gms | German Medical Science

Objectives: Experimental studies reported that the effects of mechanostimulation by low-magnitude high-frequency vibration (LMHFV) on fracture healing are highly dependent on the estrogen status. LMHFV significantly improved fracture healing in ovariectomized (OVX), estrogen-deficient mice, whereas bone formation was significantly reduced in non-OVX, estrogen-competent mice. On a molecular level, expression of estrogen receptors (ER) a and b was differentially regulated. We hypothesized that ERs may mediate vibration-induced effects on fracture healing. Therefore, the aim of this study was to investigate the role of ERa/b during LMHFV using ER-KO mice in vivo and to analyze molecular targets of vibration in osteoblasts in vitro.

Methods: Female homozygous ERa-, ERb-KO and WT mice were provided by Charles River. When aged 12 weeks, half of the mice were subjected to OVX, whereas the other half was sham-operated (non-OVX). After 4 weeks, all mice underwent standardized unilateral femur osteotomy. Half of the mice were treated with LMHFV for 20 min/day, 5 days/week (0.3g, 45Hz). Mice were sacrificed 21 days after surgery and fractured femurs were subjected to bending test, mCT analysis and histology. MC3T3-E1 osteogenic cells were subjected to LMHFV in vitro. Effects or ERa were tested by using ERa-siRNA. Differentiation and proliferation were assessed by qPCR, MTT and BrdU test. Statistical analysis was performed by ANOVA/LSD post-hoc test (p<0.05; n=6-9).

Results and conclusion: LMHFV disturbed fracture healing in non-OVX WT mice, as indicated by reduced bone formation and flexural rigidity in the fracture callus. In contrast, fracture healing was improved by LMHFV in OVX WT mice, as indicated by increased bone formation and callus flexural rigidity. Osteoblast number was reduced by LMHFV in non-OVX but increased in OVX WT mice. A lack of ERa completely abolished the vibration effects in both non-OVX and OVX mice. In contrast, ERb-KO mice displayed similar effects of vibration as WT mice. In vitro, osteogenic cells displayed increased proliferation and Cox-2 gene expression after LMHFV in the absence of estrogen. These effects were abolished by adding estrogen to the medium or by knockdown of ERa.

Our data confirmed previous findings, that bone formation in the fracture callus is disturbed in non-OVX mice but enhanced in OVX mice by LMHFV. On a cellular level, distinct effects of LMHFV on osteoblast numbers may be responsible for the respective changes in the callus bone content. A lack of ERa completely abolished LMHFV-induced effects on bone formation and osteoblast numbers, whereas the absence of ERb did not alter these effects. This suggests a critical role of ERa, but not ERb, during mechanotransduction of vibration in the fracture callus, both under estrogen-sufficient and -deficient conditions. On a molecular level, direct effects of vibration on proliferation and the mechanoresponsive Cox-2 gene in osteoblasts, mediated by ERa, may account for the influence of LMHFV on bone formation.