Wound healing using ionic silver dressing and nonocrystalline silver dressing in rats

PURPOSE: To investigate the results of the healing process on surgical wounds in the back of Wistar rats using nanocristaline and ionic silver dressing. METHODS: Sixty rats Wistar were submitted to surgical wounds with punch of 8mm in diameter. In 30 animals (groups PN nanocristaline and AD control) two surgical wounds were done diametrically opposite on the upper back side. On the right side was used nanocristaline (PN) silver dressing and on the left side, distilled water dressing (AD). On the other group of 30 rats, only one wound was made with the punch, on the right side, and was used ionic silver dressing. So, the groups were divided into three subgroups, according to the day of death (7th, 14 th and 21st day). In each of these days the wounds diameter were measured to evaluate the wound contraction. Microscopic data were analyzed using the H&E staining to verify the inflammatory process and neovascularization. The Masson trichrome staining was used to verify the fibrosis. RESULTS: Macroscopically only the subgroup of 21st day showed statistical significance; between the groups AD and PI inflammatory process appeared in the 7th day subgroup in 90% of the cases. In neovascularization there was statistical significance between the groups PN and AD in the subgroup of 7th day. Fibrosis did not show statistical significance in the studied groups. CONCLUSIONS: In relation to wound contraction, PN and PI groups showed better results than the AD group. In regard to histological analysis, H&E staining showed that there was presence of inflammation in all groups, and at the end, the control group (AD) on 7th day, was superior to PN and PI groups. In relationship to fibrosis, no differences were obtained among groups.


Introduction
Tissue loss can reach the dermis partial or fully and also include the subcutaneous tissue.Based on this, Mandelbaum et al. 1 defined two types of wounds: partial thickness and full thickness.
When the wound is in partial, to it is given the name of the dermis incomplete, and the repair is made by reepithelialization of the epithelium or epithelium derived from the unaffected adjacent skin.The final result is almost imperceptible scar.When the wound is of full thickness, dermis is said to be fully extended or in the subcutaneous tissue.There is the need to form a new tissue, granulation tissue, when the epithelization is the basis of healing of partial thickness wounds 2 .
The healing also depends on many local and general factors, as anatomical location, skin type, race and surgical technique 3 .Different classifications are used to facilitate the understanding of the dynamic process and stages.The healing process is divided into five main phases: coagulation, inflammation, proliferation, wound contraction and remodeling.
During the formation of granulation tissue fibroblasts and endothelial cells proliferate and move into the wound, producing extracellular matrix, connective tissue fibers and neovascularization 4,5 .Many fibroblasts acquire some morphological and biochemical properties of smooth muscle cells, giving rise to myofibroblasts.This factor participate in extracellular matrix synthesis and production of mechanical power, affecting the reorganization of the matrix and wound contraction.
The products for wound treatment can be divided into two major groups: topical agents and dressings.The topical agents are those applied directly to the wound bed, or intended to clean or protect the area around 6 .Dressing, also called coverage, is the feature that covers a wound, in order to protect it from external aggression, keeping it moist and preserving the integrity of its region.
Silver has medicinal properties and has been used for over 2000 years.It is a metal that is mostly a byproduct of the mining of lead and is often associated with copper.The antimicrobial property of silver and its compounds is the main base of its medical application since the nineteenth century.Since silver has many uses and has great potential for the treatment of injuries.
The popularity of the use of coatings with silver in wound has been progressively increasing 7,8 .Currently, it is present in a wide range of coverages and can present in two forms: 1) compounds / complexes when associated with a salt and produces the ionic silver (Ag +) when in contact with wound fluids or solutions; and 2) elementary silver in metallic form (Ag), also described as colloidal silver or silver nanoparticles.
The nanocrystalline silver was introduced as dressing wounds in 1998 with the claim that it reduces the occurrence of infection and provides opportunity to improve clinical practice in the treatment of wounds 9 .
This study aims comparatively evaluate the results of the process of wound healing with the use of nanocrystalline silver dressing with ionic silver and in the dorsal region of rats.Occlusive dressings were used consisting of: 1) absorbing foam, soft silicone (Safetac ® ) and vapor permeable film impregnated with ionic silver coated (Mepilex ® Ag) forming one group called Silver Ion (PI); 2) coated polyester mesh with nanocrystalline silver (Acticoat 7 Flex ® ), elastic adhesive bandage traditional BSN Medical GmbH group forming nanocrystalline Wound healing using ionic silver dressing and nonocrystalline silver dressing in rats Acta Cirúrgica Brasileira -Vol.27 (11) 2012 -763 silver (PN); and 3) sterile distilled water dressing control group, called distilled water group (AD) (Figure 1).The dressings were changed each seven days, with sedation.The wounds were cleaned with distilled sterile a water and then, each lesion received the connected dressing group.

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On death day the animals were subdivided into three subgroups, namely 7º, 14º and 21 days.They died using solution of Thiopental ® , in concentration of 1g diluted in 40ml of physiological solution, intraperitoneally.Wound diameter measurements were done with graduate scale in cm (Figure 3) and scalpel blade 20 did skin excision at previously demarcated areas (1.5cm out of injuries) (Figure 4).The specimens were preserved in bottles containing 10% formol solution for histology.

Results
The diameter of injury, when compared the types of treatment, were significant (p<0.05) in 7 th and 21 st days between groups PNxAD and ADxPI (Table 1).When compared the days of evaluation between two groups within each treatment, all groups presented significance (p<0.05)(Table 2).Intensity of inflammation was more pronounced on group AD on subgroup 7 th day (Figure 5A and B, Table 3); there was statistic significance only between the groups ADxPI on 7 th days.In relationship to days of evaluation, two by two within each treatment, the three groups showed significance when compared the days 7x21 (Table 4).In microscopic evaluation of neovascularization only had significance on 7 th day in relationship to groups PN and AD (Table 5).
Wound healing using ionic silver dressing and nonocrystalline silver dressing in rats Acta Cirúrgica Brasileira -Vol.27 (11) 2012 -765 Also in neovascularization, there was statistic significance (p<0.05)only on group AD when compared the days of evaluation, two by two within each treatment, between the days 7x14 and 7x21 (Table 6).Other important finding on microscopy was the presence of macrophages responsible by gigantocellular reaction, that were observed in PI and PN at 7 th and 14 th days (Figure 6).The lymphocytes appeared on group PN on 14 th day (two cases) and on group PI on 7 th day (two cases) and one case on 14 th day; however, on control group it did not appear.
In fibrosis evaluation through Masson staining (Figure 7), there was no difference statistically significant between the groups (Table 7).However, in fibrosis analysis comparing the days of evaluation, two by two within each treatment, there was significance on group AD between days 7x21 (Table 8).

Discussion
Contrary to Leaper et al. 10 that claimed to be irrational the use of silver dressings in non infected wounds, this study proved that there was improvement in healing in groups PI and PN, when compared to the control group (AD).
The macroscopic evaluation consisted on verification of contraction of wound in relation to the demarcated area in surgical procedure day, strategy also used by Araujo 11 .This verification needs a time since the process of contraction of wound -fourth phase of healing process -consists on centripetal movement of the edges 1,12 .The phase that precedes the wound contraction is the proliferation, responsible for the "closing" of lesion properly said, and is divided in three subphases -reepithelialization, fibroplasia and angiogenesis.At the end, the remodeling phase follows the contraction, the last moment of wound healing.In this research only on subgroup 21 days appeared significant differences between the groups AD and PI, but when compared the days of evaluation two by two within each treatment, all the comparisons between the subgroups indicated significance, more pronounced in comparison between 7x21days.
In microscopic evaluation with H&E staining -intensity of inflammation -, was found more pronounced form only on seventh day between the groups AD and PI; in group control it appeared in 90% of the cases; however, when are compared the days of evaluation, two by two within each group of treatment, the groups control and PN showed values statistically significant, like findings reported by Pundek et al. 13 .
Other important observation was the presence of macrophages responsible by gigantocellular reaction 1 .These cells appear in wound in about one week and within this period in this study were observed two cases in groups PI and PN; also, another two were noticed in the same groups in two weeks.However, control group (AD) had no case in the same time.
The neovessel formation begins with tissue aggression, activation of macrophages and substances produced at this moment.The proteases (plasmin and collagenases) digest the basal membrane and allow that the endothelial cells, stimulated by angiogenic cytokines, form a new vascular site that invades the wound; this is essential to supply oxygen and nutrients for the healing.The angiogenesis ceases by apoptosis 14,15 .In this research, the comparison PN and AD on first sacrifice (7 th day) presented significance, as well as control group when compared the days of evaluation, two by two within each treatment; this fact was also related by Balbino et al. 16 .
Another technique of coloring used was the Masson staining to observe the formation of collagen (fibrosis).The formation of tissue of granulation depends of fibroblasts, critical cell in matrix formation, in production of elastin, fibronectin, glicosaminogicans and proteases, all responsible for physiological debridement and remodeling of injury.Within this analysis only the control group presented statistic significance in comparison between the days of evaluation, two by two within each treatment.

Conclusions
In relation to wound contraction, nanocristaline (PN) and ionic silver (PI) groups showed better results than the AD group.
In regard to histological analysis, H&E staining demonstrated that there was presence of inflammation in all groups, and at the end, the control group (water dressing) on 7 th day, was superior to PN and PI groups.In relationship to fibrosis, no differences were obtained among groups.
Institute (IPEM) of the Post-Graduate Program in Principles of Surgery of Evangelical Faculty of Parana/Evangelical Hospital of Curitiba and was approved by the Ethics Committee in Research of Beneficent Evangelical Society of Curitiba, PR, Brazil.

FIGURE 1 -
FIGURE 1 -Dressing with nanocrystalline silver in A and ionic silver in B.

FIGURE 2 -
FIGURE 2 -Sample of two injuries with punch of 8mm.

TABLE 2 -
Diameter of injury comparison between the days of evaluation, two by two within each treatment.

TABLE 3 -
Evaluation of inflammatory process with H&E staining.

TABLE 4 -
Comparison of inflammatory processbetween the days of evaluation, two by two within each treatment.

TABLE 5 -
Assessment of neovascularization between the groups PN, AD and PI.

TABLE 6 -
Comparison between the days of evaluation, two by two within of each treatment.

TABLE 7 -
Fibrosis analysis by Masson staining.

TABLE 8 -
Comparison between the days of evaluation of fibrosis, two by two within each treatment.