Low-intensity pulsed ultrasound promotes cell viability of hUSCs in volumetric bioprinting scaffolds via PI3K/Akt and ERK1/2 pathways

The study aimed to investigate the impact of low-intensity pulsed ultrasound (LIPUS) on human urinary-derived stem cells (hUSCs) viability within three-dimensional (3D) cell-laden gelatin methacryloyl (GelMA) scaffolds. hUSCs were integrated into GelMA bio-inks at concentrations ranging from 2.5% to 10% w/v and then bioprinted using a volumetic-based method. Subsequent exposure of these scaffolds to LIPUS under varying parameters or sham irradiation aimed at optimizing the LIPUS treatment. Assessment of hUSCs viability employed Cell Counting Kit-8 (CCK8), cell cycle analysis, and live&dead cell double staining assays. Additionally, Western blot analysis was conducted to determine protein expression levels. With 3D bio-printed cell-laden GelMA scaffolds successfully constructed, LIPUS promoted the proliferation of hUSCs. Optimal LIPUS conditions, as determined through CCK8 and live&dead cell double staining assays, was achieved at a frequency of 1.5 MHz, a spatial-average temporal-average intensity (ISATA) of 150 mW cm^-2, with an exposure duration of 10 min per session administered consecutively for two sessions. LIPUS facilitated the transition from G0/G1 phase to S and G2/M phases and enhanced the phosphorylation of ERK1/2 and PI3K-Akt. Inhibition of ERK1/2 (U0126) and PI3K (LY294002) significantly attenuated LIPUS-induced phosphorylation of ERK1/2 and PI3K-Akt respectively, both of which decreased the hUSC viability within 3D bio-printed GelMA scaffolds. Applying a LIPUS treatment at an ISATA of 150 mW cm^-2promotes the growth of hUSCs within 3D bio-printed GelMA scaffolds through modulating ERK1/2 and PI3K-Akt signaling pathways.