3D-Bioprinting of Stromal Vascular Fraction for Gastrointestinal Regeneration

Abstract

Intestinal disorders such as inflammatory bowel diseases (IBDs), Crohn's disease, malabsorption syndromes, and gastrointestinal fistulae (GIFs) are often characterized by chronic inflammation, epithelial barrier disruption, impaired stromal remodeling, and defective angiogenesis. These multifactorial alterations hinder tissue repair and contribute to poor clinical outcomes, with limited efficacy from current therapeutic options. Despite recent advances in surgical and endoscopic techniques, current treatment options remain limited and are frequently accompanied by high morbidity and costs. In this context, regenerative medicine offers a promising avenue to support tissue repair and improve patient care Regenerative medicine offers a promising avenue to restore intestinal homeostasis using advanced biomaterials and cell-based therapies. In this study, we developed a 3D-bioprinted model based on patient-derived stromal vascular fraction (SVF) embedded in a GelMA hydrogel, designed to promote intestinal tissue regeneration. To identify the most suitable hydrogel for bioprinting, we initially evaluated the mechanical properties and biocompatibility of four distinct matrices using bone marrow-derived mesenchymal stromal cells (BM-MSCs). Among the tested formulations, GelMA demonstrated optimal support for cell viability, low oxidative stress, and structural stability in physiologically relevant conditions. Based on these results, GelMA was selected for subsequent bioprinting of freshly isolated SVF. The resulting bioprinted constructs enhanced key regenerative processes across multiple compartments. The SVF-laden constructs significantly enhanced intestinal epithelial cell viability and tight junction formation, as shown by increased trans-epithelial electrical resistance (TEER). Co-culture with fibroblasts accelerated wound closure, while endothelial cells exhibited increased tube formation in the presence of SVF. Together with VEGF secretion, indicating strong paracrine and angiogenic effects. By supporting epithelial, stromal, and vascular regeneration, this approach provides a versatile and translational platform for treating a broad spectrum of intestinal pathologies.

Keywords: 3D-bioprinting; GelMA; intestinal regeneration; regenerative medicine; stromal vascular fraction.

Figure 1

Mechanical characterization of hydrogels. (A) Force–indentation curves of the four different hydrogels. (B) Frequency distribution of the Young’s Modulus. The histograms represent measured values, while the continuous line represents the Lorentzian fit. (C) Peak of the Lorentzian fit for the four different hydrogels. (D) Elastic modulus over time of GelMA.

Figure 2

Biocompatibility of hydrogels on BM-MSCs. (A) Cell viability over time of BM-MSCs bioprinted with GelMA, GelMA A, Alginate, and Cellink-RGD. Results are expressed as % of day 0 for each bioprinted model. (B) Production of ROS over time for BM-MSCs bioprinted with the different hydrogels. Results are normalized by the number of viable cells and reported with respect to day 0 for each hydrogel. (C) Cytotoxicity of the four different hydrogels on BM-MSCs over time. Results are normalized by day 0 for each model. *** p < 0.001; ** p < 0.01; * p < 0.05 one-way ANOVA and Tukey post-hoc test.

Figure 3

Growth and stability of bioprinted SVF. (A) Viability of SVF bioprinted in GelMA over time. Results are expressed as % of day 0. (B) Production of reactive oxygen species over time from SVF. Results are normalized to day 0. (C) OD variation over 14 days. Results are reported with respect to the initial OD. (D) Area variation over 14 days. Results are reported with respect to the initial area of each bioprinted model ± SD. *** p < 0.001; ** p < 0.01 one-way ANOVA and Tukey post-hoc test.

Figure 4

Intestinal epithelial regeneration stimulated by SVF. (A) Schematic representation of the experimental setup carried out for the viability of epithelial cells and for intestinal barrier formation. (B) Cell viability of intestinal cells over time with or without the presence of SVF. Results are expressed as % of day 0. (C) Production of reactive oxygen species over time from intestinal cells with or without the presence of SVF. Results are normalized by the number of viable cells and reported with respect to day 0. (D) Increase in the electrical resistance of the intestinal barrier over time. Results are reported with respect to day 0 of control (untreated) cells. ** p < 0.01; * p < 0.05 one-way ANOVA and Tukey post-hoc test.

Figure 5

Stimulation of wound healing through bioprinted SVF. (A) Images of wound closure at 0 and 24 h. Scalebar 250 μm. (B) Quantitative analysis of the migration of fibroblasts toward the wound region over time. Results are normalized to 1 h for both conditions. ** p < 0.01 one-way ANOVA and Tukey post-hoc test.

Figure 6

Angiogenesis augmented by bioprinted SVF. (A) Bright-field and fluorescence images of the tube formation assay for endothelial cells in the presence of SVF. Scalebar 300 μm. (B) Quantitative analysis of tube formation in endothelial cells with and without SVF. Examples of nodes are represented with yellow dots surrounded by red circles, while examples of branches are represented by red arrows. (C) Expression of VEGF over time with or without SVF. * p < 0.05 and ** p < 0.01 one-way ANOVA and Tukey post-hoc test.