The Effects of Low-Intensity Pulsed Ultrasound Exposure on tissue regeneration

Objectives: The purpose of the present study was to elucidate the effect of low-intensity pulsed ultrasound (LIPUS) on tissue regeneration.
Methods: Periodontal ligament-derived fibroblasts (HPLF), human ovarian cancer-derived fibroblastic cell line HeLa and oral squamous cell carcinoma-derived cell line KB were used. HPLF and KB cells were cultured in RPMI 1640 medium, and HeLa cells were cultured in Dulbecco's Modified Eagle Medium at 37 ^oC with 5 % CO₂. The cells were plated on a 35mm dish. The plates were set on a Ultrasound Transducer and exposed to LIPUS (3.0 MHz, pulse cycle 20 %, 30 mW/cm²) from the back of the dish for 15 min. After stimulation, the cells were cultured for 0, 15, 30, 45, 60 and 180 min for RNA purification or 1, 3, 6, 12 h for cell lysate collection. The expression level of connective tissue growth factor (CTGF) and heat shock protein 70 (HSP70) and the phosphorylation of ERK1/2(p-ERK1/2) were examined by real-time PCR or Western blotting.
Results: Real-time PCR revealed that both CTGF and HSP70 mRNA were augmented by LIPUS stimulation in HPLF (5 times) and HeLa cells (1.5times), but not in KB cells. The peak induction was observed at 45 min in HPLF or right after stimulation in HeLa cells, respectively. HSP70 expression in HeLa was further examined by Western blotting and peak at 3 h of stimulation. Based on these observations, the signaling pathway was further examined using HeLa cells. The results indicated that LIPUS stimulation induced p-ERK1/2 in a time-dependent manner. The prominent phosphorylation was observed after 30 min of stimulation.
Conclusions: All these results indicated that LIPUS stimulation augmented the tissue regeneration factors such as CTGF and HSP70 in a cell-type specific manner. The clear induction was observed in fibroblastic cells, but not in epithelial cells, through ERK1/2-dependent signaling pathway.