Analysis of Exosomes and molecular biomarkers in saliva and the gingival crevicular fluid in patients with periodontal disease.
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Abstract Aim: The aim of this pilot study is to evaluate if biomarker signature(s) of exosomal miRNAs, IL-1β and IL-6 isolated from GCF and saliva exist and can be used as feasible biomarkers of periodontal treatment. Methods: In this pilot study, 12 randomly selected patients were included. The patients were divided in three groups (4 patients per group) and would receive either a repeated non-surgical periodontal treatment (NSPT) or two different types of periodontal surgery techniques-Resective (RPFO) or a Conservative flap (Simplified Papillae Preservation Flap (SPPF)) At baseline and prior to any hygiene instructions given, or commencement of their periodontal treatment, saliva and gingival crevicular fluid (GCF) were collected. The samples were immediately stored at -80°C and processed when required for analysis. GCF samples were processed with a centrifugation protocol for exosomes and microRNA (miRNA) extraction and analysis. Saliva samples were also processed for exosome isolation and miRNA extraction using the same centrifugation protocol. Also, the saliva supernatants of these samples were analysed using enzyme-linked immunosorbent assay method (ELISA) for detection of IL-1β and IL-6 cytokines. Data was analysed and linked to the clinical parameters; dental plaque, periodontal probing depth (PPD), gingival recession (REC), bleeding on probing (BoP) and clinical attachment loss (CAL) obtained at baseline for these patients. Results: Nanoparticle tracking analysis showed that extracellular vesicles isolated from GCF showed multiple peaks, indicating the presence of vesicles of different sizes and low concentration. Processing of saliva produced vesicles with a homogeneous size (close to that reported for exosomes) and a relatively good concentration. However, quality control analysis revealed that neither GCF nor saliva produced enough amounts of good quality RNA for miRNA expression analysis. Analysis of saliva samples allowed detection of IL-1β, (but not IL-6) across the three different groups of patients’ samples. Furthermore, the level of IL-1β was not significantly different in the three patients’ groups and did not correlate with any clinical parameters that define periodontitis. Conclusions: At the protein level, at least by analysis of IL-1β expression (a cytokine closely associated with periodontitis), the randomly selected samples from patients at baseline time point, suggests a homogenous set of samples that would allow detection of molecular changes related to treatment intervention. Our results suggest sampling, storing and preservation protocols used in this study, might be inadequate for the investigation and evaluation of RNA related biomarker signatures from saliva and GCF samples.
Authors
Anastasiadou, ICollections
- Theses [4125]