De Luca, Ilenia (2016) Fluoride release kinetic from dental restorative materials affects Dental Pulp Stem Cells behavior. [Tesi di dottorato]

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Fluoride-releasing restorative dental materials can be beneficial to remineralize dentin and help prevent secondary caries. However, commercialized fluoride-restorative materials (F-RMs) exhibit a non-constant rate of fluoride release depending mainly on the material composition and fluoride content. Here we investigate whether different fluoride release kinetics from new dental resins could influence the behavior of human dental pulp stem cells (hDPSCs). The innovation consists in using as dental composites fillers modified hydrotalcite intercalated with fluoride ions (LDH-F). The fillers were prepared via ion exchange procedure and the LDH-F inorganic particles (0.7, 5, 10, 20 wt.%) were mixed in a commercial light-activated restorative material (RK), provided by Kerr s.r.l. (Italy) to obtain the final resins (RK-F). The physical-chemical characteristics, the release profile and the biological effect on proliferation of hDPSCs of RK-F 0.7, 5, 10 were analyzed. Since RK-F10 and a commercial fluoride-glass filler (RK-FG10) contain the same concentration of fluoride, RK-F10 was chosen to investigate the regenerative capability induced by fluoride-controlled release. To evaluate the difference between RK-F10 and RK-FG10 in inducing cellular migration and differentiation, it was isolated the human dental pulp stem cell subpopulation (STRO-1 positive cells) known for its ability to differentiate towards an odontoblast-like phenotype. The release of fluoride ions was determined in physiological medium and artificial saliva medium using an ion chromatograph. The cell migration assay was performed in presence of transforming growth factor β1 (TGF-β1) and stromal cell-derived factor-1 (SDF-1) using a modified Boyden Chamber method on STRO-1+ cells cultured for 7 days on RK, RK-F10 and RK-FG10 materials. The expression patterns of dentin sialoprotein (dspp), dentin matrix protein 1 (dmp1), osteocalcin (ocn), and matrix extracellular phosphoglycoprotein (mepe) were assessed by quantitative RT-PCR. The incorporation of LDH-F in commercial-dental resin significantly improved the mechanical properties of the pristine resin, in particular at 37°C. Long-term exposure of STRO-1+ cells to a continuous release of low amount of fluoride by RK-F10 increases their migratory response to TGF-β1 and SDF-1, both important promoters of pulp stem cell recruitment. Moreover, the expression patterns of dspp, dmp1, ocn, and mepe indicate a complete odontoblast-like cell differentiation only when STRO-1+ cells were cultured on RK-F10. On the contrary, RK-FG10, characterized by an initial fluoride-release burst and reduced lifetime of the delivery, did not elicit any significant effect both on STRO-1+ cell migration and differentiation. Taken together our results demonstrated that STRO-1+ cell migration and differentiation into odontoblast-like cells was enhanced by the slower fluoride-releasing material (RK-F10) compared to RK-FG10, which showed a more rapid fluoride release, thus making LDH-F a promising filler for evaluation in clinical trials of minimally invasive dentistry.

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