Introduction: The secretome of stem cells represents a powerful paracrine tool for regenerative medicine. Amniotic epithelial cells (AECs), owing to their embryonic origin, secrete a unique combination of neurotrophic and immunomodulatory factors. While their role in inflammation and ECM remodeling is established, their influence on neuro-mediated tendon regeneration remains poorly defined.

Methods: AECs were isolated from ovine amniotic membranes and cultured in vitro. Expression of neurogenic markers (NGF, CGRP, GAL, NPY) was assessed at passages P0 and P3. NGF secretion was quantified by ELISA. To assess functional neurotrophic activity, AEC-conditioned media (CM) were applied to chick embryo dorsal root ganglia (DRG): neurite radial outgrowth was used as a readout for bioactive NGF signaling. In vivo, we used an ovine Achilles tendon injury model comparing spontaneous healing (CTR) to AEC-treated tendons. Histological, gene expression, and morphometric analyses were performed at 14 and 28 days post-injury. PKH26-labeled AECs were tracked for localization and co-expression of neural markers. Tendon organization was evaluated via cell alignment, angle deviation, and COL1/COL3 ratio.

Results: AECs exhibited consistent expression of NGF, CGRP, GAL, and NPY, with NGF and CGRP showing marked upregulation at P3, revealing significantly increased levels of NGF secretion. NGF-rich CM induced robust radial neurite outgrowth from chick embryo DRGs, confirming its neurogenic efficacy. In vivo, AEC-treated tendons showed transient expression of NGF, CGRP, and GAL at day 14, which became spatially restricted by day 28, unlike CTR tendons where diffuse expression persisted. Pearson’s matrix analysis revealed significant positive correlations between NGF, CGRP, and GAL expression, and between these markers and angle deviation. Notably, in AEC-treated tendons, lower levels of NGF, CGRP, and GAL were positively correlated with improved ECM organization, suggesting their potential as predictive biomarkers for regenerative success. Structural analyses confirmed better ECM alignment and a favorable COL1/COL3 ratio in the treated group.

Conclusion: AECs secrete a neurotrophic secretome that exerts a dual regenerative effect on neural elements and tendon ECM. NGF, a key secreted factor, promotes neurite outgrowth in vitro and modulates in vivo healing dynamics. NGF, CGRP, and GAL emerge as predictive biomarkers for tendon regeneration, supporting the clinical potential of AEC-derived secretomes as a cell-free therapy for neuromodulated tissue repair.