Inflammatory bowel disease (IBD) is an umbrella term for a group of disorders hallmarked by chronic and relapsing inflammation of the gastrointestinal tract (GIT). The incidence of IBD is increasing globally, thereby turning it into a major global public health problem. One subtype of IBD, Crohn’s disease (CD), is characterised by a patchy, transmural inflammation, usually occurring at the terminal ileum and/or colon, although the whole GIT can be involved. Despite improving therapies, surgical intervention remains common in CD management due to loss of response to therapy or complications such as strictures and fistulae. Approximately half of the CD patients will require a surgical resection of a bowel segment within their lifetime, and many of them will relapse. With the recent developments in intestinal organoid culture and tissue engineering, there is an increasing interest in using biomaterials and patient derived intestinal epithelial cells for the regeneration of damaged intestinal tissue. Exploiting this approach, mucosal healing can be enhanced and the complete removal of bowel parts can be avoided.
To achieve this goal, the Polymer Chemistry and Biomaterials Group led by prof. dr. Sandra Van Vlierberghe and the IBD Research Unit led by prof. dr. Debby Laukens of Ghent University started a scientific collaboration to establish hydrogels with digital light processing (DLP) that mirror the villi and crypts of the small intestine, exhibit physiological relevant stiffness (G’ ~1.52 kPa) and support proliferation and differentiation of intestinal epithelial cells. Two biomaterial inks were developed based on gelatin-methacryloyl-aminoethyl-methacrylate (gel-MA-AEMA) and gelatin-methacryloyl-norbornene (gel-MA-NB) and were compared in DLP processability. Two different computer aided designs (CAD) were utilized, one with only villi-like structures and one encompassing crypts and villi-like structures (Figure 1).
Workflow towards digital light processing (DLP) of gel-MA-AEMA and gel-MA-NB constructs with cryo-scanning electron microscopy (SEM) top and side view images. White arrows point at the crypts. The scale bars represent 1 mm in white and 100 µm in blue.
Both hydrogels exhibited an outstanding crosslinking capacity, while maintaining physiologically relevant stiffness. Due to the higher amount of photo-crosslinkable side groups present in gel-MA-AEMA, the CAD/CAM (computer aided design/computer aided manufacturing) mimicry of the biomaterial ink outperformed the gel-MA-NB-based one and resulted in successful processing into scaffolds exhibiting only villi-like projections, versus crypts and villi-like projections. Gel-MA-NB was only processable into villi-like projections exclusively. All three constructs contained physiologically accurate dimensions, but more physiologically relevant villi heights could be obtained with gel-MA-AEMA.
The hydrogel constructs showed excellent biocompatibility upon combining them with a Caco-2/HT29-MTX co-culture. All three hydrogel constructs supported the formation of a confluent monolayer (Figure 2A), expressing tight junction proteins, as evidenced by immunofluorescence (Figure 2B). A functional barrier was formed, characterized by a decrease in TEER compared to cells cultured in 2D, which resembled more physiological barrier properties (Figure 2C). TEM and comparative qPCR measurements after 21 days of culture showed that differentiation of the cells towards enterocytes was improved by the 3D architecture of the constructs compared to 2D controls and not by the hydrogel material itself (Figure 2D/E). Although both hydrogels promoted functional barrier formation and enterocyte differentiation, taking into account DLP processability, gel-MA-AEMA appeared to be more suited for DLP than gel-MA-NB as biomaterial to establish a 3D biomimetic model for small intestinal tissue regeneration.
(A) Fluorescent labeling of F-actin (red), nuclei (blue) of Caco-2/HT29-MTX cells cultured for 21 days on Gel-MA-AEMA and Gel-MA-NB hydrogel constructs. Z-stacks were taken with a confocal microscope to obtain a 3D side view. Villi are indicated with white and crypts with green arrows. (B) Maximum intensity projections localizing tight junction protein 1 (TJP1, green) on the 3D hydrogel constructs with DAPI (nuclei, blue) and phalloidin rhodamine (F-actin, red) as counterstains. White arrows indicate villi-like projections while green ones indicate crypts. Scale bars, 50 µm. (C) Transepithelial electrical resistance (TEER) measurements. (D) Cell height in µm, measured with transmission electron microscopy. Scale bars, 10 µm. (E) RT-qPCR analysis after 7 and 21 days of culture of Caco-2/HT29-MTX cells cultured in 2D and 3D hydrogel constructs. ALPI: alkaline phosphatase.
Article:
L. Maes*, A. Szabó*, J. Van Haevermaete, I. Geurs, K. Dewettinck, R. E. Vandenbroucke, S. Van Vlierberghe, D. Laukens, Digital light processing of photo-crosslinkable gelatin to create biomimetic 3D constructs serving small intestinal tissue regeneration, Biomaterials Advances 171 (2025) 214232. https://doi.org/10.1016/j.bioadv.2025.214232
Contact:
Polymer Chemistry and Biomaterials Group, Ghent University
Prof. Dr. Sandra Van Vlierberghe
Sandra.VanVlierberghe@UGent.be
IBD Research Unit, Ghent University
Prof. Dr. Debby Laukens
Debby.Laukens@UGent.be
Laure Maes
Laure.Maes@UGent.be
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20 Mar 2025