Objectives: Osteocytes, which reside in bone matrix, are thought to play crucial role in response to mechanical load. However, the effects of mechanical repetitive stimulation on osteocytes are unclear. The aim of this study was to investigate the effect of mechanical repetitive stimulation on cultured osteocyte-like cells in rats.
Materials and Methods: Female Wistar rats (12-week-old) were used. Osteoblasts were isolated from rat calvariae. The cells were cultured in silicon chamber with and without collagen-based matrix (2D culture and 3D culture, respectively). The application of mechanical repetitive stimulation started at 24 hours after the onset of 3D culture. The stimulation was applied at the frequency of 1 Hz and extension rate of 8 %. Histomorphological analyses and quantitative polymerase chain reaction (qPCR) were performed at 0, 24 and 96 hours after the application of mechanical repetitive stimulation. Kruscal Wallis test and Mann-Whitney’s test were used for statistical analyses.
Results: 3D-cultured osteoblasts significantly changed their morphology compared with 2D-cultured osteoblasts with cell process-like structures as well as osteocyte processes. Moreover, gene expressions of Dmp1, FGF23, Tnfsf11, and SOST, which are specific osteocyte markers, significantly increased in 3D-cultured osteoblasts. Hence, 3D-cultured osteoblasts were designated as osteocyte-like cells in this study. Mechanical repetitive stimulation significantly suppressed the decrease number of osteocyte-like cells on time dependent manner. Moreover, mechanical repetitive stimulation significantly upregulated the expression of Bcl2ｌ11, Bak1, Casp3, Casp7 and Ulk1 (autophagy-associated gene) compared with unloaded condition.
Conclusion: Numeral and morphological change of osteocyte-like cells by mechanical repetitive stimulation may be associated with alteration of apoptotic/anti-apoptotic gene and autophagy-related gene expressions.