3D Electrospun Scaffolds Functionalized with Amniotic Epithelial Stem Cell Secretome as Immune-Responsive Platform for Cell-Free Tendon Regeneration

Tendon repair is often compromised by chronic inflammation and disrupted immune balance, which limit current treatment outcomes and call for advanced regenerative approaches [1]. To address this, an immune-informed system was developed by functionalizing validated 3D tendon-mimetic PLGA scaffolds [2] with immunomodulatory conditioned media (CMINF ) derived from ovine amniotic epithelial stem cells (AECs), which are well recognized for their paracrine and immunoregulatory properties [3]. This cell-free approach was explored through three functionalization techniques: physical adsorption, HCl and NaOH pre-treatments. FT-IR spectroscopic and protein adsorption analyses identified NaOH treatment as the most effective method, promoting enhanced scaffold reactivity and improved retention and release of Amphiregulin (AREG), a key immunomodulatory factor a molecule implicated in tissue repair and M2 macrophage polarization [4]. Release studies demonstrated that 3D-CM INF scaffolds ensured a bioactive, sustained delivery of AREG over a 7-day period. Functionally, 3D-CM INF scaffolds inhibited significantly T-cell activation and suppressed PBMC proliferation. Moreover, conditioned media released from 3D-CM INF (CMR ) displayed timedependent immunomodulatory profiles, with early inhibition of T cells (6–72 h) and delayed suppression of PBMC proliferation (48 h–7 d). Importantly, macrophage polarization assays revealed a shift toward pro-regenerative M2 phenotype, with increased expression of M2 over M1 markers in 3D-CMINF -adherent cells. Flow cytometry showed a preferential induction of regulatory M2b macrophages, accompanied by reduced proportions of pro-inflammatory M1 and pro-fibrotic M2a subsets. Overall, these findings show that 3D-CM INF scaffolds can actively regulate immune responses by orchestrating both inflammatory resolution and tissue-regenerative signals. This integrative platform, merging structural and paracrine immunomodulation, offers a promising, cellfree, and clinically relevant immunoengineering strategy for tendon regeneration. Keywords: Amniotic Epithelial Stem Cells; Conditioned Media; Scaffold Functionalization; Immunomodulation; Macrophages Polarization; Tendon regeneration. Funding Acknowledgment: This work was supported by Progetti di Rilevante Interesse Nazionale (PRIN) – Smart Biomimetic Device for Tendon Tissue Engineering – CUP C53D23005440006.