Salivary Small Extracellular Vesicles Associated miRNAs in Periodontal Status—A Pilot Study

Abstract

This pilot study aims to investigate whether salivary small extracellular vesicle (sEV)-associated microRNAs could act as potential biomarkers for periodontal disease status. Twenty-nine participants (10 who were healthy, nine with gingivitis, 10 with stage III/IV periodontitis) were recruited and unstimulated whole saliva samples were collected. Salivary sEVs were isolated using the size-exclusion chromatography (SEC) method and characterised by morphology, EV-protein and size distribution using transmission electron microscopy (TEM), Western Blot and Nanoparticle Tracking Analysis (NTA), respectively. Ten mature microRNAs (miRNAs) in salivary sEVs and saliva were evaluated using RT-qPCR. The discriminatory power of miRNAs as biomarkers in gingivitis and periodontitis versus healthy controls was evaluated by Receiver Operating Characteristics (ROC) curves. Salivary sEVs were comparable to sEVs morphology, mode, size distribution and particle concentration in healthy, gingivitis and periodontitis patients. Compared to miRNAs in whole saliva, three significantly increased miRNAs (hsa-miR-140-5p, hsa-miR-146a-5p and hsa-miR-628-5p) were only detected in sEVs in periodontitis when compared to that of healthy controls, with a good discriminatory power (area under the curve (AUC) = 0.96) for periodontitis diagnosis. Our study demonstrated that salivary sEVs are a non-invasive source of miRNAs for periodontitis diagnosis. Three miRNAs that are selectively enriched in sEVs, but not whole saliva, could be potential biomarkers for periodontal disease status.

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

“…Another study compared saliva sEV isolation by UC and EQ and it showed that CD9 and CD81 expression was increased in EQ-sEV compared to UC-sEV [20]. Our very recent research demonstrated that SEC-sEV associated miRNAs (hsa-miR−140-5p, hsa-miR−146a−5p, and hsa-miR−628-5p) were only detected in sEV in periodontitis when compared to that of healthy controls [12].…”

“…Commercially available SEC columns (miniPURE, HansaBioMed, Lonza) were used to fractionate the saliva sample according to the manufacturer's protocol (Figure 1b), as described previously [12]. Briefly, following 1:1 dilution with PBS, the samples were centrifuged at 300× g for 15 min, 1600× g for 15 min and 16,000× g for 20 min at 4 • C. The supernatant was loaded on an SEC column and 100 µL fractions were collected as indicated.…”

“…Currently, two main methods have been reported to isolate salivary sEV, namely the 'gold-standard' ultracentrifuge (UC) and a commercial ExoQuick (EQ) precipitation kit [10]. Alternatively, size exclusion chromatography (SEC) has been proposed for sEV isolation with potential higher functionality [11] and was recently used to isolate salivary sEV [12]. Thus, evaluating the different techniques (UC vs. SEC) is of great importance for establishing an optimal method for salivary sEV isolation and understanding salivary sEV characteristics.…”

A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

“…This fact has popularized the use of SEC amongst its competitors for blood-based exosome-associated biomarker discovery research [52], [58]. SEC has been used successfully to isolate, purify and enrich exosomes from a variety of biological fluids including plasma [68]- [73], serum [74]- [76], bovine and human milk [77], [78], urine [79]- [82], saliva [83], [84], tears [83], cerebrospinal fluid [85], [86], synovial fluid [87], nasal lavage [89], seminal fluid [88] and stromal vascular fraction (SVF) from adipose tissue [63], [64] (Table 3). Recent findings reported at the ISEV Virtual Conference 2020 suggest SEC is superior to other techniques in isolation of pure exosomes from human body fluids (unpublished findings).…”

Biosensor and Lab-on-a-chip Biomarker-identifying Technologies for Oral and Periodontal Diseases

“…In search for functional and regulatory periodontitis biomarkers small extracellular vesicles (sEV) in saliva related to miRNAs were recently investigated. One study reported that three miRNAs, hsa-miR-140-5p, hsa-miR-146a-5p and hsa-miR-628-5p in sEV, may have potential as diagnostic biomarkers for periodontitis (Han et al, 2020).…”