Conservative endodontic treatments allow the maintenance of teeth in functional conditions.Mineral trioxide aggregate is composed of tricalcium silicate, dicalcium silicate, and tricalcium aluminate phases, and bismuth oxide is used as a radiopacifier agent. The first commercially available formulation of this material was ProRoot MTA (Dentsply). Bismuth has been shown to migrate locally to the skin of test animals after subcutaneous implantation. Regarding endodontics procedures, migration has been reported as occurring to the adjacent dentin and resulting in dentinal staining due to the reactiveness of this compound, especially when in contact with dentinal collagen and blood. Bismuth migration can be of serious concern due to its cytotoxicity, and very little is known regarding its leaching process and how can this leachate affects the host. To evaluate the cytotoxicity of bismuth-containing materials, neural, bone marrow, and human dental pulp cell cultures will be used. Disks of ProRoot MTA, tricalcium silicate, and hydroxyapatite replaced with 20 and 80% of bismuth oxide will be prepared and tested directly in contact with cell cultures for cell number and viability assessment. For the indirect test, the material' disks will be immersed in a mixed protein solution (Fetal Calf Serum) with the culture medium solution for 30 and 180 days, and the leachates added into cell culture medium before evaluating viable and non-viable cells to establish cell-growth curves up to 7 days. Additionally, the materials' influence on gene expression of human dental pulp cells, will be examined using the reverse transcription-polymerase chain reaction (RT-PCR) for the mineralization markers:alkaline phosphatase, osteocalcin, osteopontin, dentin sialophosphoprotein, dentin matrix protein1, bone sialoprotein, transforming growth factor-², and collagen-1±. The RT-PCR results will be expressed in percentage in comparison to the controls. Alizarin Red staining will also be used in order to evaluate the mineralization by human dental pulp cells. All tests will be performed at least twice in triplicates. The results will be submitted to appropriate statistical analysis with a significance level of 5%.
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