Development of Amniotic Epithelial Stem Cells Secretome-Loaded In Situ Inverse Electron Demand Diels-Alder-Cross-Linked Hydrogel as a Potential Immunomodulatory Therapeutical Tool

Amniotic epithelial stem cells (AEC) hold potential for tissue regeneration, especially through their conditioned medium (AEC-CM) due to their immunomodulatory and regenerative effects. Nevertheless, advanced drug delivery systems such as hydrogels are needed to enable clinical applications. Herein, an in situ gellable hyaluronic acid and polyethylene glycol-based iEDDA-cross-linked hydrogel was developed for the encapsulation and controlled release of AEC-CM. The developed system was formed by norbornene-modified hyaluronic acid and tetrazine-modified polyethylene glycol functionalized with heparin. The hydrogel was formed by mixing both precursor polymers, displaying fast cross-linking kinetics and showcasing a highly porous inner structure and low swelling properties. Moreover, the heparin-functionalized system allowed the sustained release of predominant growth factors from AEC-CM over 14 days. In vitro studies in peripheral blood mononuclear cells (PBMCs) showed an enhanced suppression efficacy and a significant shift toward the M2 macrophage phenotype in comparison with nonencapsulated AEC-CM. Therefore, this work provides a suitable alternative for the encapsulation of AEC-CM in a hydrogel formulation, highlighting its potential as an alternative immunomodulatory therapeutic tool for tissue regeneration.