Cellular metabolomics reveals the osteogenic mechanism of MC3T3-E1 cells in porous Ti6Al4V scaffolds under low-intensity pulsed ultrasound

Introduction Porous titanium alloy scaffolds offer multiple advantages in treating large-area bone defects; however, their poor bioactivity limits their application. Low-intensity pulsed ultrasound (LIPUS) serves as a non-invasive method to enhance bone regeneration in porous titanium-6aluminium-4vanadium (Ti6Al4V) alloy scaffolds. This study aimed to examine the biological behavior and metabolism of murine osteoblastic MC3T3-E1 cells in porous Ti6Al4V (pTi64) scaffolds under LIPUS. Methods MC3T3-E1 cells were incubated in pTi64 scaffolds and stimulated with LIPUS. Cell morphology, viability, and osteogenic-related gene expression were evaluated in vitro . Intracellular metabolites of MC3T3-E1 cells incubated in pTi64 scaffolds and stimulated with LIPUS for 7 days were analyzed using an ultraperformance liquid chromatography coupled with a triple quadrupole-linear ion trap mass spectrometer (UPLC-QTRAP-MS)-based cellular metabolomics approach. Results In vitro , MC3T3-E1 cells in the pTi64+LIPUS group exhibited enhanced morphology, viability, and osteogenic-related gene expression compared with the pTi64 group, suggesting that LIPUS enhances cell morphology, reduces apoptosis, and promotes osteogenic differentiation in pTi64 scaffolds. LIPUS treatment altered the cellular metabolic network; 86 differential metabolites were identified. Alterations in metabolic pathways were associated with the one-carbon pool by folate, pantothenate and coenzyme A biosynthesis, as well as the metabolism of amino acids, glycerolipids, and arachidonic acid. Discussion These findings offer novel insights into the osteogenic mechanisms of MC3T3-E1 cells in pTi64 scaffolds under LIPUS.