Tendon injuries pose a clinical challenge due to tendons’ limited recovery. Emerging evidence points to the nervous system’s critical role in tendon healing, with neural markers NGF, NF-200, NPY, CGRP, and GAL modulating inflammation, cell proliferation, and extracellular matrix (ECM) remodeling. This study investigates the predictive role of selected neural markers in a validated ovine Achilles tendon injury model, comparing spatio-temporal expression patterns in regenerating tendons transplanted with amniotic epithelial stem cells (AECs) versus spontaneous healing (CTR) 14 and 28 days post-injury (p.i.). AEC-treated tissues showed a spatio-temporal modulation of NF-200, NGF, NPY, CGRP, GAL, and enhanced ECM remodeling, with greater cell alignment, lower angle deviation, and accelerated collagen maturation, with a favorable Collagen type 1 (COL1) to Collagen type 3 (COL3) ratio. Pearson’s matrix analysis revealed significant positive correlations between NGF, CGRP, and GAL expression, along a positive correlation between the three neural markers and cell alignment and angle deviation. As opposed to CTR, in AEC-treated tendons, lower levels of NGF, CGRP, and GAL correlated positively with improved tissue organization, suggesting these markers may predict successful tendon regeneration. The findings highlight the neuro-mediated activity of AECs in tendon regeneration, with NGF, CGRP, and GAL emerging as key predictive biomarkers for tendon healing.

Unsuccessful tendon healing leads to fibrosis and occasionally calcification. In these metaplastic drifts, the mouse AT preclinical injury model represents a robust experimental setting for studying tendon calcifications. Previously, calcium deposits were found in about 30% of tendons after 28 days post-injury. Although a neuromediated healing process has previously been documented, the expression patterns of NF200, NGF, NPY, GAL, and CGRP in mouse AT and their roles in metaplastic calcific repair remain to be explored. This study included a spatiotemporal analysis of these neuromarkers during the inflammatory phase (7 days p.i.) and the proliferative/early-remodelling phase (28 days p.i.). While the inflammatory phase is characterised by NF200 and CGRP upregulation, in the 28 days p.i., the non-calcified tendons (n = 16/24) showed overall NGF, NPY, GAL, and CGRP upregulation (compared to 7 days post-injury) and a return of NF200 expression to values similar to pre-injury. Presenting a different picture, in calcified tendons (n = 8), NF200 persisted at high levels, while NGF and NPY significantly increased, resulting in a higher NPY/CGRP ratio. Therefore, high levels of NF200 and imbalance between vasoconstrictive (NPY) and vasodilatory (CGRP) neuromarkers may be indicative of calcification. Tendon cells contributed to the synthesis of neuromarkers, suggesting that their neuro-autocrine/paracrine role is exerted by coordinating growth factors, cytokines, and neuropeptides. These findings offer insights into the neurobiological mechanisms of early tendon healing and identify new neuromarker profiles predictive of tendon healing outcomes.

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 CM_INF 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 CM_INF-functionalized scaffolds (3D-CM_INF), preserving bioactivity. Functionally, 3D-CM_INF scaffolds significantly suppressed T-cell activation and peripheral blood mononuclear cell (PBMC) proliferation. The released CM from 3D-CM_INF (CM_R) 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-CM_INF-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-CM_INF 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.