# Immuno-Instructive 3D Tendon Biomimetic Scaffolds Functionalized with Amniotic Epithelial Stem Cell Secretome for Controlled Inflammation and Targeted Macrophage Polarization

## TL;DR

This paper introduces a cell-free therapeutic scaffold that modulates inflammation and promotes tendon healing by controlling macrophage behavior.

## Contribution

A novel 3D tendon scaffold functionalized with amniotic stem cell secretome to control inflammation and direct macrophage polarization is presented.

## Key findings

- NaOH pre-treatment enhanced the retention and release of the immunomodulatory protein Amphiregulin from the scaffold.
- The scaffold suppressed T-cell activation and PBMC proliferation while promoting pro-regenerative M2 macrophage polarization.
- The secretome-based scaffold demonstrated time-dependent immunomodulation, balancing early anti-inflammatory and later reparative effects.

## Abstract

Tendon healing is often hindered by unresolved inflammation and dysregulated immune responses, highlighting the need for innovative regenerative strategies. This study developed an immune-informed platform by functionalizing validated 3D tendon-mimetic poly(lactide-co-glycolide) (PLGA) scaffolds with immunomodulatory conditioned media (CM), referred to as CMINF to emphasize its anti-inflammatory and immunomodulatory properties, derived from ovine amniotic epithelial stem cells (AECs), offering a potential cell-free therapeutic solution. Three functionalization methods were compared: physical adsorption, and hydrochloric acid (HCl) or sodium hydroxide (NaOH) pre-treatments. FT-IR spectroscopy and protein adsorption analyses identified NaOH as the most effective method, enhancing retention and release of Amphiregulin (AREG), an AEC key immunomodulatory protein. Kinetic studies revealed a sustained, controlled release of AREG over 7 days (d) from CMINF-functionalized scaffolds (3D-CMINF), preserving bioactivity. Functionally, 3D-CMINF scaffolds significantly suppressed T-cell activation and peripheral blood mononuclear cell (PBMC) proliferation. The released CM from 3D-CMINF (CMR) exhibited time-dependent immunomodulatory effects: early T-cell inhibition (6–72 h) and delayed suppression of PBMC proliferation (48 h–7 d). Macrophage polarization analysis revealed a shift towards the pro-regenerative M2 phenotype, with increased expression of M2 over M1 markers in 3D-CMINF-adherent cells. Flow cytometry confirmed a preferential induction of regulatory M2b macrophages alongside reductions in pro-inflammatory M1 and pro-fibrotic M2a subsets. These results demonstrate that 3D-CMINF scaffolds can finely modulate immune responses, balancing inflammatory and reparative cues relevant to early tendon healing processes. This platform, integrating structural and immunomodulatory elements, presents a promising, cell-free, and translational immunoengineering strategy to control inflammation and support tendon repair.