Model characterization to study dental pulp stem cell differentiation

Purpose: The purpose of this study was to develop and characterize a method to investigate the differentiation of Dental Pulp Stem Cells (DPSCs) into odontoblasts. Methods: Dentin disks (1 mm)from the dentin-enamel junction were prepared from recently extracted human third molars. Biodegradable highly porous PLLA scaffolds were prepared in the pulp chamber of this tooth slices using salt as porogen. 5 x 104 cells DPSC were seeded in the scaffolds and cultured in 24-well plates. After 7-28 days in culture, RNA was extracted with Tryzol and RT-PCR was used to evaluate expression of DSPP, DMP1 and MEPE, considered markers of odontoblast differentiation. Cell proliferation was evaluated using the WST1 assay, and cell morphology was evaluated under SEM and Confocal Laser Microscopy. Alternatively, 6 x 105 DPSC were seeded in scaffolds/tooth slices and implanted subcutaneously in immunodeficient mice. After 28 days, scaffolds were retrieved and prepared for histological evaluation and immunostaining with DSP and Factor VIII. Also, from some specimens the RNA was extracted soon after scaffolds were retrieved and differentiation was evaluated by RT-PCR. Results: In vitro, DPSCs seeded in the scaffolds/tooth slices expressed all odontoblast differentiation markers starting on day 14, which was not detected in the scaffolds without tooth slices. Cell proliferation in the scaffold/tooth slices decreased after 14 and 21 days, compared to scaffolds alone (p<0.05). Confocal microscopy showed cells growing in the interface tooth slice/scaffold, and SEM showed cells most of the scaffold after 28 days. Histological analysis showed that DPSCs seeded in tooth slices showed a tissue structure that resembles that of a normal dental pulp. Expression of odontoblast markers was observed for cells in the scaffold/tooth slice after 28 days in vivo. Conclusion: The method presented allowed for studies of processes involved on the differentiation of DPSC within the tooth structure.

Funding: CAPES 0532/06-1