Digital light processing of photo-crosslinkable gelatin to create biomimetic 3D constructs serving small intestinal tissue regeneration

Abstract

Regeneration of small intestinal mucosal tissue could offer a promising strategy for Crohn's disease patients suffering from chronic inflammatory damage. Here, we aimed to develop hydrogels that mirror the villi and crypts of the small intestine and exhibit a physiological stiffness of G' ~ 1.52 kPa. For this purpose, we developed gelatin-methacryloyl-aminoethyl-methacrylate (gel-MA-AEMA)-, and gelatin-methacryloyl-norbornene (gel-MA-NB)-based biomaterial inks to fabricate 3D hydrogels (“villi only” versus “crypts and villi”) with digital light processing (DLP) and co-cultured Caco-2/HT29-MTX cells. Gel-MA-AEMA was selected for its higher amount of methacrylates which was hypothesized to provide superior photo-crosslinking kinetics and hence superior DLP fabrication potential while gel-MA-NB was evaluated for its selective functionalization potential with thiolated bioactive compounds following DLP processing, resulting from its incorporated NB moieties which remain unreacted during the DLP process. Both gel-MA-AEMA-, and gel-MA-NB-based hydrogels exhibited a physiologically relevant stiffness, but only the gel-MA-AEMA-based biomaterial ink could be successfully utilized for printing hydrogels encompassing villi and crypts. Paracellular permeability of small sized marker molecules in combination with transepithelial electrical resistance measurements showed the formation of a functional barrier over time on all hydrogel constructs. Transmission electron microscopy and enterocyte differentiation marker genes' expression levels revealed the superior differentiation of Caco-2 on the 3D constructs compared to 2D hydrogel sheets. In summary, while both hydrogels enhanced functional barrier formation and enterocyte differentiation, gel-MA-AEMA proved more conducive to DLP compared to gel-MA-NB. Furthermore, our study underscored the benefits of cultivating intestinal cells on soft 3D constructs, enhancing cell barrier properties and differentiation, thus providing added value over traditional 2D supports.