Immune-Informed 3D Electrospun Scaffolds Functionalized with Amniotic Epithelial Stem Cell-secretome: A Cell-Free Immunomodulatory Strategy for Tendon Regeneration

Tendon healing remains a clinical challenge, often due to the inability to properly modulate inflammation and immune responses following injury. Immune-informed scaffold approaches represent promising solutions by modulating these crucial responses [1]. Thus, this study proposes a novel immuno-informed strategy that combines 3D tendon biomimetic poly(lactide-co-glycolide) (PLGA) scaffolds [2] with conditioned media enriched in immunomodulatory factors (CMINF) derived from amniotic epithelial cells, known for their strong paracrine and immunomodulatory activity [3]. However, the effective delivery and retention of secretome-derived molecules at injury sites remain critical limitations. To address this, three scaffold functionalization strategies: physical adsorption, acid (HCl), and alkaline (NaOH) pre-treatments, were evaluated. NaOH treatment significantly enhanced protein adsorption (~53%) and enabled a more sustained release of bioactive molecules, including Amphiregulin (AREG), a molecule implicated in tissue repair and M2 macrophage polarization [4]. In vitro, CMINF-functionalized scaffolds markedly inhibited peripheral blood mononuclear cell (PBMC) proliferation and suppressed NFAT activation in Jurkat T cells over a 7-day period. These scaffolds also modulated macrophage polarization toward an anti-inflammatory M2-dominant phenotype (CD206⁺) on scaffold-adherent cells, with a specific shift toward the M2b (CD86⁺CD80⁻) subpopulation. Altogether, these findings validate NaOH-pretreated 3D tendon biomitetic scaffolds as potent immune-modulatory platforms and establish a robust foundation for the development of advanced, cell-free immunoengineering strategies to orchestrate inflammation resolution and support tendon tissue regeneration. This research was funded by NextGenerationEU under MUR grant ECS00000041-VITALITY and PRIN–Smart Biomimetic Device for Tendon Tissue Engineering CUP C53D23005440006.