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Svrha: Željelo se pronaći optimalne uvjete i metode za identifikaciju maksimalnog broja proteina u uzorcima tkiva gingive pacijentice s agresivnim parodontitisom. Pritom smo se koristili tehnologijom Label-free kvantifikacije, tekućinske kromatografije i spektrometrije masa (LC –MS/MS), te usporedili proteom zdravih i bolesnih uzoraka tkiva gingive. Materijali i metode: Četiri uzorka gingivnog tkiva (dva zdrava i dva bolesna) uzeta su od pacijentice, inače nepušačice, koja boluje od teškog oblika generaliziranog agresivnog parodontitisa. Proteini tkivnog lizata razdvojeni su 1D gel-elektroforezom nakon koje je slijedila digestija u gelu te mjerenje proteina nanoljestvicom HPLC-sistema preko nanoelektro spreja, ionizacijskog izvora i spektrometra mase. Dobiveni podaci obrađeni su u programu MaxQuant. Rezultati: Label-free kvantifikacija i LC-MS/MS analiza, zajedno s pripremom uzoraka, 1D gel-elektroforezom i digestijom u gelu pokazali su se kao učinkovita metoda za analizu proteoma gingivnog tkiva. Ukupan broj identificiranih i kvantificiranih proteina iznosio je 2429. Proteini koji su pokazali pojačanu ekspresiju u tkivu zahvaćenom agresivnim parodontitisom bili su: imunoglobulinski gama-1 lanac regije C, imunoglobulinski kapa lanac regije C i imunoglobulinski gama-3 lanac regije C (Ig gamma-1 chain C region, Ig kappa chain C region i Ig gamma-3 chain C region), a desmoplakin, aneksini, proteini S100-A8/A9 i keratini pokazali su sniženu ekspresiju. Zaključak: Ovo eksperimentalno istraživanje omogućuje novi uvid u proteomski profil zdravog i bolesnog gingivnog tkiva, što bi moglo pridonijeti napretku u dijagnostici i prognozi te boljem razumijevanju patogeneze agresivnog parodontitisa. Tehnologija LCMS/ MS pokazala se učinkovitom kad je riječ o proteomskoj analizi gingivnog tkiva., Aim: The aim of this study was to find an optimal method to identify maximal number of proteins from human gingival tissue samples in a patient with aggressive periodontitis using label-free quantitative liquid chromatography mass spectrometry technology (LC-MS/MS), and to compare proteome of healthy and diseased gingival tissue samples. Materials and methods: Four gingival tissue samples (2 healthy, 2 diseased) were obtained from a nonsmoking female patient suffering from severe generalized aggressive periodontitis. Proteins from the tissue lysate were separated by 1D gel electrophoresis, in gel digested, measured by nano-scale HPLC system coupled to a mass spectrometer through a nano-electrospray ionization source. Raw data were processed by MaxQuant software. Results: Label-free quantitative LC-MS/MS analysis together with sample preparation, 1D gel electrophoresis and in-gel digestion showed to be an effective method for the proteome analysis of the gingival periodontal tissues. A total of 2429 proteins were identified and quantified. Among proteins upregulated in disease were: Ig gamma-1 chain C region, Ig kappa chain C region, Ig gamma-3 chain C region, and among downregulated proteins in the disease were: Desmoplakin, annexins, S100-A8/A9 proteins, and keratins. Conclusions: This pilot study is providing a novel insight into the protein profiles of gingival healthy and diseased tissues, which may contribute to the improvements in diagnosis, prognosis and better understanding of aggressive periodontitis pathogenesis. LC-MS/MS technology proved to be an effective method for the proteome analysis of the gingival tissues.

Formation of root cementum is a crucial moment in the development of the periodontium. Cells that produce the cementum are named cementoblasts and they posses some unique characteristics, which differentiates them from osteoblasts. Bone morphogenetic proteins (BMPs) are crucial regulators of both bone and tooth formation. In animal studies BMPs have shown to induce periodontal regeneration, however the molecular mechanism as how BMP-7 induces cementogenesis is largely unknown. We have investigated how BMP-7 regulates gene expression of BMP-4, Dentin matrix protein- 1 (DMP-1), Insulin-like growth factor-I (IGF-I) and –II (IGF-II) in cementoblasts. BMP-7 induced proliferation, and mineralized nodule formation of cementoblasts. Our results show that gene expression was influenced by the BMP-7 concentration used, with 75 ng/mL generally down regulating gene expression at 6 hours and then up-regulating after 24 hours. The 300 ng/mL concentration had an opposite effect while the 150 ng/mL concentration generally up-regulated gene expression after 6 hours and then after 24 hours maintained this up-regulation or had no effect compared to control, depending on the examined gene. The results show that BMP-7 down-regulated BMP-4 expression in cementoblasts but still up-regulated DMP-1 gene expression suggesting that BMP-7 can, in a paracrine manner, functionally substitute for BMP-4. Furthermore, it seems that BMP-7 exerts its effect more through the IGF-II than the IGF-I pathway as shown by an up-regulation of IGF-II and down-regulation of IGF-I. These results suggest that a combination of BMP-7/IGF-II could have a potential therapeutical significance in inducing cementogenesis and periodontal regeneration.