Micropatterned hydrogels and cell alignment enhance the odontogenic potential of stem cells from apical papilla in-vitro

Abstract

Introduction: An understanding of the extracellular matrix characteristics which stimulate and guide stem cell differentiation in the dental pulp is fundamental for the development of enhanced dental regenerative therapies. Our objectives, in this study, were to determine whether stem cells from the apical papilla (SCAP) responded to substrate stiffness, whether hydrogels providing micropatterned topographical cues stimulate SCAP self-alignment, and whether the resulting alignment could influence their differentiation towards an odontogenic lineage in-vitro.

Methods: Experiments utilized gelatin methacryloyl (GelMA) hydrogels of increasing concentrations (5, 10 and 15%). We determined their compressive modulus via unconfined compression and analyzed cell spreading via F-actin/DAPI immunostaining. GelMA hydrogels were micropatterned using photolithography, in order to generate microgrooves and ridges of 60 and 120μm, onto which SCAP were seeded and analyzed for self-alignment via fluorescence microscopy. Lastly, we analyzed the odontogenic differentiation of SCAP using alkaline phosphatase protein expression (ANOVA/Tukey α=0.05).

Results: SCAP appeared to proliferate better on stiffer hydrogels. Both 60 and 120μm micropatterned hydrogels guided the self-alignment of SCAP with no significant difference between them. Similarly, both 60 and 120μm micropattern aligned cells promoted higher odontogenic differentiation than non-patterned controls.

Significance: In summary, both substrate mechanics and geometry have a statistically significant influence on SCAP response, and may assist in the odontogenic differentiation of dental stem cells. These results may point toward the fabrication of cell-guiding scaffolds for regenerative endodontics, and may provide cues regarding the development of the pulp-dentin interface during tooth formation.

Keywords: GelMA; Mechanotransduction; Micropatterning; Odontogenic differentiation; Regenerative dentistry.

Fig. 1 –

(a) Stress-strain curves and (b) elastic modulii of 5%, 10% and 15% (w/v) GelMA hydrogels show an increase in mechanical properties of the hydrogels with increased polymer concentration. Representative images of SCAPs cultured on (c) 5%, (d) 10% and (e) 15% GelMA hydrogels for 7 days and immunostained for F-actin (green) and DAPI (blue) shows increased substrate coverage due to apparent enhanced proliferation on stiffer hydrogels as indicated by increased number of cell nuclei. *p < 0.05, **p < 0.01 and ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 2 –

Schematic depiction of the fabrication of micropatterned hydrogels where (a) a volume of GelMA precursor is placed on a silanized substrate and then (b) covered with a photomask before being (c) exposed to UV light for photopolymerization. (d) Uncrosslinked hydrogel is then rinsed with PBS before (e) removal of the photomask (f) to retrieve the micropatterned hydrogel.

Fig. 3 –

The photolithography based micropatterning method was successfully used to form the (a) OHSU logo, (b) 60 μm and (c) 120 μm wide grooves in rhodamine stained GelMA hydrogels.

Fig. 4 –

SCAPs cultured on (a) unpatterned, (b) 60 μm- and (c) 120 μm-patterned GelMA hydrogels showed a (d) tendency to self-align along the substrate geometry within 3 days with (e) no significant difference in aligned populations between 60 and 120 μm patterned groups.

Fig. 5 –

SCAPs cultured on (a) unpatterned, versus (b) 60 μm- and (c) 120 μm- patterned hydrogels showed (d) increased ALP expression in patterned substrates within 7 days in culture, indicating an increased tendency to an odontogenic phenotype in these hydrogels.