Study of Effectiveness of Curcumin-Chitosan-Aloe Vera Healing Agent with Mupirocin for the Treatment of Aseptic Wounds in Dogs

The wound healing is an active, complicated and a series of closely related processes which includes haemorrhage, coagulation and starting of an acute inflammatory response to the original injury. In order to restore the disrupted anatomical continuity and disturbed functional status of the affected part, it is essential to minimize tissue damage, supply proper tissue perfusion and oxygenation, proper nutrition, moist wound healing environment (Bowler et al., 2001; Gosain and DiPietro, 2004; Mani et al., 2002 and Dinesh et al., 1997).
       
Wounds of animals treated with curcumin showed early re-epithelialization, better neo-vascularization, a higher collagen content with enhanced migration of different cells including dermal fibroblasts, myo-fibroblasts and macrophages into the site of wound bed (Sidhu et al., 1999). Chitosan, a cationic natural polymer, has a number of biomedical uses and is considered as an innovative substance in wound healing for its antibacterial, haemostatic and hypocholesterolemic effect (Shakeel and Saiqa, 2015). It also promotes formation of granulation tissue during proliferative stage of wound healing process. It maintains sterile wound exudates under a dry scab, restraining dehydration and wound contamination, to enhance the environment for wound healing. Similarly, aloevera has been found as a promoter of wound healing for its antibacterial and antifungal activities; it also increases synthesis of collagen and elastin in the wound bed (Rajeswari et al., 2012).
       
Basing on above context, a preparation was made using curcumin, chitosan and aloevera as a nano-based topical healing agent. To know its effect, a comparative study with that of Mupirocin ointment was carried out for treatment of aseptic surgical wounds in 57 clinical cases of dogs.
 

The study was conducted for research purpose varying for a period of 13 months on canine surgical cases (castration and ovario-hysterectomy) presented to the Department of Veterinary Surgery and Radiology, CVSc and AH, OUAT, Bhubaneswar from different geographical locations of the state. A total of 57 numbers of dogs of different breeds, sex and various age groups were studied for this purpose. The animals were divided into two groups depending on local application of the healing agents on surgical wounds i.e. test group (application of Curcumin-Chitosan-Aloevera based healing agent) and control group (Mupirocin ointment application; Staphban, Savavet). Out of total 57 numbers of cases, 44 were kept under test whereas 13 under control group. The details of preparation of the test agent has been kept secret.
       
Before initiating surgical treatment, complete history and other clinical observations like rectal temperature, respiration rate (breaths/ minute), heart rate (beats/ minute) and status of visible mucous membrane were recorded. With the consent of owner, blood was collected for haemato-biochemical examination and as per condition required, surgery were done in the presented cases and sutured (Fig 1). The characteristics of wounds like type of exudates, extent of exudates, rate of wound contraction and swelling were also recorded (Fig 2) in scale point basis (Table 1) following the method of Gopinathan et al., (2007) with slight modification. Tissue samples from wound margin on 0 and 14^th day were collected in 10% buffered neutral formalin then processed routinely to obtain 6 µm thick tissue sections. The tissue sections were stained with Hematoxylin and Eosin (H and E) stain for studying the histo-architecture, Masson’s trichrome for demonstration of collagen fibers and Verhoeff’s stain for elastic fibers (Bancroft and Stevens, 1996). After operation and suturing, the curcumin-chitosan-aloevera based topical gel was applied twice daily on the animals of Test group (Fig 3). Likewise the standard therapeutic regimen i.e. Mupirocin ointment was applied twice daily in Control group.
 

 

 

 

       
Haematological observations like estimation of haemoglobin, total erythrocyte count (TEC), total leukocyte count (TLC), packed cell volume (PCV) and differential leukocyte count (DLC) were done. Biochemical parameters like blood glucose, total protein, albumin, globulin, SGPT, SGOT, phosphorous and calcium were estimated by biochemical auto analyser (TURBOCHEM-100) on day 0 and 14^th day.
       
The mean parametric observations were compared by analysis of variance (ANOVA) as per the procedure described by Snedecor and Cochran (1989) while the non- parametric observations were compared by Kruskal-Wallis one way ANOVA. 

In the present study the clinical parameters like respiration (per minute), pulse rate (beats per minute), rectal temperature (°F) were recorded during post treatment which showed no significant differences between the groups during healing of wounds which corroborates with the findings of Borena et al., (2009). It indicates the body physiology during healing period remains normal irrespective of treatment regimen.
       
The findings of hematological parameters like Hb, TLC, TEC, PCV and DLC on 0 and 14^th day were in good agreement with that of Gopinathan et al., (2007) in horses and in dogs by Borena et al., (2009) which showed no significant differences between test and control groups.
       
The estimation of biochemical parameters like glucose, total protein and albumin were done in horses by Gopinathan et al., (2007) and in dogs by Borena et al., (2009). But in all the above cases there was no significant differences existed in their reports. Likewise in this pilot study also the parameters like glucose, SGPT, SGOT, total protein, albumin, globulin, calcium and phosphorus were estimated during treatment period which showed no significant differences and all those data remained within the normal physiological limits. This indicates that the impact of wound on the whole body system was negligible in comparison to total body area and there were normal liver and renal functioning in the body of patients.
       
Wound assessment was studied by parameters of gross observation of the wound viz. types of exudates, extent of exudates, swelling and percentage of wound contraction. The evaluation of wound was done by Bose et al., (2006) in bovines on 0-3 scale basis. The similar facts were also recorded by other researchers (Archibald and Blackley, 1974; Stegman and Swaim, 1990 and Curtis, 1993). The wound healing was assessed by subjective scores by Gopinathan et al., (2007) on 1-3, 1-5, 1-4, 1-3, 1-4 and 1-4 for color of wound, type of exudates, extent of exudates, peripheral swelling, pain and warmth respectively in horses. In the present study, the wounds in both groups were studied on 0-3 scale basis. Basing on the above observations the mean±SE values of the score for extent of exudates, type of exudates, swelling and percentage of wound contraction, significant difference (P<0.05) was found on 14^th day of observation in both the test and control group (Table 2). It revealed that the process of wound healing was going on in enhanced manner in test group which might be due to more collagen formation by the application of curcumin based healing agent.
 
 
       
In test group, the basal mean ± SE of duration of healing of wound was 8.77±0.63 with minimum days of 7 and maximum of 14 days. On the other hand in control group, the basal mean±SE of same parameter was 13.38±2.13 with minimum of 10 and maximum of 31 days (Table 3). But, the less duration of healing period in test group might be due to early neo-vascularisation, adequate collagen formation and attracting different types of wound healing factors within short span of time. The work is being supported by Panchatcharam et al., (2006) while experimenting the effects of curcumin for wound healing in rats. They interpreted that curcumin based healing agent promotes wound healing process by enhancing granulation tissue, increasing biosynthesis of transforming growth factor (TGF)-β1 and proteins in extra cellular matrix.
 

       
Kirubanandan and Sehgal (2010) stated that the collagen which is one of the prominent proteins in the extracellular matrix enhances keratinocytes and fibroblast proliferation which are important factors in wound healing. In the present study, the wound samples on 14^th day of topical application of test agent discerned clear evidence of the phenomena i.e. neo-vascularization, neo-epithelialization, fibro-collagen deposition and remodelling of epidermis were clearly evident. The epidermis had normal morphology with 10-11 numbers of cells arranged in layers starting from stratum basale to stratum corneum (Fig 5). The blood capillaries were profused in deeper and superficial part of dermis (Fig 6). Also profuse capillary bed was observed around the sebaceous and sweat glands. The collagen fibres were abundant in the dermis with large numbers of fibroblasts as well as fibrocytes. Isolated patches of skeletal muscles were reinforced in between the collagen fibre bundles (Fig 6). Elastic fibers of moderate thickness were noted in the walls of some of sebaceous glands and they were continuous with those of the hair follicular wall (Fig 7). Some of the capillary walls were noted to have sparse amount of fine elastic fibers (Fig 8). There was less formation of collagen and reticular fibres on 14th day in control group than test group (Fig 9). The control group showed scanty blood vessels with moderate fibrosis, less connective tissue, less reticular fibers, less epithelialization and less rebuilding of dermal tissue due to disintegration of collagen bundles (Fig 10 and 11). There was infiltration of inflammatory cells in the dermis in the control group (Fig 12). The frequency distribution of hair follicles was comparatively less in control group than test group at 14^th day of treatment (Fig 13). It was indicative of faster wound healing process (Fig 4) in test group of animals than control group as because curcumin, chitosan and aloevera have more potential wound healing capacity with different mechanism of action.
 

 

 

 

 

 

 

 

 

 

Application of Chitosan-Curcumin-Aloe vera based healing agent on the wound margins of test groups resulted in early and significantly higher percent of wound contraction and early complete healing process than mupirocin. Hitological and histo-chemical observations furthermore revealed that healing activity is enhanced by increasing cellular proliferation, formation of granulation tissue, synthesis of collagen, epithelialization, neovascularization and early histological maturation in test groups than control groups. Therefore, it is concluded that application of chitosan-curcumin based healing agent is economical, risk free; hence have great potential for use in wound healing in routine clinical cases.

The authors are thankful to the Dean, College of Veterinary science and Animal Husbandry, OUAT and Vice Chancellor, KIIT University, Bhubaneswar for providing necessary support during the research.

None.