located between cementum and alveolar bone has important role in regeneration of periodontal tissues damaged by mechanical or biochemical factors (Ozer et al., 2013). In order to maintain PDL homeostasis in both normal and pathologic conditions, resident PDL multipotent progenitors, or stem cell-like cells (PDLSCs) act depending on local tissue demands, since PDLSCs can differentiate into major cellular constituents of PDL, such are osteoblast, fibroblast, and cementoblasts, thus maintaining PDL structure and function (Bassir et al., 2016;Han, Menicanin, Gronthos, & Bartold, 2014;Liu et al., 2015;Mao, Robey, & Prockop, 2012;Zhu & Liang, 2015).Oral cavity presents host niche for pathogenic bacteria which may provoke host tissue inflammation, damage dental tissues homeostasis, and finally lead to gingivitis or periodontitis (Chatzivasileiou, Kriebel, Steinhoff, Kreikemeyer, & Lang, 2015), as well as various systemic diseases (Nagpal, Yamashiro, & Izumi, 2015;Seymour, Ford, Cullinan, Leishman, & Yamazaki, 2007 bovine serum (FBS; Capricorn-Scientific, Germany), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Thermo Fisher Scientific), and incubated at 37°C in humidified atmosphere containing 5% CO 2 (standard conditions). The medium was replaced twice a week, and after reaching the 90% of confluence, the cells were detached using 0.25% solution of trypsin/EDTA (Gibco). All experiments were performed using PDLSCs between passage two and six.Human peripheral blood mononuclear cells (MNCs) were isolated from fresh heparinized blood collected from 15 healthy voluntary unrelated donors by density gradient using lymphocyte separation medium Lymphocyte Separation Medium 1.077 g/ml (CapricornScientific). Purified MNCs were cultivated in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin in standard conditions.Human endothelial cell line (EA.hy926) was purchased from The American Type Culture Collection (ATCC, CRL-2922), MD, and cultured in GM in standard conditions. antibodies (Thermo Scientific) and CD14 (Sigma-Aldrich). The percentage of nonspecific binding was determined by using the appropriate FITC-and PE-conjugated isotype control antibodies (R&D Systems). Flow cytometry was performed using flow cytometer Cytomics FC 500 (Beckman Coulter).
| Phenotype analysis
| Multilineage differentiationFor evaluation of multilineage differentiation capacity PDLSCs were cultivated in GM or corresponding differentiation medium (DM) with orwithout LPS (1000 ng/ml, purchased from Sigma-Aldrich) in standard conditions. Osteogenic DM contained DMEM supplemented with 5% FBS, 100 U/ml penicillin/streptomycin, 50 µM ascorbic acid-2-phosphate (Sigma-Aldrich), and 10 mM β-glycerophosphate (SigmaAldrich). For osteogenic differentiation detection, alkaline phosphatase (ALP) staining has been followed after 7 days of cultivation, while calcium in DMEM, 100 U/ml penicilin/streptomycin, 100 µg/ml isobutylmethylxanthine (IBMX; Sigma-Aldrich),...
