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Bull Vet Inst Pulawy 48, 311-316, 2004 INFLUENCE OF HONEY-BALM ON THE RATE OF SCARE FORMATION DURING EXPERIMENTAL BURN WOUND HEALING IN PIGS AGATA KABAŁA-DZIK1, RAFAŁ STOJKO1, EWA SZAFLARSKA-STOJKO1, IRENA WRÓBLEWSKA-ADAMEK1, ARTUR STOJKO2, JERZY STOJKO2 AND BARBARA STAWIARSKA-PIĘTA1 1 2 Department of Pathology, Department of Bioanalysis, Sanitary Sciences and Environmental Studies, Faculty of Pharmacy in Sosnowiec, 41-200 Sosnowiec, Silesian Medical University in Katowice, Poland e-mail: [email protected] Received for publication January 15, 2004. Abstract The pharmacological activity of honey balm, its influence on wounds healing processes and the rate of scar formation in comparison with control groups were assessed. Clinical and histopathological studies showed that honeybalm not only shortened the period of wound healing, but also had a positive impact on the general health condition of the animals. Key words: pig, burn wounds, honey-balm, healing. Wound healing is a complex process influenced by a number of factors. An important factor in the wound healing is correct blood supply. Tissue ischaemia caused by blood vessels coagulation leads to the development of a protein scab which is a perfect ground for microorganisms development. Also, impaired blood flow in wound tissues (especially in the 1st and 2nd degree wounds) may lead to further damage and may cause the infection of a wound by bacteria living in soft tissues (10, 12, 13). Judging from the processes taking place during wounds healing, it follows that the basic conditions for damaged tissue restoration and healing are: wound cleaning, proper macrophages accumulation (they provide the growth factors), proliferation of fibroblasts producing extracellular matrix and the formation of new blood vessels (neoangiogenesis). The activity of these processes may be a marker of the reconstruction process occurring in damaged tissues. The current progress made in the analytical studies and in the field of biotechnology has led to a renewed interest in pharmacotherapy based on biogenic components. More and more attention has been given to apipharmacotherapy which utilizes the healing properties of active fractions of bee products. The great physician and philosopher Hippocrates, who documented bee products’ therapeutical properties, used strained honey or honeycombs with the crust in the treatment of wounds difficult to cure (5). Clinical and research centers – which are becoming better and better equipped in modern analytical equipment – have enriched the knowledge known for generations as far as to the composition and properties of bee products. Their studies have led to the isolation of active substances in bee products and then to the standardization of their pharmacological properties (18). The pharmacological activity of obtained fractions in relation to human organisms arises from the fact that bee products – to a greater or lesser extend – include in their composition all basic substances necessary for human body homeostasis (17, 18). One of the newly discovered apitherapeutics is the honey crust. Polish scientists have isolated in it the active substance which has anti-bacterial and regenerative properties, activating the immunological system through the stimulation of properdine and the complement activity. 312 Substances in the honey have a positive effect on the correct functioning of an animal organism and are also well-assimilated by it. They also determine a number of honey's actions: antibacterial, regenerative, detoxic, nutritious, immunomodulative, preservative, anti-allergic as well as cardioprotective and soothing (2, 3, 6 ,7, 9, 15, 16, 21, 23). Honey bioelements are the activators of a number of immunological mechanisms (7, 18, 22). Having in mind the difficulties experienced during the treatment of burn wounds as well as the described properties of bee products, the aim of the present study was to assess the healing rate and the process of scar formation in burn wounds under the influence of honey balm in pigs. Material and Methods The experimental part of the study was conducted in the Central Experimental Animal Quarters of the Silesian Medical University according to the standards arising from the European Conference and the standards of Good Laboratory Practice. The experimental model consisted of identical after-burn wounds of skin done according to the Hoekstr model. This model is in accordance with the Dutch Act of Animal Testing and the currently in force experimental protocol of the Committee of Charity operating at the University of Amsterdam (4, 11). The tested pigs were of the "white lop-eared" breed, aged 15-16 weeks, with weight 30-40 kg. Throughout the experiment the animals lived in identical zoohygienic conditions and were fed, according to their age, the standard fodder. In order to produce the burn wounds a hot brass block (size: 1.5 x 3 cm, temperature: 170°C) was placed for 10 s on the skin of premedicated and then generally anaesthetized animals. The wounds were performed on the sides, along the spine of each animal (Fig.1). Depending on the substance used, the burn wounds were divided into the following groups: Group I (control): - subgroup IA: 9 wounds of pig no 1 - not treated but only washed once a day with 0.9% NaCl solution; - subgroup IB: the remaining 9 wounds of pig no 1 treated with a thin layer of Dermazin (SSD-silver sulphadiazine) applied once a day; Group II (experimental): - 9 wounds of pig no 2 treated with honey-balm consisting honey crust. It was applied once a day as a thin layer to each of the wounds. Fig. 1. A pig after burn wounds execution. Burn wounds after therapeutics application and providing with sterile dressing. 313 The clinical assessment was done throughout the duration of the study and covered the general health conditions as well as the reaction to environment. The process of burn wound healing was especially carefully assessed. The appearance and processes taking place in the tissue surrounding the wounds were evaluated as well as the granulation tissue formation and the process of scar formation. The histopathological assessment of the healing process was done on the 3rd, 5th, 10th and 15th d of the study. Before material samples were taken for the histopathological tests, animals were subjected to general anesthesia. The samples were collected from the middle of the wounds, from their borders as well as from the skin in the wound area. Paraffin sections were stained with the standard haematoxylin – eosin method. The study was accepted by the Regional Ethical Commission of Animal Experiments in Katowice, Poland. The study was performed as part of the NN-2-047/01. Project financed by the Scientific Research Committee. Results Clinical observations: Day 1 3 5 10 15 Honey-balm NaCl Dermazin The clinical pictures of all the subgroups were similar. Along the necrotic changes, there were inflammatory reactions with a large amount of exudate and a skin oedema around the wound. The wounds were smaller with a No change in the clinical picture. formed scab. Oedema and reddening at the wound were visually smaller. The wounds were clean with no Wounds were covered with thin, Wounds were covered with non-fully formed scab. exudate with a correctly developed poorly-formed scab. Oedema and reddening on scab. skin around wounds. Wounds were covered with smaller No changes when compared to Dry wounds. Formation of scabs, and on the skin border on the day 5. dry scabs, no changes in surface of the wound there was a skin around wounds. pink scar, what suggests presence of advanced epidermalization. The wounds were covered with an Wounds covered with dry scabs Wound cavity filled with fresh granulation tissue. organized scar, and they could be of the same size. Wound granulation process treated as healed completely. on the edges. During palpation the animals did not react defensively. The clinical evaluation showed visible differences in the process of wound healing after applying the above mentioned medications. In the case of the apitherapeutic treatment, the differences in wound appearance and animal behaviour were visible after 48 h. Wounds were more wet, skin at the wounds was more elastic. Oedema and reddening at the wound surroundings were smaller. On the 10th d of the experiment the wound surface became visually smaller. The scabs started to come off at the boundaries of the wound surface, and a light-pink scar was visible underneath. This suggested an advanced epidermalization process. The wounds developed after taking small segments to the histopathological tests could be treated as healed completely. The applied apitherapeutics not only shortened the period of wound healing, but also has a positive impact on the general health condition of the animals. During the time of the experiment the animal did not show any signs of excitation and did not react defensively. After applying the honey balm, the signs of granulation on the surface of the wound were observed on the 5thday. The inflammatory infiltration with a considerable amount of granulocytes was found. Their presence in the wound surrounding indicated the stimulation of cell migrating processes, intensified wound cleaning and favourable conditions for the epithelium migration. On the 10th d, the loss of 314 epithelium was observed in a part of the wound. In the remaining part, the regenerating epithelium with the mature granulation underneath was still visible. On the 15th d the defect was completely covered with a great amount of collagen fibers, which suggested the process of scar formation (Fig.2). Histopathological study: Da y 3 5 10 15 Honey balm NaCl Dermazin Loss of stratified squamous epithelium, extensive coagulative necrosis and thick inflammatory infiltration. First signs of granulation both in Loss of epithelium with Loss of epithelium and bottom and on rims of wound. coagulative necrosis. coagulative necrosis, under which Inflammatory infiltration on the there was inflammatory deeper layers. infiltration. A regenerating epithelium was Scab visible on the surface of Inflammatory infiltration seen seen on the edge of the wound. epithelium. First phases of “fresh” under necrotic masses. Signs of In the middle part of the wound granulation seen on the bottom granulation seen on the bottom there was a granulating tissue and edges of wound. and edges of wound. filing the whole defect. The skin defect filled completely Wound cavity filled with fresh Wound cavity filled with fresh with granulation with a granulation. Granulation granulation. Granulation considerable amount of collagen processes seen on the wound processes seen on the wound fiber (Fig.2). edges (Fig. 3). edges (Fig. 4). Fig. 2. The 15th d after burn execution. Honey-balm. Granulation tissue with dominance of collagen fibers. (Area 100x, HE). 315 Fig. 3. The 15th d after burn execution. NaCl. A wound covered with crust. On border a epithelium visible. (Area 100x, HE). Fig. 4. The 15th d after burn execution. Dermazin. Granulation filling a wastage. (Area 100x, HE). 316 Discussion The histopathological study revealed that pathological process in each of the sub-groups was similar, the time from the moment the thermal wound was made to the begining of repair processes in the wound was different depending on the applied remedy. In the case of honey-balm it was the 5th d, after applying Dermazin (a standard medication used in burn healing) the repairing process was observed at day 15 after its application. The therapeutic effectiveness of an apitherapeutic, whose pharmacological activity results from the physico-chemical characteristics of honey or propolis, confirmed the previous report (14). The effects of apitherapeutics on wound healing are well known in the cases when the conventional methods of healing are not effective (8). The favourable effect of honey was also substantiated in healing of the infected wounds encontered after the cesarean section and histerectomy. The application of honey not only shortens the healing period. The hospitalization period in female patients, who were treated with honey was 10 d shorter than in those treated with the conventional methods (1). Honey treatment influences scar formation and its cosmetic appearance. Subrahmanyam (19, 20) in his study showed that healthy granulation tissue was observed earlier in patients treated with honey. Of the wounds treated with honey 87% healed within 15 d versus 10% in group treated with silver sulfadiazine (SSD). Other histological and clinical studies of wound healing showed satisfactory epithelialization after 7 d in 84% and after 21 d in 100% of wounds treated with honey. In wound treated with SSD epithelialization occurred by the 7th d in 72% of patients and by the 21st d in 84% of patients. 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