Abstract Periodontal regeneration is a challenge, and tissue engineering based on periodontal ligament stem cells (PDLSCs) has been shown to be a promising alternative to this process. However, the need for scaffolds has limited the therapeutic use of PDLSCs. In this context, scaffold-free tissue engineering using the cell sheet (CS) technique has been developed as an alternative approach to improve tissue regeneration. Previously, we showed that Protease-activated receptor-1 (PAR1) can regulate PDLSCs. Herein, we evaluate whether PAR1 influences osteogenesis in CSs produced from PDLSCs, without the use of scaffolds. PDLSCs were isolated and immunophenotyped. Then, CSs were obtained by supplementing the culture medium with ascorbic acid (50 µg/mL), and PAR1 was activated through its agonist peptide (100 nM). Scaffold-free 3D CSs were successfully produced from PDLSCs, and they showed higher proliferation potential than isolated PDLSCs. Also, PAR1 activation decreased senescence and improved osteogenic differentiation of CSs by increasing mineralized nodule deposition and alkaline phosphatase concentration; PAR1 also modulated osteogenic markers at the gene and protein levels. We further demonstrated that this effect was regulated by Wnt, TGF-βI, MEK, p38 MAPK, and FGF/VEGF signaling pathways in PDLSCs (p < 0.05%). Overall, PAR1 activation increased osteogenic activity in CSs, emerging as a promising scaffold-free therapeutic approach for periodontal regeneration. challenge (PDLSCs process However context scaffoldfree scaffold free CS (CS Previously Proteaseactivated Protease receptor1 receptor 1 receptor- PAR (PAR1 Herein immunophenotyped Then 50 (5 µg/mL, µgmL µg/mL , µg mL µg/mL) 100 (10 nM. nM . nM) Scaffoldfree Scaffold D Also concentration levels Wnt TGFβI, TGFβI TGF βI, βI TGF-βI MEK p p3 MAPK FGFVEGF FGF VEGF 0.05%. 005 0.05% 0 05 0.05%) Overall (PAR 5 ( 10 (1 00 0.05 0.0 0.

Abstract Protease-activated receptor-2 (PAR2) is associated with the pathogenesis of many chronic diseases with inflammatory characteristics, including periodontitis. This study aimed to evaluate how the activation of PAR2 can affect the osteogenic activity of human periodontal ligament stem cells (PDLSCs) in vitro. PDLSCs collected from three subjects were treated in osteogenic medium for 2, 7, 14, and 21 days with trypsin (0.1 U/mL), PAR2 specific agonist peptide (SLIGRL-NH2) (100 nM), and PAR2 antagonist peptide (FSLLRY-NH2) (100 nM). Gene (RT-qPCR) expression and protein expression (ELISA) of osteogenic factors, bone metabolism, and inflammatory cytokines, cell proliferation, alkaline phosphatase (ALP) activity, alizarin red S staining, and supernatant concentration were assessed. Statistical analysis of the results with a significance level of 5% was performed. Activation of PAR2 led to decreases in cell proliferation and calcium deposition (p < 0.05), calcium concentration (p < 0.05), and ALP activity (p < 0.05). Additionally, PAR2 activation increased gene and protein expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) (p < 0.05) and significantly decreased the gene and protein expression of osteoprotegerin (p <0. 05). Considering the findings, the present study demonstrated PAR2 activation was able to decrease cell proliferation, decreased osteogenic activity of PDLSCs, and upregulated conditions for bone resorption. PAR2 may be considered a promising target in periodontal regenerative procedures. Proteaseactivated Protease activated receptor2 2 receptor- PAR (PAR2 characteristics periodontitis (PDLSCs vitro 7 14 0.1 01 0 1 (0. U/mL, UmL U/mL , U mL U/mL) SLIGRLNH2 SLIGRLNH SLIGRL NH2 NH (SLIGRL-NH2 100 (10 nM, nM nM) FSLLRYNH2 FSLLRYNH FSLLRY (FSLLRY-NH2 nM. . RTqPCR RT qPCR (RT-qPCR ELISA (ELISA factors metabolism cytokines (ALP staining assessed 5 performed p 0.05, 005 0.05 05 0.05. Additionally kappaΒ kappa Β RANKL (RANKL <0 05. 05) findings resorption procedures (PAR 0. (0 (SLIGRL-NH 10 (1 (FSLLRY-NH 00 0.0 (