Experimental Evaluation of Repair Process of Burn-Wounds Treated with 15 UMF Manuka Honey

MATERIALS AND METHODS

Animals
Healthy male and female SD rats weighing between 250-350 g were used in this investigation. The rats were kept according to the type of treatments, one rat in one cage and given regular standard feed and water ad libitum.

Burn wound model
The hairs on the back and flanks on both the left side and right side of the rats were shaved. This area was disinfected with 70% ethanol before inducing of wound. Partial thickness burn wounds were inflicted on animals by modified commercial soldering iron method (Yam Mun Fei et al., 2002) under inhaled anaesthesia using diethyl ether.

Experimental protocol
Animals bearing the partial thickness burn wounds were divided into 2 groups with 15 animals each.

Determination of tensile strength
The tensile strength determination was done on day 5, 10, 15 and 20 post-burn. The skin was cut into dump-bell shape (wounded part lying in the center) by a special cutter. Instron tensiometer was used to measure the tensile strength of the skin with 15 mm min^-1 of crosshead speed (Yam Mun Fei, 2002).

Histopathology
Skin (containing both normal and healing sides) was collected on day 0, 5, 10, 15 and 20 for histological studies. Paraffin sections of 4Fm thickness were stained by hematoxylin and eosin (H&E) and examined under light microscopy. The sections were scored on the fibroblast using a numerical grading system 0 to 4 (where 0 represents an undetectable infiltrate with increasing number of cells to massive infiltrate as 4).

RESULTS

Histological studies
Control
These are typified by the illustration in Fig. 1. On day 0 post-burn, entire epidermis and upper portion of dermis were damaged. The dermal appendages were also abnormal. No sign of epithelialization was observed (Fig. 1a), very small collection of fibroblasts was found in the dermis (score 0). By day 5 post-burn, there was no sign of epidermis regeneration. Moderate numbers of polymorphonuclear leucocytes dispersed in the dermis (Fig. 1b) and few fibroblasts was observed (score 1). During day 10, debridement occurred, dermis was covered by granulated tissue and there was no evidence of epidermal regeneration. Abundant fibroblasts (score 4) were found in the dermis (Fig. 1c). Epidermis has completely regenerated on day 15 and moderate fibroblasts (score 3) accumulated in the dermis (Fig. 1d). By day 20, stratified epithelium (epidermis) has formed (Fig. 1e).

Fig. 1:	Mean tensile strength ± SEM of control and 15 UMF manuka honey treated wounds on various days

Fig. 1a:	Control day 0, entire epidermis, upper region of dermis, hair follicle and sebaceous gland are damaged (arrow). There is no evidence of epidermal bridging. H&E stain x 200

Fig. 1b:	Control day 5. There is complete epidermal loss, no sign of epithelial regeneration and no dermal appendages persist. Note the polymorphonuclear leukocytes

Fig. 1c:	Control day 10. Debridement has occurred. The dermis is covered by granulation tissue (white arrow). There is no evidence of epidermal regeneration. Note the presence of polymorphonuclear leukocytes (black arow) H&E stain x 200

Fig. 1d:	Control day 15. There is complete epithelialization and moderate number of fibroblasts dispersed in dermis. H&E stain H 200

Fig. 1e:	Control day 20. Shows complete regeneration of epidermis (arrow). H&E stain H 200

Fig. 2a:	15 UMF manuka honey treatment, day 0. Surface necrosis is found (black arrow). Moderate number of Polymorphonuclear leukocytes (white arrow) has accumulated in the upper part of the dermis (H&E stain H 100)

Fig. 2b:	15 UMP manuka honey treatment day 5. Epithelialization has occurred. Arrector pili muscle develops (back arrow). Note the active cells (white arrow) in basal layer of epidermis, sweat gland and hair follicle. There is no sign of inflammatory cells (H&E stain x 200)

Fig. 2c:	15 UMP manuka honey treatment day 10. There is complete epithelialization and well identified epidermal strata (arrow). H&E stain x 400

Fig. 2d:	15 UMP manuka honey treatment day 15. Note the complete regeneration and orderly stratification of the epidermis with basal layers of columnar cells (arrow) (H&E stain x 400)

Fig. 2e:	15 UMF manuka honey treatment day 20. There is complete epithelialization and well identified epidermal strata (arrow) (H & E stain x 400)

Treatment using 15 UMF manuka honey
On day 0 (Fig. 2a), moderate number of polymorphonuclear leucocytes has accumulated in the upper part of the dermis and very little fibroblasts are observed (score 0). There is no epithelialization over the injured site (Fig. 2a). On day 5, epithelialization has occurred and the injury is covered by new epidermal layers, arrector pili muscle develops (Fig. 2b). This epidermis might have been formed and migrated from the basal layers of near-by uninjured epidermis or from the undamaged hair follicle papillae, or from the sweat glands. Moderate number of fibroblasts (score 2) is observed in the dermis. On day 10 (Fig. 2c), a lot of fibroblasts (score 3) are clearly observed in the dermis and the process of epidermal regeneration is complete (Fig. 2c). On day 15 (Fig. 2d) and day 20 (Fig. 2e), epithelialization is complete having well-defined epidermal strata. Abundant fibroblasts (score 4) are also dispersed in the dermis.

Tensile strength
Treatment group always shows higher tensile strength (mean±SEM) compared to control during the experimental period. On day 5 post-injury, tensile strength in the treatment group is 5.668±1.139 MPa while in the control group the tensile strength is 4.746±1.893 MPa. On day 10 post-injury, tensile strength in the treatment group has increased to 8.008±2.702 MPa but in the control group this is slightly decreased having 4.170±1.332 MPa. On day 15 post-injury the tensile strength in the control and treatment groups are 1.664±0.648 and 2.511±0.485 MPa, respectively. Tensile strength of both treatment and control groups on day 20 post-injury are 1.345±0.85 and 2.799±0.5 MPa, respectively.

Discussion
The rats have been kept in good condition throughout the experimental period. There is no sign of infection and no pustule in all the wounds during experimental period. The wounds treated with 15 UMF manuka honey have healed faster compared to that in the control. Scars have formed between days 6-9 in treated group compared to the control’s 8-12 days.

Tensile strength
Tensile strength of 15 UMF manuka honey treated wounds has increased between day 5 and day 10 while in the controls, the tensile strength has decreased during this period. The results in the present study is similar to that of Kenyon and Michaels (1983) and Lee at el. (1986) that showed that the tensile strength increased during the fibroblastic phase (day 3 to day 10 post-injuries) and decreased during the differentiation phase (day 15 to day 20 post injuries). However, there is no significant difference (P<0.05) of tensile strengths in these two groups during various intervals of experimental period.

Histology
This investigation shows that wounds treated with 15 UMF manuka honey has healed faster than the controls. Epithelialization of treated wounds has occurred as from day 5 and is almost complete by day 10 compared with that in the control rats that showed epithelialization only on day 15. This clarifies that the honey (manuka honey) provides a healing environment to the wounds. Its antibacterial properties and its viscosity provide a barrier to cross-infection of wounds (Cooper et al., 1999; Molan, 1999 and Fox, 2002). Therefore, during the whole period of this investigation, the wounds treated with 15 UMF manuka honey looked healthier compared to the control. Furthermore the manuka honey not only contains hydrogen peroxide (produced by a heat and light sensitive enzyme in honey), which has a mild antibacterial activity (Molan, 1999); but also has a second antibacterial agent which is stable through the entire manufacturing process.

Acidity of the honey not only assists in the antibacterial actions of the macrophages (Ryan and Manjo, 1977); but also prevents the toxic unionized form of ammonia from being released by the bacteria (Nychas et al., 1988). The high concentration of glucose in the honey will be used by the infecting bacteria rather than the amino acids in dead tissue and serum (Nychas et al., 1988) and this would produce lactic acid instead of ammonia, amines, or sulfur compounds which would cause odor in the wounds.

Granulation is an essential process before epithelialization in the wound healing process. Based on the results, debridement has been found earlier (in day 5) in 15 UMF manuka honey treated wound than the control (day 10). Manuka honey that has a high osmolarity (Greenwood, 1993 and Lawrence, 1999) is believed to induce outflow and the lifting of dirt and debris from the bed of wounds causing debridement (Molan, 1999) occurring earlier than those without treatment. Thus, the high osmolarity of honey will also cause an outflow of lymph that serves to provide nutrition for regenerating tissue. Usually healing of wound is delayed if the circulation of an area is poor. Some studies suggested that the application of honey might also increase oxygenation of tissues thus causing faster tissue regeneration (Efemsee, 1988).

Honey contains a wide range of nutrients including amino acids, vitamins and trace elements (White, 1975). These nutrients can be expected to have direct effects in regenerating tissue (Molan, 1999). Besides that, low levels of hydrogen peroxide can stimulate angiogenesis and fibroblasts. The present histological observation clearly showed that fibroblasts dispersed in the dermis of treatment wounds have higher scores than the controls on day 5, day 15 and day 20. This investigation clearly shows that the 15 UMF manuka honey helps to promote epithelial regeneration and increases wounds tensile strength of rats.