TiO₂ nanoparticles synergize with substrate mechanics to improve dental pulp stem cells proliferation and differentiation

Multiple studies exist on the influence of TiO₂ nanoparticle uptake on cell behavior. Yet little is known about the lingering influence of nanoparticles accumulation within the external environment which is particularly important to stem cell differentiation. Herein, dental pulp stem cells were cultured on hard and soft polybutadiene substrates, where 0.1 mg/mL rutile TiO₂ nanoparticles were introduced once, 24 h after plating. In the absence of TiO₂, the doubling time on soft substrate is significantly longer, while addition of TiO₂ decreases it to the same level as on the hard substrate. FACS analysis indicates particle uptake initially at 25% is reduced to 2.5% after 14 days. In the absence of TiO₂, no biomineralization on the soft and snowflake-like hydroxyapatite deposits on the hard substrate are shown at week 4. With the addition of TiO₂, SEM/EDAX reveals copious mineral deposition templated on large banded collagen fibers on both substrates. The mineral-to-matrix ratios analyzed by Raman spectroscopy are unremarkable in the absence of TiO₂. However, with addition of TiO₂, the ratios are consistent with native bone on the hard and dentin on the soft substrates. This is further confirmed by RT-PCR, which showed upregulation of markers consistent with osteogenesis and odontogenesis, respectively.