Mechanical Loading Role in Mitochondrial Function/CypD Expression During Osteogenesis
Objectives: Mitochondrial function (MF) plays a fundamental role in osteoblast differentiation and bone formation, providing the most significant energy source through oxidative phosphorylation. Mitochondrial dysfunction (MD) could decrease osteogenic differentiation, trigger mitochondrial DNA release, inflammation, catabolic pathways, and cell death. MD may open a pore in the inner matrix, decreasing its membrane potential (MP) and increasing reactive oxygen species, defined as mitochondrial permeability transition pore (MPTP). The expression of the mitochondrial protein Cyclophilin D (CypD) is directly correlated to MPTP activity. The objective of this study was to investigate the role of mechanical loading on MF and CypD expression during osteogenesis. Methods: For the experimental methods, calvaria preosteoblasts cells (MC3T3-e1) were used for in vitro studies. MC3T3-e1 were cultured in alpha-MEM media for 48 hours and subjected to fluid shear stress for 1 hour in a parallel plate flow chamber using a 1.2 Pa closed flow loop and collected after 24 hours. RT-PCR was performed to determine gene expression of Ppif (CypD) and Runx2 (osteogenesis), and cell staining was done to assess MF. For the in vivo model, transgenic mice overexpressing CypD were stained using the Whole Mount Staining Technique to evaluate osteogenesis. Two-way ANOVA was employed to determine differences between the in vitro sample measurements and their interaction. A power analysis was performed on preliminary biomechanical data for n=14, 7 per group: α=0.05 and β ≤10%. T-test was used for normal distribution differences and a non-parametric test for other distributions. Results: The results for the in vitro studies were an upregulation of Runx2 gene (p=0.0232), and downregulation of Ppif (p=0.0471) with a significant difference in MP of the mitochondria. A greater presence of cartilage was observed for the in vivo samples with overexpression of CypD. Conclusions: In conclusion, mechanical loading stimulates MF due to the downregulation of CypD expression during osteogenesis.