A hydrogel scaffold that maintains viability and supports differentiation of dental pulp stem cells

Abstract

Objectives: The clinical translation of stem cell-based Regenerative Endodontics demands further development of suitable injectable scaffolds. Puramatrix™ is a defined, self-assembling peptide hydrogel which instantaneously polymerizes under normal physiological conditions. Here, we assessed the compatibility of Puramatrix™ with dental pulp stem cell (DPSC) growth and differentiation.

Methods: DPSC cells were grown in 0.05-0.25% Puramatrix™. Cell viability was measured colorimetrically using the WST-1 assay. Cell morphology was observed in 3D modeling using confocal microscopy. In addition, we used the human tooth slice model with Puramatrix™ to verify DPSC differentiation into odontoblast-like cells, as measured by expression of DSPP and DMP-1.

Results: DPSC survived and proliferated in Puramatrix™ for at least three weeks in culture. Confocal microscopy revealed that cells seeded in Puramatrix™ presented morphological features of healthy cells, and some cells exhibited cytoplasmic elongations. Notably, after 21 days in tooth slices containing Puramatrix™, DPSC cells expressed DMP-1 and DSPP, putative markers of odontoblastic differentiation.

Significance: Collectively, these data suggest that self-assembling peptide hydrogels might be useful injectable scaffolds for stem cell-based Regenerative Endodontics.

Figure 1

Proliferation of DPSC in Puramatrix™ over time and cell density. DPSC cells were plated in 0.2% Puramatrix™ at a cell density of 1-8×10⁵ cells/ml and allowed to grow for up to 72 hours. WST-1 assay was used to determine relative cell densities at indicated time points.

Figure 2

Proliferation of DPSC according to the concentration of Puramatrix™. DPSC cells were plated in 0.05-0.25% Puramatrix™. WST-1 was used to determine relative cell densities at indicated time points.

Figure 3

Confocal microscopy images of DPSC cells cultured in Puramatrix™. In the top row, DPSC were observed after 24 hours in culture (red: actin; blue: nuclear staining). In the bottom row, DPSC were evaluated after 72 hours in culture (green: actin; blue: nuclear staining).

Figure 4

Expression of putative markers of odontoblastic differentiation by DPSC cells. DPSC were cultured in Puramatrix™ only, or Puramatrix™ casted within a human tooth slice. RT-PCR was used to evaluated the expression of DMP-1 and DSPP after 21 days in culture. Primary human odontoblasts scrapped from extracted teeth were used as positive controls for DMP-1 and DSPP.