In vitro and in vivo anti-periodontitis effects of combination treatment of photodynamic and antibiotic therapies

Abstract

Objective: To investigate the effects of combination treatment of photodynamic therapy (PDT) based on photosensitizer chlorin e6 (Ce6) and antibiotic agent tinidazole (TNZ) against periodontitis both in vitro and in vivo. Methods: The Sprague-Dewley (SD) rat periodontitis model was constructed using the method of orthodontic wire ligation. After successful modeling, SD rats were randomly divided into the following 6 groups (3 rats in each group): positive control (Ctrl+), Ce6, TNZ, a mixture of Ce6 and TNZ (Ce6/TNZ), Ce6 with laser irradiation (Ce6+L), a mixture of Ce6 and TNZ with laser irradiation (Ce6/TNZ+L). Methyl thiazolyl tetrazolium (MTT) assay was used to assess the cytotoxic activities of Ce6 (concentration range: 0-20 mg/L), TNZ (concentration range: 0-16.6 mg/L) and their mixture (Ce6/TNZ) in mouse fibroblast L929 cells. Fluorescence probe method was applied to measure the production of reactive oxygen species in the dental plaque biofilms after various treatments with and without 5-minute laser irradiation at 635 nm at a power density of 0.5 W/cm2 (Ce6+L and Ce6/TNZ+L groups), thus to evaluate the PDT performances. Cell counting kit-8 (CCK-8) and live/dead staining were used to assess the antibacterial activity in each of the groups and the combination index (CI) of PDT combined with TNZ was calculated subsequently. Flow cytometry was utilized to detect the apoptosis-inducing effects of these treatments in macrophage RAW264.7 cells after processing with the apoptosis detection kit. The inhibitory effects of various treatments on the absorption of alveolar bone of SD rats were further evaluated in the periodontitis rats by using the micro-CT. Results: The survival rates of L929 cells in the preset concentration range were all above 90% in Ce6, TNZ and Ce6/TNZ groups. Upon laser irradiation, the plaque biofilms in Ce6 and Ce6/TNZ groups showed significant green fluorescence, indicating that large amounts of reactive oxygen species were triggered and generated significantly in the dental plaque biofilms. However, the survival rates of dental plaque microorganisms in 5 Ce6/TNZ concentrations were (85.4±5.5)%, (76.0±8.9)%, (61.7±0.6)%, (56.3±2.6)% and (43.5±0.6)% respectively, which were significantly lower than that in Ce6 only and TNZ only groups (P<0.01). The CI levle of each drug concentration group was less than 1.0, which showed a significant synergistic antibacterial efficiency. Stronger apoptotic activities were observed in Ce6+L and Ce6/TNZ+L groups compared with those in Ce6 only and Ce6/TNZ only groups (P<0.01). In periodontitis rats, Ce6/TNZ combined laser irradiation could effectively inhibit the absorption of alveolar bone. The alveolar bone volume and the ratio of bone volume and tissue volume were (1.49±0.07) mm3 and (47.08±0.71)%, respectively. The distances between cementoenamel junction to alveolar bone crest on buccal and palatal sites decreased to (2.13±0.07) mm and (1.94±0.10) mm respectively, showing a high inhibition efficiency. Conclusions: Ce6-mediated PDT combined with TNZ possessed notable synergistic effects against periodontitis, reflecting in the efficient antibacterial effect, the apoptosis-inducing action on macrophages, and the inhibitory efficacy on the alveolar bone absorption in vivo.