Engineering injectable alginate hydrogels for mesenchymal stem cell delivery: comparative evaluation of ionic crosslinking strategies and early in vitro outcomes.

Injectable hydrogels are promising platforms for minimally invasive delivery of mesenchymal stem cells (MSCs), yet the effects of ionic crosslinking mechanisms on their injectability, mechanical behavior, and early cell responses remain insufficiently defined. Here, we systematically investigate how three ionic crosslinking strategies-internal gelation (CaCO₃/GDL), external gelation (CaCl₂), and combined internal-external gelation-govern the material performance of alginate hydrogels engineered for human adipose-derived MSC (hAd-MSC) delivery. Comprehensive physicochemical characterization, including microstructural analysis, swelling behavior, rheological profiling, and protein diffusion, revealed distinct structure-property relationships dictated by the gelation pathway. Internal gelation produced softer and more deformable networks with broader linear viscoelastic regions, whereas external and combined gelation resulted in stiffer, more brittle hydrogels with restricted deformation and reduced injectability. Biological evaluations-including metabolic activity, DNA content, and post-injection viability-demonstrated that crosslinking strategy markedly modulates early cell fate. Internally gelled hydrogels supported significantly higher viability, metabolic function, and post-injection survival, whereas combined gelation compromised both injectability and early cell performance. Overall, this comparative study offers design-oriented insights that link ionic crosslinking mechanisms to mechanical behavior and early biological outcomes, providing practical guidelines for engineering alginate-based injectable systems for MSC delivery.