Biological Responses of Calcium-Silicate-Based Cements on a 3D Pulp Analogue

Objectives: Aim of this study was to evaluate biological responses of two bioactive calcium silicate-based cements (MTA and Biodentine™) on a three-dimensional, tissue-engineered organotypic pulp analogue.
Methods: For the establishment of the pulp analogue, Human Umbilical Vein Endothelial Cells (HUVEC) and Stem Cells from Human Exfoliated Deciduous teeth (SHED) were first co-cultured in 2D conventional culture flasks at a ratio of HUVEC:SHED=3:1. After 24h, the co-cultures were trypsinized and the pellet incorporated into a collagen I/fibrin hydrogel (total protein content 3.5mg/ml), at a concentration of (2.5-6.5)×10⁵cells/hydrogel, depending on the experimental procedure. Biodentine™ and MTA cylindrical specimens (6.5×2mm) were prepared and placed in direct contact with the cell-seeded hydrogels 48h later, whereas hydrogels without specimens served as control. Live/dead staining, MTT assay and Scanning Electron Microscopy (SEM) were used to evaluate cell viability/proliferation and morphology. Real-time PCR to evaluate expression of angiogenic markers (PECAM-1, VEGFa, VEGFR1, VEGFR2, ANGPT-1, Tie-2).
Results: Live/dead staining at day 3 after treatment showed that viable- dominated over non-viable cells (not exceeding 10% for both materials). Cells were evenly distributed inside the hydrogels exposed to the bioactive cements without any differences compared to the control (untreated) cultures. MTT analysis at day 1 and 3 showed that viability/proliferation inside the cell-seeded hydrogels was not influenced by the presence of the bioactive cements. Real-time PCR at day 3 showed upregulation of VEGF and ANGPT-1 in both treated and untreated hydrogels, whereas the respective receptors VEGFR2, VEGFR1 and Tie-2 were downregulated. These alterations were more pronounced in the silica cement-treated groups.
Conclusions: Both calcium silicate-based materials are biocompatible and exert a positive angiogenic effect on the organotypic pulp analogue, validating further application in vital pulp therapy of deciduous teeth.