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

Abstract

Injectable hydrogels are widely investigated as carriers for mesenchymal stem cells (MSCs); however, the influence of ionic crosslinking strategy on injectability-relevant properties and early in vitro cell responses remains insufficiently defined from a design perspective. In this study, alginate-based hydrogels were prepared using three ionic crosslinking approaches-internal (CaCO3/ glucono-delta-lactone (GDL)), external (CaCl2), and combined gelation-and comparatively evaluated as delivery matrices for human adipose-derived MSCs. The hydrogels were characterized in terms of microstructural features, weight variation behavior, viscoelastic properties, protein diffusion, and early biological performance. Rheological analysis revealed a broad range of mechanical responses, with storage moduli varying from approximately 102–103 Pa depending on the crosslinking mechanism. Internally-gelled hydrogels exhibited a softer, more deformable network and a wider linear viscoelastic region, while externally and double-crosslinked hydrogels showed increased stiffness but more brittle behavior. Microstructural observations indicated larger and more interconnected pores in internally crosslinked systems compared to the denser networks formed via external gelation. In vitro biological assessments, including metabolic activity, DNA quantification, and post-injection viability, demonstrated that the crosslinking strategy significantly affected early cell fate. Notably, internally-crosslinked hydrogels supported higher cell viability and metabolic activity while maintaining superior post-injection cell survival (>95%), whereas double-crosslinked hydrogels showed lower viability following syringe delivery consistent with their higher stiffness and lower deformation tolerance. Overall, this comparative study highlights the trade-offs between mechanical integrity, handling during syringe delivery, and early biological performance in ionically-crosslinked alginate hydrogels. The findings provide practical design-oriented guidance for selecting alginate crosslinking strategies in injectable MSCs delivery systems where minimally invasive administration and early cell survival are critical considerations.