Effects of ultrasound in presence of microbubbles on cartilage tissue regeneration in three-dimensional printed scaffolds

Abstract

Gas-filled microbubbles encapsulated with lipids and other surfactants are highly responsive to ultrasound, which has led to their effective role as ultrasound contrast agents (UCA). In this study, for the first time, we used lipid-coated microbubbles (MB) prepared in-house in order to better harness the beneficial effects of ultrasound stimulation on proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs) within a novel 3D printed poly (ethylene glycol) diacrylate (PEG-DA) hydrogel scaffolds. A significant increase in cell number (p <0.001) was observed with low intensity pulsed ultrasound (LIPUS) treatment in the presence of 0.5 % (v/v) MB after 1, 3, and 5 days of culture. MSC proliferation enhanced up to 40% after 5 days of culture in the presence of MB and LIPUS while this value was only 18% when excited with LIPUS alone. We investigated the effects of acoustic parameters such as excitation intensity, frequency, and pulse repetition period on MSC proliferation rate. Our 3-week chondrogenic differentiation results demonstrated that combining LIPUS with MB significantly enhanced both Glycosaminoglycan (GAG) and type II collagen production. Therefore, integrating LIPUS and MB appears to be a promising strategy for enhanced MSC growth and chondrogenic differentiation.