Effects of Three Types of Honey on Cutaneous Wound Healing

Hatice Ozlem Nisbet, DVM, PhD; Cevat Nisbet, DVM, PhD; Murat Yarim, DVM, PhD; Ahmet Guler, PhD; Ahmet Ozak, DVM, PhD

Disclosures

Wounds 

In This Article

Evaluation Parameters

Planimetry

Planimetry was performed on days 0, 7, 14, and 21 on anesthetized animals (the anesthesia protocol used to create the wounds was repeated). The wound area of each lesion on each evaluation day was obtained by tracing the perimeter of the wound onto a sterile piece of clear acetate film with a special marking pen. The acetate was placed on the wound surface, smoothed, and held flat and immobile by an assistant, while the tracing was made by the examiner (Dr. Nisbet) wearing 2.5x loupes. The examiner traced the margin at the leading edge of the advancing epithelium. The area within the margin of the advancing epithelium was defined as the "unhealed wound area" (Figure 1). Wound tracings were digitized using digital scanning software and hardware (Sigma Scan® Pro 5.0, SPSS Science, Chicago, IL).[14] The unhealed wound area was recorded at each day of measurement and was used for statistical analyses.

Figure 1.

The area within the margin of the advancing epithelium (white arrow) was defined as the "unhealed wound area" (black arrow). A) Pure chestnut honey treatment group on day 7. B) Pure chestnut honey treatment group on day 14.

Healing Time

The time between wound creation and the day that each wound was covered with epithelium was evaluated to compare the time to healing among the four study groups.

Histopathological Evaluation

A 5-mm punch biopsy instrument was used to take skin specimens from the wound edge of each rabbit on days 7, 14, and 21 immediately following measurement. The specimens were fixed in 10% neutral buffered formalin and processed routinely for histopathological examination. Five-micrometer sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome. Although several histopathological parameters could be used to assess the progression of healing from the inflammatory to the repair stage, the progressive decrease in macrophages, fibrosis, and progressive increase in angiogenesis, epithelization, and collagen level were selected.

The four selected areas were examined under 400x magnification. The Abramov's histological scoring system (modified Greenhalgh's scoring system) was used for scoring epithelization, fibrosis, angiogenesis, and collagen level; the number of macrophages under this system was modified.[15,16] While the Greenhalgh's scoring system compiled several histological parameters simultaneously to create a single score, the Abramov's system assessed each parameter independently and gave a score of 0–3. The collagen level was graded as: 0 (none), 1 (scant), 2 (moderate, [Figure 2]), and 3 (abundant, [Figure 3]). Epithelization was graded as either: 0 (none, [Figure 4]), 1 (partial), 2 (complete, but immature or thin), and 3 (complete and mature, [Figure 5]). Angiogenesis was graded as either: 0 (none), 1 (up to 5 vessels per highpower field [HPF]), 2 (6 –10 vessels per HPF, [Figure 7]), and 3 (more than 10 vessels per HPF, [Figure 6]). Fibrosis was graded as: 0 (none to minimal fibroblasts), 1 (few fibroblasts), 2 (more fibroblasts, [Figure 3]), 3 (predominantly fibroblasts, [Figure 2]). The number of macrophages was scored as 0–25 = 1, 26–50 = 2 (Figure 8) and > 51 = 3 (Figure 9).

Figure 2.

PBH treatment group on day 7. Dermis showing mild collagen levels and dense fibrosis (Masson's trichrome stain).
Bar = 200 μm.

Figure 3.

PBH treatment group on day 21. Dermis showing abundant collagen levels and mild fibrosis (Masson's trichrome stain).
Bar = 200 μm.

Figure 4.

PCH treatment group on day 7. Epithelization was not seen (H&E stain).
Bar = 200 μm.

Figure 5.

PCH treatment group on day 21. Third level epithelization is seen (H&E stain).
Bar = 200 μm.

Figure 6.

PRH treatment group on day 14. Dense angiogenesis (arrows) was present in dermis (H&E stain).
Bar = 200 μm.

Figure 7.

PRH treatment group on day 21. Mild angiogenesis (arrows) was present in dermis (H&E stain).
Bar = 200 μm.

Figure 8.

PBH treatment group on day 7. Mild macrophage infiltration is seen (H&E stain).
Bar = 200 μm.

Figure 9.

PRH treatment group on day 14. Abundant macrophage infiltration is seen (H&E stain).
Bar = 200 μm.

The following criteria were used to compare normal tissue morphology in different regions of the trunk. All the histological sections were blindly evaluated by the same investigator (Dr. Yarim).

Biochemical Evaluation

The biopsy specimens were taken from the wound edges of each rabbit on days 7, 14, and 21. The samples were freeze-dried and stored at −80°C until use. Hydroxyproline was measured using the Bergman's spectrophotometric method.[17]

Statistical Analysis

Data are presented as mean ± standard deviation (SD). First, the normality was investigated using the Kolmogorov-Smirnov test. Data on planimetry, biochemical measurements, and histopathological changes were considered to be nonparametric data; therefore, they were analyzed using the Kruskal-Wallis H-test. The differences between the groups were determined using the Mann-Whitney U-test. P < 0.05 was considered statistically significant.

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