There are multiple habitats in the oral cavity with bacteria, fungi, viruses, and protozoa residing in, which together constitute the oral micro‐ecosystem. These microflorae in the oral cavity primarily include saliva, supragingival dental plaque, subgingival dental plaque, submucosal plaque around implants, plaque in root canals, and plaque on the mucosal surface. The interest and knowledge of the microbiome have dynamically increased with the advancement of technology. Therefore, a reliable, feasible, and practical sampling strategy for the oral microbiome is required for the investigation. This paper introduced the sampling strategy of oral microorganisms, consisting of sample collection, transport, processing, and storage. The materials and devices involved in this study are all commonly used in clinical practice or laboratory. The feasibility and reliability of the sampling methods described in this paper have been verified by multiple studies.

Mitochondria have their own mitochondrial DNA (mtDNA). Aberrant mtDNA is associated with inflammatory diseases. mtDNA is believed to induce inflammation via the abnormal mtDNA release. Periodontitis is an infectious, oral inflammatory disease. Human gingival fibroblasts (HGFs) from patients with chronic periodontitis (CP) have shown to generate higher reactive oxygen species (ROS) that cause oxidative stress and have decreased mtDNA copy number. Firstly, cell-free mtDNA was identified in plasma from CP mice through qRT-PCR. Next, we investigated whether mtDNA efflux was maintained in primary cultures of HGFs from CP patients and the possible underlying mechanisms using adenovirus-mediated transduction live cell imaging and qRT-PCR analysis. Here, we reported that mtDNA was increased in plasma from the CP mice. Additionally, we confirmed that CP HGFs had significant mtDNA efflux from mitochondria compared with healthy HGFs. Furthermore, lipopolysaccharide (LPS) from Porphyromonas gingivalis can also cause mtDNA release in healthy HGFs. Mechanistically, LPS upregulated ROS levels and mitochondrial permeability transition pore (mPTP) opening by inhibition of pyruvate dehydrogenase kinase (PDK)2 expression, resulting in mtDNA release. Importantly, mtDNA efflux was even persistent in HGFs after LPS was removed and cells were passaged to the next three generations, indicating that mtDNA abnormalities were retained in HGFs in vitro, similar to the primary hosts. Taken together, our results elucidate that mtDNA efflux was maintained in HGFs from periodontitis patients through abnormal ROS/mPTP activity. Therefore, our work indicates that persistent mtDNA efflux may be a possible diagnostic and therapeutic target for patients with periodontitis.