Growth Factor Gradients in Migration-Permissive Hydrogels for Salivary Gland Assembly

Objectives: Engineered 3D morphogen gradients in hydrogels can direct salivary gland progenitor cell differentiation into ductal and acinar cell phenotypes for gland replacement. Morphogen gradients direct tissue organization during embryogenesis/tissue repair. Heparin-binding growth factor (HBGF) gradients form by release from matrix heparan sulfate proteoglycans such as perlecan at sites on heparan sulfate chains decorating N-terminal domain I (PlnD1). HBGFs including the FGF family elicit both cell differentiation and motility. Gradients of HBGFs, PlnD1, and matrix components can be introduced into migration-permissive hydrogels made using our custom 3D-printed gradient maker device.
Methods: The hydrogel system consisted of thiol-modified hyaluronate (HA-SH), a matrix metalloproteinase (MMP)-cleavable crosslinker (acrylate-PEG-KGGGPQGIWGQGK-PEG-acrylate), and an adhesive sequence (acrylate-PEG-GRGDS), providing a support for hS/PC expansion, self-organization, and motility. hS/PCs were encapsulated in hydrogels at three crosslinking densities. The extent of cell spreading and motility was assessed. hS/PCs, acrylated PlnD1, and acrylated laminin peptides were incorporated into hydrogels of optimal crosslinking density where PlnD1 was introduced as a gradient using the 3D-printed gradient maker device at flow rate 0.9mL/min. After gradient hydrogels were crosslinked, FGF-7/10 were ionically complexed with PlnD1 to promote directional cell movement. Morphogenesis was assessed using confocal microscopy.
Results: Human hS/PCs remained viable and assembled into acinar-like structures at each crosslinking density, defined as molar ratios of thiols on HA-SH and acrylates (AC) on the crosslinker at 6:1, 11:1, and 16:1 SH:AC, with the size and shape of acini determined by extent of crosslinking. Cell motility was greatest at a SH:AC ratio 11:1. PlnD1/HBGF gradients permitted greater hS/PC cell motility over time than PlnD1 gradients lacking growth factors.
Conclusions: HA-based hydrogel with enzymatically-degradable crosslinkers for selective degradation and cell motility allow for gland assembly. Growth factor gradients in PlnD1-modified migration-permissive hydrogels support salivary gland cell motility, and can serve as instructive matrices for salivary gland tissue engineering.