Three-dimensional modelling of lymphangiogenesis in-vitro using bioorthogonal click-crosslinked gelatin hydrogels

Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing vessels, is crucial for maintaining tissue homeostasis and immune function. Despite recent advances in understanding the molecular mechanisms regulating lymphangiogenesis, most in vitro studies rely on traditional two-dimensional (2D) cell cultures, with limited replication of the complex microenvironment that governs lymphangiogenesis in vivo. Here, we present a three-dimensional (3D) lymphangiogenesis model using gelatin hydrogels modified with click-chemistry motifs (tetrazine and norbornene, GelTN), providing a biomimetic and mechanically tunable extracellular matrix (ECM) for lymphatic endothelial cells. By encapsulating human dermal lymphatic endothelial cells (HDLEC) spheroids in GelTN, we established a robust and reliable in vitro sprouting assay (<48 h duration) to investigate the effects of GelTN stiffness on lymphangiogenesis. HDLEC encapsulated in low GelTN concentrations exhibited enhanced sprouting in response to vascular endothelial growth factor (VEGF)-C stimulation, compared to HDLEC encapsulated in higher GelTN concentrations. We also provide evidence for the involvement of β3 integrin in lymphangiogenesis. The reduced sprout length upon β3 integrin inhibition further decreased with combined inhibition of α5β1, suggesting a synergistic interaction of the integrin subunits in controlling HDLEC-ECM mechanotransduction. GelTN hydrogels were also evaluated for their translational potential, demonstrating sustained release of VEGF-C in vitro and supporting cellular infiltration and neo-vessel formation following subcutaneous injection in an in vivo mouse model. Overall, these findings highlight the versatility of GelTN hydrogels as a platform for studying lymphangiogenesis and their potential use for therapeutic applications that require controlled growth factor delivery in tissue engineering and regenerative medicine.