emulsions 3. Weimann et al. reported that the turnover of coenzyme A in damaged skin is relatively high, and therefore increases the need for pantothenic acid during wound healing. Therefore, it is likely that additional quantities of pantothenic acid are needed locally to optimise the many cellular processes involved in wound healing 16. Ebner et al. showed that dexpanthenol caused the dermatologic effects of increased fibroblast proliferation as well as accelerated reepithelialisation in wound healing 3. Presto et al. reported that dexpanthenol improved epidermal regeneration and significantly accelerated wound healing in in vivo models of skin injuries. They also showed that dexpanthenol stimulates epithelialisation and granulation 17. Many authors, including Ebner et al., Romitti et al., Schmuth et al. and Baschong et al. have reported the anti‐inflammatory effects of dexpanthenol in various clinical situations 3, 18, 19, 20. Weimann et al., Oztürk et al. and Wiederholt et al. reported that dexpanthenol had positive effects on fibroblast proliferation during the wound healing process 16, 21, 22. Similar to these previously reported studies, in our study, the wound healing ratios, epithelialisation ratios and the mean fibrosis scores were higher in the dexpanthenol group than in the control group. Based on these data, we hypothesise that the improvement in wound healing ratios is a result of increased fibroblast proliferation as well as accelerated epithelialisation. In addition, we saw significantly less inflammation in the dexpanthenol group than in the nebivolol group and the control group. We believe that this was because of the anti‐inflammatory effects of dexpanthenol.

Nitric oxide has been shown to play a major role in wound healing 4. Conditions associated with impaired wound healing, such as protein/calorie malnutrition, diabetes and steroid use have all been shown to be associated with reduced nitric oxide expression 23. In addition to the role of endogenous nitric oxide in wound healing, improvements in wound healing following its application have also been reported. Bauer et al. and Masters et al. reported that nitric oxide‐releasing polymers applied topically improved wound repair 24, 25. In addition, Mandeep Kaushal et al. reported that nitric oxide acts as a chemoattractant for fibroblasts and promotes fibroplasia 5. Jamshidzadeha et al. reported that the topical administration of sildenafil improved wound healing by increasing the release of nitric oxide 6. Nebivolol is a third generation β1 blocker and is an agonist for β3 receptors 7, 26, 27. Rozec et al. reported that nebivolol increases nitric oxide release by stimulating β3 receptors, but that this release is blocked in the presence of β3 blockers 28. Gulcan et al. reported that the topical administration of nebivolol increased wound healing rates 8. In our study, the nebivolol group had better wound healing ratios and epithelialisation ratios than the control group, and the mean fibrosis score was significantly higher in the nebivolol group than in the control group.

Although the dexpanthenol group and the nebivolol group had better wound healing ratios and epithelialisation ratios than the control group, there were no statistically significant differences between these two groups.

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

The limitation of this study is that we used a small number of animals. More experimental and clinical studies should be performed in order to evaluate the clinical benefits of topical nebivolol administration on wound healing.

Conclusion

Dexpanthenol has been shown to improve wound healing and is widely used for various types of wounds. In addition, nebivolol has also been shown to improve wound healing. This study showed that the effects of nebivolol on wound healing are comparable to those of dexpanthenol, and both substances can be used to improve wound healing.