Porphyromonas gingivalis (Pg), a keystone pathogen of chronic periodontitis, releases outer membrane vesicles (OMVs) that act as nanoscale vehicles to disseminate virulence factors within periodontal tissues and systemically beyond the oral cavity. Although Pg-OMVs are increasingly recognized as critical mediators of host–pathogen interactions, their effects on the differentiation and function of monocyte–macrophage/osteoclast lineage cells remain unclear. Here, we examined the impact of Pg-OMVs on the differentiation of RAW264.7 monocyte/macrophage-like cells into osteoclasts (OC) and/or macrophages (MΦ) in the presence of receptor activator of nuclear factor-κB ligand (RANKL). OMVs were isolated from Pg W83 and applied to RANKL-primed RAW264.7 cells using three distinct stimulation schedules: (1) simultaneous treatment with Pg-OMVs and RANKL at Day 0; (2) RANKL priming at Day 0 followed by Pg-OMV stimulation at Day 1; and (3) RANKL priming at Day 0 followed by Pg-OMV stimulation at Day 3. In all schedules, cells were cultured for 7 days from the initial RANKL exposure. Remarkably, simultaneous exposure to Pg-OMVs and RANKL (Schedule 1) markedly suppressed osteoclastogenesis (OC-genesis) while promoting M1 macrophage polarization. In contrast, delayed Pg-OMV stimulation of RANKL-primed cells (Schedules 2 and 3) significantly enhanced OC-genesis while reducing M1 polarization. These schedule-dependent effects were consistent with altered expression of osteoclastogenic markers, including dc-stamp, oc-stamp, nfatc1, and acp5. Importantly, a monoclonal antibody against OC-STAMP counteracted the Pg-OMV-induced upregulation of OC-genesis in Schedules 2 and 3. Furthermore, levels of Pg-OMV phagocytosis were inversely correlated with osteoclast formation. Finally, co-stimulation with RANKL and Pg-OMVs (Schedule 1) enhanced macrophage migratory capacity, whereas delayed stimulation with Pg-OMVs (Schedules 2 and 3) did not. Collectively, these findings indicate that Pg-OMVs exert stage-specific effects on the OC/MΦ lineage: stimulation at early stages of RANKL priming suppresses OC-genesis and promotes M1 polarization, whereas stimulation at later stages enhances OC-genesis without inducing M1 differentiation. Thus, Pg-OMVs may critically influence the fate of the OC/MΦ unit in periodontal lesions, contributing to disease progression and tissue destruction.

Objectives: Residual bacteria at the time of root canal obturation significantly influence endodontic treatment outcomes in teeth with apical periodontitis, and confirmation of bacterial elimination from the root canal prior to obturation may improve long-term prognosis. However, the contribution of root canal bacterial culture to treatment outcomes in teeth with infected root canals is debated. This study aimed to retrospectively evaluate treatment outcomes in cases in which root canal obturation was performed after confirming negative bacteriological test results.Methods: The medical records of patients with apical periodontitis or pulp necrosis in whom root canal obturation was performed after confirming a negative bacteriological test result using an anaerobic culture system at the Department of Endodontics and Operative Dentistry at Hiroshima University Hospital between April 1, 2015, and September 30, 2023, were retrospectively reviewed. Treatment outcomes were assessed by comparing dental radiographs obtained before treatment with those obtained during the follow-up, ranging from 3 to 24 months after root canal obturation.Results: This retrospective study included 284 teeth from 218 patients, and the success rate was 89.1%. Tooth type, number of root canals, type of treatment, preoperative radiolucency around the root apex, preoperative root canal obturation material, dentist experience, and postoperative extent of root canal obturation material did not significantly affect treatment outcomes.Conclusions: The findings of this retrospective study suggest that infection control incorporating bacteriological examination of root canals using an anaerobic culture system may contribute to a high success rate of nonsurgical endodontic treatment, regardless of other prognostic factors.