Effect of Topical Application of Medihoney on the Healing of the Maxillary Second Molar Tooth Socket in Wistar Rats

Introduction. While ancient civilizations reportedly used honey for wound management, making it one of the oldest known agents in medicine used for wound dressings, there are limited studies regarding the effects of honey in treating oral wounds and the effects of honey on repairing bony tissue. This experimental study compared treatment with Medihoney (Derma Sciences Inc, Princeton, NJ) with no treatment on 48 Wistar rats in Rafsanjan University of Medical Sciences (Rafsanjan, Iran). The aim was to determine the effects of honey on the healing process of hard and soft tissue of a second molar tooth socket following extraction. Materials and Methods. Twenty four rats were in the experimental group and 24 in the control (no treatment) group. An iris scissor was used as an elevator to pull out the second molar tooth of both groups’ samples. In the experimental group, 1 drop of the honey product was administrated into the socket of the tooth in the rats. The day of surgery was considered day 0, and the examinations were carried out on postoperative days 3, 7, and 14. The amount of inflammation and wound healing was assessed with a light microscope. Wound healing was assessed by evaluating the formation of granulation tissue, the amount and type of inflammation, angiogenesis, number of fibroblasts, and formation of bone trabeculae. The mean number of new blood vessels, neutrophils, lymphocytes, macrophages and fibroblasts, and the extent of the granulation tissue in the experimental group were higher at days 3 and 7. However, these differences were not significant (P < 0.05). On days 7 and 14, the extent of bone trabeculae was more pronounced in the experimental group. This difference was significant (P = 0.03, P = 0.006, respectively). Conclusion. This study showed honey can promote wound healing, but the authors recommend additional randomized clinical trials on humans to further assess the effects of honey on the wound healing process.

Tooth extractions cause open wounds in soft and bony tissue,¹ and various drugs and materials may be used to accelerate wound healing in this tissue. Ancient Persians, Greeks, and Egyptians reportedly used honey for wound management, making it one of the oldest known agents in medicine used for wound dressings.² While unprocessed honey has been used for years, one of the first medically certified products in Europe and Australia was Medihoney (Derma Sciences Inc, Princeton, NJ).³ Honey has also been shown to be a non-toxic and non-irritating agent.⁴

  A number of animal studies have provided histological data for wound healing, and reported that honey can help wounds heal faster.^5-8 The bioactivity of honey promotes wound healing by stimulating cytokine production, which starts the healing process.^9,10 Some studies have demonstrated that the amount of angiogenesis, epithelialization, and granulation tissue increases with the administration of honey.³ In addition, honey does not adhere to wounds and can be easily removed from the wound without damage to new tissue or pain for the patient.²

  Animal studies have shown that honey decreases inflammation in wounds,^1,10 and can destroy microbes by dehydrating the bacteria and holding back their growth, thereby decreasing the inflammatory process, amount of exudates, and edema.^2,3,12,13 It also decreases the pH level of the environment, which provides an inhospitable setting for bacterial growth and colonization.³

  Honey can manage many wounds when treatment with modern wound-treatment agents have not shown good results.² It has also been reported to be beneficial in managing burn wounds.⁴

  The use of honey as a wound dressing agent is increasing worldwide; however, more evidence is needed to confirm its effectiveness in the wound healing process.^14-18 The authors of the present study found nothing in the literature about the effects of honey on human bone. It would be difficult to conduct a double-blind randomized clinical trial in this matter, as human studies might be affected by placebo effect. One of the benefits of animal studies is that the results are not affected by the placebo effect.^{2, 3}

  There are limited studies regarding the effects of honey in treating oral wounds and the effects of honey on repairing bony tissue. The objective of the study was to assess the effects of honey on the healing process of hard and soft tissue of the second molar tooth socket after extraction in Wistar rats.

This experimental study was conducted at the Rafsanjan University of Medical Sciences. Forty-eight female Wistar rats weighing 120 g to 180 g were kept in solitary sterile cages with a clean environment and free access to water and food. Procedures were completed according to the regulations of Institutional Animal Care and Use Committee. This study was approved by the ethics committee of Rafsanjan University of Medical Sciences. All rats were examined by a veterinarian who confirmed their general health.

  While similar studies used 10-15 animals in each group, the current study used 24 rats per group to increase the power of the results. Sterile honey (Medihoney, Derma Sciences Inc, Princeton, NJ) was used on the wound dressings in the experimental group and no substances were used to dress the wounds of the control group. All rats underwent general anesthesia using 100 mg/ml of ketamine and 100 mg/ml of xylazine (both from Iran Daru Pharmaceutical Co, Tehran, Iran) with a ratio of 9.1 -0.6 cc/10 g. The drugs were administered intraperitoneally using an insulin syringe.

  An iris scissor was used as an elevator to pull out the second molar tooth of both groups’ samples. In the experimental group, 1 drop of sterile honey was administrated into the socket of the tooth and left uncovered. The rats were maintained in a warm environment and after recovery they were transferred to their separate cages with free access to water and food. All the samples were maintained in similar circumstances.

  The day of surgery was considered as day 0 with examinations carried out on days 3, 7, and 14. On each examination day, one third of the rats of each group were sacrificed using a guillotine and according to all the protocols and recommendations provided by the Institutional Animal Care and Use Committee. Samples were fixed in 10% formalin with a pH of 7.

  After 1 week, the samples were taken out of formalin and inserted into 4% ethylene diamine tetra-acetate solution for 3 weeks to decalcify the bone tissue. The solution was changed twice per week and at the end of the third week, the bone tissues were examined. Afterwards, the samples were placed into a tissue processor device (DidehSabz Company, Tehran, Iran). After 19 hours, the samples were immersed in paraffin and 5 micron cuts were prepared using a microtome. Hematoxylin and eosin staining was performed on the samples.

  A pathologist who was blinded to the groups from which the samples were taken assessed the amount of inflammation and wound healing by using a light microscope. Wound healing was assessed by evaluating the amount of granulation tissue, the amount of inflammation, angiogenesis, and extent of bone trabeculae.

  The inflammatory cells (neutrophils, lymphocytes, macrophages, and fibroblasts) were counted in 5 different fields (x 1000) using an ocular micrometer and the mean number of these 5 fields was recorded. The extent of the granulation tissue and bone trabeculae was measured by an ocular micrometer. The number of blood vessels was counted in 10 different fields (x400) and the mean number was recorded. The data was entered into software (SPSS for Windows, version 16, SPSS Inc, Chicago, IL) and descriptive statistics (ie, frequency, relative frequency, mean, and standard deviation) and t test were used for analysis. A P-value ≤ 0.05 was considered as significant.

  In this study, there were 48 rats total, randomly divided in 2 groups – experimental and control. The difference between the mean number of new blood vessels, neutrophils, lymphocytes, macrophages, and fibroblasts of the control and experimental groups were not significant, although the amount of fibroblasts on day 7 in the experimental group were significantly higher than the control group (Table 1). Pathologic view of the tissues of the control and experimental groups are shown in Figure 1. The mean extent of bone trabeculae of the experimental group was significantly higher than the control group on days 7 and 14 (Table 2).

This study was carried out to evaluate the effects of topical honey on the healing process of a wound to a tooth socket in rats. There was no significant difference in the number of new blood vessels, lymphocytes, neutrophils, macrophages, and the amount of the granulation tissue between the 2 groups on days 3 and 7. However, on day 7, the extent of bone trabeculae and fibroblasts of wounds in the experimental group were significantly larger than the control group (P = 0.05 and P = 0.03, respectively). Extent of bone trabeculae was also significantly larger in the experimental group rats on day 14 compared to the control group (P = 0.006).

  There are few studies that assess the effects of honey on oral wounds and bony tissue. Malavazzi et al¹⁹ compared the effects of honey and 20% autologous serum on epithelial corneal ulcers of 40 rabbits. The results of their study showed there was no significant difference between the wound healing qualities of the 2 groups.¹⁹ Osuagwu et al⁷ compared the effects of honey and saline in 20 wistar rats and showed that oral open wounds treated with honey were significantly more healed than wounds treated with saline (P = 0.002). However, unlike the current study, the amount of granulation tissue was significantly greater with honey (P = 0.002).⁷ Oladejo et al⁶ conducted the same study on 20 rats and showed that wounds treated with honey were 80% contracted while saline-treated wounds were 55% contracted (P = 0.001).

  Gupta et al²⁰ conducted a retrospective study on 108 patients. They compared the effects of honey and silver sulfadiazine on patients with first or second degree burns. They reported that patients’ wounds treated with honey recovered in 18.16 days while the other group was healed in 32.68 days. This difference was significant (P < 0.05).²⁰

  Biglari et al²¹ also conducted a study on 121 patients with chronic infected wounds with various aetiologies treated with a medical grade honey product and reported that the overall wound size decreased in a significantly shorter period of time and the healed wounds showed less necrosis.

  A limitation of this study was that it was not a randomized clinical trial. Also, since this experiment was conducted on animal samples, its results cannot be extended to humans. However, it showed that honey can accelerate the healing of oral wounds and the repair of bony tissue. As discussed above, many studies have confirmed our findings and reported different kinds of honey to have noticeable benefits in healing human or animal wounds.^{20, 22-27} Thus, it is recommended to conduct randomized clinical trials on human and assess the effects of honey on wound healing process.

The authors would like to thank the staff of the Physiology Department of Rafsanjan University of Medical Sciences for their help during the study, as well as the Farzan Institute for Research and Technology for their technical assistance.

Affiliations: The authors are from the Rafsanjan University of Medical Sciences, Rafsanjan, Iran.

Address correspondence to:
A. Peimani, MD
Alipeimani2001@yahoo.com

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