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This in vitro study evaluated cell viability and metabolism, nitric oxide release and production of two chemokines and one cytokine by cultured human dental pulp fibroblasts (HDPF) in contact with two glass ionomer cements (Ketac Molar-KM and Vitrebond-VB), Single Bond (SB) and calcium hydroxide (Dycal-DY). Cultures of HDPF were established by means of an explant technique. The specimens were prepared under sterile conditions and in disks measuring 5 mm x 2 mm obtained from a prefabricated mold and placed on a permeable membrane to avoid direct contact with the cells. Cytotoxicity was assessed by Trypan Blue exclusion method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide release in cell supernatant was detected by the Griess Method whereas stromal derived factor-1 alpha (SDF-1α or CXCL12), chemokine (C-X-C motif) ligand 8 [Interleukin 8 (IL-8 or CXCL8)] and interleukin-6 (IL-6) were detected by ELISA. RT-qPCR was employed for gene expression analysis. Statistical analyses were performed by One-way ANOVA followed by Tukey's post hoc test for materials independent of the time, and Two-way ANOVA followed by Bonferroni correction test for the comparisons between materials and experimental time (p<0.05). Cytotoxic tests showed significant differences only for DY. Protein levels and mRNA expression were significantly increased for IL-8 for both periods of time. IL-6 production increased when fibroblasts were stimulated by KM. SDF-1α protein production and mRNA expression were not affected by any of the materials. There was a decrease in nitrate/nitrite levels only for KM. Although DY caused intense cell death and did not stimulate the production of the inflammatory mediators evaluated in this work, it is known that this event seems to be fundamental for the process of repair of the pulp tissue and formation of mineralized barrier. KM and VB increased production of proteins related to the inflammatory process, thus favoring tissue repair. Therefore, although these glass ionomer cements did not lead to large cell death, they should be used with caution.

This study evaluated in vitro cell viability and metabolism, nitric oxide release and production of chemokines by cultured human dental pulp fibroblasts (DPF) under contact with HEMA and Single Bond. Cultures of DPF were established by means of an explant technique. Once plated, cells were kept under contact with increasing concentrations of HEMA (10, 100 and 1000 nM) or Single Bond (SB) [10-fold serially diluted in culture medium (10-4, 10-3 and 10-2 v/v)] and also with polymerized SB components. Cytotoxicity was assessed by Trypan Blue exclusion method and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Nitric oxide release on cell supernatant was detected by Griess Method whereas chemokines (CXCL12 and CXCL8) were detected by ELISA. RT-qPCR was employed for chemokines gene expression analysis. Cytotoxic tests showed significant differences for SB 10-2. None of the tested materials significantly altered NO levels. Protein levels of CXCL12 were significantly decreased only by HEMA. On the other hand, while CXCL12 mRNA remained unaltered, gene expression of CXCL8 had significant decrease with all materials, except for polymerized SB. In conclusion, Single Bond and HEMA at various concentrations, decreased expression and production of molecules involved in inflammatory processes and, therefore, the use of adhesive systems such as pulp capping materials must be viewed with caution due to its large cytotoxic effect when in close contact with the pulp.

Introduction: Production of chemokines by tissue resident cells is one of the main mechanisms involved in the inflammatory infiltrate formation during inflammation. The specific ability of fibroblasts from different oral tissues such as gingiva, periodontal ligament, and dental pulp from permanent and deciduous teeth in producing the chemokines CCL3 and CXCL12 under stimulation by bacterial products commonly found in endodontic infections was investigated., Methods: Cultures of fibroblasts from gingiva and periodontal ligament as well as from dental pulp from permanent and deciduous teeth were established by using an explant technique and stimulated with increasing concentrations of Escherichia coli lipopolysaccharide (EcLPS) and Enterococcus faecalis lipoteichoic acid (EfLTA) for 1, 6, and 24 hours. Supernatants were tested for CCL3 and CXCL12 by enzyme-linked immunosorbent assay., Results: In general, CCL3 production was induced by EcLPS in the 4 fibroblast subtypes and by EfLTA in fibroblasts from gingiva and periodontal ligament. Constitutive CXCL12 synthesis decreased in all fibroblast subtypes especially under stimulation with EcLPS. Fibroblast from permanent deciduous teeth was the cell type presenting the most expressive reduction in CXCL12 release by both stimuli. On the basis of computational matching of CXCL12 mRNA with the microRNAs miR-141 and miR-200a, their expression was also investigated. Although detected in the fibroblasts, these molecules remained unaltered by bacterial by-product stimulation., Conclusions: EcLPS and EfLTA induced the production of CCL3 and unbalanced the synthesis of CXCL12 in a manner dependent on the specific tissue origin of fibroblasts., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS)., Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0-10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results., Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF., Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation.