Ultrasound-enhanced osteogenesis of human mesenchymal stem cells encapsulated in collagen meshwork

Bone marrow mesenchymal stem cells (MSCs) are well-regarded as potential cell sources for regenerative medicine. An osteoinductive way of implanting these MSCs is to first encapsulate them into larger particles through collagen fiber meshes and then injecting these particles to the treatment site, but these particles´ osteogenic differentiation is known to be slow for human MSCs (hMSCs). In this work, we investigated the possibility of using ultrasound to stimulate the osteogenesis of hMSCs in collagen-hMSC particles (6^th passage; entrapped with type-I collagen; 2mm radius; 1mm thickness; 500,000 cells/ml concentration). The in-vitro calcium deposition level was studied over an 18-day period for collagen-hMSC particles that were routinely exposed to ultrasound (1MHz frequency; 30mW/cm² intensity (SATA); 20% duty cycle; 30min daily exposure). Our phase contrast images showed that ultrasound-exposed collagen-hMSC particles had a darker appearance than the unexposed ones because of the increased presence of calcium deposits. Also, in our calcium assay, particles in the ultrasound-exposed group all showed increase in their calcium mass (N=6). A similar impact was observed for the collagen-hMSC particles that were only exposed to ultrasound in the first six days, thereby suggesting that ultrasound stimulation makes early impact on the osteogenic differentiation of these particles. These initial evidences indicate that ultrasound stimulation has potential in improving the efficacy of injectable bone tissue engineering by enhancing the osteogenic process of collagen-hMSC particles.