An Optimized Injectable Hydrogel Scaffold Supports Human Dental Pulp Stem Cell Viability and Spreading

Abstract

Introduction. HyStem-C™ is a commercially available injectable hydrogel composed of polyethylene glycol diacrylate (PEGDA), hyaluronan (HA), and gelatin (Gn). These components can be mechanically tuned to enhance cell viability and spreading. Methods. The concentration of PEGDA with an added disulfide bond (PEGSSDA) was varied from 0.5 to 8.0% (w/v) to determine the optimal concentration for injectable clinical application. We evaluated the cell viability of human dental pulp stem cells (hDPSCs) embedded in 2% (w/v) PEGSSDA-HA-Gn hydrogels. Volume ratios of HA : Gn from 100 : 0 to 25 : 75 were varied to encourage hDPSC spreading. Fibronectin (Fn) was added to our model to determine the effect of extracellular matrix protein concentration on hDPSC behavior. Results. Our preliminary data suggests that the hydrogel gelation time decreased as the PEGSSDA cross-linker concentration increased. The PEGSSDA-HA-Gn was biocompatible with hDPSCs, and increased ratios of HA : Gn enhanced cell viability for 14 days. Additionally, cell proliferation with added fibronectin increased significantly over time at concentrations of 1.0 and 10.0 μg/mL in PEGDA-HA-Gn hydrogels, while cell spreading significantly increased at Fn concentrations of 0.1 μg/mL. Conclusions. This study demonstrates that PEG-based injectable hydrogels maintain hDPSC viability and facilitate cell spreading, mainly in the presence of extracellular matrix (ECM) proteins.

Figure 1

Gelation time as a function of PEGSSDA concentration fit to a nonlinear logarithmic regression model. Diamonds represent experimental data. The curved line is theoretical data.

Figure 2

Immunofluorescence results for live/dead staining of human dental pulp stem cells embedded in 2.0% (w/v) PEGSSDA and varying (v/v) ratios of HA : GN. Green = live cells. Red = dead cells. Magnification = 20x. Scale bar = 100 μm.

Figure 3

(a-b) The average number of live and dead hDPSC per field of view as a function of (v/v) ratio of HA : GN. (c) The percentage of live hDPSC in the 1 : 1 and 1 : 3 (v/v) ratios of HA : GN. ^∗ p < 0.05.

Figure 4

Proliferation of hDPSC in PEGDA-HA modified hydrogels with different concentrations of fibronectin. n = 3 samples. WST-1 was used to determine the relative cell densities at days 1 and 4. ^∗ p < 0.05.

Figure 5

(a–j) Immunofluorescence staining results of human dental pulp stem cells seeded on the surface of polyethylene glycol diacrylate-based hydrogels. Data represents a sample of a random field of view from 4 random fields of view per hydrogel. Green = live cells. Magnification = 20x. Scale bar = 50 μm.

Figure 6

Effect of fibronectin concentration on hDPSC rounding. Data represents an average of 40 cells/field of view at 20x magnification. n = 3 samples. ^∗ p < 0.05.

Figure 7

Effect of fibronectin concentration on hDPSC spreading. Data represents an average of 40 cells/field of view at 20x magnification. n = 3 samples. ^∗ p < 0.05.