Identification of osteogenic gene response under ultrasound exposure in vitro

Objectives: Low-intensity pulsed ultrasound (LIPUS) has been utilized to accelerate bone fracture healing. In a previous study, we reported that LIPUS enhanced differentiation and mineralization of osteoblasts derived from rat bone marrow stromal cells, although the mechanisms were still unclear. The aim of this study was to explore the genetic mechanisms of the enhancement of osteogenesis with LIPUS exposure using cDNA microarray analysis.
Methods: Bone marrow cells were obtained from femora of 8-week-old male Sprague-Dawley rats and were suspended in an osteogenic medium. LIPUS exposure was performed for 15 min/day from day 3 to 9 after cell suspension in culture plates (LIPUS group). LIPUS signals were transmitted at a frequency of 3 MHz with a spatial-average intensity of 40 mW/cm² from the bottoms of the cell culture plates. The cultures without LIPUS exposure were harvested in the same manner (Control group). Total RNA was extracted from the both group cell cultures on day 10. From each sample, total RNA was hybridized on the whole rat genome oligo microarray kit. Sirius red staining was performed to quantify collagen deposition on day 4, 7 and 10. Alizarin red-S staining was performed to detect calcified areas of cell cultures on day 10, 14 and 21.
Results: A total number of 45 at least a 2-fold up-regulated genes and 514 at least a 2-fold down-regulated genes in LIPUS group were found in the microarray analysis. The up-regulated genes included cell adhesion-, osteoblast-, collagen- and ossification- related genes. Optical densities with sirius red staining were significantly denser on day 14 in the LIPUS group. The stained areas with alizarin red-S were significantly larger in the LIPUS group than in the control group on each day of the experiments.
Conclusions: The genes, related enhanced osteogenesis under LIPUS exposure, were identified.