Ultrasound-Responsive Engineered Tissue Constructs for Remote Manipulation of Cell Signaling

The controlled presentation of cell signaling proteins in time and space is critical for coordinating biological processes that regulate tissue regeneration. There is a need to recreate and harness these complex dynamic processes within 3D cell scaffolds to better understand their biological roles and provide new regenerative therapies. Manipulating gene expression of targeted cells at specific times and locations within these scaffolds enables controlled protein presentation within a 3D tissue construct. This is a critical tool to control cell behavior and influence cell interactions, including those involved in tissue vascularization and wound repair. These genetic manipulations are difficult to achieve in 3D using traditional transfection methods due to diffusional barriers created by the scaffold itself. To address this challenge, our team has pioneered the development of ultrasound-responsive cell culture platforms (SonoScaffolds) for noninvasive and spatiotemporally-controlled genetic manipulation of cells in 3D scaffolds. Here, focused ultrasound interacts with integrated echogenic particles within hydrogel matrices to locally deliver nucleic acids to cells to manipulate protein expression and secretion. Ultrasound has distinct advantages as a triggering stimulus as it can be focused to small volumes with multi-centimeter tissue penetration depth [1-2].<br/><br/>Using this approach, we have successfully demonstrated ultrasound-induced transfection of cells embedded in SonoScaffolds, including localized expression of vascular endothelial growth factor (VEGF). A dual expression VEGF-GFP fusion plasmid was coupled to lipid-based ultrasound-responsive microparticles and incorporated into a cell-seeded collagen hydrogel. Focused ultrasound (1 mm³ focal zone) was applied noninvasively within the hydrogel matrix. Transfection of matrix-embedded cells was localized to the ultrasound-exposed region. The cells showed a punctate distribution of VEGF-GFP consistent with packaging of VEGF into secretory vesicles. Creating user-defined 3D spatiotemporal patterns of growth factor expression can be leveraged to elucidate how spatial presentation and timing of protein expression affect 3D cell migration and to inform therapeutic strategies for tissue repair.<br/><br/>Pushing the boundaries even further, we are creating ultrasound stimuli-responsive scaffolds with complex 3D architectures by integrating our echogenic gene delivery particles into 3D-bioprintable inks. Transfection was selectively activated by ultrasound within printed constructs, demonstrating the first cell-seeded bioprinted structure designed for ultrasound-controlled gene delivery. We are further extending this technique to 3D-printed spheroids and organoid cultures. Together, this work demonstrates a new class of 3D-programmable cell culture materials that enable remote, spatiotemporally-defined genetic perturbation of embedded cells and multicellular structures. These systems will provide new insights into coordinated cell processes and how growth factor presentation can be leveraged to direct healing in implantable biomaterials, with important applications in tissue construct maturation, vascularization, and tissue regeneration.<br/><br/>[1] Gelmi A and <u>Schutt CE.</u> <i>Advanced Healthcare Materials</i>. 10(1): 2001125, 2021.<br/>[2] Chapla R., Huynh KT, <u>Schutt CE.</u> <i>Pharmaceutics. </i>14(11): 2396, 2022.