Introduction: Tendon injuries still represent a medical challenge due to their limited healing capacity and the risk of chronic inflammation and fibrosis. Tissue engineering represents a promising strategy to support tendon regeneration. Bioinspired scaffolds mimicking native tendon architecture may offer a functional solution by supporting tenogenic differentiation and modulating immune responses. This study investigated the regenerative potential of electrospun poly(lactide-co-glycolide) (PLGA) 3D tendon biomimetic scaffolds engineered with amniotic epithelial stem cells (AECs), in vitro and in vivo.

Methods: PLGA scaffolds were fabricated via electrospinning to mimic tendon hierarchical architecture. AECs were engineered on 3D scaffolds and the resulting conditioned media (CM) were collected and characterised. In vitro, CM effects were tested on angiogenesis (HUVECs), immune modulation (PBMCs and Jurkat cells), and tenogenesis (co-cultured naïve AECs). Expression of tendon markers (SCX, COL1, TNMD, THBS4) was assessed at gene and protein levels. For in vivo validation, the AEC-engineered 3D scaffolds were allotransplanted into a validated sheep Achilles tendon defect model. Explanted tissues (n=10) at day 14 (d14) were evaluated for scaffold biocompatibility, engraftment, teno-differentiation, and local immune modulation.

Results: AECs cultured on 3D scaffolds exhibited enhanced secretion of VEGF-D, PDGF-BB, b-FGF, and RANTES compared to control groups (p < 0.0001). CM significantly reduced PBMC proliferation and Jurkat NF-κB activation (p < 0.001), indicating a strong immunosuppressive effect, without promoting HUVECs proliferation and tubule formation. Naïve AECs co-cultured with AEC-3D scaffolds exhibited robust teno-differentiation, with significant upregulation of COL1, TNMD, and THBS4 (p < 0.01) and TNMD protein expression (p < 0.01). In vivo, the scaffolds were well tolerated, successfully engrafted, and induced in situ teno-differentiation of the transplanted AECs. Histological and molecular analyses, differently from control tendons, revealed an organised COL1 deposition, modulation of local inflammation and preferential M2 macrophages recruitment, indicating a regenerative response.

Conclusion: AEC-engineered 3D PLGA scaffolds embody a next-generation approach to tendon regeneration, offering a smart, biomimetic platform that actively instructs stem cell fate and orchestrates immune modulation. By simultaneously guiding tenogenic differentiation and modulating the immune response, these constructs shift from passive supports into dynamic drivers of tissue regeneration. Their efficacy, demonstrated in vitro and in a large-animal tendon injury model, paves the way for their application in regenerative medicine. Beyond structural integration, they established a pro-regenerative niche that promoted organized extracellular matrix deposition and M2 macrophage recruitment. Future preclinical studies will focus on long-term functional recovery as a proof of concept for translational feasibility and clinical scalability in tendon therapies.