Enhancing Tendon Regeneration: The Role of 3D PLGA Scaffolds in Modulating Ovine Amniotic Epithelial Stem Cell Paracrine Activity

Tendon disorders present an arduous challenge for regenerative medicine, mostly due to the inherently poor healing capabilities of the tissue. Achieving successful tendon healing necessitates a harmonious integration of angiogenesis, immune modulation, and tenogenesis. Thus, an efficient tendon engineering (TE) strategy must carefully regulate the interactions among these systems to reach tissue regeneration. In this context, ovine amniotic epithelial stem cells (oAECs) represent an attractive stem cell source to accelerate tendon regeneration due to their low immunogenicity, high immunomodulatory properties, and tenogenic differentiative ability, as demonstrated through in vitro [1] and in vivo[2] studies. Moreover, these properties can be modulated and boosted when oAECs are engineered on validated tendon biomimetic PLGA 3D electrospun scaffolds with highly aligned #bers, resembling tendon macrostructure, hierarchical microarchitecture and biomechanics [3].The present research represents a step forward, delving into the paracrine effects exerted by oAECs cultured on 3D scaffolds on the target cell types involved in tendon repair: HUVECs for angiogenesis, PBMCs/Jurkat for immune response, and oAECs for tenogenic differentiation.Findings from the study highlight the significant impact of scaffolds' topography and topology on the paracrine signaling of oAECs. Specifically, taking advantage of a protein microarray analysis, it was shown that cells engineered on 3D scaffolds enhanced their basal secretion of key bioactive molecules, notably VEGF-D, b-FGF, RANTES, and PDGFBB, among other 40 cytokines, indicating a marked increase compared to control media (p<0.0001). Furthermore, biological assays demonstrated the 3D scaffolds' ability to amplify the paracrine-mediated suppression of PBMCs proliferation (p<0.001 vs. CTR) and to mitigate LPS-induced Jurkat activation (p<0.01 vs. CTR) without promoting pro-angiogenic activities in HUVECs. Moreover, the paracrine teno-inductive ability of oAECs seeded on 3D scaffolds was evaluated on co-cultured ones, which formed tendon-like structures. These newly formed structures exhibited the expression of tendon-specific genes (SCX, THBS4, COL1, and TNMD) and proteins (TNMD and COL1) with respect to naïve AECs, which normally do not express these markers, underscoring the potential of this approach for tendon regeneration.Overall, this research emphasizes the crucial role of PLGA 3D scaffolds’ topography and topology in influencing oAECs behavior, underscoring the strategic significance of in vitro models in predicting the interplay between engineered scaffolds and somatic/ immune/blood vessels, essential for tendon regeneration. Additionally, the study suggests that the secreted molecules could serve as a valuable source of factors for potential cell-free therapy in tendon repair.