Low-intensity pulsed ultrasound enhances differentiation of osteolineage-committed human mesenchymal stem cells

Abstract

Even after committing to osteolineage, human meschenchymal stem cells (hMSCs) tend to develop into osteoblasts at a slow pace that may not lead to significant bone mineral deposits after weeks. In attempt to accelerate this process, we have investigated how hMSCs can be stimulated towards osteogenesis in-vitro through low-intensity pulsed ultrasound (LIPUS) exposures. The hMSCs (500000cells/ml) were seeded within a collagen meshwork that served as an osteo-conductive platform and were cultured in the presence of osteolineage supplements. Each experimental group (N>3) was treated with LIPUS for seven consecutive days (frequency: 1MHz; duty cycle: 20%; PRF: 1kHz; duration: 30min; peak-negative pressure: 0, 60, 80, 90, 105, 120, 180kPa), and in one group 1% microbubbles was added before the daily LIPUS expsoure. Results show that: 1) LIPUS did not affect the seven-day viability of hMSCs (confirmed by fluorescent live-dead assay); 2) LIPUS has transiently increased the secretion of the BMP-2 protein that is involved in the osteoinduction process; 3) LIPUS has led to increase in the seven-day calcium deposit level, with maximum detected at 60-120mW/cm2 intensities; 4) observations were similar for the group with microbubbles, and thus stable cavitation may not be a contributing factor to osteogenesis.