Ultrasound and Lipid-Coated Microbubbles Effect on Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells in 3D Printed Tissue Scaffold


With increasing incidence of bone disorders in a rapidly aging, sedentary, and overweight population, engineered bone tissues promise to be a better alternative for conventional bone grafts. Human mesenchymal stem cells (hMSCs) extracted from a patient’s bone marrow can be cultured to create bone tissue via osteogenic differentiation of the stem cells. The engineered bone tissue may then be implanted into the injury site. However, bone tissue engineering currently suffers due to the inability to form mechanically strong, porous structures that can be grown quickly and efficiently. Low intensity pulsed ultrasound (LIPUS) stimulation has proven to enhance bone healing and recovery rates. In this study, the beneficial effects of LIPUS stimulation are harnessed to improve bone tissue engineering methods and are further improved with the addition of in-house prepared, lipid-coated microbubbles (MBs). Bone marrow hMSCs were seeded on 3D printed poly(lactic acid) (PLA) porous scaffolds and cultured to study the effects of LIPUS in the presence of MBs on proliferation and osteogenic differentiation. For proliferation studies, the samples were treated with LIPUS in the presence of 0.5% (v/v) for 3 minutes a day at 30 mW/cm2 with a frequency of 1.5 MHz and a duty cycle of 20%. A significant increase in cell number was observed after 1, 3, and 5 days of culture as compared to control. For osteogenic differentiation studies, the samples were treated with LIPUS in the presence of 0.5% (v/v) MBs for 3 days for 3 minutes a day with the same parameters as the proliferation studies. The samples were then cultured for 1, 2, and 3 weeks for osteogenic differentiation to occur. Total protein, alkaline phosphatase (ALP) activity, and total calcium content were found to increase with LIPUS with and without the presence of MBs. Integrating LIPUS and MB appears to be a promising strategy for bone tissue engineering and regeneration therapies.

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