Effects of low-intensity pulsed ultrasound of different acoustic parameters on viability of cultured chondrocytes and ex vivo growth of mouse embryonic metatarsals

Abstract

Objective To investigate the effects of low-intensity pulsed ultrasound (LIPUS) on the viability, proliferation, differentiation and apoptosis of cultured chondrocytes and ex vivo development of embryonic mouse metatarsals. Methods Chondrocytes isolated from neonatal mice (3-5 days old) and metatarsals from mouse embryos (18 days of gestation) were both divided into control group and 3 LIPUS treatment groups for LIPUS exposures with acoustic intensity and duty ratio combinations of 30 mW/cm2-20% (LIPUS Ⅰ), 30 mW/cm2-40% (LIPUS Ⅱ), and 50 mW/cm2-20% (LIPUS Ⅲ); all the other ultrasound parameters were identical (central frequency of 1.5 MHz, repetition frequency of 1 kHz and exposure time of 20 min per day). After LIPUS treatments, the chondrocytes were examined for changes in cell viability, proliferation and apoptosis using cell counting kit-8, EdU assay and TUNEL assay, respectively. The mRNA expression levels of cartilage matrix-related genes were examined by qRT-PCR. The changes in the total length and cartilage length of the metatarsals were measured after the treatments. Results Exposure to LIPUS significantly enhanced the viability and proliferation and reduced the apoptotic rate of the chondrocytes, and the effect was the most obvious in LIPUS Ⅱ group. LIPUS treatment significantly up-regulated the mRNA expressions of Col Ⅱ and proteoglycan and reduced the mRNA expression of Col X in the chondrocytes (P < 0.05). As for the metatarsals, the growth rates of the total length and cartilage length were significantly greater in LIPUS Ⅱ and LIPUS Ⅲ groups than in the control group (P < 0.001), but no significant difference was found between LIPUS Ⅰ group and control group. Conclusion LIPUS treatment can promote bone growth by stimulating chondrocyte proliferation and extracellular matrix synthesis while inhibiting cell apoptosis. These effects are related with the acoustic parameters of LIPUS, and the parameter setting with an acoustic intensity of 30 mW/cm2 and a duty ratio of 40% can be optimal for LIPUS treatment.

Keywords

• low-intensity pulsed ultrasound
• intensity
• duty ratio
• chondrocytes
• bone culture
• mice