A Bilayered Poly (Lactic-Co-Glycolic Acid) Scaffold Provides Differential Cues for the Differentiation of Dental Pulp Stem Cells

Regenerative endodontics (RE) is a clinical procedure that aims to regenerate the dentin-pulp complex (DPC). Current clinical outcomes of RE are unpredictable, and the regenerated tissue lacks the spatial organization observed in normal DPC. The purpose of this study was to develop and characterize in vitro a bilayered scaffold with distinct porosities on each side that supports directional cell penetration and differential odontoblastic differentiation of cultured human dental pulp stem cells (DPSCs). Materials and Methods: Bilayered scaffolds were manufactured from poly (lactic-co-glycolic acid) (PLGA) using diffusion-induced phase separation. The layers were generated separately from 12% and 20% (w/v) PLGA, and combined by lamination. Scaffold morphology was assessed through scanning electron microscopy. Human DPSCs were cultured on either side of the scaffold. Cell proliferation, viability, and penetration into the scaffolds were analyzed biochemically and by confocal imaging. Odontoblastic differentiation of the DPCSs and mineralization were analyzed by quantitative real-time polymerase chain reaction, quantification of Alizarin red staining, energy-dispersive X-ray spectroscopy, and Fourier-transform infrared spectroscopy. Results: The bilayered scaffold (thicknesses 277 ± 15 μm) contained continuous channels with gradual taper. Channel diameters ranged from 45 to 10 μm on the open side (20% side) and 10-5 μm on the closed side (12% side). While proliferating equally on either scaffold surfaces, DPSCs penetrated into the open side and through the entire thickness of that layer in 14 days. By contrast, the closed side limited cell penetration into the scaffold but significantly promoted dentinogenic differentiation in the absence of any dentinogenic induction medium. Conclusions: Bilayered scaffolds provide spatial control of differential DPSC penetration and dentinogenic differentiation, thus providing a potential scaffold for DPC regeneration. In this article we used an FDA-approved biodegradable biomaterial, poly (lactic-co-glycolic acid) (PLGA 75:25) to generate a bilayered scaffold with the capacity to induce differential, layer-specific dentinogenic differentiation of dental pulp stem cells (DPSCs) in vitro. We surmise that such a scaffold can be used in conjunction with current regenerative endodontic procedures to help regenerating a physiologic dentin-pulp complex in vivo. We hypothesize that our scaffold in conjunction with DPSCs will advance current regenerative endodontics by restoring dentin and initiating the innervation and revascularization of the pulp.