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.
