Top down proteomic analysis of gingival crevicular fluid in deciduous, exfoliating and permanent teeth in children
Graphical abstract
Introduction
Human Gingival Crevicular Fluid (GCF) is a plasma-derived exudate present in the gingival crevice of the periodontal pocket, between teeth and the surrounding gingival tissue [1]. The constituents of the fluid arise from the host as well as from microorganisms resident in the subgingival and supragingival plaque. Contribution from the host includes molecules deriving from blood and from cells and tissues of the periodontium. In fact, the junctional epithelium is infiltrated by neutrophils, mononuclear cells and lymphocytes [2]. GCF composition varies between healthy and diseased periodontium conditions [3]. Important host-derived constituents in GCF are markers of inflammation including enzymes, cytokines, and interleukins. Further, products of tissue breakdown can also be detected [4].
The major attraction of GCF for its possible diagnostic use is the site-specific nature of the sampling. Indeed, analysis of GCF collected from distinct sites/teeth could offer the potential of diagnostic information for individual sites and teeth. In previous studies, our group has been involved in the analysis of the GCF fluid in adults with low-resolution top-down proteomic platform [5,6]; while in this study we have deepened the analysis of proteins and peptides in children's GCF with a high resolution top-down pipeline. GCF from deciduous and permanent teeth has been already analyzed in children [1,7] and biomarkers of bone and tissue metabolism associated with orthodontic tooth movement, neutrophil-related proteins and immunoglobulins were identified. However, we have a limited understanding of the physiological changes occurring in GCF biochemical composition when deciduous teeth are replaced by permanent teeth, and little information is available on GCF of exfoliating teeth [8].
Previous studies reported the changing of gingival tissues surrounding permanent and deciduous teeth during the replacement [9,10]. Tooth eruption is a complex process, and little is known about the mechanism of its control [11]. The main hypothesis is that the periodontal ligament promotes eruption through the shrinking and cross-linking of collagen fibers and the contraction of the fibroblasts [12].
Areas of tension and compression are generated in the soft tissues surrounding unerupted teeth by the distribution of bite forces through the jaws. These patterns of tension and compression are further proposed to result in patterns of bone resorption and deposition that lift the tooth into the mouth [13]. This theory is based on Wolff's Law, which is the long-established idea that bone changes shape in accordance with the forces applied [14]. In deciduous dentition, all these processes are active especially in 10 yrs. old children where deciduous dentition is near to be replaced by permanent dentition. The aim of this study was to analyze GCF protein composition in deciduous, exfoliating and permanent teeth in children and check if variations occur during the exfoliating process by a top-down strategy based on a high-resolution nano-HPLC-ESI-MS platform.
High-resolution mass spectrometry equipped with micro/nano-ESI source and LC devices is an important tool for the in deep and accurate molecular characterization of biological fluids available in few amounts such us GCF. Furthermore, the top-down proteomics approach that explores the intact proteins and the peptides naturally occurring in the sample, minimizing, as much as possible, any alteration during pretreatment [[15], [16], [17]] is a powerful analytical platform for investigating both the qualitative and quantitative variations of GCF proteins/peptides composition linked to physiological or pathological conditions and for disclosing proteoforms and PTMs possible alterations [18,19].
Top-down approach, as demonstrated in papers previously published by our group [16,17], provides univocal information and instantaneous images on the protein content and modification of the fluid under examination at the moment in which it is withdrawn, and allows a direct qualitative-quantitative comparison in samples originated from different sites as described in this study.
Section snippets
Materials and methods
Chemicals- All chemicals and reagents were of LC-MS grade and were purchased from Sigma Aldrich (St. Louis, MI) and Merck (Damstadt, Germany).
Results
The proteins and peptides identified in GCF are reported in Table 1, and their elution peak is shown in the enlarged view of the chromatographic profile of Fig. 1. The level of peptides and proteins under study, established in GCF based on the eXtracted Ion Current (XIC) peak area, is reported in Table 2 and showed in Fig. 2.
The nano-HPLC high resolution ESI-MS-MS allowed the top-down characterization of several proteins and peptides. Among them members of the S100A family (S100A8, S100A9
Discussion
GCF is a serum exudate that originates from the periodontal sulcus or pocket and is regarded as a promising biological fluid for the detection of periodontal composition in physiological and pathological conditions [4]. In this study, we focalized our attention on the proteomic characterization of GCF deriving from deciduous, exfoliating and permanent teeth in children by high-resolution mass spectrometry following a top-down approach.
In a previous proteomic study, our group highlighted the
Conclusion
GCF, an exudate, harnessed from the sulcus or periodontal pocket, is a promising medium for detection of periodontal disease activity because its composition reflects the status of surrounding tissues in a specific and localized site. In this study, we propose for the first time a comparison between three different sampling sites in children that are changing their dentition from deciduous to permanent teeth. It is important to consider that to reduce the complexity of the sample we worked on
Author contributions
Conceptualization: FI, AO, MC.
Data curation: FV, FI.
Formal analysis: FI, MC, TC.
Methodology: RP, PG, LDT, MC.
Writing original draft:FI, AO, BM, TC.
Supervision: CD, IM, AU, MC.
Funding acquisition: MC, MC. Project administration: FI. Resources MC, IM. Software: IF. Validation : IF, AO. Visualization: BM, TC. Writing review &editing: IF, AO.All authors approved of the final version of the manuscript.
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgment
The authors acknowledge the financial support of the Catholic University of Rome, and the Nando Peretti Foundation
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These authors contributed equally to this work.