Intensity-related differences in collagen post-translational modification in MC3T3-E1 osteoblasts after exposure to low- and high-intensity pulsed ultrasound.

Abstract

Low-intensity pulsed ultrasound (LIPUS) has distinct effects on biologic mineralization at intensities of <100 mW/cm2. Intensity-dependent differences in the pattern of accelerated mineralization may be due to different alterations in regulation of collagenous matrix formation. However, little is known about the influence of LIPUS on collagen metabolism in the context of mineralization processes. Therefore, we attempted to evaluate differential effects of two intensities of pulsed ultrasound (30 vs. 120 mW/cm2) on collagen post-translational modification and mineralization in osteoblastic MC3T3-E1 cells. Murine osteoblastic MC3T3-E1 cells were exposed to pulsed ultrasound (1.5-MHz, 200-ms burst sine wave at 1.0-kHz frequency, either 30 or 120 mW/cm2 SATA, for 20 min/day from Day 14 to Day 35 postconfluence). Expression patterns of lysyl oxidase (LO), procollagen-lysine, 2-oxyglutarate, 5-dioxigenase 1 (PLOD1, LH1), and 2 (PLOD2, LH2) was examined using quantitative PCR. Quantitative analysis of reducible immature cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible mature cross-links (pyridinoline and deoxypyridinoline) as well as analysis of the maturation of immature to mature cross-links were performed. Exposure to 30 mW/cm2 LIPUS upregulated LH2 mRNA expression and enzyme activity compared to controls. It was associated with increased relative amounts of telopeptidyl hydroxylysine (Hyl)-derived cross-links beginning on Day 14, upregulated LO mRNA expression, increased total reducible and nonreducible cross-links, and increased ratios of newly formed nonreducible to reducible cross-links. Similarities in the pattern of cross-link formation and calcium deposition in matrices between 30 mW/cm2 LIPUS-treated MC3T3-E1 cultures and bone suggest that 30 mW/cm2 LIPUS may promote the maturation of collagenous matrix as a scaffold for calcification. In contrast, exposure to 120 mW/cm2 ultrasound increased calcium accumulation compared to control at Day 35, but increases were delayed until Day 25. No differences in the extent and pattern of cross-links were observed compared to controls. These results suggest that the promotion of mineralization induced by 120 mW/cm2 may be attributed to other factors involved in mineralization process rather than cross-link pattern. Our results demonstrated the existence of differential effects of lower versus higher intensities of ultrasound on mineralization processes in vitro.