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

Abstract

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.

Integration of microbubbles with biomaterials in tissue engineering for pharmaceutical purposes

“…They proved that MBs with a size of 200 μm in diameter results in the best chondrogenesis results. Similarly, Lipid-coated MBs demonstrated that it is capable of enhancing the osteogenic differentiation of mesenchymal stem cells in 3D printed scaffolds under low-intensity pulsed ultrasound [84]. Furthermore, highly ultrasound responsive Gas-filled MBs encapsulated with lipid revealed robust potential in increasing the number of human mesenchymal stem cells in a 3D printed poly (ethylene glycol) diacrylate scaffold [85].…”

Inhibition of Human Breast Cancer Cell Proliferation by Low‐Intensity Ultrasound Stimulation

“…Unlike previous studies by other groups, to study the effects of varying the different parameters of LIPUS, a custom‐designed US exposure system was used (Figure ). The same setup has been successfully used in our past investigations . Briefly, a programmable function generator (33250A; Agilent Technologies, Palo Alto, CA) produced sinusoidal pulses in burst or continuous modes.…”

“…Ultrasound (US), best known for its application in medical diagnostic imaging, can also deliver high‐frequency mechanical energy to stimulate thermal and nonthermal bioeffects in cells and tissues and act as a therapeutic tool . Ultrasound stimulation of varying intensities and waveforms is currently under investigation for therapy of wide‐ranging ailments: fracture healing, painless transdermal insulin delivery, wound healing, enhancing chondrogenesis and osteogenesis of mesenchymal stem cells, and treatment of glaucoma . Relatively high‐intensity (on the order of 100–1000 W/cm 2 ) focused US has been used in thermal ablation of solid tumors .…”

Process hybridization schemes for multiscale engineered tissue biofabrication

“…RF processes utilizing electrical (Boccaccini et al, 2010; Liu et al, 2019), optical, and magnetic fields (Armstrong & Stevens, 2020; Lim et al, 2011) enable manipulation of nanoparticles, while the ones employing acoustic fields are more suitable for manipulation of microparticles due to the size constraints associated with the viscous penetration depth (Drinkwater, 2016). Notably, low‐intensity ultrasound has been used toward cell functionalization, including aiding differentiation (Aliabouzar, Lee, Zhou, Zhang, & Sarkar, 2018; Osborn et al, 2019), proliferation (Subramanian et al, 2013) and ECM production (Min, Choi, & Park, 2007). In general, ON processes excel in processing efficiency, but are often limited in resolution or control over distribution of cells and bioadditives (Roseti et al, 2017).…”