Effect of low-intensity pulsed ultrasound on scaffold-free ectopic bone formation in skeletal muscle.

Background: Low-intensity pulsed ultrasound (LIPUS) is reported to have the effects of rapid appearance and early maturation of ossification in animal models.

Method: We examined the influence of LIPUS on bone formation in C57BL/6J mouse muscle induced by gene transfer of BMP-4 expression plasmid. Electroporation was employed to transfer plasmid DNA. First, an in vitro study was carried out to confirm that LIPUS has no effect on the forced expression of BMP-4 gene transferred by electroporation into C2C12 cells. Next, the BMP-4 plasmids were injected into mouse calf muscles, and transcutaneous electroporation was applied. LIPUS (30 mW/cm(2)) exposure was performed daily for 20 minutes on one side of hind limbs (LIPUS side). The contralateral limbs were not exposed to LIPUS (control side). Muscle samples were collected at 7, 10, 14, and 21 days after electroporation. Soft X-ray films of muscles were taken, and areas of bone formation were measured. After pepsin solubilization of the muscles, calcium and total collagen content were measured.

Results: Radiographical measurements showed significantly more bone formation in the LIPUS side at Day 10. The area of bone was the maximum in both sides at Day 14. The LIPUS side exhibited significant increase in the calcium content at Day 10. The total collagen content with LIPUS exposure was increased significantly over control at Day 10 and 21.

Conclusions: According to these results, accelerated maturation of ectopic bone formation by LIPUS was confirmed at Day 10. Moreover, our results showed that LIPUS increases the total collagen content during osteogenesis.