ラット骨髄由来の骨芽細胞様細胞への低出力超音波パルスの影響

抄録

Objective: Acceleration of bone and titanium integration is a critical concern in dental implantation. Low-intensity pulsed ultrasound (LIPUS) has been widely used for accelerating healing of bone fracture in orthopedic practice. Some studies demonstrated that LIPUS accelerated bone formation around a titanium implant in vivo animal model. We hypothesized that LIPUS would enhance osteoblastic differentiation. To test the hypothesis, we examined the effects of exposure to LIPUS on the behaviors of osteoblastic cells derived from rat bone marrow cells.
Materials and Methods: A LIPUS exposure system that could generate a 2 msec burst of 3.0 MHz sine waves repeated at 100 Hz was constructed for the cell culture study model. Bone marrow cells, obtained from the femurs of 8-week male SD rats, were suspended in α-MEM supplemented with 15% fetal bovine serum, 10^-8M dexamethasone, 10 mM Na-β-glycerophosphate and 50 μg/ml L-ascorbic acid 2-phosphate. The suspension was poured into 12-multiwell cell culture plates and cultured at 37℃ in an atmosphere supplemented with 5% CO₂ . The treatment group was exposed to LIPUS underneath culture plates with a coupling gel for 15 min/day from day 3 after primary seeding (LIPUS group). The intensity of LIPUS was 40 mW/cm². The control group without ultrasound treatment was cultivated in the same manner. Cell proliferation was observed by using the WST-8 assay. Collagen synthesis was measured by using a colorimetric method of sirius red dye. von Kossa staining was performed to analyze the calcification nodule formation, and the orthocresolphthalein complexone method was performed to detect the calcium deposition in each cell culture tissue. Total RNA was extracted from each group and realtime quantitative polymerase chain reaction analysis after reverse transcriptase reaction was performed to quantify the osteoblastic gene expressions.
Results: Cell proliferation in the LIPUS group was more suppressed than that in the control group on day 7 (p＜0.01). Collagen synthesis in the LIPUS group was further developed than that in the control group on day 14 and 21 (p＜0.01). The total area of bone nodule in the LIPUS group was wider than that in the control group on days 14 and 21 (p＜0.01) and calcium concentration in the culture of the LIPUS group was thicker than in the control group on days 7, 14 and 21 (p＜0.01). Osteocalcin and osteopontin gene expressions were more upregulated in the LIPUS group than in the control group on days 7 and 14 (p＜0.01).
Conclusion: LIPUS suppressed the proliferation of osteoblastic cells, but enhanced osteoblastic differentiation and calcification in the in vitro cell culture model.