Biomimetic PLGA 3D Scaffold Potentiate Amniotic Epithelial Stem Cells Biological Capability for Tendon Tissue Engineering Applications

INTRODUCTION: Tendon tissue engineering represents a promising solution to deal with tendinopathies and aims to develop effective implantable 3D biomimetic scaffolds with ideally native tissue’s physical, mechanical, biological, and functional qualities. These constructs can be engineered with stem cells to potentiate their teno-inductive and immunomodulatory properties (El Khatib, Mauro, Di Mattia, et al., 2020; El Khatib, Mauro, Wyrwa, et al., 2020; Russo et al., 2020). In this context, amniotic epithelial stem cells (AECs) have recently received much attention in the field of regenerative medicine due to their capacity to differentiate into the tenogenic lineage and to their immunomodulatory profile (Barboni et al., 2012, 2018; Mauro et al., 2016). The focus of this research was to create bundle tendon-like PLGA 3D scaffolds, which mimic tendon macro and micro-architecture and biomechanics, and to assess their impacts on AECs’ biological potential. METHODS: PLGA fleeces, with highly aligned fibers, were fabricated via electrospinning technique through a rotatory collector. The obtained fleeces were then wrapped manually to form 3D tendon-like scaffolds, which were evaluated in terms of structure, mechanical characteristics, and biological influence on AECs by conducting in vitro experiments. Indeed, ovine AECs, seeded on the PLGA 3D scaffolds and fleeces, were compared for their morphological changes and for the cytoplasmic expression of TNMD, a mature tendon protein, respect to cells cultured on Petri dishes (CTR), after 48h and 7d of culture through a confocal microscope. Moreover, the teno-differentiative potential and immunomodulatory properties of the produced constructs were assessed by analyzing the gene expression of tendon related markers (early: SCX, late: COL1 and TNMD) and of anti- (IL10) and pro- (IL12) inflammatory cytokines respectively. Moreover, the present research evaluated YAP protein activation in the engineered AECs through immunofluorescence assay by assessing its cellular localization. RESULTS: The produced PLGA 3D scaffolds, analyzed though a scanning electron microscope, showed high fiber alignment, which closely resemble the architecture, both macroscopically and microscopically, and the biomechanical properties of native tendon tissue. AECs seeded on the produced constructs exhibited an elongated tenocyte-like morphology already after 24 hours, while AECs cultivated on petri dishes (CTR) retained their characteristic polygonal morphology. The engineered AECs' phenotypic change was also confirmed by visualizing the cytoplasmic expression of TNMD protein and supported by tendon-related genes (SCX, COL1, and TNMD) upregulation at 7-day culture respect to CTR cells (p<0.05), which showed no TNMD protein expression or significant increase in tendon-related genes. Moreover, AECs seeded on 3D PLGA scaffolds showed an anti-inflammatory profile, with a significant higher IL10/IL12 ratio respect to the CTR (p<0.05). Finally, 3D scaffolds with highly aligned fibers stimulated AECs in terms of cell cytoskeleton stress, activating their mechanosensitive YAP pathway by significantly increasing YAP nuclear localization compared to the CTR (p<0.05), in which YAP was instead localized in the cytoplasm. DISCUSSION & CONCLUSIONS: Overall, these results support the biomimicry of the fabricated scaffolds in terms of structure and biomechanics and reveal their great teno/immuno-inductive potential and mechanosensing stimulus on AECs, thus standing biomimetic PLGA 3D scaffolds as a potential candidate for tendon regeneration

Insight into Hypoxia Stemness Control

Recently, the research on stemness and multilineage differentiation mechanisms has greatly increased its value due to the potential therapeutic impact of stem cell-based approaches. Stem cells modulate their self-renewing and differentiation capacities in response to endogenous and/or extrin- sic factors that can control stem cell fate. One key factor controlling stem cell phenotype is oxygen (O2). Several pieces of evidence demonstrated that the complexity of reproducing O2 physiological tensions and gradients in culture is responsible for defective stem cell behavior in vitro and after transplantation. This evidence is still worsened by considering that stem cells are conventionally incubated under non-physiological air O2 tension (21%). Therefore, the study of mechanisms and signaling activated at lower O2 tension, such as those existing under native microenvironments (referred to as hypoxia), represent an effective strategy to define if O2 is essential in preserving naïve stemness potential as well as in modulating their differentiation. Starting from this premise, the goal of the present review is to report the status of the art about the link existing between hypoxia and stemness providing insight into the factors/molecules involved, to design targeted strategies that, recapitulating naïve O2 signals, enable towards the therapeutic use of stem cell for tissue engineering and regenerative medicine

Tendon Healing Response Is Dependent on Epithelial–Mesenchymal–Tendon Transition State of Amniotic Epithelial Stem Cells

Tendinopathies are at the frontier of advanced responses to health challenges and sectoral policy targets. Cell‐based therapy holds great promise for tendon disorder resolution. To verify the role of stepwise trans‐differentiation of amniotic epithelial stem cells (AECs) in tendon regeneration, in the present research three different AEC subsets displaying an epithelial (eAECs), mesenchymal (mAECs), and tendon‐like (tdAECs) phenotype were allotransplanted in a validated experimental sheep Achilles tendon injury model. Tissue healing was analyzed adopting a comparative approach at two early healing endpoints (14 and 28 days). All three subsets of transplanted cells were able to accelerate regeneration: mAECs with a lesser extent than eAECs and tdAECs as indicated in the summary of the total histological scores (TSH), where at day 28 eAECs and tdAECs had better significant scores with respect to mAEC‐treated tendons (p < 0.0001). In addition, the immunomodulatory response at day 14 showed in eAEC‐transplanted tendons an upregulation of pro‐regenerative M2 macrophages with respect to mAECs and tdAECs (p < 0.0001). In addition, in all allotransplanted tendons there was a favorable IL10/IL12 compared to CTR (p < 0.001). The eAECs and tdAECs displayed two different underlying regenerative mechanisms in the tendon. The eAECs positively influenced regeneration mainly through their greater ability to convey in the host tissue the shift from pro‐inflammatory to pro‐regenerative responses, leading to an ordered extracellular matrix (ECM) deposition and blood vessel remodeling. On the other hand, the transplantation of tdAECs acted mainly on the proliferative phase by impacting the density of ECM and by supporting a prompt recovery, inducing a low cellularity and angle alignment of the host cell compartment. These results support the idea that AECs lay the groundwork for production of different cell phenotypes that can orient tendon regeneration through a crosstalk with the host tissue. In particular, the obtained evidence suggests that eAECs are a practicable and efficient strategy for the treatment of acute tendinopathies, thus reinforcing the grounds to move their use towards clinical practice

Mammal comparative tendon biology: advances in regulatory mechanisms through a computational modeling

There is high clinical demand for the resolution of tendinopathies, which affect mainly adult individuals and animals. Tendon damage resolution during the adult lifetime is not as effective as in earlier stages where complete restoration of tendon structure and property occurs. However, the molecular mechanisms underlying tendon regeneration remain unknown, limiting the development of targeted therapies. The research aim was to draw a comparative map of molecules that control tenogenesis and to exploit systems biology to model their signaling cascades and physiological paths. Using current literature data on molecular interactions in early tendon development, species-specific data collections were created. Then, computational analysis was used to construct Tendon NETworks in which information flow and molecular links were traced, prioritized, and enriched. Species-specific Tendon NETworks generated a data-driven computational framework based on three operative levels and a stage-dependent set of molecules and interactions (embryo–fetal or prepubertal) responsible, respectively, for signaling differentiation and morphogenesis, shaping tendon transcriptional program and downstream modeling of its fibrillogenesis toward a mature tissue. The computational network enrichment unveiled a more complex hierarchical organization of molecule interactions assigning a central role to neuro and endocrine axes which are novel and only partially explored systems for tenogenesis. Overall, this study emphasizes the value of system biology in linking the currently available disjointed molecular data, by establishing the direction and priority of signaling flows. Simultaneously, computational enrichment was critical in revealing new nodes and pathways to watch out for in promoting biomedical advances in tendon healing and developing targeted therapeutic strategies to improve current clinical interventions