The necessary permissions for animal experiments were obtained from the Gaziantep University Animal Experiments Local Ethics Committee on June 9, 2020, under approval number 6 and protocol number 149. This specialization thesis was supported by the Gaziantep University Scientific Research Projects Unit (project number DHF.UT.21.01). In line with the recommendations of the Gaziantep University Animal Experiments Local Ethics Committee, the purchase and maintenance fees for the rats were covered by the Gaziantep University Experimental Animal Research Center.

The study involved 24 adult male Wistar rats, which were randomly selected and evenly divided into four groups. Throughout the experiment, the rats had unrestricted access to tap water and were fed standard rat chow containing 21% protein. During the study period, they were kept in a room equipped with a specialized ventilation system, maintained at a temperature of 23 ± 3 °C, and subjected to a 12-hour light/dark cycle. Post-surgery, each rat was housed in an individual plastic cage.

A veterinarian conducted pre-procedure health assessments for each rat. All surgeries were performed on the same day by the same surgeon at the Gaziantep University Experimental Animal Research Center. Histopathological examinations were carried out at the Histology and Embryology Department of the Gaziantep University Basic Sciences. The experimental procedures were carried out following the standards outlined in the “Guide for the Care and Use of Laboratory Animals” [8] as well as the ARRIVE guidelines [9].

Four groups of 6 rats each were formed: Control 1, Ozone 1, Control 2, and Ozone 2. A 6 mm diameter full-thickness soft tissue graft was harvested from the parietal bone of all rats using a 6 mm diameter soft tissue punch. Additionally, a circular block of autogenous bone graft was extracted from the underlying skull area without damaging the dura mater, using a 5 mm diameter trephine bur under physiological serum cooling.

The harvested block autogenous bone graft was ground into particles using a mortar and placed back into the prepared skull area. This was then covered with a free tissue graft obtained from the same rat and sutured using 5/0 vicryl sutures.

In the Control 1 group, the rats were sacrificed after 2 weeks without any additional procedures. The Control 2 group was sacrificed after 4 weeks, also without any further interventions. In the Ozone 1 group, the rats were observed for 2 weeks, with ozone treatment applied to the graft site 3 times a week for 120 s during the observation period. At the end of the 2 weeks, the rats were sacrificed. The Ozone 2 group was observed for 4 weeks, with ozone treatment applied to the graft site 3 times a week for 120 s during the first 2 weeks. These rats were sacrificed at the end of the 4th week. The flow-chart diagram of the study is shown in Fig. 1.

Flow-chart diagram of the study

Prior to the operation, all rats were placed under general anesthesia through intraperitoneal administration of 75 mg/kg ketamine hydrochloride and 10 mg/kg xylazine hydrochloride solution. The calvarial areas were shaved and cleansed with an antibacterial povidone-iodine solution. The rats were then secured in a head immobilization device to ensure a stable and comfortable procedure environment (Fig. 2). The operation area was covered with a sterile drape, exposing only the surgical site. All instruments used were sterilized before the procedure, and strict adherence to asepsis and antisepsis protocols was maintained. A 6 mm diameter full-thickness free soft tissue graft was harvested from the soft tissue on the parietal bone of the rats’ heads using a 6 mm diameter soft tissue punch (Fig. 3). A circular block of autogenous bone graft was extracted from the skull at the site of the procedure using a 5 mm diameter trephine bur attached to a contra-angle handpiece. The dura mater was carefully preserved, and physiological serum was used for cooling throughout the procedure to prevent thermal damage (Fig. 4). The block autogenous bone graft taken was ground in a mortar and turned into particles (Fig. 5), and placed in the same area we prepared on the skull (Fig. 6), and covered with a free tissue graft taken from the same living creature using 5/0 vicryl sutures (Fig. 7). At the end of the procedure, in order to protect the rats from hypothermia, they were placed on a heating plate and kept for a while. While no other procedure was applied to the control groups, 120 s of ozone was applied to the ozone groups using an ozone generator (Apoza-Ozone Dta, 1982, APOZA Enterprise Co., LTD, TAIWAN) at the end of the procedure (Fig. 8).

Obtaining free tissue graft with soft tissue punch

Creation of a 5 mm calvarial defect

The block graft taken is crushed in a mortar to make it particulate

Filling the calvarial defect with autogenous bone

Suturing the free flap tissue graft

Topical ozone application to the operation area

Although some protocols have been established by institutions, there is no known definitive or standardized dose for topical ozone therapy in the literature. Researchers have conducted studies with a wide range of durations and frequencies. While ozone application times vary from 10 s to 360 s in the literature, shorter applications are generally aimed at observing lesion healing, pain reduction, and decrease in trismus, whereas longer ozone applications tend to support findings with histological results. Regarding the frequency of ozone application, different protocols have been observed, ranging from a single application to five applications per week. The most commonly used method is three applications per week for two weeks, starting on the day of surgery [10,11,12]. To allow for better comparison with other studies, we chose to apply ozone three times a week for two weeks following surgery, with each application lasting 120 s.

The ozone groups were treated with ozone for 120 s, three days a week for two weeks following surgery. The appropriate application tip was positioned close enough to cover the entire operation area, maintained at a 1 mm distance from the site, as recommended by the manufacturer. The ozone application device does not work at distances longer than 1 mm. Ozone gas was applied to the wound area as seen in Fig. 9. The power setting was set to “9,” and ozone was applied at an 80% concentration. The control groups underwent a simulated ozone application, administered for 120 s, three days a week, simultaneously with the ozone groups, for two weeks post-surgery. The image of the wound site of the rat that received ozone treatment for 28 days is shown in Fig. 10.

To prevent possible infections, a total of three doses of 25 mg/kg ceftriaxone were injected into the gluteal muscle of each rat: one immediately after the operation and one dose per day for the following two days. To alleviate postoperative pain, 4 mg/kg ketoprofen was administered intramuscularly as an analgesic, with one dose given immediately after the operation and one dose daily for the next two days, for a total of three doses.

Rat treated with ozone for 28 days

The rats were euthanized with the cervical dislocation technique at the end of the 2nd and 4th weeks. For histopathological analyses, the soft tissue to which a free tissue flap was applied and the calvarial bone defect area to which an autogenous graft was applied were resected with intact tissue surrounding them. The soft tissue and calvarium samples of each subject were fixed in 10% formalin for at least 24 h. After the fixation process, the calvarial bone sample of each subject was decalcified in 15% nitric acid solution for 72 h. The fixed soft tissue samples and the fixed and decalcified calvarial bone samples were taken from the vertical sections containing the defect area and placed in cassettes. The sampled tissues were embedded in paraffin blocks after being subjected to a follow-up process that lasted approximately 14 h. 4–5 μm thick sections were taken from each paraffin block obtained and stained with Hematoxylin & Eosin (H&E). In each subject, Masson Trichrome staining was applied to the sections where the healing process was best observed. Histopathological and histochemical evaluation was performed with a Nikon Eclipse Ni brand microscope. Photography was taken in digital environment with a Nikon DS-Fi2 brand camera connected to the same microscope device. The images obtained by taking photographs were transferred to the computer. Measurements of wound surface areas were performed using the ImageJ (ImageJ and NIH Image Software, National Institutes of Health, Bethesda, Md) program.

In the histological analysis, mature bone tissue was identified as dense bone areas without internal spaces, whereas unhealed bone tissue was defined as immature bone with internal spaces and a spongy structure. Bone trabeculae, osteoblasts, osteoclasts, and occasional osteocytes were observed within these areas. Connective tissue elements and vascular regions were prominent in the procedure site.

Histologically, tissue integrity and vascularization were observed in the soft tissue, and epithelialization was complete. Areas lacking skin appendages, such as hair follicles, were considered unhealed. In these regions, granulation tissue containing epithelialization, neovascularization, connective tissue elements, fibrosis, and inflammatory cells was present.

SPSS 26.0 Mac version (Statistical Package for the Social Sciences Inc.) was used for the statistical analysis. Continuous variables are presented as mean ± standard deviation. Mann-Whitney U test was used to compare different groups. A p value of < 0.05 was considered statistically significant. Power analysis was performed for sample size calculation using the G*Power software (latest ver. 3.1.9.7; Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany). When calculating the sample size, the study of Olğun et al. was evaluated [4]. According to the power analysis we performed, when p < 0.05, power of 0.80, enrollment ratio = 1, and effect size of 0.5 were accepted. In order to find a significant difference in terms of bone and soft tissue healing values ​​between the control and ozone groups, the required number of rats in each group was calculated as at least five. p < 0.05 accepted as statistical significance value.