Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation.

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-ßI, 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.

Protease-activated receptor type 2 activation downregulates osteogenesis in periodontal ligament stem cells.

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.

Protease-activated receptor type 1 (PAR1) increases CEMP1 gene expression through MAPK/ERK pathway.

PAR1 is a G-coupled protein receptor that regulates several cellular metabolism processes, including differentiation and proliferation of osteogenic and cementogenic related cells and our group previously demonstrated the regenerative potential of PAR1 in human periodontal ligament stem cells (hPDLSCs). In this study, we hypothesized that PAR1 regulates the cementogenic differentiation of hPDLSCs. Our goal was to identify the intracellular signaling pathway underlying PAR1 activation in hPDSLC differentiation. hPDLSCs were isolated using the explant technique. Cells were cultured in an osteogenic medium (OST) (α-MEM, 15% fetal bovine serum, L-glutamine, penicillin, streptomycin, amphotericin B, dexamethasone, and beta-glycerophosphate). The hPDLSCs were treated with a specific activator of PAR1 (PAR1 agonist) and blockers of the MAPK/ERK and PI3K pathways for 2 and 7 days. The gene expression of CEMP1 was assessed by RT-qPCR. The activation of PAR1 by its agonist peptide led to an increase in CEMP1 gene expression when compared with OST control. MAPK/ERK blockage abrogated the upregulation of CEMP1 gene expression induced by PAR1 agonist (p < 0.05). PI3K blockage did not affect the gene expression of CEMP1 at any experimental time (p > 0.05). We concluded that CEMP1 gene expression increased by PAR1 activation is MAPK/ERK-dependent and PI3K independent, suggesting that PAR1 may regulate cementogenetic differentiation of hPDLSCs.

Therapeutic potential of periodontal ligament stem cells.

Inflammatory periodontal disease known as periodontitis is one of the most common conditions that affect human teeth and often leads to tooth loss. Due to the complexity of the periodontium, which is composed of several tissues, its regeneration and subsequent return to a homeostatic state is challenging with the therapies currently available. Cellular therapy is increasingly becoming an alternative in regenerative medicine/dentistry, especially therapies using mesenchymal stem cells, as they can be isolated from a myriad of tissues. Periodontal ligament stem cells (PDLSCs) are probably the most adequate to be used as a cell source with the aim of regenerating the periodontium. Biological insights have also highlighted PDLSCs as promising immunomodulator agents. In this review, we explore the state of knowledge regarding the properties of PDLSCs, as well as their therapeutic potential, describing current and future clinical applications based on tissue engineering techniques.

Protease-Activated Receptor Type 1 Activation Enhances Osteogenic Activity in Human Periodontal Ligament Stem Cells.

Protease-activated receptor 1 (PAR1) has been associated to tissue repair and bone healing. The aim of the present study was to evaluate the effect of PAR1 activation on the osteogenic activity of human periodontal ligament stem cells (PDLSCs). PDLSCs were cultured in the presence of PAR1-selective agonist peptide (100 nM), thrombin (0.1 U/mL), or PAR1 antagonist peptide (100 nM). Calcium deposits, calcium concentration (supernatant), alkaline phosphatase activity (ALP), cell proliferation, and gene (qPCR) and protein expression (ELISA assay) of osteogenic factors were assessed at 2, 7, and 14 days. PAR1 activation led to increased calcium deposits (p < 0.05), calcium concentration (p < 0.05), ALP activity (p < 0.05), and cell proliferation (p < 0.05). Further, PAR1 activation may increase gene and protein expression of Runx2 (p < 0.05) and OPG (p < 0.05). In conclusion, PAR1 activation increases osteogenic activity of PDLSCs, providing a possible new strategy for periodontal regenerative therapies.

Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation

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.

Protease-activated receptor type 2 activation downregulates osteogenesis in periodontal ligament stem cells

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.

Protease-activated receptor type 1 (PAR1) increases CEMP1 gene expression through MAPK/ERK pathway

Abstract: PAR1 is a G-coupled protein receptor that regulates several cellular metabolism processes, including differentiation and proliferation of osteogenic and cementogenic related cells and our group previously demonstrated the regenerative potential of PAR1 in human periodontal ligament stem cells (hPDLSCs). In this study, we hypothesized that PAR1 regulates the cementogenic differentiation of hPDLSCs. Our goal was to identify the intracellular signaling pathway underlying PAR1 activation in hPDSLC differentiation. hPDLSCs were isolated using the explant technique. Cells were cultured in an osteogenic medium (OST) (α-MEM, 15% fetal bovine serum, L-glutamine, penicillin, streptomycin, amphotericin B, dexamethasone, and beta-glycerophosphate). The hPDLSCs were treated with a specific activator of PAR1 (PAR1 agonist) and blockers of the MAPK/ERK and PI3K pathways for 2 and 7 days. The gene expression of CEMP1 was assessed by RT-qPCR. The activation of PAR1 by its agonist peptide led to an increase in CEMP1 gene expression when compared with OST control. MAPK/ERK blockage abrogated the upregulation of CEMP1 gene expression induced by PAR1 agonist (p < 0.05). PI3K blockage did not affect the gene expression of CEMP1 at any experimental time (p > 0.05). We concluded that CEMP1 gene expression increased by PAR1 activation is MAPK/ERK-dependent and PI3K independent, suggesting that PAR1 may regulate cementogenetic differentiation of hPDLSCs.

Periodontitis as a risk factor for head and neck cancer / Periodontitis como factor de riesgo de cáncer de cabeza y cuello

Background: Periodontitis may be associated with the development of head and neck cancer (HNC). A literaturereview was conducted to understand the possible association between them.Material and Methods: Articles published in the PubMed database from January 1999 and May 2020 were re-trieved. Limitations of the studies and biological mechanisms were discussed.Results: A total of 4,232 articles were found. Of these, 13 were analyzed according to inclusion criteria. Most pa-pers found some association between periodontitis and HNC, although differences in periodontal evaluation, sam-ple size, study design and tumor sites were observed. Porphyromonas gingivalis appears to increase the chanceof both diseases, and it may be one of their main potential risk factors. Genetic predisposition is increased byexposure to environmental factors which can directly induce epigenetic changes that contribute to these diseases.Conclusions: Understanding the mechanisms related to periodontitis and HNC has increased, however, well-de-signed clinical studies are needed for better conclusions. Furthermore, the advent of multiple "omic" technologieswill help comprehend their possible association.(AU)