Bio-orthogonally double cross-linked alginate-gelatin hydrogels with tunable viscoelasticity for cardiac tissue engineering

Growing evidence has shown that cells respond to the viscoelastic properties of the extracellular matrix (ECM), particularly its stress-relaxation, which influences their spreading, proliferation, and remodeling. Since cardiac tissue viscoelasticity plays a key role in modulating cellular mechanosensing, the development of biomimetic viscoelastic hydrogels is highly needed in cardiac tissue engineering (CTE). This work presents bio-orthogonal double cross-linked alginate-gelatin hydrogels with tunable viscoelasticity, designed to replicate the dynamic mechanical properties of cardiac ECM. Alginate and gelatin were functionalized with azide groups and cross-linked by a 4-arm-dibenzocyclooctyne (DBCO) crosslinker using strain-promoted azide-alkyne cycloaddition (SPAAC) with 0.5:1 (AG_Click(R0.5)) and 1:1 (AG_Click(R1)) DBCO:azide molar ratios. Calcium ions were also introduced to obtain double cross-linked hydrogels (AG_DC(R0.5) and AG_DC(R1)). Rheology showed that hydrogels exhibited tunable stiffness and stress relaxation, closely mimicking the properties of native cardiac tissue. The behavior of human cardiac fibroblasts (HCFs), seeded on hydrogels, was analyzed. When compared to purely elastic polyacrylamide (pAAm) hydrogels with comparable stiffness, soft stress-relaxing hydrogels (AG_Click(R0.5) and AG_DC(R0.5)) were found to promote cell spreading area, while stiffer stress-relaxing hydrogels (AG_Click(R1) and AG_DC(R1)) enhanced asymmetric cell elongation, reflecting substrate-mediated mechanosensing. Additionally, HCFs showed high viability when cultured in 3D hydrogels over 7 days. Overall, rapid gelation, biocompatibility, and tunable viscoelastic properties of bio-orthogonal double cross-linked alginate-gelatin hydrogels support their use as injectable formulations or engineered cardiac tissues for CTE.