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Comparison between the Effect of 810 nm and 940 nm Diode Laser Irradiation on Histopathological Changes in Iatrogenic Oral Ulcers: an Animal Study

Statement of the Problem: Considering the relatively high prevalence of oral mucosal ulcers, their fast healing is of significance. Purpose: This study aimed to histopathologically compare the effects of 810 nm and 940 nm diode laser on the healing of iatrogenic oral ulcers in rabbits. Materials and Method: In this single-blind experimental study, mucosal ulcers measuring 3mm in diameter and 1mm in depth were bilaterally created in the buccal mucosa of 18 rabbits using a biopsy punch. The defects were irradiated with 810 nm diode laser on the right side and 940 nm diode laser on the left side. Biopsy samples of the same depth were obtained from the ulcers on days 3 and 7 followed by histopathological analysis. The intensity of inflammation was determined on hematoxylin-eosin-stained sections using a four-point scale. Data were analyzed employing the Wilcoxon signed rank test. Results: The degree of inflammation was not significantly different between the 810nm and 940nm diode laser groups on day 3; but on day 7, animals receiving 810 nm experienced a significantly lower degree of inflammation compared to those treated with 940 nm laser (p= 0.028). Conclusion: When comparing 810- and 940-nm diode lasers, 810 nm irradiation significantly decreased the severity of inflammation in oral wounds created on the buccal mucosa of rabbits in a time-dependent manner.


Introduction
Ulcers are well defined and usually depressed characterized by an epithelial defect covered with a fibrin clot, which often has a yellow-white appearance. Oral mucosal wounds have a high prevalence and their painful nature interferes with normal oral function [1]. These lesions may develop due to a variety of reasons such as nutritional deficiency of iron and folate, infections, stress, trauma, gastrointestinal problems, deep fungal infection, cutaneous diseases with oral manifestations, medications and allergens [2].
Oral ulcers are associated with tissue inflammation and it causes pain. Thus, any mechanism that can subside wound inflammation will play a role in pain relief of oral ulcers. Conventionally, wound healing in the oral cavity involves several phases including hemostasis, inflammation, proliferation and maturation. In addition to platelets, neutrophils, monocytes, macrophages, lymphocytes, fibroblasts, collagen and a series of mediators and cytokines such as growth factors, including platelet derived growth factor; vascular endothelial growth factor and transforming growth factor-beta play a role in wound healing. In this regard, growth factors stimulate neutrophils and macrophages, have a mitogenic effect on fibroblasts and cause angiogenesis in the process of wound healing and tissue remodeling. Neutrophils and lymphocytes play a primary role in healing of ulcers, and neutrophils are the dominant cells in the first 48 hours following injury. In addition to wound healing, lymphocytes play a role in cellular immunity [3][4][5]. Oral tissue healing is challenging; thus, various methods are recommended to accelerate this process and inhibit the impeding factors involved in its delay [6].
Any mechanism that can reduce wound inflammation can play a role in pain relief of oral ulcers. Several studies have evaluated the application of lasers in the healing of oral ulcers like aphthous stomatitis, considering their analgesic, anti-inflammatory and wound healing properties as well as the fact that they cause immune system stimulation and affect physiological tissue status [7][8]. Laser therapy has advantages such as costeffectiveness, faster formation of granulation tissue, improvement in bone repair, wound closure, and faster re-epithelialization [9][10].
Low-level laser therapy (LLLT) is an adjunct modality in many dental fields. It has positive effects on oral hard and soft tissues with minimal complications.
Moreover, LLLT is used for alleviation of chronic pain and healing of chronic wounds (such as lichen planus) [11]. It has been documented that LLLT can efficiently decrease inflammation; however, controversy exists regarding the preferred wavelength of laser for this purpose. Thus, the present study aimed to compare the antiinflammatory effects of 810 nm and 940 nm diode lasers on oral mucosal wounds.

Materials and Method
The present study was designed based on the Ethi- Both treatments were used for single session. The probe tip had 1cm diameter and 2.5 J/cm 2 energy density.
Next, biopsy samples were obtained from the wounds of 9 rabbits after 3 days using the same punch. All samples were immersed in 10% formalin and sent to the pathology laboratory for histopathological processing followed by hematoxylin and eosin staining ( Figure 3).
The same was done for the remaining rabbits after 7 days ( Figure 4) [12].

Statistical analysis
The degree of inflammation on the right and left sides was compared using the Wilcoxon signed rank test.

Results
Mean data in the 810 nm and 940 nm laser groups at day 3 and day 7, were separately calculated and categorized to determine the degree of inflammation. The results are presented in Tables 1 and 2. After assessment of inflammation cells, the maximum and minimum average percent inflammation cells in day 3, for laser 940nm was 98.9% and 23.5% and for laser 810nm was 100% and 37.2% and also the maximum and minimum average percent of inflammation cells in day 7, for laser 940nm was 92% and 28.7% and for laser 810nm was 52.8% and 29%.
The degree of inflammation was not significantly different between the laser groups on day 3 (p= 0.441), but it was significantly lower in animals receiving 810 n m compared to those irradiated with 940 nm laser (p= 0.028).

Discussion
The effect of LLLT on wound healing is mediated These cells synthesize collagen following activation by transforming growth factor beta, which is also increased by LLLT. Another element upregulation via irradiation is the vascular endothelial growth factor with stimulatory effects on neovascularization [13][14]. The application of LLLT for management of mucosal wounds has been previously investigated with results supporting anti-inflammatory, analgesic and biostimulatory impacts on oral mucosal wounds [15][16]; however, the optimum irradiation specifications has not been established. The present study demonstrated decreased inflammatory cell count at the site of ulcers, following LLLT application. Although there are some studies that did not compare different laser types, our study exclusively showed the effect of diode laser wave   According to the results obtained in the present study, a significant difference in the degree of inflammation was observed between the two laser groups on day 7, but not on day 3. We obtained lower inflammatory cell count in animals receiving 810 nm compared to those irradiated with 940 nm. This was in contrast to another study who found a significant continuous reduction in inflammation during days 3, 7 and 14 using 658nm [12]. This finding appears to be due to the different biological behavior of various wavelengths of laser and their subsequently different effects on inflammation.
A systematic review on the effects of LLLT on human periodontal ligament fibroblasts concluded that LLLT has positive effects on proliferation of fibroblasts and different types of osteogenic cells and also modulates cellular inflammation [17].
Studies on periodontitis and recurrent aphthous ulcers using 940 nm similar to our setting, also demonstrated reduced inflammation and even reported that one-time irradiation of this wavelength can have immediate lasting analgesic effect [23].
In the current study, two different wavelengths of diode laser were evaluated. Since the biological behavior of LLLT and light changes with different wavelengths, it is expected that the number of inflammatory cells may also change following alterations in this fac- week. There was no significant difference between the two wavelengths on day 3 of treatment.

Conflict of Interest
None declared.