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Fırat Üniversitesi Sağlık Bilimleri Veteriner Dergisi
2009, Cilt 23, Sayı 3, Sayfa(lar) 135-139
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Ratlarda Yanık Yaralarının İyileşmesi Üzerine Kollagenaz ve Silver Sulfadiazine'in Etkilerinin Karşılaştırılması
Ali Said DURMUŞ1, Mehmet Cengiz HAN1, İhsan YAMAN2
1Fırat Üniversitesi, Veteriner Fakültesi, Cerrahi Anabilim Dalı, Elazığ, TÜRKİYE
2Fırat Üniversitesi, Sivrice Meslek Yüksekokulu, Elazığ, TÜRKİYE
Anahtar Kelimeler: Kollagenaz, silver sulfadiazine, yanık, rat
Özet
Bu çalışma ratlarda yanık yaralarının iyileşmesinde kollagenaz ve silver sulfadiazine'in (SSD) karşılaştırmasını yapmak için gerçekleştirildi. Altmış Wistar albino rat üç eşit gruba bölündü. Bütün ratların sırtlarında yanık modeli oluşturuldu. Yanık bölgeler günlük olarak, birinci, ikinci ve üçüncü gruplarda sırasıyla kollagenaz, SSD deri kremi ve cold cream (kontrol) ile kaplandılar. On ve 21 gün sonra histopatolojik muayeneler için ratlar anestezi altına alınarak her gruptan 10 hayvandan yanık uygulanan deri örnekleri alındı. Sonuç olarak, kollagenaz merhem uygulamasının rat modelinde deri yanıklarının iyileşmesi üzerinde anlamlı derecede etkili olduğu belirlenmiştir (P< 0.0001).
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    One of the important characteristics of burns is the formation of an eschar, resulting from burned and traumatized tissue1. Necrotic tissue covering the burned wound has a negative influence on wound healing and provides a medium for the growth of microorganisms and is therefore a source of infection, contamination and sepsis2. Wounds do not heal as long as dead tissue remained on its via natural collagen fibers3. The first aim in the initial treatment of burned wounds is the complete removal of necrotic tissue and other contaminants4-8. An ideal surgical technique should remove all necrotic tissue in a single procedure leaving behind viable undamaged tissue7,9.

    Enzymatic debridement of necrotic tissue has gained interest in the recent years providing an efficient alternative treatment to replace the standard of care surgical debridement.5,10-13.

    Enzymatic agents are also relatively safe and effective12,14. Preparations including enzymes such as collagenases, fibrinolysin, deoxyribonuclease, or streptokinase are seen as alternatives to surgical debridement2,3. For example, Mekkes et al.6, reported that krill enzyme preparation, applied twice daily in a concentration of 3.0 or more casein units per ml, appears to be an effective product for necrotic wound debridement in the pig model, its debriding properties.

    One of the exogeneous enzymes used for burn wound debridement is collagenase clostridipeptidase A (CCA), derived from Clostridium hystolyticum2,3. Collagenase has a high affinity for all major collagen types and is useful in debridement, increase of granulation tissue formation, and prevention of abnormal scar formation3,15.

    Collagenase is best used for digesting collagen and elastin, but it does not decrease fibrin. This agent should be applied only to the nonviable tissue within the wound and not to the surrounding normal tissue12,16.

    ilver sulfadiazine (SSD) is the topical agent of choice in severe burns and is used almost universally today in preference to compounds such as silver nitrate and mafenide acetate. SSD cream, while being effective, causes some systemic complications which include neutropenia, erythema sultiforme, crystalluria and methaemoglobinemia17-21.

    The purpose of this study was to compare healing rates of wounds treated with collagenase dressing and with silver sulfadizine clinically and histologically in rat model to produce likely an alternative for the treatment of thermal wound in domestic animals, i.e. dog, cat, cattle and horse

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    Animals: This study was carried out in sixty male Wistar albino rats weighing between 250 and 300 g. Animals were housed at 21ºC with a day/night cycle of 12 h. During the study these animals were fed ad libitum standard rodent feed.

    Anaesthesia: The rats were anaesthetized with single intramuscular injection of 6 mg/kg xylazine hydrochloride (Rompun, Bayer, 23.32 mg/ml) and 85 mg/kg ketamine hydrochlorure (Ketalar, Parke-Davis, 50 mg/ml).

    Test drugs: Collagenase ointment (Novuxol, Abbott, Collagenase SF, clostridiopeptidase A, 1.2 IU/1g), silver sulfadiazine (Silverdin, Deva, Silver sulfadiazine, 10mg/g) skin cream and cold cream (Botafarma, 12.5 % spermaceti + 12 % white wax + 56 % liquid paraffin + 0.5 % borate of soda + 19 % distilled water) were used in this study.

    Thermal injury: The backs of the rats were shaved and prepared with 10 % antiseptic povidone-iodine solution (Kim-Pa, Poviiodeks, % 10 povidone-iodine) and burns of 1 cm in diameter were established.

    Skin burns were made as described by Hoşnuter et al.22. Animals were subjected to a 1 cm diameter surface area of full-thickness second-degree skin burns by brass probe. The brass probe, was immersed in boiling (100 ºC) water until thermal equilibrium was achieved, then it was placed without pressure for 20 s on the back of the rats. All animals were resuscitated immediately with lactated Ringer's solution (2 ml/100 g body weight) intraperitoneally. Following the burning, each animal was placed in a separate cage.

    Experimental protocol: The animals were divided randomly into three equal groups. Immediately after burn, the burned areas were cleaned by sterile saline solution, and these areas in the first (n = 20), second (n = 20) and third groups (n = 20) were covered with 2 mm thickness collagenase ointment, SSD skin cream and cold cream (control) respectively. These applications were repeated every day. Any topical antibacterial agent was not combined with these applications. The wounds in all groups were observed clinically every day.

    Histopathological examination: Ten and 21 days following injury, ten rats in each group were anaesthetized and the burned skin tissue samples were collected for histopathological examinations. This tissue samples were fixed in 10% neutral-buffered formalin solution and embedded in paraffin wax, and were cut into 5 µm-thick sections and stained with hematoxylin and eosin (H&E) and Mason's trichrome, and examined by light microscopy.

    Statistical analyses: The thickness of granulation tissue at the center of each wound was examined and recorded. Statistically, all data are expressed in milimeters as mean ± standard error of the mean. The differences between 10 and 21th days were compared using the Mann-Whitney U test. The differences between groups were compared by analysis of Kruskal-Wallis and Mann-Whitney test. P values of less than 0.05 were considered as statistically significant. These analyses were accomplished by using statistical analysis system configured for computer (SPSS, Relase 10.0, SPSS. Inc).

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    No mortality was seen in the animals during the study. Burn wounds treated with SSD appeared to display a greater degree of inflammation as notable by the three clinical signs of the inflammatory process such as heat, redness, and swelling which appeared to be lessened in wounds treated with collagenase.

    The results clearly indicated that early removal of the injured tissues with collagenase ointment accelerated the rate of burn wound healing compared the other groups. Treatment with collagenase resulted in significantly shorter time to achieve a clean wound bed than SSD and control groups. Collagenase debridement had promoting effect compared to SSD treated and control group.

    Histopatologic examinations, on the 10th day showed that burn healing was better in collagenase than the other groups. Regenerative and reparative attempts were started in epidermal layer. No epithelium was present in the SSD and control groups, however inflammatory cell infiltration was observed beneath the scab in the all groups. Also, few capillaries and sparse collagen deposition were noticable especially at wound centers. The capillaries were hyperemic in the dermis with no hair follicularis, sebace and sweat glands in the all groups (Figure 1).


    Büyütmek İçin Tıklayın
    Figure 1: Microscopic appearance of burned skin on the 10th day.

    A. Collagenase group a) Scab b) Epidermis c) Dermis, and infiltration of mononuclear cells (arrow)
    B. SSD group a) Scab c) Dermis
    C. Control group a) Scab c) Dermis, and infiltration of mononuclear cells (arrow), (H E x 50).

    On the 21th day, development of the epidermis was observed in all groups. But, this development was better in collagenase group. While wound healing was completed in this group it remained incomplete in the SSD and control groups. Also, in the SSD and control groups the scab is clearly vissible on the 21th day (Figure 2).


    Büyütmek İçin Tıklayın
    Figure 2: Microscopic appearance of burned skin on the 21th day.
    A. Collagenase group a) Scab b) Epidermis c) Dermis
    B. SSD group a) Scab b) Epidermis c) Dermis
    C. Control group a) Scab b) Epidermis c) Dermis, (H E x 50).

    Wound healing was significantly different in each groups at 10 and 21th days (p<0.0001). Thickness of granulation tissue was significantly different between each groups (p<0.0001). The mean values of thickness of granulation tissue in the center of the wounds for collagenase cream, SSD and control groups are shown in Table 1.


    Büyütmek İçin Tıklayın
    Table 1: Thickness (mm) of granulation tissue in the center of the wound.

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    Dead tissue covering the burn wound also serves as a medium for bacterial growth, reduces the host's resistance to infection, and delays the formation of granulation tissue and the re-epithelialization. Therefore, wound debridement is an imperative to the healing of burns. Surgical tangential excision of dead tissue, the repeated application of moistened dressings, hydrocolloid or semiocclusive dressings, dextranomers, intracavity gels, hydrosurgery, or various enzyme preparations can be used of burn debridement4,6,7,23.

    While effective, surgical tangential excision has several major disadvantages. This method is non-selective and it is technically difficult to control the amount of tissue to be removed, often converting a partial thickness burn into a full thickness defect. In addition, tangential excision may be associated with pain and bleeding1,5,6,12.

    Different test animals such as pig6,24, Guinea pig5,13 and rat14,19,22 were used for various studies. We used rat model in the present study similar to some investigators14,19,22. The model used here is simple and repeatable. The burned wound healing model provides an in vivo approach for studying the healing of burned wounds in domestic animals.

    Topical agents with benefits only as antimicrobials include silver nitrate, sulfamylon and a combination of a sulfonamide and SSD. Sulfamylon has wide spectrum activity, but it is easily absorbed systemically and can result in toxic complications. SSD has become the standard topical treatment for burn wounds17. Thus, we have chosen SSD in our study.

    A wide range of enzymatic debridement have been used in the treatment of burn injury both clinically and experimentally. Among them; trypsin5, bromelain1,5, collagenase2,3,13, krill enzymes6, and papain11,14 are advocated. These agents offer the advantage of easy application with additionally being relatively safe and effective12.

    Collagenase is an essential component in the wound healing process. Collagenase is best used for digesting collagen and elastin, but it does not degrade fibrin. This enzyme is responsible for the breakdown of collagen and its presence is vital to maintain the dynamic equilibrium between the breakdown of old collagen and new collagen formation in the scar-remodeling phase16. This agent should be applied only to the nonviable tissue within the wound and not to the surrounding normal skin or tissue12. Therefore, in this study, collagenase ointment was applicated only on the burned tissue.

    Detergents, soaps, antiseptic solutions, and heavy metal ions are decreased efficacy of collagenase8. When using enzymatic debridement, the main problem is infection as is reported by Klasen15 and Özcan et al.2. In the present study topical antiseptic solutions and antimicrobial agent were not used with collagenase, and no infections were observed.

    Alaçam et al.10 treated chronic wounds located between udder and hind extremities using collagenase in cows. They reported that collagenase had a beneficial effects on the healing of these wounds. The results of current study showed that collagenase ointment plays a role in healing of burned skin wounds possible via its enzymatic debridement effect.

    The present study showed that early nonsurgical removal of injured tissues is an effective treatment for thermal burns. Collagenase debridement accelerated burn wound healing as determined by the rate of lesion area closure and histopathological evidence. Histopathological comparison of the three groups indicated that healing of burned skin wounds was best in the collagenase group (p<0.0001).

    Consequently, the data and observations collected in this study indicated that collagenase could be applied to treatment of thermal burns and it may be viable and desirable alternative to the use of SSD. Acknowledging the limitations of rat model of this experimental animal study, we believe that further studies are warranted to investigate whether collagenase is useful in domestic animals with burn wounds.

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    1) Rosenberg L, Lapid O, Bogdanov-Berezovsky A et al. Safety and efficacy of a proteolytic enzyme for enzymatic burn débridement: a preliminary report. Burns 2004; 30: 843-850.

    2) Özcan C, Ergun O, Çelik A et al. Enzymatic debridement of burn wound with collagenase in children with partial-thickness burns. Burns 2002; 28: 791-794.

    3) Tıraş U, Erdeve O, Karabulut AA et al. Debridement via collagenase application in two neonates. Pediatr Dermatol 2005; 22: 472-475.

    4) Cubison TCS, Pape SA, Jeffery SLA. Dermal preservation using the Versajet® hydrosurgery system for debridement of paediatric burns. Burns 2006; 32: 714-720.

    5) Eldad A, Weinberg A, Breiterman S et al. Early nonsurgical removal of chemically injured tissue enhances wound healing in partial thickness burns. Burns 1998; 24: 166-172.

    6) Mekkes JR, Le Poole C, Das PK et al. Efficient debridement of necrotic wounds using proteolytic enzymes derived from Antarctic krill: a double-blind, placebo-controlled study in a standardized animal wound model. Wound Rep Reg 1998; 6: 50-58.

    7) Rennekampff HO, Schaller HE, Wisser D et al. Debridement of burn wounds with a water jet surgical tool. Burns 2006; 32: 64-69.

    8) Soroff HS, Sasvary DH. Collagenase ointment and polymyxin B sulfate/bacitracin spray versus silver sulfadiazine cream in partial thickness burns: a pilot study. J Burn Care Rehabil 1994; 15 (1): 13-17.

    9) Durgun T, Durmuş AS, Kaplan M. Effects of local diphenylhydantoin on wound healing in clinical and experimental studies. Firat Universitesi Saglik Bilimleri Dergisi 1999; 13(2): 151-157.

    10) Alaçam E, Küplülü S, Salmanoğlu R et al. The use of collagenase in the treatment of skin wounds located between udder and hind extremities of cows. Veteriner Cerrahi Dergisi 1996; 2(1): 29-32.

    11) Starley IF, Mohammed P, Schneider G et al. The treatment of paediatric burns using topical papaya. Burns 1999; 25: 636-639.

    12) Steed DL. Debridement. Am J Surg 2004; 187 (Suppl to May): 71S–74S.

    13) Webster ME, Altieri PL, Conklin DA et al. Enzymatic debridement of third-degree burns on guinea pigs by Clostridium histolyticum proteinases. J Bacteriol 1962; 83: 602-608.

    14) Hebda PA, Lo CY. The effects of active ingredients of standard debriding agents-papain and collagenase-on digestion of native and denatured collagenous substrates, fibrin, and elastin. Wounds 2001; 13: 190-194.

    15) Klasen HJ. A review on the nonoperative removal of necrotic tissue from burn wounds. Burns 2000; 26: 207-222.

    16) Beckenstein MS, Kuniaki T, Matarasso A. The effect of scarguard on collagenase levels using a full-thickness epidermal model. Aesthetic Surg J 2004; 24: 542-546.

    17) Gracia CG. An open study comparing topical silver sulfadiazine and topical silver sulfadiazine-cerium nitrate in the treatment of moderate and severe burns. Burns 2001; 27: 67-74.

    18) Gregory RS, Piccolo N, Piccolo MT et al. Comparison of propolis skin cream to silver sulfadiazine: A naturopathic alternative to antibiotics in treatment of minor burns. J Altern Complem Med 2002; 8: 77-83.

    19) Han MC, Durmuş AS, Karabulut E et al. Effects of Turkish propolis and silver sulfadiazine on burn wound healing in rats. Revue Méd Vét 2005; 156(12): 624-627.

    20) Karabulut E, Durgun T, Durmuş AS. Use of honey in treatment of cornea alkali burns. Indian Vet J 2004; 81: 993-994.

    21) Subrahmanyam M. A prospective randomised clinical and histological study of superficial burn wound healing with honey and silver sulfadiazine. Burns 1998; 24: 157-161.

    22) Hoşnuter M, Gürel A, Babuccu O et al. The effect of CAPE on lipid peroxidation and nitric oxide levels in the plasma of rats following thermal injury. 2004; Burns 30: 121-125.

    23) Mekkes JR, Westerhof W. Debridement of necrotic venous leg ulcers using absorbing polysaccharide pads. J Drug Ther Res 1992; 17: 253-258.

    24) Henze U, Lennartz A, Hafemann B et al. The influence of the C1-inhibitor BERINERT® and the protein-free haemodialysate ACTlHAEMYL20%® on the evolution of the depth of scald burns in a porcine model. Burns 1997; 23(6): 473-477.

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