Spontaneous repair versus Amniotic Epithelial Cells-Induced tendon regeneration in an Ovine Achilles Tendon Model: can neural markers predict tendon healing outcome?

Introduction/Objectives Recent evidence highlights the nervous system's role in tendon healing through neural markers Nerve Growth Factor (NGF), Neurofilament 200 (NF-200, marker of nerve ingrowth), Neuropeptide Y (NPY), Calcitonin Gene-related peptide (CGRP) and Galanin (GAL), which influence inflammation, cell proliferation and extracellular matrix (ECM) remodeling 1. This study explored the predictive role of these neural markers in tendon healing using a validated ovine Achilles tendon injury model2,3. The spatiotemporal expression pattern of these markers was compared in tendons undergoing amniotic epithelial stem cells-induced regeneration (AECs) vs. spontaneously healing tendons, 14 and 28 days post-injury (p.i.) 2,3. Methods An ovine Achilles tendon injury model was used under two conditions: AECs treatment and spontaneous healing (CTR). Neural markers (NGF, NF-200, NPY, CGRP, GAL) were analyzed for spatiotemporal expression patterns. Histological assessment of tendon healing and ECM remodeling analyzed tendon morphometric parameters (cell alignment, angle deviation) and collagen maturation, notably the ratio of Collagen type 1 (COL1) to Collagen type 3 (COL3) mRNA expression. Statistical analyses were conducted to assess relationships between neural marker expression and tissue organization. Results AECs-treated tendons showed enhanced ECM remodeling, with lower angle deviation at 28 days p.i. (AECs vs. CTR p<0.05), and better COL1/COL3 ratio at 14 and 28 days p.i. (AECs vs. CTR p<0.01). Spatiotemporal modulation of neural markers was observed in treated tendons compared to CTR. CTR tendons showed significant nerve ingrowth at 14 days (p<0.05); in treated groups, NF-200 expression remained lower (p<0.05), suggesting AECs regulate nerve ingrowth during tendon regeneration. NGF expression increased over time in CTR (14 vs. 28 days p<0.05) but stayed constant in AECs-treated tendons (p<0.05 vs. CTR 14 days). Unlike CTR, neuropeptides expression was modulated in AECs-treated groups. NPY significantly increased in treated tendons from 14 to 28 days (p<0.01). CGRP and GAL significantly decreased in treated groups over time (p<0.0001); at 14 days their expression was higher than CTR (p<0.05). Pearson’s correlation analysis revealed strong positive correlations between NGF, CGRP and GAL expression. In AECs-treated groups lower levels of these markers positively correlated with better organization, unlike CTR, where higher neural markers levels were linked to delayed, disorganized healing. Conclusions The study highlights the predictive value of neural markers NGF, CGRP, and GAL in tendon healing, emphasizing their role as indicators of effective regeneration. AECs treatment showed a neuro-mediated mechanism, improving ECM remodeling, collagen maturation, and overall tissue organization. These results offer novel neurobiological insights into tendon regeneration, supporting the potential of biomarker-driven strategies to enhance therapeutic outcomes for tendinopathies.