Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and Low Intensity Pulsed Ultrasound on 3D Printed Scaffolds

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 µm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm-2 , 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.