AECS ENGINEERED ON VALIDATED ELECTROSPUN 3D PLGA SCAFFOLDS: AN IN VITRO MODEL FOR TENDON REGENERATION

Amniotic epithelial stem cells (AECs) are considered promising candidates for musculoskeletal regeneration due to their low immunogenicity, immunomodulatory properties, and intrinsic plasticity toward tenogenic differentiation both in vitro [1] and in vivo [2]. Investigating AECs in tendon engineering (TE) strategy is therefore crucial to fully unlock their regenerative potential and design effective cell-based strategies. This study explores in vitro the paracrine influence of AECs engineered on validated 2D and 3D electrospun PLGA scaffolds, produced using electrospinning technique to replicate tendon-like macroarchitecture [3], on angiogenesis, immune response, and tenogenesis, a key processes for tendon regeneration. In particular, AEC morphology, expression of tenogenic markers (SCX, COL1, TNMD), and TNMD protein were evaluated in the experimental conditions. Contextually, Array analysis of AECs derived- conditioned media (CM) proteins’ content was performed and then their influence on in vitro angiogenesis was evaluated on HUVECs cells by in vitro Angiogenesis Assay. CM’s immunomodulatory effect was evaluated on PBMCs proliferation and Jurkat reporter cells activation. Moreover, Yes-associated protein (YAP) activation was assessed to investigate scaffold-induced mechanotransduction. Finally, the teno-inductive paracrine effect of AECs’ engineered constructs was assessed with longterm co-cultures with freshly isolated AECs (up to 14 days) conducted by using a trans-well system [1]. Results revealed that both 2D and 3D scaffolds promoted AECs alignment, acquisition of an elongated, tenocyte-like morphology, and significant upregulation of tenogenic genes including SCX, COL1, and TNMD (p<0.05 vs. CTR). However, AECs cultured on 3D scaffolds exhibited a significantly greater increase in gene expression levels and stronger TNMD protein expression compared to 2D scaffolds (p<0.05), indicating that the 3D configuration provides a more potent teno-inductive environment. These results were supported by enhanced activation in cells of mechanosensitive YAP pathways in response to fiber alignment and 3D scaffold architecture. Moreover, data obtained revealed the role of scaffold’s topology and topography in significantly modulating the paracrine profile of the cells. AECs basal release of bioactive molecules was boosted in the cells engineered on 3D scaffolds, in particular VEGF-D, b-FGF, RANTES, and PDGF-BB (p<0.0001 vs. CM CTR ). Additionally, biological tests demonstrated 3D scaffolds’ proactive role in potentiating AECs’ paracrine inhibition on PBMCs proliferation (CM 3D vs. CTR, p<0.001) and LPS-mediated respect to controls (CM 3D and CM Jurkat activation with 2D vs. CTR, p<0.01 and p<0.05, respectively), without exerting any in vitro proangiogenic role in promoting HUVECs proliferation and tubule formation. Teno-inductive paracrine ability of AECs engineered on 3D scaffolds was assessed on co-cultured ones, which formed tendon-like structures. These latter demonstrated an upregulation of tendon-related genes (SCX, THBS4, COL1, and TNMD) and the expression TNMD and COL1 proteins. Overall, this study highlights the pivotal role of the 3D topology and topography of PLGA scaffold architecture in AECs stem cell fate and their paracrine mechanisms. The 3D scaffold provided the most biologically effective microenvironment for instructing AEC teno-differentiation reinforcing its potential in tendon-focused stem cell and cellfree therapy.