"Angiogenesis, the process by which new blood vessels sprout from existing surrounding ones, is essential for the survival of implanted tissue engineered constructs. In fact, the lack of proper re-vascularization and core necrosis is the main pitfall of many developed biomaterials (1). In this study, we describe a highly tunable hydrogel system for the growth of capillaries based on alginate.
Alginate was chemically functionalized with norbornene. Norbornene forms a covalent bond with a thiol group in the presence of a photoinitiator upon exposure to UV light. The degree of functionalization obtained was 4,63%, as measured with NMR. Mechanical properties were analyzed by rheology and hydrogels ranged 10 – 1000 Pa, depending on final polymer concentration and concentration of cross-linker. Photoinitiator LAP was used at 2 mM. To introduce biodegradability to the system, we used matrix metalloproteinases (MMP)-cleavable sequences flanked by two thiol groups as cross-linkers. The speed of degradation can be tuned by modifying the sequence specificity to a range of MMPs. Non-degradable hydrogels were fabricated by using 2000 dalton PEG di-thiol. Degradable hydrogels dissolved when exposed to collagenase, while non-degradable gels remained unaltered.
In a one-pot-synthesis manner, alginate-norbornene, thiolated-RGD, dithiol MMP-cleavable peptide, cells, VEGF165 and photoinitiator were mixed. Solution was casted onto siliconized glass-slides and irradiated with UV for 30 seconds. Human Umbilical Vein Endothelial Cells (HUVECs) and Mesenchymal Stromal Cells (MSCs) were encapsulated in hydrogels in a 1:10 ratio (HUVEC:MSC). Cells were viable after cross-linking and were able to fuse and sprout, recapitulating the process of angiogenesis. Cell elongation was apparent already after 24 hours of culture. Hydrogels were stained against CD31 and imaged with confocal microscopy. Then, networks were analyzed and quantified using Amira and WinFiber3D (2). Vessel parameters were superior in hydrogels with lower degree of cross-linking, and thus softer, and with higher concentration of RGD. Initial studies have shown no major differences depending on the cell-binding peptide used: RGD (derived from fibronectin), YIGSR (a sequence found in laminin) and GFOGER (a collagen-derived peptide).
In conclusion, we have developed a platform that allows the study of the influence of stiffness, matrix degradability and ECM binding motifs on angiogenesis. Further work will focus on the use of these hydrogels as bioinks for 3D printing vascularized tissues and organoids.
1. Lesman et al. Mechanical regulation of vascular network formation in engineered matrices. Adv Drug Deliv Rev. 2016.
2. Bonda et al. 3D Quantification of Vascular-Like Structures in z Stack Confocal Images. STAR Protocols. 2020."