Annunziata Mauro

Barbara Barboni

Giuseppe Prencipe

Maria Rita Citeroni

Matthias Schnabelrauch

Mohammad El Khatib

Paolo Berardinelli

Valentina Russo

English

Archivio della Ricerca - Università degli Studi di Teramo

Tendon Biomimetic PLGA 3D Scaffold Enhances Amniotic Epithelial Stem Cells Biological Capability for Tissue Engineering Applications   Giuseppe Prencipe a, Mohammad El Khatib a, Maria Rita Citeroni a, Annunziata Mauro a, Matthias Schnabelrauch b, Paolo Berardinelli a, Valentina Russo a and Barbara Barboni a  a Unit of Basic and Applied Biosciences, Faculty of Bioscience and Agro-Food and Environmental Technolo-gy, University of Teramo, 64100 Teramo, Italy; b Department of Biomaterials, INNOVENT e. V., 07745 Jena, Germany. *e-mail: gprencipe@unite.it  Keywords: tendon; stem cells; tissue engineering; scaffold; mechanotransduction.  Introduction Tendon tissue engineering (TE) represents a promising treatment option for tendinopathies, which to date do not have a resolutive conventional solution. TE aims at creating successful implantable 3D biomimetic scaffolds with the physical, mechanical, biological, and functional properties of the natural tissue. These constructs can be engineered with stem cells to enhance their teno-differentiative properties [1–3]. In this context, amniotic epithelial stem cells (AECs), collected from the placenta, represent an attractive stem cell source due to their low immunogenicity, high immunomodulatory properties and tenogenic differentiative ability, as demonstrated through in vitro [4] and in vivo [5] studies, in which they were able to accelerate tendon regeneration. However, in the case of tendon ruptures, in order to rebuild the damaged tissue, it is necessary to engineer these cells within a scaffold. Thus, the focus of this research was to develop bundle tendon-like PLGA 3D scaffolds resembling tendon macro and microarchitecture and biomechanics, and to evaluate their direct impact on AECs' biological potential in terms of teno-differentiation. Moreover, to acquire more insights into the biological results, the present research aimed to investigate the possible involvement of Yes-associated protein (YAP) activation, which represents a mechanotransducer implicated in the regulation of stem cell fate [6], and in particular in their tenogenic differentiation [7]. Finally, to simulate the in vivo microenvironment, in this study the AECs engineered scaffolds were tested, by using the in vitro co-culture system, for their teno-inductive potential on co-cultured cells.  Methods PLGA fleeces with highly aligned fibers were produced via an electrospinning process through a rotatory collector. The obtained fleeces were then hand bundled to produce 3D tendon-like scaffolds, which were characterized in vitro in terms of ultrastructure (SEM analysis). Then, the constructs were tested for their teno-inductive influence on AECs. In detail, cells engineered on PLGA fleeces and 3D scaffolds were compared with respect to cells cultured on Petri dishes (CTR) through a confocal microscope after 48 h of culture for their morphological changes and for the cytoplasmatic expression of TNMD, a mature tendon protein, by using immunofluorescence assay (IF). In addition, AECs teno-differentiation on scaffolds has been also assessed by analyzing the gene expression of tendon related markers (early: SCX and late: COL1 and TNMD) by means of real time-PCR.  Moreover, the present research evaluated YAP protein activation in the seeded cells through an IF by assessing its localization within the cells’ nuclei. Finally, the AECs engineered fleeces and 3D scaffolds were co-cultured with AECs for 14 d to evaluate, through a morphological investigation and TNMD protein western blot quantification, if the differentiated cells engineered on scaffolds exerted a paracrine action on the co-cultured AECs.  Results and Discussion The produced PLGA 3D scaffolds closely resemble tendon architecture, both macroscopically and microscopically, in fact, analyzed through scanning electron microscopy, these showed high fiber alignment.  AECs seeded on the constructs exhibited an elongated tenocyte-like morphology already after 48 h of culture, with a lower aspect ratio with respect to CTR, which 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) significant upregulation already after 48 h of culture respect to CTR cells (p<0.05), which showed no TNMD protein expression or significant increase in tendon-related genes. Interestingly, AECs cultured on 3D scaffolds showed a more elongated phenotype and a significant upregulation of all the analyzed genes with respect to cells seeded on fleeces (p<0.05), thus standing 3D scaffolds as greater teno-inductive stimuli for AECs.  Moreover, the highly aligned fibers of both fleeces and 3D scaffolds stimulated AECs in terms of cell cytoskeleton stress through the mechanosensitive YAP pathway. Indeed, the constructs significantly induced YAP nuclear translocation, even if 3D scaffolds significantly exhibited a higher impact on YAP activation respect to fleeces (p<0.05), and CTR (p<0.05), in which YAP was exclusively localized in the cytoplasm.  The in vitro co-culture system allowed to verify that the microenvironment established by the engineered constructs with AECs were able to teno-differentiate the co-cultured cells through a paracrine stimulus. Indeed, by co-culturing the AECs engineered fleeces and 3D scaffolds with AECs, these last differentiated into tenocyte-like cells and produced also tendon-like structures that were not found in CTR. The co-cultured AECs' phenotypic change was also confirmed by quantifying the expression of TNMD through western blot analysis, showing a significantly higher protein expression in the teno-differentiated cells co-cultured with fleeces and even significantly greater with 3D scaffolds with respect to the CTR (p<0.05). This result is probably the effect of stimulating factors released by the teno-differentiated cells contained within the scaffolds’ fibers.  Conclusion Overall, these findings corroborate the biomimicry of 3D scaffolds in terms of macro and microstructure, as well as their significant teno-inductive potential on AECs. This boosted effect could be probably associated to YAP activation which recently raised to be related to the alignment of the scaffolds’ fibers, which act through a mechanosensing stimulus on the AECs aligned along the construct [8]. Indeed, these latter not only differentiated into a tenogenic lineage, but also stimulated indirectly the surrounding cells to teno-differentiate even if not in contact with the scaffolds, potentially mimicking the in vivo microenvironment in which progenitor/stem cells should be stimulated to differentiate.  Thus, biomimetic PLGA 3D scaffolds stand as potential candidates for tendon regeneration. Acknowledgments This research was funded by Perspective for Future Innovation in Tendon Repair H2020MSCA-ITN-EJD-P4 FIT—Grant Agreement ID: 955685.  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Tendon Biomimetic PLGA 3D Scaffold Enhances Amniotic Epithelial Stem Cells Biological Capability for Tissue Engineering Applications

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

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