In recent years, nanotechnology has been emerging as a rapidly growing field with various applications in science and technology for the purpose of new material production; it is defined as the utilization of structures with at least 1 nm size dimension in the construction of materials, devices, or systems with improved properties. ¹ Since the 1980s, nanotechnology has been attending much attention and has been adapted into many engineering fields, such as electronics and mechanics, as well as significant progress in the biomedical fields. ² Nanomedicine is referred to the application of nanotechnology for achieving breakthrough in healthcare and improving physical, chemical, and biological properties of materials at the nanometer scale. ² Silver nanoparticles (AgNPs) are useful prophylactic and therapeutic agents for the prevention of wound colonization by organisms that impede the healing. ³ The main rationale for using AgNPs on open wounds is the prevention and treatment of infection to increase the healing rate. ⁴ An ulcer is a breakdown in the continuity of the skin or mucous membrane, disintegration, and necrosis of the epithelial tissue. Oral ulcers usually are accompanied by pain and discomfort during eating, drinking, or talking. ⁵ , ⁶ There are many treatment protocols for relieving pain and accelerating oral ulcer healing; however, the most usual treatment is triamcinolone acetonide paste. ⁷ This medication is a medium-strength corticosteroid and is used to relieve symptoms of mouth sores and to reduce the swelling and pain of the ulcers. ⁸ , ⁹ Thus, this study was designed to evaluate the therapeutic effect of AgNPs gel on oral ulcers in comparison with triamcinolone as the traditional treatment of these cases.

Animals

Seventy-two adult male Sprague–Dawley rats (weighing 250 ± 50 g) were used in this experimental study. The rats maintained under standard condition (22°C ± 3°C temperature, 50% ± 5% relative humidity, and 12/12 h light-dark cycles) and had free access to standard diet of soft food stuff and water.

Silver nanoparticle gel preparation

AgNPs embedded topical gel were prepared in Pharmacy Faculty of SUMS by the polyol process. Briefly, ethylene glycol was heated in an oil bath to 100°C. After 1 h, a solution of silver nitrate (3 ml, 2.5 mM) and a solution of polyvinyl pyrrolidone in ethylene glycol (3 ml, 25 mM) were added gradually under vigorous stirring. The mixture was precipitated by adding acetone. The final silver concentration in the dispersion was determined by ICP-AES (Inductively coupled plasma-atomic emission spectrometry). The average particle size and zeta potential were studied by dynamic light scattering. For gel preparation, 200 mg of Carbopol ^®934 was dissolved in 20 ml of AgNP dispersion in distilled water (200 ppm). Then, 200 μl of triethylamine was added gradually while stirring to reach the neutral point. Gel containing AgNPs was stored in a sealed container.

Surgical procedure

After an adaptation period of 10 days, oral traumatic ulcers were induced according to Alsadat Hashemipour et al.'s procedure. ¹⁰ All the 72 animals were anesthetized by intraperitoneal (IP) injection of a mixture of xylazine (5 mg/kg) and ketamine hydrochloride (40 mg/kg); then, a buckle-shaped clamp was fastened on the upper and lower incisor teeth to keep the mouth open Figure 1a and Figure 1b. Punch biopsy tools of 4 mm in diameter were used to create a standard size circular wound, outlined posterior to second transverse palatine fold of mucoperiosteum of the hard palate midline Figure 1c. Soft tissues were removed by sharp dissection to expose the underlying bone. Hemostasis was done by pressure of sterile cotton gauze on the wound. Analgesia was provided by the administration of a single dose of flunixin meglumine (1 mg/kg, intramuscular (IM)) postoperatively. The dead rats were replaced by new rats. Figure 1

(a) Handmade buckle shape clamp made by stainless steel wire. (b) Keeping rat mouth open during surgery by buckle clamp. (c) Surgical procedure and hard plate punching posterior to second transverse palatine fold of mucoperiosteum. (d) Medicine application by rotational movement of sterile swap at days after ulcer induction.

Wound size

Although the effect of different medications on wound size varies in each group, the wound size alterations in all groups had nearly a same trend of reduction Figure 2. No significant differences were detected between the groups at different days (P > 0.05); except the AgNPs group at 14 ^thday, which had significantly the smallest wound size than other groups and days. In control group at 14 ^thday, the wound size was significantly smaller than the other days (P = 0.002); however, this size reduction was not significant between 3 ^rdand 7 ^thdays. In placebo group, the wound size was significantly smaller at 7 ^thday than 3 ^rdday (P = 0.004); however, the reduction was not significant between 7 ^thand 14 ^thdays. In triamcinolone group, the trend of wound size reduction was not significant at different days (P > 0.05). Moreover, in AgNPs group, the wound size was significantly smaller at 14 ^thday than the other days (P < 0.001); however, the difference between 3 ^rdand 7 ^thdays was not significant. Figure 2

The bar chart showing the mean ± standard deviation of wound size (mm) of different groups at different days.

Healing of ulcers and surgical wounds is the most desirable procedure in all types of surgeries. During the healing phase, several factors are needed to inhibit the infections and suppress inflammation to induce cellular proliferation. ¹³ , ¹⁴ The use of antimicrobial agents at these sites may benefit the healing process as they prevent microorganisms to be colonized. ¹⁵ The antibacterial and antifungal activities of AgNPs have been reported in the literature. ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ This study was designed to evaluate the therapeutic effect of AgNPs gel on oral ulcers and wounds in comparison with triamcinolone as the traditional treatment of these situations.

The finding of this study may indicate that although AgNPs had delayed effect, they showed a better healing rate according to the number of cases with complete healing. Indeed, daily application of AgNPs gel could be more effective than triamcinolone paste on healing of oral ulcers. Furthermore, their therapeutic effects were almost the same and AgNPs have the option of microbiocidal activity. Both agents had no side effects, too.

At the 3 ^rdday after induction of the ulcer, clinical and histological examination of the specimens of AgNPs group revealed the highest healing rate among other groups. Although it was nonsignificant, AgNPs group had higher degree of re-epithelialization compared to triamcinolone group at 3 ^rdday, despite the equal degree of inflammation. In addition, it was better than control and placebo groups in all the criteria. In this group, histopathology showed submucosal band-like inflammatory infiltrate (approximately ½ of power field) and re-epithelialization more than edges of the wound and less than half of it. However, in control group, histopathology revealed submucosal band-like inflammatory infiltrate (more than ½ of one power field) with tissue necrosis and re-epithelialization at the edges of the wound. These findings were in agreement with Aziza et al., who reported the healing response in nanosilver group, ³ which was enhanced by rapid proliferation and migration of the epithelial basal cells over the ulcerated area and decreased number of inflammatory cells in the connective tissue beneath the ulcer. In control group, they observed infiltration of many inflammatory cells to subepithelial connective tissues, and thinner appearance and loose arrangement of collagen fibers ^3]Moreover, Sibbald et al. stated that 3 days after ulcer induction and treatment with nanocrystalline silver, it had lessened the number of inflammatory cells, in return reduced the wound inflammation, and enhanced the healing process of chronic venous leg ulcer patients. ²⁰

At the 7 ^thday in this study, triamcinolone group showed a superior healing effect than all the other groups. In this group, the degree of re-epithelialization and inflammation increased with a steady slope that was not significant. Further, it had a significant difference in wound size compared with the 3 ^rdday. It seems that triamcinolone causes constant trend in healing process. However, at 7 ^thday, AgNPs had the lowest healing rate among the other groups. At this day, clinical study of AgNPs specimens showed only a little decrease in wound size and histopathology demonstrated a deterioration in ulcer condition as mild increase in degree of inflammation and low reduction in re-epithelialization, although these changes was not statistically significant. In addition, at 7 ^thday, the control and placebo groups had mild nonsignificant improvement in clinical and histopathological scores compared with AgNPs group. These results were in contrast with the result of Aziza et al. in 2015 that reported significant decrease in inflammation and re-epithelialization on 7 ^thday. ³ This variation might be related to the type of wound (chemical burn), deepness of ulcer, medication type (nanosilver solution), or methods of evaluation in their study. ³

At the 14 ^thday, this study demonstrated that AgNPs group had the most cases of complete healing and also showed the highest improvement of inflammation, re-epithelialization, and wound size compared with other groups although it was not significant. AgNPs caused healing by a more sloping than triamcinolone. This steady slope trend of improvement was seen especially in re-epithelialization of triamcinolone group that was not significant between 3 ^rdand 7 ^thdays and between 7 ^thand 14 ^thdays. These findings were in agreement with previous studies that stated nanosilver is an effective treatment. ³ , ¹³ The main rationale for applying AgNPs on open wounds is the prevention and treatment of infection and increasing the healing rate. ¹³ , ²¹ The accelerated healing rate also recorded in the AgNPs group of the present study; this could be related to the extremely large surface area of nanosilver crystals that led to increase contact with bacteria and inhibit their replication. ¹⁷ Khorshidi et al. reported that proper concentration of nanosilver increases the contact with large number of bacteria in inflamed wound, leading to adverse effects on cell metabolism and transport of substances through the microbial cell membrane, which inhibits multiplication and growth of bacteria. ¹⁷ Other studies also evaluated the toxicity of similar product but not in topical form. They had shown dose-dependent accumulation of silver content in all the tissues. ²²

Overall, AgNPs gel had a good healing effect on oral ulcers, similar to previous studies in all the body skin wounds. ²³ It also reported by similar study in rats that their results on the excision wounds using nanoparticles which could be a novel therapeutic way of improving wound healing in clinical practice. ²⁴ It seems that AgNPs have delayed effect on oral ulcer healing. It might be due to its antibacterial effect or its slow absorption to the tissue.

Here, the gel was produced in mucoadhesive form, hence evaluating the effect of other type of AgNPs (such as mucoadhesive pill and oil-based gel), in addition to more advanced methods of immunohistochemistry, and also, clinical trials on humans are suggested.

The results showed that AgNP oral gel was effective on wound healing both clinically and histopathologically. Although it had delayed effect, it showed a better healing rate. Hence, daily application of AgNP oral gel on mouth sores may be more effective than triamcinolone acetonide paste and can be an alternative treatment in these cases.

Acknowledgments

We thank the Vice Chancellor of Research, Shiraz University of Medical Sciences for financial support of this work (Grant No. 15988). This manuscript is based on the thesis written by Alireza Bonyani (DMD).

Financial support and sponsorship

This work was supported financially by the Vice Chancellor of Research, Shiraz University of Medical Sciences (Grant No. 15988).

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.