To explore differential biological responses of cementoblasts versus osteocytes to mechanical vibration
Mouse osteocyte cell line MLO-Y4 and cementoblatic cell line OCCM.30 cells were cultured in α-MEM supplemented by 2.5% FBS and 2.5% CS (for MLO-Y4), and 10% FBS (for OCCM.30). The cells (up to the 10th passage) were seeded in 6-well plates coated with type I collagen and grew to 70% (for MLO-Y4) and 100% (for OCCM.30) confluency. Completely filled up with α-MEM with 0.5% FBS and sealed, the 6-well plates were subjected to a low-magnitude (0.3g) and high-frequency (30, 60 and 90Hz respectively) mechanical vibration for 1 hour. To explore the possible involvement of MAPK signaling pathway, a specific MEK1 inhibitor - PD98059 (15μM) was added in designated groups during the vibration. After vibration, the cells were lysed to collect total RNA. Total RNA was extracted and reverse-transcribed to synthesize cDNA. For the genes of RANKL, OPG, and 18S, quantitative PCR was used to amplify the cDNA of the samples using gene-specific primers and SYBR Green I. The copy numbers of RANKL and OPG for each experimental group were normalized to 18S (housekeeping gene) rRNA. Each experiment was repeated at least 3 times, and 3-6 samples for each group. One-way ANOVA was used to compare the results among the groups, with p value being set at 0.05.
Result: Among the 4 experimental groups (0, 30, 60, 90Hz), LMHF mechanical vibration significantly reduced the RANKL/OPG ratio specifically at 60Hz for MLO-Y4 cells (45% down, p<0.01, n=6) and 30Hz for OCCM.30 cells (35% down, p<0.05, n=5). When PD98059 was added during vibration, the vibration-induced reduction of RANKL/OPG ratio was obliterated only in MLO-Y4 cells.
Conclusion: LMHF mechanical vibration induces differential biological responses in osteocyte and cementoblasts by different specific frequencies, which is partially mediated by MAPK signaling pathway.
Keywords: Biophysics, Bone, Cell culture, Loading and Orthodontics