Abstract
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 β1 mRNA levels were assessed by reverse transcription polymerase chain reaction analysis after LIPUS stimulation. Blocking antibody against integrinβ1 was used to assess the effects of integrinβ1 inhibitor on LIPUS-induced ALP activity, osteocalcin production as well as calcium deposition. Our data showed that LIPUS at the intensity of 90 mW/cm2 with 20 min/day was more effective. The ALP activities in lysates and supernatants of LIPUS-treated 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 β1, while a significant decline when the integrinβ1 inhibitor was used. Moreover, ALP activity, osteocalcin production as well as calcium nodules of cells treated with both daily LIPUS stimulation and integrinβ1 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 β1, which may thus provide therapeutic benefits in periodontal tissue regeneration.
Highlights
Congenital malformations, trauma or periodontal disease and other factors often lead to different degrees of alveolar bone defects, injuring to tooth supporting tissues, causing damages to periodontal attachment loss, eventually leading to loss of tooth [1]
The alkaline phosphatase (ALP) activity of Human periodontal ligament cells (hPDLCs) markedly changed with both intensity and exposure time of low-intensity pulsed ultrasound (LIPUS)
The StudentNewman-Keuls test showed that hPDLCs exposed to LIPUS at an intensity of 90 mW/cm2 for 20 min per day had a significantly (P,0.05) greater increment in ALP activity, compared with any of the other LIPUS-treated groups (Fig. 3)
Summary
Congenital malformations, trauma or periodontal disease and other factors often lead to different degrees of alveolar bone defects, injuring to tooth supporting tissues, causing damages to periodontal attachment loss, eventually leading to loss of tooth [1]. Regeneration of periodontal tissue is derived from ancestral cells in periodontal ligament and bone. Like bone marrow stromal cells, PDLCs have the ability to give rise to mesoderm cell lineages, such as alveolar bone, cementum, and periodontal ligament for periodontal tissue regeneration [4], [8]. New bone formation is critical for maintaining the structural stability and physiological function of the dentition [10], so promoting osteogenic differentiation of PDLCs during wound healing and regeneration is significative. Most PDLCs in periodontal lesion tissues, require the inflammatory-free microenvironments and biological activity for periodontal wound healing. It is of significance to develop new approaches to improve the osteogenic potential of PDLCs
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