The comparison of the proteomic profile of periodontal pocket and of corresponding gingival crevicular fluid to study periodontal disease biomarkers: feasibility study. biomarkers: feasibility study

Aim: Periodontitis is a set of inflammatory disorders characterized by periodontal attachment loss by periodontal pocket development, leading to tooth loss if remain untreated. The etiology and progress of periodontal disease is complex and remains mostly unknown. So, periodontal disease therapy has considerable limitations. The easy, reliable and correct early detection and control of the disease, markedly reduces biological and social costs. However, the diagnosis of periodontitis is established exclusively by clinical criteria based on probing to assess periodontal pockets, which are the pathognomonic expression of periodontal disease. The -omic sciences acquired substantial significance of late years and, in particular, proteomic seemed to be the more promising in this initial stage. Most proteomic analysis regarding periodontal diseases have been performed on saliva, crevicular fluid samples, peripheral blood or periodontal plaque samples which are more easily to harvest than the tissue of the periodontal pocket. However, they failed to provide reliable results for clinical applications. On the contrary, very few studies were directly performed on the periodontal pocket. So, the aim of this study was to compare the proteomic profile of interproximal pocket tissues with that of GCF, and to analyze if they show a significant similarity in the proteomic profile. Methods: in this preliminary study, we enrolled 3 healthy subjects affected by severe periodontitis needing of periodontal surgery. Immediately before the surgery, GCF samples were taken by means of filter paper strips positioned in the gingival sulcus correspondent to periodontal pockets. Then, periodontal pocket tissue, harvested during surgery, was adequately stored for proteomic analyses. All samples were immediately frozen at –80°C and maintained until further analysis. Tissue samples were mechanically disrupted and incubated in lysis buffer, while GCF was obtained incubating the collecting paper in phosphate buffered. In both cases, after centrifugation, the supernatant was precipitated in cold acetone overnight and protein content were pelleted by centrifugation and then dissolved in a rehydration buffer. Mono-dimensional gel electrophoresis was used to separate protein content. After staining gel images were acquired and compared. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) analysis was performed to allow protein spot identification. Results: 1-DE gels from periodontal pocket tissue and the correspondent GCF was analyzed by software Quantity One. Almost the same qualitative protein expression profile in pocket tissue and GCF was found from each patient. However, no statistical significant correlation between the quantitative proteomic profile of pocket tissue and GCF was found. Only one band (that of K immunoglobulin) resulted statistically significant between GCF and pocket tissue proteome in all patients. Conclusions To date, this is the first study comparing the proteome of periodontal pocket tissue and corresponding GCF. The periodontal pocket and the GCF are similar as proteomic networks, but the protein network of the periodontal pocket does not influence significantly the GCF protein network and the other way around. So, with the limitations of this study, the preliminary results seem to indicate that the GCF does not seem suitable to study on the pathogenesis of periodontal disease explaining the reason for the failure of studies based only on GCF to control the periodontal disease in real-time.