Anisotropic Hydrogel Degradation Enhances 3D Collective Cell Alignment, Mechanotransduction and Osteogenic Differentiation
Authors: Garrido Claudia A. , Garske Daniela S. , Bucher Christian H. , Amini Shahrouz , Duda Georg N. , Schmidt-Bleek Katharina , Cipitria Amaia 
Published: April 2024

Tissue engineering involves assembling cells and mimicking the complex anisotropic architecture of biological tissues to perform specific functions. This study explores the impact of patterned hydrogels with anisotropic degradation compared to single-phase materials on the potential of enhancing cell spreading, collective alignment, mechanotransduction and osteogenic differentiation of encapsulated human mesenchymal stromal cells (hMSCs). Spatial patterns of degradable (Deg) and non-degradable (noDeg) subregions are formed by photolithography: UV-triggered thiol-ene crosslinking with matrix metalloprotease (MMP) sensitive peptides form Deg phases, while non-UV exposed regions result in Diels-Alder spontaneous click crosslinking and noDeg phases. 3D patterns in hydrogel degradation enhance hMSC spreading and allow collective cell alignment in Deg areas, while cells remain rounded with no alignment in noDeg regions. In addition, we observe a boosted osteogenic differentiation when compared to single-phase materials, as mid osteogenic markers (osteocalcin) are expressed at day 14 in anisotropic gels, whereas in single-phase only early osteogenic markers are found (osterix). Mechanosensing pathways were evaluated using the expression and localization of YAP. Deg sections in patterned materials have an enhanced nuclear translocation and higher YAP expression compared to single-phase Deg materials and noDeg sections. This effect is lost and no patterns in YAP expression and localization emerge when using an MMP-scramble peptide or no-RGD materials. These findings demonstrate that 3D patterns in alginate hydrogel degradation guide hMSC spreading, collective alignment, enhance YAP nuclear translocation and osteogenic differentiation. Mimicking tissue anisotropy in 3D patterned hydrogels could have broad applications in biofabrication and tissue engineering.

Statement of significance

Patterned materials integrate multiple characteristics within a single material, closely mimicking the anisotropy found in tissues. This research goes further by demonstrating how anisotropic degradation of cell-laden hydrogels leads to emerging patterns in mechanics. As a consequence, anisotropic hMSC morphology and collective alignment are shown in 3D patterned materials compared to single-phase counterparts. Additionally, we show enhanced and spatially guided hMSC osteogenic differentiation in patterned materials. Furthermore, anisotropic mechanosensing via YAP/TAZ signaling is shown to mediate this enhanced and spatially guided mechanotransduction and osteogenic differentiation. Finally, we explore how additional biochemical stimuli can further boost the spatially guided hMSC osteogenic differentiation. These findings advance our understanding of cell response in anisotropic microenvironments, with broad applications in biofabrication and tissue engineering.

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Graphical abstract