Ultrasound-integrated biofabrication of aligned piscine muscle tissue

The directional alignment of highly mature muscle fibers is a crucial hallmark of successful muscle tissue regeneration in vitro, both in regenerative medicine and cultured meat. While 3D bioprinting enables precise fabrication of tissue architecture, it inherently lacks the capacity to control the spatial distribution and growth trajectories of cells embedded within the bioink. Here, we integrated ultrasound with 3D bioprinting to leverage its precision control capabilities for engineering highly aligned cellular organization and induce muscle fiber formation. Both computational simulations and experimental validation confirmed that exposure to 4000 kHz ultrasonic frequency yielded distinct cell alignment, concomitant with an enhanced proliferation rate. Intermittent low-intensity ultrasound (LIUS) stimulation effectively activated the mechanosensitive Piezo1 channels in piscine satellite cells (PSCs), promoting Ca²⁺ influx. Ca²⁺ activates ERK5 in the downstream MAPK pathway, which up-regulates Myogenin and MHC, and finally promotes PSCs differentiation and fusion into myotubes. This non-invasive ultrasound physical stimulation method has great potential for application in the construction of edible muscle tissue.