Amniotic epithelial cells promote tendon regeneration through nerve-mediated tenogenic signaling

Amniotic epithelial cells (AECs) secrete a distinct combination of neurotrophic and immunomodulatory factors. While their role in inflammation and extracellular matrix (ECM) remodeling is established, their contribution to nerve-mediated tendon regeneration remains largely unexplored [1]. This study examined the impact of AECs-based therapy on the expression of neural markers during tendon regeneration in a validated ovine Achilles tendon injury model [2,3], highlighting the neuromodulatory effects of stem cell intervention compared to spontaneous repair (CTR). Methods AECs were isolated from ovine amniotic membranes and expanded in vitro. Neural marker expression (NGF, CGRP, GAL, NPY) was assessed at passages P0 and P3, and NGF secretion was quantified by ELISA. A validated ovine Achilles tendon injury model was used to compare the spatiotemporal expression of neural markers in AECs-treated versus control (CTR) tendons at 14 and 28 days post-injury. Expression levels and localization of NF-200, NGF, NPY, CGRP, and GAL were evaluated by immunohistochemistry (IHC). PKH26-labeled AECs were tracked using confocal microscopy to assess their localization and co-expression of neural markers. Structural reorganization was assessed through key morphometric parameters cell alignment and angle deviation. Gene expression profiling of ECM markers (COL1, COL3) and tenogenic markers (SCX, THBS4, TNMD) was performed to evaluate ECM deposition and tendon maturation. Results AECs maintained expression of all neurogenic markers in vitro, with significant upregulation of GAL and CGRP at p3, alongside increased NGF secretion. In vivo, AECs-treated tendons showed transient upregulation and localized expression of NGF, CGRP, and GAL at day 14, which declined and became more spatially confined by day 28. In contrast, CTR tendons exhibited sustained, diffuse expression. Pearson correlation analysis revealed that elevated neuralmarker levels were associated with greater ECM disorganization, while lower levels of NGF, CGRP, and GAL correlated with improved structural alignment. Gene expression analysis showed early SCX upregulation at 14 days in AECs-treated tendons, followed by a decrease at 28 days. THBS4 and TNMD remained consistently elevated at both time points, indicating enhanced tenogenic maturation. ECM gene profiling revealed favorable COL1 and COL3 expression patterns, with a higher COL1/COL3 ratio in treated tendons. Consistently with gene expression levels, morphometric analysis further confirmed improved cell alignment and reduced angle deviation in AECs treated tendons. Conclusion AECs-derived secretomes support tendon regeneration through nerve-mediated tenogenic modulation. The dynamic expression of tendon and ECM-related genes, combined with improved tissue organization, highlights the potential of AECs-based strategies for tendon regeneration, with NGF, CGRP and GAL emerging as potential predictive biomarkers of tendon healing outcome. 1 Ackermann et al. Frontiers in Bioscience Landmark 19:1251–1278 2014. doi: 10.2741/4280 2 Barboni et al. Cell Transplant 21:2377–2395 2012. doi: 10.3727/096368912X638892 3 Russo et al. Biomedicines 2022 19;10(5):1177. doi: 10.3390/biomedicines10051177