Acoustic Assembly of Collagen Hydrogels in Petri Dishes: The Distinct Roles of Traveling and Standing Waves

Abstract

Anisotropic biomaterials containing oriented collagen fibers are critical for mimicking the native extracellular matrix (ECM) architectures, showing great potential for various biomedical research areas, such as wound dressing, corneal grafting, and the study of cancer cell invasion in biomimetic microenvironments. While previous studies have utilized electric, microfluidic, magnetic, and mechanical methods to align collagen fibers, these conventional approaches often suffer from significant limitations, including complex fabrication setups, prolonged processing times, potential cell toxicity, or the requirement of specialized micro-chambers that hinder high-throughput applications.

To address these challenges, this thesis puts forward a novel, non-contact, and rapid fabrication approach utilizing traveling and standing acoustic waves to arrange collagen fibers dynamically. This method enables the rapid in-Petri-dish fabrication of anisotropic biomaterials without the need for chemical additives or complex modifications. To develop these approaches, we systematically investigated the physical effects of acoustic waves on collagen self-assembly kinetics and the resulting micro/nanoscale architectures of the fabricated biomaterials using confocal fluorescence microscopy. Our mechanistic investigations reveal a dual-action acoustic effect. Specifically, traveling acoustic wave-induced fluid streaming actively transports collagen molecules, thereby accelerating and spatially directing the collagen self-assembly process. Concurrently, standing acoustic waves generate acoustic radiation forces that accumulate self-assembled collagen fibers, increasing their localized concentrations and aligning them into periodically distributed acoustic potential wells. This combined acoustic approach achieved a high degree of fiber alignment along the fluid flow within just 5 min. Using our acoustics-assisted approach, we successfully manufactured anisotropic collagen hydrogels containing highly aligned collagen fibers, which provide structurally biomimetic and anisotropic microenvironments for cell growth and development. To validate the biological functionality of these fabricated constructs, in vitro cell culture studies were conducted using U251 cells. Quantitative morphological analysis demonstrated that cells cultured in these hydrogels exhibited significant contact guidance, facilitating pronounced cell elongation and directional alignment along the acoustically arranged collagen fibers.

Compared to previous methods, our acoustics-based approaches are remarkably easy to operate. They eliminate the requirement for customized microfluidic chambers for loading collagen and are capable of rapidly fabricating anisotropic collagen hydrogels directly in commercial Petri dishes. This unique advantage allows our approach to be readily integrated into existing standard laboratory workflows and seamlessly combined with other test protocols. In the long run, we expect this work to inspire the development of versatile, high-throughput tools that will significantly benefit biomedical researchers working in tissue engineering, regenerative medicine, biomaterials, and bioprinting.