HPDLCs were characterized morphologically under an inverted microscope and examined immunohistochemically using anti-vimentin antibody and anti-keratin antibody.

<p>(A) HPDLCs were long-shuttle in shape after generation (×100). (B) Positive immunohistochmical staining for Vimentin on hPDLCs (×100). (C) Negtive immunohistochmical staining for Keratin on hPDLCs (×100).</p

Ostogenic differentiation-related gene expression: product size of Runx2 and Intergrin β1.

<p>Ostogenic differentiation-related gene expression: product size of Runx2 and Intergrin β1.</p

Time course of ALP activity in hPDLCs treated with LIPUS for 13 days.

<p>The cells were treated with or without LIPUS and ALP activities in the cultured cells and supernatants were determined at 3, 5, 7, 9, 11, and 13 days of culture. 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, and the ALP activity was significantly higher in the LIPUS stimulation group than that in the control group at each time point. The data are presented as the mean±SD of three separate experiments. ^*<i>P</i><0.05 vs. the control group.</p

Effect of different daily dosages of LIPUS on ALP activities of hPDLCs.

<p>HPDLCs were treated with LIPUS at an intensity of 30, 60, or 90/cm² for 10, 20, or 30 min/day for 5 consecutive days. ALP activity was determined by colorimetric assay using a commercially available ALP kit. LIPUS treatment caused a significant increase in the ALP activity, as compared to non-treated control group and LIPUS at the intensity of 90 mW/cm² with 20 min/day was more effective. The data are presented as the mean±SD of three separate experiments. ^*<i>P</i><0.05 vs. the control group; ^#<i>P</i><0.05 vs. any of the other LIPUS-treated group.</p

Detection of calcium nodules by Alizarin red staining in hPDLCs.

<p>HPDLCs were assigned into LIPUS-treated group, non-treated control group and the combination group treated wtih LIPUS and integrinβ1 inhibitor, cultured with the osteoblast inducing conditional media for up to 21 days. The mineralized matrix deposition was determined using alizarin red staining. (A) The formation of calcium nodules in non-treated control cells. (B) The formation of calcium nodules in LIPUS-treated cells. (C) The formation of calcium nodules in cells treated wtih both LIPUS and integrinβ1 inhibitor. (D) LIPUS stimulation promoted formation of calcium deposition in hPDLCs, while a significant decline when the specific integrinβ1 inhibitor was used. The data are presented as the mean±SD of three separate experiments. ^*<i>P</i><0.05 vs. any of the other two groups.</p

Effects of integrinβ1 inhibitor on LIPUS-induced ALP activity.

<p>ALP activities in cell lysates were measured at 7 and 14 days. ALP activity was significantly higher in the LIPUS-treated cells than that in the control group and the combination group treated wtih both LIPUS and integrinβ1 inhibitor. The data are presented as the mean±SD of three separate experiments. ^*<i>P</i><0.05 vs. any of the other two groups.</p

Schematic illustration of ultrasound device and procedure.

<p>(A) Schematic view of the LIPUS exposure device used in this study, which consists of an array of 6 transducers. (B) Schematic illustration showing that a culture plate is placed on the ultrasound transducer array with a thin layer of ultrasonic coupling gel, and the distance between the transducer and cells was less than 2 mm.</p

Effects of integrinβ1 inhibitor on LIPUS-induced osteocalcin production.

<p>Total osteocalcin production in the stored culture media were measured. LIPUS stimulation promoted the production of osteocalcin, while a significant decline when the integrinβ1 inhibitor was used. The data are presented as the mean±SD of three separate experiments. ^*<i>P</i><0.05 vs. any of the other two groups.</p

Effects of LIPUS on Runx2 and integrin β1 mRNA expression.

<p>HPDLCs were assigned into LIPUS-treated group, non-treated control group and the combination group treated wtih LIPUS and integrinβ1 inhibitor, cultured for up to 7 days. The abundance of Runx2 and integrin β1 transcripts was determined by semi-quantitative RT-PCR assay. (A) Representative agarose gels showing gene expression changes between the three groups. (B) Densitometric analysis of gel images normalized against the GAPDH gene. LIPUS caused a significant increase in the mRNA expression of Runx2 and integrin β1, while a significant decline when the integrinβ1 inhibitor was used. The data are presented as the mean±SD of three separate experiments. ^*<i>P</i><0.05 vs. any of the other two groups.</p

Focused ultrasound treatment system.

<p>Focused ultrasound treatment system.</p