Periodontitis is a chronic multifactorial inflammatory disease driven by the dysbiotic relationship between the host immune-inflammatory response and the commensal oral microbiota in plaque biofilms that results in the progressive destruction of the tooth-supporting apparatus (Papapanou et al., 2018). Several modifiable and non-modifiable factors including genetic, environmental, and systemic factors can influence its pathogenicity and alter patient's susceptibility to the development and severity of the periodontal disease (Kinane et al., 2017).

According to the 2017 World Workshop classification, stages III or IV, grade C periodontitis, formerly defined as aggressive periodontitis, is characterized by the rapid progression of a particularly severe form of periodontal disease (Papapanou et al., 2018). According to the EFP S3 Clinical Treatment Guidelines, in patients with deep periodontal pockets (≥ 6 mm) step 2 of periodontal therapy (subgingival instrumentation, with or without adjunctive therapies) may be insufficient, and step 3 of surgical periodontal therapy incorporated with regenerative procedures may be required to eliminate deep residual pockets and reconstruct the intrabony part of the defects, as well as prevent tooth loss (Sanz et al., 2020). Despite of some limitations of access flap periodontal surgeries such as increased risk of gingival recession, increased postoperative dentin hypersensitivity, and morbidity, regenerative surgical procedures have been shown to be efficacious for the treatment of intrabony defects in patients with severe periodontitis, with the rapid rate of progression (Diaz-Faes et al., 2020). To improve the outcomes of therapy in severe periodontitis patients, adjunctive interventions such as local or systemic antibiotics, and antimicrobial photodynamic or photobiomodulation therapies using lasers or light-emitting-diodes (LED) have been explored in non-surgical and surgical treatment approaches (Collins et al., 2022; Andere et al., 2022; Giannelli et al., 2018).

Within the visible red or near-infrared (NIR) range of the spectrum (600 to 700 nm and 780 to 1100 nm), low-level lasers (LLL) or LEDs have been widely utilized as an adjunct therapy for periodontitis treatment based on their photobiomodulation and decontamination effects, which primarily occur at the level of the cellular respiratory chain (Zhao et al., 2021). These modalities promote mitochondrial activity and activate the mechanisms, i.e., inducing intracellular metabolic changes, enhancing the cellular resuscitation system, increasing adenosine triphosphate (ATP) and extracellular matrix (ECM) production (Heidari et al., 2018). Through the biological characteristics of these approaches, various studies have shown to increase angiogenesis, promote modulation of the immunoinflammatory response, stimulate the processes of regeneration and epithelialization, and reduce postoperative symptoms. The use of LLLs or LEDs in conjunction with photosensitizing agents (optical absorption-dye) has been referred as antimicrobial photodynamic therapy (aPDT). This procedure stimulates the dye to form free radicals of singlet oxygen that will act as toxic to the target cells or bacteria mainly as a result of deterioration to the cytoplasmic membrane and DNA, thereby demonstrating anti-microbial activity at periodontal pathogenic bacteria in combination with periodontal treatment (Katsikanis et al., 2020). Similar to the biostimulatory features of LLLs or LEDs, adjunctive gaseous ozone therapy applications have been recently exploited because of their analgesic, immunomodulatory, and anti-inflammatory effects, which may have the ability to provide significant added benefits in both step 2 and 3 of the periodontal therapy due to its analgesic, immunomodulatory, and anti-inflammatory effects (Rapone et al., 2022). All these adjunct therapies have also been shown to have a positive effect on the proliferation and osteogenic differentiation of undifferentiated periodontal tissue cells or the direct stimulation of osteoblasts in several studies. However, little information exists regarding the efficacy of these modalities adjunct to the periodontal regenerative therapies on the processes and sequences of the healing and consequently, in the postoperative expression levels of biomarkers of inflammation, angiogenesis, and osteogenesis. Therefore, the objectives of the present study were: (i) to investigate the additional influence of multiple sessions of aPDT, LED photobiomodulation, and topical gaseous ozone therapy applications associated with surgical regenerative treatments by using an allogenic bone graft in combination with a collagen membrane on clinical and patient-centered outcomes in patients with stage III, grade C periodontitis, and (ii) to analyze mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin -6 (IL-6), runt-related transcription factor 2 (RunX2), NEL-like 1 (Nell-1), and osterix in gingival crevicular (GCF) samples at baseline and the 1-, 3- and 6-month follow-ups after the treatment procedures.