CLINICAL AND HISTOLOGICAL ANALYSIS OF THE EFFECT OF OZONE OIL AND LASER PHOTOBIOMODULATION ON THE TISSUE REPAIR OF TRAUMATIC ULCER ON THE RAT ORAL MUCOSA
Description
Tissue repair is a complex event that begins at the time of injury, resulting in an attempt to restore fisiological balance. In an attempt to find alternatives that accelerate healing and consider these promising therapies for modulating this process, as well as to address the identified gap in the current literature regarding the combined use of LPBM with ozonized oil during tissue repair, no studies have addressed the combination of these agents. Thus, the aim of this study was to evaluate the effect of ozonized oil, with or without LPBM, on the healing of traumatic ulcers on the dorsal surface of the rat tongue. A total of 60 male Wistar rats underwent tongue ulceration on the dorsal surface using a six-mm diameter punch. Animals were divided into four experimental groups: control, LPBM, Ozonized oil, and LPBM + Ozonized oil. Euthanasia was performed on days three, seven, and 14 after surgery. Clinical analysis of the wound size and histomorphological evaluation using hematoxylin and eosin and Sirius red staining were conducted to identify edema, inflammatory infiltrates, fibroblastic cellularity, reepithelialization, and collagen characterization. Descriptive and exploratory analyses were performed and inferential statistics were calculated using Fisher's exact tests. Analyses were performed using R, with a significance level of 5%. LPBM and Ozonized oil positively modulated the repair phases clinically (p<0.05) and histologically, showing reepithelialization, fibroblastic population, and thick collagen fiber deposition from the 3rd day of the experiment. LPBM + Ozonized oil exhibited a significant difference from control in the clinical parameters (p<0.05), but did not demonstrate superiority in histomorphological terms of tissue repair. Isolated use of LPBM and Ozonized oil improved tissue repair in rat tongues.
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The animals were randomly assigned to four experimental groups, as described below. G1 - Negative Control Group: application of 0.9% saline with a flexible-tipped applicator and cotton tips (Cotton Swabs®; Johnson & Johnson, São Paulo, Brazil). G2 - LPBM Group: application of LPBM with low-power laser (LPL) (DMC®, São Carlos, São Paulo, Brazil), at a wavelength of 660 nanometers (nm), power of 100 milliwatts (mW), spot size 0.028 cm², a single and focal application of 1 joule (J) at the wound center for 10 seconds (s), and energy density of 35.714 J/cm2. G3 - Ozonized Oil Group: topical application of one drop (0.05 mL) of ozonized olive oil (Philozon®, Santa Catarina, Brazil) with a flexible-tipped applicator and cotton tips (Cotton Swabs®; Johnson & Johnson, São Paulo, Brazil) for 30 s. G4 - LPBM + Ozonized Oil Group: combination of protocols from groups G2 and G3. First, LPBM was applied to LPL, followed by the immediate application of ozonized olive oil. Applications began immediately after the standardized ulcer was performed on day zero (D0), every 24 h in the morning, until the 14th day of the experiment. After the application, all animals remained without food or water for 30 min. They were anesthetized with a mixture of Ketamine® and Xylazine®, 90 mg/kg and 5 mg/kg, respectively. A circular portion of the tongue dorsum was removed using a circular punch-type scalpel with a diameter of six mm. Ulcers were created in all the animals on day zero (D0) of the morning experiment. Euthanasia was performed on days three, seven, and 14 using a mixture of 70% carbon dioxide (CO2) and 30% oxygen (O2). The tongue was removed to obtain two fragments, the surgical wound margin and subcutaneous tissue, which were fixed in 4% buffered formalin (pH 7.4) for 24 h. After this period, routine histological processing was initiated until obtaining histological sections of 5 µm, which were sequentially stained with hematoxylin and eosin (HE) and Sirius red. Clinical measurements of the ulcers were performed in mm using a universal analog caliper (Starrett, Athol, MA, USA) on the D0 and on the D3, D7, and D14. The histomorphological analyses were conducted blindly and in triplicate to avoid bias using slide masks. Motic Images Plus 2.0 Software (Motic Asia, Hong Kong, China) and a Quimis BA410 light microscope (Quimis, São Paulo, Brazil) coupled with a Moticam 5.0 camera were used. Histological sections stained with HE were subjected to histomorphological analyses using a 10x objective (epithelium and the connective tissue). Characterization was semi-quantitative and categorized as described by Medrado et al. (2008). The predominance of polymorphonuclear or monomorphonuclear inflammatory infiltrates was verified using a 40x objective. The degree of reepithelialization was evaluated and classified according to the description by Dantas et al. (2023). The deposition of the collagen fibers was semi-quantitatively assessed (Alvarenga et al., 2020).