Abstract: Appropriate treatment and wound care accelerate the healing process and prevent infection. The aim of present study was to evaluate the effect of HESA-A (a drug of marine-plant origin) on the wound healing process. The effect of HESA-A at concentrations of 2.5% (mixture of 2.5% drug and 97.5% chow), 5 and 10% on the healing process of a 35 mm long full-thickness wound was prepared dorsally on rats, evaluated through measuring the length of the healed region on different days and conducted tensiometery experiments after complete wound healing. The mean percentage of wound healing on days 12, 14, 16 and 18 in control group changed in the group treated with 2.5% HESA-A from 62.5, 71.1, 86.7 and 100-75.5, 91.2, 100 and 100 (p<0.001), respectively; in the group treated with 5% HESA-A to 88.5, 98.6, 100 and 100 (p<0.001), respectively and in the group treated with 10% HESA-A to 92.8, 100, 100 and 100 (p<0.001), respectively. The stress changed from 16.5±0.9 Newton (N) in the control group to 19.2±1.5 N, 24.2±1.3 N (p<0.001) and 32.1±0.7 N (p<0.001) in the groups treated with 2.5, 5 and 10% HESA-A, respectively. The strain increased from 14.8±0.6 millimeter (mm) in the control group to 16.5±0.8, 25.7±0.4 (p<0.001) and 35.7±1.1 mm (p<0.001) in the groups treated with 2.5, 5 and 10% HESA-A, respectively. Our findings suggest, that HESA-A may have accelerated the skin wound healing process in rat in a dose-dependent manner and it seems increased tissue strength through stimulating collagen formation.

INTRODUCTION

Wound healing is a homeostatic mechanism for restoration of physiological balance and is triggered by the interruption of the connection between adjacent cells (Brunicardi and Schwartz, 2005). Appropriate treatment and wound care accelerate the healing process and prevent infection (Leaper and Harding, 1998; Choucair and Phillips, 1997). Thus far, different methods and approaches have been used to achieve shorter healing times. Patino et al. (1996) used wound area and circumference measurements to evaluate the wound healing process (Patino et al., 1996). Another highly valuable measure for evaluating the wound healing process is the tissue tensile strength. There is evidence of increased collagen synthesis in the hours immediately following injury. Multiple bonds and special arrangement of collagen fibers are responsible for tissue strength; hence tensiometry can be used to assess the tensile strength of the healed wound (Watson, 1995; Bargeson, 1987).

HESA-A is a drug of herbal-marine origin (patented by Iranian researchers) consisting of an array of various non-organic elements. HESA-A includes mineral constituents (50%), organic constituents (45%) and water (5%). The mineral constituents are a mixture of calcium carbonate, magnesium sulfate, potassium sulfate, sodium sulfate, magnesium phosphate, potassium phosphate and sodium phosphate. Low percent of other elements such as Br, Sr, Ti, Mn, Ni, As, Ag, Cu, Tm, Lu TI, Er, V, Cs, Ba, Te and so forth are found in salt or complex forms in HESA-A mixture (Ahmadi et al., 2001, 2003) given their physiological properties, these components may be able to accelerate the healing process and form collagen (Rizoiu et al., 1996; Simoes et al., 2002; Bang and Dashti, 1995; Gumustekin et al., 2004). Our previous study showed hepatoprotective effect of HESA-A against hepatic damage in rabbits (Ahmadi et al., 2005) and anti-inflammatory effect (in press). Hence, The present study, was conducted to evaluate the effect of HESA-A on the wound healing process in rat.

MATERIALS AND METHODS

Male Wistar rats, weight 250-300 g (supplied from Razi Vaccine and Serology Research Center) were caged individually in a controlled environment at 23-25°C and 50% humidity with a 12 h light-12 h dark cycle and allowed free access to food and water. The NIH guide for the care and use of laboratory animals was used for animal study. Two groups of rats were studied, control and case groups. The control group (n = 6) was given normal chow after incision to create wound until the end of the study. The case group divided into three subgroups; the 2.5% group (n = 6) was given a mixture of 2.5% drug (powdered) and 97.5% normal chow after incision until the end of the study (daily weigh), the 5% group (n = 6) was given a mixture of 5% drug and 95% normal chow after incision until the end of the study (daily weigh) and the 10% group (n = 6) was given a mixture of 10% drug and 90% normal chow after incision until the end of the study (daily weigh).

Prior to incision, the rats were anesthetized by intraperitoneal injection of ketamine (50 mg kg^-1) and Xylazine 2% (5 mg kg^-1). Then the animals were shaved on the back and the skin was disinfected using cotton and alcohol wipes. Using sterile surgical scalpels, full-thickness incisions, 35 mm in length were prepared 2 mm from midline dorsally. The wounds were left open. After incision, the wound was thoroughly disinfected using cotton and alcohol and injected gentamicin (5 mg kg^-1 daily, 3 days) as antiseptic (Dana et al., 1993).

Evaluation of the healing process:

Statistical methods: Data pertaining to the duration of healing and tissue strength were evaluated using one-way ANOVA and Tukey post test. Significance level was set at 0.05. All results were reported as Mean±SEM.

RESULTS

The mean percentage of healing on days 8, 10, 12, 14, 16 and 18 in the control group increased from 39.4, 51.3, 62.5, 71.1, 86.7 and 100 to 47.5, 59.3, 75.5 (p<0.001), 91.2 (p<0.05) and 100 in the 2.5 group, to 65.3 (p<0.001), 77.5 (p<0.001), 88.5 (p<0.001), 98.6 (p<0.001) and 100 in the 5% group and to 66.5 (p<0.001), 81.7 (p<0.001), 92.8 (p<0.001) 100 in the 10% group (Table 1).

Table 1:	The mean percentage of wound healing in the control and drug groups of length changes

Wound healing in the drug groups has occurred at a faster pace than in the control group and a significant difference was seen between the 2 groups in the percentage of healing on different days. Acceleration of healing in the drug subgroups seems to be dose-dependent. Values are Means±SEM. Sample size is 6 rats in each group. Tests: One-way ANOVA, Tukey. ^ap<0.05 Compared to control. ^bp<0.01 Compared to control. ^cp<0.001 Compared to control. ^dp<0.01 Compared to 2.5% group. ^ep<0.01 Compared to 5% group

Fig. 1:

The stress in control and drug (2.5, 5 and 10%) groups at the end of study. The tensile strength of the skin tissue from rats receiving HESA-A increased compared to the control group and a significant difference is seen between the groups. Acceleration of healing in the drug subgroups seems to be dose-dependent. Tests: One-way ANOVA, Tukey. N: Newton. *** p<0.001 compared to control group. ###p< 0.001 compared to 2.5% group. #p<0.05 compared to 2.5% group. ††Hp<0.001 compared to 5% group

Fig. 2:

The strain in control and drug (2.5, 5 and 10%) groups at the end of study, The tensile strength of the skin tissue from rats receiving HESA-A increased compared to the control group and a significant difference is seen between the groups. Acceleration of healing in the drug subgroups seems to be dose-dependent. Tests: One-way ANOVA, Tukey. mm: millimeter. ***p<0.001 compared to control group. ###p<0.001 compared to 2.5% group. ††Hp< 0.001 compared to 5% group

The stress changed from 16.5±0.9 Newton (N) in the control group to 19.2±1.5 N, 24.2±1.3 N (p<0.001) and 3.12±0.7 N (p<0.001) in the groups treated with 2.5, 5 and 10% HESA-A, respectively (Fig. 1).

The strain increased from 14.8±0.6 millimeter (mm) in the control group to 16.5±0.8, 25.7±0.4 (p<0.001) and 35.7±1.1 mm (p<0.001) in the groups treated with 2.5, 5 and 10% HESA-A, respectively (Fig. 2).

DISCUSSION

Table 1 show the effect of HESA-A at different concentrations on wound healing in rats treated with the drug compared to control rats and also shows the percentage of healing based on changes in wound length on different days compared to the first day (0 day) of the experiment. Statistical analysis, including ANOVA and Tukey show a significant difference in the percentage of healing on days 4, 6, 8, 10, 12, 14 and 16 of the study. In the present study, HESA-A apparently led to a notable decrease in length of the wound, as demonstrated by the significant difference in the mean percentage of healing on different days of the study as compared to the control group. In view of the results (Table 1), it can be concluded that HESA-A significantly accelerated the wound healing process. The percentage of healing increased with increased drug concentration, hence, it can also be concluded that the effect of HESA-A on healing is dose-dependent.

A number of studies conducted by Grzesiak and Pierschbacher (1995) showed that elements such as magnesium cause migration of keratinocytes and mediators of the healing process such as E-cadherin and integrin to the wounded area (Kuwahara et al., 2001). The role of elements such as strontium in the movement and immigration of non-muscle cells in the tissue healing process has also been shown (Oore and Pastan, 1979). Obtaining counts of fibroblasts, capillaries and polymorphonuclear leukocytes, as well as measurement of immunoglobulin G levels on the surface of wound using the immune-centrifuge technique has shown the notable effect of selenium on the wound healing process (Gumustekin et al., 2004). Also spectrophotometric studies have demonstrated the role of trace elements in healing of burn wounds (Selmanpakoglu and Cetin, 1994).

Measurement of tissue strength after complete healing of the wound was among the parameters assessed by the present study (Fig. 1 and 2). Another highly valuable measure for evaluating the wound healing process is the tissue tensile strength. There is evidence of increased collagen synthesis in the hours immediately following injury. Multiple bonds and special arrangement of collagen fibers are responsible for tissue strength; hence tensiometry can be used to assess the tensile strength of the healed wound (Watson, 1995; Bargeson, 1987). Experiments conducted by Grzesiak and Pierschbacher (1995) showed that the amount of magnesium and calcium increases in wound exudates. Analysis of wound exudates showed that elements such as magnesium promote tissue adhesion, migration of macrophages, keratinocytes, fibroblasts and production of type I collagen, all of which contribute to the wound healing process (Bang and Dashti, 1995; Wayne, 1998; Jensen, 1997; Witt and Thornton, 2000). As shown in Fig. 1 and 2, the tensile strength of the skin tissue from rats receiving HESA-A increased compared to the control group. It has been demonstrated that the tensile strength of the skin is related to the number of collagen fibers and how they are connected. HESA-A may has positive effects on the maturation, deposition and correct orientation of collagen fibers. Neovascularization is an important factor in wound healing. Povies and colleagues studied the metabolism of minerals in the healing arterial walls of rats by measuring the accumulation of some radioisotopes. Full thickness incisions were made in the aorta of rats injected with radioisotopes. A significant accumulation of radioisotopes such as selenium and chromium was seen in the healing area compared to the control group (Pories et al., 1979). The role of vanadium, another trace element in the healing process has also been studied (Urban and Antonowicz, 2001). Separate studies have investigated the role of trace elements in healing (Agren and Stromborg, 1986; Stanisstreet, 1982).

CONCLUSION

It can be concluded, from the obtained evidence that HESA-A may have accelerated the skin wound healing process in the rat in a dose-dependent manner and seems increased tissue strength through stimulating collagen formation.

Study limitation: The main limitation of this study was obtaining a fixed chow/drug percentage ratio. To achieve this, the appropriate chow/drug ratios were supplied to the Razi Vaccine and Serology Research Center to produce plates easily consumable by rats.