3D bone printing via primed differentiation of stem cells with ultrasound (3DBonUS)

Abstract

Bone disorders and skeletal defects represent a significant clinical challenge. Tissue engineering and regenerative medicine (TERM) strategies using 3D bioprinting have emerged as promising alternatives, but still limited by the inability of directing stem cell differentiation in a controlled and reproducible manner. Advancing beyond this, we are proposing 3D bone printing via primed differentiation of stem cells with ultrasound (referred to here as 3DBonUS). This approach synergistically integrates low-intensity pulsed ultrasound (LIPUS) with a microfluidic-assisted 3D bioprinting platform enabling the biophysical stimulation of human bone marrow stromal cells (HBMSCs) during extrusion, promoting osteogenic differentiation without the need for post-fabrication treatments. Moreover, the incorporation of microbubbles enhanced the effects of LIPUS by amplifying mechanical signals at the cellular level. 3DBonUS was found to significantly upregulate key osteogenic markers (RUNX-2, ALP, COL1A1, BMP-2, OCN, OPN) as confirmed by immunofluorescence and RT-qPCR analysis. Furthermore, the LIPUS-treated constructs showed a significant increase in alkaline phosphatase activity and calcium deposition, indicating enhanced mineralisation. The 3DBonUS strategy represents a new modality in skeletal biofabrication, harnessing targeted minimally-invasive mechanical stimulation, with potential for manufacturing scalability and clinical application. Future studies will aim to validate 3DBonUS in vivo to assess the ultimate regenerative potential with enhanced osteogenic properties.