Detection of Salivary Small Extracellular Vesicles Associated Inflammatory Cytokines Gene Methylation in Gingivitis

Abstract

Salivary small extracellular vesicles (sEV) are emerging as a potential liquid biopsy for oral diseases. However, technical difficulties for salivary sEV isolation remain a challenge. Twelve participants (five periodontally healthy, seven gingivitis patients) were recruited and salivary sEV were isolated by ultracentrifuge (UC-sEV) and size exclusion chromatography (SEC-sEV). The effect of UC and SEC on sEV yield, DNA methylation of five cytokine gene promoters (interleukin (IL)−6, tumor necrosis factor (TNF)-α, IL−1β, IL−8, and IL−10), and functional uptake by human primary gingival fibroblasts (hGFs) was investigated. The results demonstrated that SEC-sEV had a higher yield of particles and particle/protein ratios compared to UC-sEV, with a minimal effect on the detection of DNA methylation of five cytokine genes and functional uptake in hGFs (n = 3). Comparing salivary sEV characteristics between gingivitis and healthy patients, gingivitis-UC-sEV were increased compared to the healthy group; while no differences were found in sEV size, oral bacterial gDNA, and DNA methylation for five cytokine gene promoters, for both UC-sEV and SEC-sEV. Overall, the data indicate that SEC results in a higher yield of salivary sEV, with no significant differences in sEV DNA epigenetics, compared to UC.

Salivary Outer Membrane Vesicles and DNA Methylation of Small Extracellular Vesicles as Biomarkers for Periodontal Status: A Pilot Study

“…Four published studies reporting on salivary sEV diagnosis research in periodontitis used either a precipitation-based ExoQuick kit [ 14 , 15 , 19 ] or ultracentrifuge method [ 20 ], with potential nuclear acid and protein contamination [ 30 ]. Our very recent research used the SEC method to isolate salivary sEVs [ 16 , 18 ] with enriched salivary sEV particles compared to the ultracentrifuge method [ 18 ], and this approach was applied in the current study. Our current study was consistent with our previous study [ 16 ], showing that salivary sEV mode and particle concentration were comparable between healthy, gingivitis and periodontitis groups.…”

“…Comprehensive periodontal charting was performed by two independent experienced periodontists for each participant to determine bleeding on probing (BOP) and periodontal pocket depths (PPDs). Healthy ( n = 7), gingivitis ( n = 7) and periodontitis ( n = 8) subjects were defined as described previously [ 16 , 18 ] and the new classification of periodontitis guidelines [ 2 ]: (a) healthy: no periodontal disease history; PPD < 3 mm; BOP < 15 % sites; (b) gingivitis: no periodontal pocket, PPD < 3 mm; BOP > 30 % sites; (c) stage III/IV periodontitis: > 30% of the sites with PPD ≥ 3 mm and BOP, at least five sites with PPD ≥ 5mm on at least three non-adjacent teeth. All participants had no underlying systemic diseases.…”

“…Unstimulated whole saliva samples were collected before the full-mouth periodontal charting, as described previously [ 16 , 18 , 36 ], by asking the participants to spit the saliva samples into a sterile tube. The saliva samples were collected between 9 a.m. and 12 p.m. from the participants who refrained from eating and drinking for at least 1 h (up to 2 h).…”

“…Besides the common cytokines, salivary redox, such as stress oxidants and antioxidants, has emerged as a potential biomarker for periodontitis [ 9 , 10 , 11 ], but does not have the ability to differentiate between the stages of periodontal disease. Salivary small extracellular vesicles (sEVs, also named exosomes), a group of biological nanoparticles produced by virtually every species [ 12 , 13 ], are emerging as potential biomarkers for periodontitis [ 14 , 15 , 16 , 17 , 18 , 19 , 20 ], owing to their cargos of nucleic acids (microRNAs, DNAs, etc. ), lipids and protein.…”

Molecular Mechanisms of Periodontal Disease

“…In combination with the advanced technology for proteomics analysis, Preianò et al proposed a future reliable diagnostic system using GCF [ 14 ]. Han et al reported that the size exclusion chromatography method is appropriate for salivary small extracellular vesicle isolation, which harbors DNA methylation genes and microRNAs, such as miR-146a-5p, reflecting the physiological condition [ 15 , 16 ]. Interestingly, the systematic review by Asa’ad et al indicated that miR-146a could be utilized as a fluid biomarker in periodontitis patients [ 17 ].…”