Biocompatibility Assessment of Resin-based Cements on Vascularized Dentin/Pulp Tissue-Engineered Analogues

Objectives: Two-dimensional (2D) in vitro models are extensively utilized for cytotoxicity assessment of dental materials, but with certain limitations regarding in vitro-in vivo extrapolation. Three-dimensional (3D) models seem more appropriate, recapitulating the structure of human tissues. This study developed a tissue-engineered dentin/pulp analogue for biocompatibility assessment of resin-based cements.

Methods: Stem Cells from Apical Papilla (SCAP) and Human Umbilical Vein Endothelial Cells (HUVEC) were embedded in Collagen-I/Fibrin hydrogels at 1:3 ratio within 24-well plates. Hanging culture inserts were placed over the hydrogels, housing a layer of odontoblast-like cells and a human treated dentin barrier. Eluates of representative resin-based cements [a dual-cure resin cement (Breeze^TM, Pentron =Cement-1; C1) and a self-adhesive resin cement (SpeedCEMplus^TM, Ivoclar-Vivadent= Cement-2; C2) were applied into the construct after pre-stimulation with the endotoxin LPS (1μg/ml). Biocompatibility assessment was performed by MTT assay, live/dead staining and real-time PCR for angiogenesis-related markers. Dynamic mechanical analysis was used to evaluate shear modulus of the hydrogels at two concentrations (3.5 and 5 mg/ml).

Results: The dentin/pulp analogue supported cell viability and capillary-like network formation. Both cements caused no significant toxicity. Exposure to C1 induced significant stimulation (p< 0.01) of cell metabolic activity (158.0 ±3.3% compared to control) at 72h, while pre-stimulation with LPS attenuated this effect. Exposure to C2 (± LPS) caused minor reduction of metabolic activity (approx.15-20%) at 24h, that recovered at 72h for the LPS+ group (111.1 ±22.4%). Both cements caused upregulation of VEGF, ANGP-1, while the opposite trend was observed for the respective receptors VEGFR-2 and Tie-1. Both hydrogel concentrations showed similar shear moduli to the natural pulp until day-7, while the 5 mg/ml-hydrogel substantially increased stiffness at day-14.

Conclusions: Both materials under investigation showed acceptable biocompatibility and triggered angiogenic responses within the tissue-engineered dentin/pulp organotypic analogue, indicative of initiation of pulp repair processes as response to the released xenobiotics.