Endocannabinoids Modulate Production of Osteoclastogenic Factors by Stem Cells of the Apical Papilla In Vitro.

INTRODUCTION: Many mediators are produced during pulp inflammation and necrosis, including endocannabinoids (ECbs), which might affect the function of stem cells of the apical papilla (SCAP), cells of paramount importance for root formation, and regenerative endodontic treatment. The aim of this study was to evaluate the production of osteoclastogenesis-related mediators by SCAP modulated by ECbs and lipopolysaccharide (LPS) in vitro. METHODS: SCAP were cultured and treated with ECb anandamide (AEA), 2-arachidonoylglycerol, or N-arachidonoylaminophenol. All groups were incubated in the presence of a vehicle or LPS and the antagonist of transient receptor potential cation channel subfamily V member 1, capsazepine. After 24 hours, the culture medium supernatants were collected for further quantification of tumor necrosis factor alpha, CCL2, macrophage colony-stimulating factor, osteoprotegerin, and receptor activator of nuclear factor kappa B ligand. RESULTS: Small amounts of tumor necrosis factor alpha and receptor activator of nuclear factor kappa B ligand were detected in SCAP supernatants, and none of the experimental conditions altered their production. A down-regulation in constitutive CCL2 production was observed in the AEA group compared with that in the LPS group. The production of macrophage colony-stimulating factor was significantly increased in all groups treated with AEA compared with the control and LPS-treated groups. Osteoprotegerin was significantly increased by AEA alone and by 2-arachidonoylglycerol and N-arachidonoylaminophenol in the presence of LPS and capsazepine. CONCLUSIONS: AEA modulates some of the osteoclastogenic factors produced by SCAP in a bone resorption protective fashion.

Endocannabinoids Regulate Stem Cells of the Apical Papilla via a Cannabinoid Receptor and TRPV1-Independent Mechanism.

INTRODUCTION: Endogenous cannabinoids (endocannabinoids [eCBs]) have been shown to have a multitude of functions including neurotransmission and immune modulatory effects. This study aimed to evaluate if stem cells of the apical papilla (SCAP) express the receptors and enzymes of the endocannabinoid system (ECS) and whether eCBs regulate their proliferation and mineralization potential. METHODS: Gene expression of the main components of the ECS and transient receptor potential vanilloid 1 (TRPV1) was evaluated in SCAP cultures. SCAP were treated with 2 concentrations of eCBs and/or capsazepine, a TRPV1 antagonist. SCAP viability was evaluated after 1, 4, and 7 days. Osteogenic differentiation was assessed after 14 days, and the gene expression of mineralization markers was assessed after 7 days. RESULTS: The enzymes of ECS and TRPV1 but not the cannabinoid receptors (cannabinoid receptors 1 and 2) were expressed in SCAP. Anandamide, 2-arachidonoylglycerol, and N-arachidonoylphenolamine (AM-404) reduced SCAP viability in all experimental periods at the highest concentration compared with the group with no treatment. Anandamide and AM-404 did not inhibit SCAP differentiation potential, but 2-arachidonoylglycerol at the highest concentration did. SCAP treated with AM-404 presented a down-regulation in gene expression of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSPP) compared with the proliferation medium group but not with control group. CONCLUSIONS: SCAP expressed the genes of the main components of ECS and TRPV1, and eCBs can affect SCAP viability, mineralization, and gene expression.

Cytotoxic Effect of Niobium Phosphate Glass-based Gutta-Percha Points on Periodontal Ligament Fibroblasts In Vitro.

INTRODUCTION: The outcome of root canal obturation might be affected by the chemical components of the chosen filling materials. Niobium phosphate glass-based gutta-percha (GNB) was proposed as a biomaterial-based obturation point. This study aimed to investigate the cytotoxic and cell modulation effects of GNB points on human periodontal ligament fibroblasts (PDLFs) in vitro. METHODS: Human PDLFs were cultured for the assays. Extracts of regular gutta-percha (GP) points and GNB were obtained, serially diluted (1:5, 1:10, and 1:25), and used to stimulate PDLFs. A cell viability assay was performed using alamarBlue reagent (Molecular Probes, Waltham, MA), and reverse transcription quantitative polymerase chain reaction was used to assess the gene expression for collagen type I and cementum protein 1. One-way analysis of variance followed by the Tukey post hoc test was performed (P < .05). RESULTS: Regular GP reduced cell viability only in pure extracts, whereas GNB exhibited cytotoxicity to PDLFs in pure extracts as well as 1/5 and 1/10 dilutions. The gene expression of collagen type I was down-regulated only in the GNB group (P < .05). The expression of cementum protein 1 remained unaltered by both tested materials. CONCLUSIONS: The addition of niobium phosphate glass to GP points increased cytotoxicity, affecting PDLF viability and partially disturbing physiological cell function.

Prostaglandin E2 Affects Interleukin 6 and Monocyte Chemoattractant Protein 1/CCL2 Production by Cultured Stem Cells of Apical Papilla.

INTRODUCTION: Root canal treatment of immature necrotic teeth is a major challenge in current endodontics. The effect of inflammatory mediators, such as prostaglandin, on the modulation of stem cells of the apical papilla (SCAP) is not completely understood. The aim of this study was to investigate the role of prostaglandin E2 (PGE2) on SCAP activation by Escherichia coli lipopolysaccharide (LPS) in vitro. METHODS: SCAP cultures were established and characterized. Increasing concentrations of lipopolysaccharide (0.1-10 µg/mL) were used to investigate cyclooxygenase-2 (COX-2/PTGS2) and PGE2 receptors (EP1-4) gene expression. Then, SCAP were treated with a COX-2 inhibitor (indomethacin) before treatment with different concentrations of LPS. The levels of the chemokine CCL2/monocyte chemoattractant protein 1 and interleukin (IL)-6 were detected in cell supernatants (24 hours) by enzyme-linked immunosorbent assay. Data analysis was performed using analysis of variance followed by the Tukey post test. RESULTS: The expression of COX-2 was up-regulated in the group treated with LPS at 1µg/mL compared with that in the control group. EP1-4 were detected in all experimental conditions at similar levels. SCAP treated with indomethacin presented a down-regulation in the production of LPS-induced CCL2 and the secretion of IL-6. CONCLUSIONS: SCAP showed increased COX-2 (PTGS2) gene expression induced by LPS and a PGE2-dependent production of IL-6 and CCL2.