Gene Expression in Response to Low-Intensity Pulsed Ultrasound Treatment of Bone Marrow Cells under Osteogenic Conditions <i>In Vitro</i>

Abstract

This study was conducted to examine the gene-expression changes that might contribute to enhanced osteogenesis after low-intensity pulsed ultrasound (LIPUS) exposure. Bone marrow cells were obtained from the femora of rats and were suspended in an osteogenic medium to prepare cell cultures. After the cultures were established, test cultures were exposed to LIPUS through the base of the cell-culture plates for 15 min/day on Days 3-9 (LIPUS group). Control cultures were not exposed to LIPUS, but were otherwise treated identically to the LIPUS group. On Day 10, total RNA was extracted from both sets of cultures and hybridized to microarray slides, and the obtained datasets were analyzed. Real-time PCR was used to confirm the microarray analysis results. Cell-proliferation assays and Sirius Red staining were performed on Days 4, 7 and 10, and Alizarin Red S staining was performed on Days 10, 14 and 21. Markers for differentiated osteoblasts and osteocytes and genes encoding collagen-related molecules and cell-adhesion factors were upregulated in the LIPUS group on Day 10. Cell proliferation was lower in the LIPUS group than in the controls on Day 7. Sirius Red staining in the LIPUS group was significantly higher than in the controls on Day 10, and the cell areas stained with Alizarin Red S were significantly larger in the LIPUS group than in the controls on each day of the experiment. Thus, LIPUS exposure increased the gene expression of extracellular matrix factors and promoted the differentiation of osteoblast-like cells into osteocytes in an in vitro cell culture model.

Low-Intensity Pulsed Ultrasound Accelerates Differentiation of Osteoblastic Cells on Roughened Titanium Surface

“…Cell proliferation was shown to be suppressed on the roughened titanium disk compared with the machined surfaced disk 23) . The cell number on the surface of polystyrene culture plates on day 7 was observed to be decreased in the LIPUS group compared with the control group 15) . In this study, the cell number on days 5 and 7 did not show any statistical difference between the LIPUS and control groups.…”

“…We noted that the density of Sirius Red staining was significantly higher in the LIPUS group compared with that in control cultures on day 14. Yamaguchi et al, using a microarray analysis, concluded that exposure to LIPUS increased the expression of collagen rerated gene molecules and promoted the differentiation of osteoblastic cells into osteocytes 15) . Collagen is a key determinant in bone for fortifying bone strength 45) .…”

“…In implant-related studies, noninvasive low-intensity ultrasound was demonstrated to significantly affect bone ingrowth into porous titanium implants in an in vivo animal model 12) and bone formation was reported to be stimulated around titanium implants in dog mandibles 13) . We demonstrated that the application of LIPUS enhanced differentiation and mineralization of osteoblastic cells cultured in polystyrene cell-culture dishes 14) and promoted the expression of genes in the extracellular matrix and the differentiation of osteoblast-like cells into osteocytes in an in vitro cell culture model 15) . In in vivo studies, LIPUS was shown to accelerate bone formation around titanium implants and enhance the strength of bone-titanium integration in rat femora 16) .…”

Gene Expression in Early Stages of Low-Intensity Pulsed Ultrasound Exposure on Bone Marrow Cells

“…Kokubu et al revealed in an in vitro study that cultured osteoblasts stimulated by LIPUS induced prostaglandin 2 via cyclooxygenase-2 production; exposure of whole bone marrow cells to LIPUS promoted differentiation more than isolated mesenchymal cells 12) . A comprehensive analysis of mRNA expression in whole marrow cells after 7 d of stimulation with LIPUS showed an increase in the gene expression of bone matrix-specific markers 13) .…”