(F1187) SOFT EXTRACELLULAR MATRIX REGULATING GLUCOSE METABOLISM AND MICROTUBULE ACETYLATION TO PROMOTE OSTEOGENIC DIFFERENTIATION IN 3D CULTURED MESENCHYMAL STEM CELLS
Abstract: The early stage of bone reforming contains low stiffness ECM. Matrix stiffness is crucial to regulating stem cell behavior. However, the mechanism of stem cells respond to matrix stiffness at the early stage of bone remodeling was still unrevealed. Our study aims to explore the effect of ECM stiffness on the differentiation of MSCs in 3D cultures and the potential mechanism. Human mesenchymal stem cells (hMSCs) and GelMA were used to construct a 3D culture system with various matrix stiffness. The osteogenic differentiation level was evaluated both in vivo and vitro. Meanwhile the energy metabolism level was detected to further investigate the mechanism of 3D matrix stiffness effects on MSCs differentiation. In our studies, it was found that the soft (5 kPa) matrix promoted osteogenic differentiation both in vitro [mRNA (~1.4-fold change), ALP staining (~2-fold change)] and in vivo, which was different from 2D culture. Compared with the stiff matrix (~ 20 kPa), the cells in the soft matrix spread faster and showed larger volume (~3000 μm3), had more filamentous-structure-like mitochondria (~80 %) with more active metabolism [ATP content (~1.8-fold change), the oxygen consumption (~2.5-fold change) and acid production rate (~2.7-fold change)]. We focused on the microtubules (MTs) cytoskeleton differences and its effects on mitochondria. Both mRNA level and protein level showed that the acetylation level of α-tubulin (~1.5-fold change) and the expression of microtubule acetylase αTAT1 were significantly up-regulated (~2.5-fold change) in the soft matrix group. The mitochondrial networking, and ALP activity was inhibited in the soft matrix group by si-αTAT1 or eliminating microtubule homeostasis with Taxol. This study revealed that the soft 3D matrix promoted MSCs spreading, cytoskeletal aggregation, mitochondrial networking, and metabolism function, thereby promoting osteogenic differentiation. The cytoskeleton, especially MTs, mediates the network of mitochondria and energy metabolism to regulate osteogenic differentiation. This study demonstrated the important role of soft ECM in the design of tissue engineering materials for bone repair and revealed the necessary effects of soft tissues such as collagen in promoting the osteogenic differentiation of cells at the early stage of bone reformation.
Funding Source: This work was supported by Beijing Natural Science Foundation [L242125, L234072, L242042]; NSFC [11972067, 32171310, 12472315, U20A20390, 12332019]; SKLOD2024OF01; Fundamental Research Funds for the Central Universities.
