Acoustic traveling wave induced shear torsion (A-TWIST) platform in biomimetic hydrogels to promote endothelial cell vascularization

Introduction: : Innovations that deliver precise and tunable physical forces on hydrogels are of great interest for generating and manipulating biomimetic cellular constructs, including mechanosensitive cell models (e.g., endothelial cells, immune cells, and stem cells). This scalable innovation is necessary, from the cellular to tissue level, for advancing research into the fundamental biology of immune cell activation for immunotherapies or for guiding stem cell fate for tissue engineering-derived therapies. Various physical-force-based methods are currently under investigation for enabling these active biomaterials. These methods employ fluidic, tensile, viscoelastic, electromagnetic, optical, thermal, and acoustic forces. However, previous in vitro physical material deformation models have been limited by the lack of precise control of the resulting 3D distribution of forces or the absence of a biologically relevant scaffolding for scaling to relevant biomimetic tissue engineered models.

In this study, we present the development of an acoustic traveling wave induced shear torsion (A-TWIST) platform to precisely control the 3D deformation distribution of a biomimetic hydrogel. We quantified and characterized, for the first time, the macromaterial shear stress response of a hydrogel to the applied surface acoustic waves (SAWs) of the A-TWIST platform via rheometry. We then investigated how different hydrogel properties, including density and crosslinking, affected the SAW shear response. Next, we numerically simulated and experimentally verified the 2D and 3D deformation distribution induced by A-TWIST. Finally, we employed A-TWIST to strain a biomimetic hydrogel loaded with endothelial colony forming cells (ECFC) to demonstrate the biocompatible nature of A-TWIST to activate mechanosensitive cell models.

Materials and

Methods: : A-TWIST device fabrication standard positive photolithography and metal e-beam evaporation techniques to create the custom focused surface acoustic waves (SAW) that would enter at different flanks of the hydrogel that was on top of the lithium niobate acoustic substrate. The working frequency of 19.73 MHz, and for biological experiments a custom PDMS chamber was fabricated to enable cell culture and maximum traveling SAW propagation. Collagen and collagen methacrylate of varying densities and crosslinking times were implemented to study how the material properties changed the propagated traveling SAWs from A-TWIST. Rheometry was utilized to quantify the shear force magnitude on the traveling SAWs. 3D displacement within the hydrogel was measured via confocal imaging and particle tracing. 2D displacement within the hydrogel was imaged via fluorescent microscopy and measured via particle image velocimetry.

Endothelial colony forming cells (ECFCs) laden collagen hydrogels were stimulated via A-TWIST on day 2 of growth for 1 hour at varying amplitudes of acoustic power on passage 8 to passage 10. Endothelial colony forming cell vascularization was quantified via immunostaining and confocal imaging and image processing from previously published methods via ImageJ. [1]

Results, Conclusions, and Discussions:: We found that with A-TWIST we could simulate and demonstrate controlled 3D deformation of biomimetic hydrogels. We also found that the different material properties of the hydrogel, density and crosslinking density, can also tune the desired shear stress response of the biomimetic matrix. Finally, we demonstrated that a 37% and 48% enhancement of surface area and volume of vascularization of ECFCs is achieved with tuned strain from A-TWIST. Overall, this manuscript presents rigorous characterization and a novel application of acoustic forces for the precise and biocompatible 3D deformation of a biomaterial loaded with mechanosensitive cells and the resulting enhancement in tissue-level vascularization via A-TWIST. We anticipate future applications of this work to investigate custom deformation distributions and the impact of these extracellular strains on cellular pathways effecting their growth and organization. This will enable precisely engineered biological functions with other mechanosensitive cell models for unique applications in the field of biomedical engineering, tissue engineering, mechanobiology, and immunology.

Acknowledgements and/or References (Optional):: We acknowledge the assistance of Duke University’s shared materials instrumentation facilities and equipment and of the Duke light microscopy core facilities and equipment. We acknowledge support from the National Science Foundation (CMMI-2104526, CMMI-2243771, and CMMI-2104295), the Department of Energy (DE-NE0009187), and the National Institutes of Health (R01GM141055, R01HD103727, R01GM143439, and R01GM145960). This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. 2139754 for J.R. and C.D.A.

[1] Hajal, C.; Offeddu, G. S.; Shin, Y.; Zhang, S.; Morozova, O.; Hickman, D.; Knutson, C. G.; Kamm, R. D., Engineered human blood–brain barrier microfluidic model for vascular permeability analyses. Nature Protocols 2022, 17 (1), 95-128.