Speaker
Description
Introduction
Tissues and organs are composed of cells embedded in an instructive 3D extracellular matrix (ECM). Changes in mechanical properties of the ECM act as dynamic cues that guide cells through different stages of development. In synthetic 2D culture systems, matrix stiffness, viscoelasticity, and their variation have been shown to influence cell spreading and differentiation by modulating intercellular traction forces.[1,2] Similar effects have been observed in 3D models [3], but our understanding of how mechanical signals are processed at subcellular level and through specific signaling pathways remains limited. Studying these mechanisms requires synthetic 3D culture systems with precise control over mechanical stimuli - something current technologies struggle to provide.[4] To address this, we introduce the adaption of Xolography volumetric 3D printing into a grayscaled bioprinting process.[5] Xolography employs intersecting light of two different wavelengths and a dual-color photoswitch-photoinitiator to locally crosslink a viscous photoresin. We hypothesize that this method will enable the fabrication of hydrogels with spatially defined mechanical properties like stiffness, offering new opportunities to study 3D mechanotransduction.
Methods
Xolography was used to fabricate hydrogel constructs based on gelatin methacryloyl (GelMA), polyethylene glycol diacrylate (PEGDA) and N-isopropyl acrylamide (NIPAAm). Local reduction of the applied energy dose was implemented by applying intermediate intensity values (i.e. grayscaling) to the visible light projection, one of the two light sources in Xolography. Mechanical properties of printed constructs were assessed by compression testing, nanoindentation, and atomic force microscopy (AFM)-based force spectroscopy. The degree of crosslinking was evaluated with Fourier-transformed infrared spectroscopy (FTIR) and tested as a mechanism to drive reversible, anisotropic shape-changes in simple thermoresponsive hydrogel geometries.
Results
For a hydrogel based on PEGDA and a given set of printing parameters, projections with full exposure of 242 mW cm-² led to constructs with stiffness of 400 ± 100 kPa on macroscale while 50 % decrease of the visible light dose resulted in a lower stiffness of 33 kPa. The same trend was observed on milli-scale: indentation measurements demonstrated a highly significant decrease in Hertzian Young’s modulus from 2.10 ± 0.30 kPa to 0.23 ± 0.03 kPa by decreasing the visible light dose by 30 %. A fivefold decrease of the modulus was observed on microscale within a scan area of 5 μm x 5 μm.
Xolography based on NIPAAm and GelMA allowed printing of shrinkable 4D geometries. Thermo-induced shrinking was found to be reversible to similar extents when compared to cast hydrogels. Anisotropic shrinking based on heterogeneous crosslinking was achieved through grayscaling in hydrogel beams.
Discussion
The capability of grayscaled Xolography to modulate the degree of crosslinking and subsequently stiffness was demonstrated. The effect was confirmed on multiple lengths scales and successfully employed to alter shrinkage spatially. Modulation of local mechanical properties can now be employed to create cell-instructive matrices and eventually unravel 3D mechanotransduction.
References
[1] Janmey et al. (2019) 695–725 doi.org/10.1152/physrev.00013.2019
[2] Yang et al. (2016) doi.org/10.1073/pnas.1609731113
[3] Huebsch et al. (2010) doi.org/10.1038/nmat2732
[4] Saraswathibhatla et al. (2023) doi.org/10.1038/s41580-023-00583-1
[5] Stoecker et al. (2025) doi.org/10.1002/adma.202410292
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