Low-Intensity Pulsed Ultrasound Stimulation Facilitates Osteogenic Differentiation of Human Periodontal Ligament Cells

Human periodontal ligament cells (hPDLCs) possess stem cell properties, which play a key role in periodontal regeneration. Physical stimulation at appropriate intensities such as low-intensity pulsed ultrasound (LIPUS) enhances cell proliferation and osteogenic differentiation of mesechymal stem cells. However, the impacts of LIPUS on osteogenic differentiation of hPDLCs in vitro and its molecular mechanism are unknown. This study was undertaken to investigate the effects of LIPUS on osteogenic differentiation of hPDLCs. HPDLCs were isolated from premolars of adolescents for orthodontic reasons, and exposed to LIPUS at different intensities to determine an optimal LIPUS treatment dosage. Dynamic changes of alkaline phosphatase (ALP) activities in the cultured cells and supernatants, and osteocalcin production in the supernatants after treatment were analyzed. Runx2 and integrin beta 1 mRNA levels were assessed by reverse transcription polymerase chain reaction analysis after LIPUS stimulation. Blocking antibody against integrin beta 1 was used to assess the effects of integrin beta 1 inhibitor on LIPUS-induced ALP activity, osteocalcin production as well as calcium deposition. Our data showed that LIPUS at the intensity of 90 mW/cm(2) with 20 min/day was more effective. The ALP activities in lysates and supernatants of LIPU-Streated cells started to increase at days 3 and 7, respectively, and peaked at day 11. LIPUS treatment significantly augmented the production of osteocalcin after day 5. LIPUS caused a significant increase in the mRNA expression of Runx2 and integrin beta 1, while a significant decline when the integrin beta 1 inhibitor was used. Moreover, ALP activity, osteocalcin production as well as calcium nodules of cells treated with both daily LIPUS stimulation and integrin beta 1 antibody were less than those in the LIPUS-treated group. In conclusion, LIPUS promotes osteogenic differentiation of hPDLCs, which is associated with upregulation of Runx2 and integrin beta 1, which may thus provide therapeutic benefits in periodontal tissue regeneration.