Fabrication of tapered fluidic microchannels conducive to angiogenic sprouting within gelatin methacryloyl hydrogels

Abstract

Introduction: The transplantation of stem cells/tissue constructs into root canal space is a promising strategy for regenerating lost pulp tissue. However, the root canal system, which is cone shaped with a taper from the larger coronal end to the smaller apical end, limits the vascular supply and, therefore, the regenerative capacity. The current study aimed to fabricate built-in microchannels with different tapers to explore various approaches to endothelialize these microchannels.

Methods: The fluidic microchannels with varying tapers (parallel, 0.04, and 0.06) were fabricated within gelatin methacryloyl (GelMA) hydrogel (with or without stem cell from the apical papilla [SCAP] encapsulation) of different concentrations (5%, 7.5%, and 10% [w/v]). Green fluorescent protein–expressing human umbilical vein endothelial cells (HUVECs-GFP) were seeded alone or with SCAPs in coculture into these microchannels. Angiogenic sprouting was assessed by fluorescence and a confocal microscope and ImageJ software (National Institutes of Health, Bethesda, MD). Immunostaining was conducted to illustrate monolayer formation. Data were statistically analyzed by 1-way/2-way analysis of variance.

Results: HUVEC-only inoculation formed an endothelial monolayer inside the microchannel without angiogenic sprouting. HUVECs-GFP/SCAPs cocultured at a 1:1 ratio produced the longest sprouting compared with the other 3 ratios. The average length of the sprouting in the 0.04 taper microchannel was significantly longer compared with that in the parallel and 0.06 taper microchannels. Significant differences in HUVEC-GFP sprouting were observed in 5% GelMA hydrogel. Encapsulation of SCAPs within hydrogel further stimulated the sprouting of HUVECs.

Conclusions: The coculture of SCAPs and HUVECs-GFP at a ratio of 1:1 in 0.04 taper fluidic microchannels fabricated with 5% (w/v) GelMA hydrogel with SCAPs encapsulated was found to be the optimal condition to enhance angiogenesis inside tapered microchannels.