Influence of Biophysical and ‐Chemical Cues of Crosslinked Gelatin Networks on Endothelial Cell Adhesion, Morphology, Proliferation, and Angiogenic Signaling

Pre-vascularization through endothelial cell seeding within 3D-bioprinted constructs holds great promise to advance tissue engineering vascularization strategies. Herein, the effect of biophysical (bulk modulus (0.50-76.09 kPa), local elasticity (3.65-53.68 kPa), roughness (421.07-858.30 nm)) and biochemical (0, 25 ng/mL VEGF-A or 3 mg/mL adenosine) cues in widely used bioinks (methacryloyl vs. thiol-norbornene modified gelatins incorporating 6.21-25.81 mM crosslinked moieties) was systematically investigated on the adhesion, morphology, proliferation and angiogenic signaling of seeded human umbilical vein endothelial cells. Chain-growth networks exhibited an enhanced roughness together with the need for a higher concentration of converted moieties to obtain a non-statistically significant local substrate elasticity compared to the step-growth substrates (5 w/v% GelNBSH DS 74/67 vs. 5 w/v% GelMA DS 67: 6.02 ± 2.94 vs. 3.85 ± 1.24 kPa with 6.21 vs. 7.33 mM crosslinked moieties). Despite bulk compressive moduli with insignificant difference (5 w/v% GelNBSH DS 74/67 vs. 5 w/v% GelMA DS 99: 13.57 ± 0.56 vs. 14.18 ± 1.59 kPa), the 5 w/v% GelNBSH DS 74/67 networks outperformed the 5 w/v% GelMA DS 99 samples, especially through enhanced early and more mature angiogenic signaling (Ang-2, HB-EGF, VEGF-C, FGF-1, IL-8, Endothelin-1) after 1 day of culture, while chain-growth networks required VEGF-A supplementation to attain similar signaling.