Dual-Frequency Ultrasound Enhances Cavitation of Microdroplets for Controlled Scaffold Porosity in Tissue Engineering

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Abstract
The development of porous scaffolds with tunable mechanical and structural properties is essential for advancing tissue engineering strategies. In this study, we present a noninvasive, adjustable method for generating porous collagen scaffolds by utilizing micron-sized phase-shift droplets in combination with dual-frequency ultrasound. These microdroplets, generated via a microfluidic chip and composed of a liquid perfluoropentane core stabilized by a phospholipid shell, were embedded within collagen hydrogels and served as ultrasound-responsive cavitation nuclei. A 3.5 MHz imaging transducer was employed to trigger acoustic droplet vaporization of the embedded microdroplets, transitioning them into microbubbles. Then, a 200 kHz therapeutic transducer induced bubble oscillation and collapse, leading to localized pore formation. This combined ultrasound strategy enabled both vaporization and bubble implosion at reduced pressure thresholds compared to conventional acoustic droplet vaporization methods. Theoretical modeling using the Marmottant model predicted microbubble dynamics and corresponding pore sizes, which were validated through scanning electron microscopy and histological analysis. Ultrasound-treated scaffolds containing droplets exhibited significantly increased porosity of 56.53 ± 3.91% compared to untreated controls, with a pore diameter of 39.42 ± 10.28 μm, observed via scanning electron microscopy. Rheological analysis revealed enhanced elasticity and structural resilience in ultrasound-treated scaffolds. Finally, in vitro studies confirmed that fibroblast viability remained high within the treated scaffolds, with cells observed in close proximity to ultrasound-generated pores. This work introduces a tunable and clinically relevant strategy for fabricating functional scaffolds that could support tissue regeneration and customizable healing environments.
Funding Information
  • HORIZON EUROPE European Research Council (101041118)
  • Israel Science Foundation (192/22)
  • Israel Cancer Research Fund (1286686)
  • Nicholas and Elizabeth Slezak Super Center for Cardiac Research and Biomedical Engineering

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