Effectiveness of Changing the Application of Japanese Honey to a Hydrocolloid Dressing in Between the Inflammatory and Proliferative Phases on Cutaneous Wound Healing in Male Mice

Introduction. The aim of this study was to investigate the effects of changing the application of Japanese honey to a hydrocolloid dressing (HCD) in between the inflammatory and proliferative phases on cutaneous wound healing in 8-week-old, BALB/cCrSlc male mice.Materials and Methods. Mice were divided into 4 groups: acacia honey followed by a HCD, buckwheat flour honey followed by a HCD, Chinese milk vetch honey followed by a HCD, and a HCD alone (control group). All mice received 2 full-thickness wounds on both sides of the dorsum using a Disposable Biopsy Punch. The wounds of the control group were covered with a HCD, whereas wounds in the other groups were treated with 0.1 mL of the relevant type of honey until day 3 post-wound and then were covered with a HCD from days 4 to 14. Results. In the experimental groups, the wound area ratio was significantly smaller in the inflammatory phase but significantly larger in the proliferative phase. Reepithelialization, collagen deposition, and wound contraction were significantly delayed compared with those in the control group. Discussion. The re-expansion of the wounds in the proliferative phase could not be prevented, and reepithelialization, collagen deposition, and wound contraction were delayed compared with those upon the use of a HCD. Conclusion. The study’s authors concluded that these methods do not promote cutaneous wound healing better than the use of a HCD alone.

Honey is a sweet, sticky substance produced by bees following the collection of nectar and honeydew.¹ Its mean composition is: 17.1% water, 82.4% total carbohydrates, and 0.5% proteins, amino acids, vitamins, and minerals.² Honey has been used to treat wounds since ancient times,³ and Russian soldiers used honey to promote wound healing in World War I.⁴ Honey is considered useful for treating wounds due to its antibacterial, anti-inflammatory, and immunostimulatory effects, which are related to its hygroscopicity, sugar content, and the fact that it contains hydrogen peroxide.⁵

Initially, researchers only used pure, non-boiled commercial honey when evaluating the effects of honey on cutaneous wound healing.^4,6,7 However, it has subsequently been demonstrated that different types of honey exhibit substantially different activities, partly dependent on the honey’s source.⁸ Researchers have evaluated the effects of numerous types of honey from around the world on cutaneous wound healing. For example, studies of manuka honey^9,10-12,14 and mixed pasture honey^9,10 from New Zealand, jelly bush honey¹⁰ from Australia, acacia honey¹³ from Pakistan, gelam honey⁸ and tualang honey¹⁴ from Malaysia, and Indonesian honey¹⁵ have been performed.

To the best of the authors’ knowledge, Ranzato et al¹⁶ are the only researchers who have reported on the effects of Japanese honey (Yamada Apiculture Center, Inc., Okayama, Japan), and they did so with an in vitro study. This study’s authors previously macroscopically and microscopically investigated the in vivo effects of 3 types of Japanese honey — acacia honey, buckwheat flour honey, and Chinese milk vetch honey — as topical therapies for promoting cutaneous wound healing.^17-  The study found the wound area ratio and the number of macrophages in the inflammatory phase were significantly decreased in mice treated with Japanese honey compared with those seen in mice treated with a hydrocolloid dressing (HCD). However, the wound area ratio — the wound area at each time point relative to the wound area on day 0 — of mice treated with Japanese honey gradually increased during the proliferative phase and gradually decreased during the remodeling phase. In addition, the application of the Japanese honey did not promote cutaneous wound healing better than the use of a HCD alone. Therefore, on the basis of these findings, the authors have created 2 treatment methods to prevent the re-expansion of the wound during the proliferative phase and to promote cutaneous wound healing to a greater extent than the use of a HCD alone. One of the methods involves the combined use of Japanese honey and a HCD, with honey being applied to the wound followed by an HCD. The other method involves changing the application of Japanese honey to a HCD in between the inflammatory and proliferative phases. For the first method, Mukai et al¹⁸ reported that the combined use of Japanese honey and a HCD did not promote cutaneous wound healing compared with the use of a HCD alone, although this method did not repeat the episodes of expansion and reduction of the wound in the proliferative phase.  As a result of previous findings,¹⁸ the study authors chose to investigate the effects of changing the application of Japanese honey to a HCD in between the inflammatory and proliferative phases on cutaneous wound healing in 8-week-old, BALB/cCrSlc male mice. Specifically, the study considered whether applying Japanese honey would reduce the inflammatory response induced at the wound site, whether changing the application of Japanese honey to a HCD at an appropriate time could prevent the re-expansion of the wound during the proliferative phase, and whether applying a HCD would promote reepithelialization, collagen deposition, and wound contraction.

Animals. This study used BALB/cCrSlc, 8-week-old male mice (Sankyo Lab Service Corporation, Inc, Toyama, Japan) with a mean weight of 23.6 g ± 2 g (N = 76). The mice were caged individually in an air-conditioned room at 25°C ± 2°C, in which the lights were on from 8:45 AM to 8:45 PM, and they were given ad libitum access to water and food. All of the animal experiments conducted in this study were reviewed and approved by the Kanazawa University Animal Experiment Committee and were carried out in accordance with the University’s Guidelines for the Care and Use of Laboratory Animals, Japan (AP-122521).

Honey. The authors used 3 different types of Japanese honey within 1 year of purchase as the experimental groups: acacia (Robinia pseudoacacia) honey, buckwheat flour (Fagopyrum esculentum) honey, and Chinese milk vetch (Astragalus sinicus) honey (Yamada Bee Farm, Okayama, Japan). The acacia honey was composed of 17.2% water, 82.5% total carbohydrates, 0.2% protein, and 0.1% lipids, with an energy density of 332 kcal/100 g. The buckwheat flour honey was composed of 17.5% water, 81.5% total carbohydrates, 0.7% protein, 0.1% lipids, and 0.2% ash, with an energy density of 330 kcal/100 g. The Chinese milk vetch honey was composed of 17.0% water, 82.7% total carbohydrates, 0.2% protein, and 0.1% lipids, with an energy density of 333 kcal/100 g (Honey contents data from the Foundation of Food Analysis Technology Center SUNATEC, Mie, Japan). These 3 types of honey were selected based on the authors’ previous study.¹⁷

Wounding. The mice were anesthetized via the intraperitoneal (IP) injection of pentobarbital sodium (0.05 mg/g weight), and their dorsal hair was shaved using an electric razor. Then, under anesthesia, 2 circular full-thickness skin wounds (4 mm in diameter), which included the panniculus carnosus muscle, were made on both sides of the dorsum using a Disposable Biopsy Punch (Kai Industries Co. Ltd., Seki, Japan). The mice were divided into 4 groups: acacia followed by a HCD (acacia  HCD), buckwheat flour followed by a HCD (buckwheat flour  HCD), Chinese milk vetch followed by a HCD (Chinese milk vetch  HCD group), and a HCD alone (control group). In the experimental groups, until day 3 post-wound each wound was treated with 0.1 mL of honey^17,18 daily, covered with gauze, and then wrapped with sticky bandages to prevent the honey and gauze from peeling off (MESHPORE, NICHIBAN Co., Ltd., Tokyo, Japan). The authors deemed 0.1 mL of honey was sufficient to cover the wound surface. From days 4–14 post-wound, each wound was covered with a HCD (Tegaderm, 3M Health Care, Tokyo, Japan) and then wrapped with sticky bandages, which were changed daily. The wounds of the control group were only covered with a HCD and then wrapped with sticky bandages, which were also changed daily.

Macroscopic examination. The day the wounds were made was designated as day 0, and wound healing was observed on days 0–14. The edges of the wounds were traced on polypropylene sheets, and photographs of the wounds were taken daily. The traces on the polypropylene sheets were captured with a scanner and transferred to a personal computer using Adobe Photoshop Elements 7.0 (Adobe System Inc, Tokyo, Japan), an image-editing program. The area of each wound was calculated using the image analysis software Scion Image Beta 4.0.2 (Scion Corporation, Frederick, MD). The wound area ratio — shown as a proportion of the wound area on day 0 — was calculated daily.

Histological procedure and immunohistological staining. The mice were euthanized via the intraperitoneal injection of a lethal dose of pentobarbital sodium on days 3, 7, 11, or 14 post-wound. The wound and the surrounding intact skin were harvested, and each sample of wound tissue and the surrounding intact skin was bisected at the center of the wound. The wound samples were stapled onto polypropylene sheets to prevent over-contraction, before being fixed in 4% paraformaldehyde for 18 hours. The samples were dehydrated in a graded alcohol series, cleaned with xylene, and embedded in paraffin. At least 6 serial sections from close to the wound’s center were obtained per wound and stained according to the following methods.¹⁷ Five-µm thick paraffin sections were subjected to hematoxylin and eosin (H&E) or azan staining, or they were immunohistologically stained with an antineutrophil antibody at a concentration of 1:100 to detect neutrophils (ab2557; Abcam Japan, Tokyo, Japan), anti-Mac-3 antibody at a concentration of 1:100 to detect macrophages (550292, Purified Rat Anti-mouse CD107b; BD Pharmingen, San Jose, CA), or anti-alpha-smooth muscle actin (α-SMA) antibody at a concentration of 1:300 to detect myofibroblasts (ab5694; Abcam Japan, Tokyo, Japan). Negative control slides were obtained by omitting each primary antibody.

Microscopic observations. Images of the wounds were taken with a digital microscopic camera (DP2-BSW; Olympus, Tokyo, Japan) and saved on a computer. Assessments of reepithelialization were performed using the software attached to the digital microscope. In these assessments, the distance between the edges of the wound and the length of the new epithelium were measured, and the latter was divided by the former (reepithelialization ratio = length of new epithelium/total wound length). The evaluations of collagen (stained blue) deposition (collagen fiber ratio = collagen pixels/total wound pixels), and the number of myofibroblasts (stained brown) (myofibroblast ratio = myofibroblast pixels/total wound pixels) were calculated using the image-editing program as follows: the wound area — ie, both wound edges, the surface of the wound, and the bottom of the wound, including the panniculus carnosus muscle — was first selected, and then the number of pixels in the selected area (the wound area) was calculated by counting the number of total pixels. Next, the assessments of collagen deposition or the number of myofibroblasts were performed by counting the number of blue- or brown-stained pixels. Finally, the number of blue- or brown-stained pixels was divided by the total number of pixels within the wound area. To analyze the numbers of neutrophils and macrophages in granulation tissue, the number of positively stained cells was counted under a light microscope using a x40 objective lens in 3 regions of granulation tissue — 2 sites near the wound edges and 1 site in the center of the granulation tissue. The area of the 3 sites was calculated using the image analysis software Image J (National Institutes of Health, Bethesda, MD), and the total number of neutrophils or macrophages found at the 3 sites was divided by the total area of the 3 sites.

Statistical analysis. Data are expressed as mean ± standard deviation and were analyzed using JMP 8.0.1 (SAS Institute Inc., Cary, NC). Comparisons of means among multiple groups were performed by one-way analysis of variance (ANOVA) followed by post hoc pairwise comparisons using the Tukey-Kramer multiple comparison test. A two-tailed P < 0.05 was considered statistically significant.

Wound area. The wound area ratio was calculated on days 1–14. In the control group, the wound area increased for 4 days and then decreased rapidly until day 11, after which it gradually shrunk until day 14 (day 14 wound area ratio: 0.09 ± 0.03). On the other hand, in the experimental groups, the wound area did not increase until day 4, after which it increased rapidly until day 6. Then, it decreased rapidly until day 11, after which it decreased gradually until day 14 (day 14 wound area ratio: 0.09 ± 0.06 in the acacia, 0.11 ± 0.04 in the buckwheat flour, and 0.09 ± 0.03 in the Chinese milk vetch groups) (Figure 1).

The wound area ratio of the acacia followed by a HCD group was significantly smaller on days 1–5 (P < 0.01) and significantly larger on day 9 (P < 0.01) than that of the control group. The wound area ratio of the buckwheat flour followed by a HCD group was significantly smaller on days 1–4 (P < 0.01) and significantly larger on days 8–11 (P < 0.05) than that of the control group. In addition, the wound area ratio of the Chinese milk vetch followed by a HCD group was significantly smaller on day 1 (P < 0.05) and days 2–4 (P < 0.01) and significantly larger on days 8 and 9 (P < 0.05) than that of the control group. There were no significant differences among the experimental groups (Figure 1).

Neutrophils and macrophages. On days 3 and 7, the large number of neutrophils were observed in the wounds of all groups, and they decreased in number until day 11. The number of neutrophils showed no significant differences among all groups on days 3–14 (Figure 2). 

On day 3, a small number of macrophages were observed in the wounds of all groups. On day 7, numerous macrophages were observed in the wounds of all groups, especially in the control group. The macrophages gradually decreased in number until day 14 and had almost disappeared in the wounds of the control group on day 14. The number of macrophages in the buckwheat flour followed by a HCD and Chinese milk vetch followed by a HCD groups was significantly smaller than that in the control group on day 7 (P < 0.01 and P < 0.05, respectively). Also, the number of macrophages in the acacia followed by a HCD and buckwheat flour followed by a HCD groups were significantly larger than that in the control group on day 14 (P < 0.05 and P < 0.01, respectively). Moreover, the number of macrophages in the acacia followed by a HCD group tended to be larger than that in the buckwheat flour followed by a HCD group on day 7 (P < 0.1) and that in the Chinese milk vetch followed by a HCD group on day 11 (P < 0.1) (Figure 2).

Reepithelialization, collagen deposition, and wound contraction. In the control group, new epithelial tissue was seen at the wound edges on day 3, and it rapidly progressed on day 7. The new epithelial tissue had completely covered the wound surface by day 11. In the experimental groups using 3 different types of Japanese honey followed by a HCD, new epithelial tissue was seen at the wound edges on day 3, but had not progressed by day 7. However, it had rapidly progressed on day 11 and had completely covered the wound surface by day 14. The reepithelialization ratio of the 3 experimental groups was significantly smaller than that of the control group on days 3 and 7 (P < 0.01). There were no significant differences among the experimental groups on days 3–14 (Figure 3). 

In all groups, collagen deposition increased as wound healing progressed. However, the collagen deposition in the experimental groups was less than that in the control group. The collagen fiber ratio of the experimental groups was significantly smaller than that of the control group on days 7–14 (P < 0.01). There were no significant differences among the experimental groups (Figure 3).

In the control group, a large number of myofibroblasts were present in the wound bed that lined the granulation tissue to bond the myofibroblasts to the wound edges, so they formed bridge-like structures across the circular wound on day 7. They then decreased rapidly until day 11, after which they decreased gradually until day 14. In the experimental groups, a few myofibroblasts were observed at the wound edges on day 7. By day 11, many myofibroblasts were present in the wound bed that lined the granulation tissue to connect the myofibroblasts on the wound edges, so they formed bridge-like structures across the circular wound in the granulation tissue and then decreased gradually until day 14. The myofibroblast ratios of the 3 experimental groups were significantly smaller on day 7 (P < 0.01) and significantly larger on days 11 and 14 (P < 0.01) than that of the control group. There were no significant differences among the experimental groups (Figure 4).

Nakajima et al¹⁷ reported the wound area ratio in mice treated with Japanese honey had significantly decreased in the inflammatory phase compared with that in mice treated with a HCD alone; this gradually increased in the proliferative phase and then decreased again in the remodeling phase. On the basis of these findings, the study authors created 2 treatment methods in order to prevent re-expansion of the wound in the proliferative phase and to promote better cutaneous wound healing than in the use of a HCD alone. One of methods involved the combined use of Japanese honey and a HCD. Although the combined use of Japanese honey and a HCD prevented repetition of the episodes of expansion and reduction of the wound in the proliferative phase, this method could not promote improved cutaneous wound healing compared with the use of a HCD alone.¹⁸ Therefore, in this study, the authors chose to change the application of Japanese honey to a HCD in between the inflammatory and proliferative phases. By applying Japanese honey to the wound, the authors hoped it would reduce the inflammatory response induced at the wound site. They also believed changing the application of Japanese honey to a HCD at an appropriate time could prevent the re-expansion of the wound in the proliferative phase, and that a HCD would promote reepithelialization, collagen deposition, and wound contraction. 

Contrary to the hypothesis, re-expansion of the wound in the proliferative phase could not be prevented.  As was reported by Nakajima et al,¹⁷ the wound area ratios of the 3 experimental groups were significantly smaller than that of the control group during the inflammatory phase. However, after changing the wound treatment from the application of Japanese honey to a HCD, the wound area ratios in the experimental groups increased and were significantly larger than that of the control group during the proliferative phase. In the authors’ laboratory, they used hydrocolloid, which is one of the most well-known dressing materials, to maintain a moist environment. The authors have found that the wounds covered with a HCD increased in size in the inflammatory phase with a large amount of exudate, and then they were rapidly reduced in the proliferative phase.^19-21 From these studies, the authors concluded that a HCD possesses a natural mechanism of healing in which wound contraction is rapidly promoted after the preparation period when wounds have expanded with a large amount of exudate. In fact, the wound area ratios of the experimental groups from days 4–14 showed the same shift as that in the control group from days 0–14. Therefore, the authors believe the re-expansion of the wound after changing the treatment from Japanese honey to a HCD is related to the natural wound healing process of healing with a HCD. However, these concepts are only speculative at this stage, so more detailed examination is required in the future.

Moreover, contrary to the study’s hypothesis, the experimental methods did not promote cutaneous wound healing better than the use of a HCD alone. The reepithelialization ratios of the experimental groups were significantly smaller than that of the control group on days 3 and 7. The collagen fiber ratios of the experimental groups were significantly smaller than that of the control group on days 7–14. Moreover, the myofibroblast ratios of the experimental groups were significantly smaller on day 7 and significantly larger on days 11 and 14 than that of the control group. The authors believe this result is also related to the typical mechanism involved in wound healing when using a HCD for treatment. They concluded that the application of Japanese honey to wounds in the inflammatory phase and covering the wounds with a HCD in the proliferative and remodeling phases could not promote  better cutaneous wound healing compared with the use of a HCD alone by delaying reepithelialization, collagen deposition, and wound contraction. 

This study had 2 major limitations. One limitation was the lack of information about the specific ingredients of each type of honey, which might have influenced cutaneous wound healing. Honey is widely known to be useful for treating wounds due to its antibacterial, anti-inflammatory, and immunostimulatory effects, which are related to its hygroscopicity, sugar content, and the fact that it contains hydrogen peroxide.⁵ However, recent research has indicated that specific ingredients — eg, the arabinogalactans in Kanuka honey,²² the 261 MW component of Jungle honey,²³ the 5.8 kDa component of Manuka honey,²⁴ the major royal protein 1 in Acacia honey,^25,26 and the apigenin and kaempferol in Honeydew honey²⁷ — are beneficial in promoting wound healing. Therefore, the authors need to identify the specific ingredients of Japanese honey that promote wound healing in their next study. The effect of species differences on the findings was the other limitation. Wound contraction is the primary mechanism responsible for wound closure in rodents. On the other hand, reepithelialization is an early and critical point during cutaneous wound healing in humans.^28,29 Therefore, future studies will need to perform an observational study in humans to definitively evaluate the effectiveness of Japanese honey.

In summary, the authors hypothesized in this study that the application of Japanese honey to wounds in the inflammatory phase and covering the wounds with a HCD in the proliferative and remodeling phases could prevent the re-expansion of wounds in the proliferative phase and might promote cutaneous wound healing to a greater extent than the use of a HCD alone. However, contrary to the authors’ hypothesis, the re-expansion of wounds in the proliferative phase could not be prevented and reepithelialization, collagen deposition, and wound contraction were delayed compared with those upon the use of a HCD alone. Therefore, it is indicated that this method could not promote cutaneous wound healing better than the use of a HCD alone.

From the Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University; Department of Clinical Nursing, Graduate Course of Nursing Science, Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University; and Department of Nursing, School of Health Sciences, Kanazawa University, Kanazawa, Japan

Address correspondence to:
Toshio Nakatani, MD, PhD
Faculty of Health Sciences
Institute of Medical, Pharmaceutical, and Health Sciences
Kanazawa University
Kanazawa, Japan
nakatosi@staff.kanazawa-u.ac.jp

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