A newly identified mechanism involved in regulation of human mesenchymal stem cells by fibrous substrate stiffness.

UNLABELLED: Stiffness of biomaterial substrates plays a critical role in regulation of cell behavior. Although the effect of substrate stiffness on cell behavior has been extensively studied, molecular mechanisms of regulation rather than those involving cytoskeletal activities still remain elusive. In this study, we fabricated aligned ultrafine fibers and treated the fiber with different annealing temperatures to produce fibrous substrates with different stiffness. Human mesenchymal stem cells (hMSCs) were then cultured on these fibrous substrates. Our results showed that annealing treatment did not change the diameter of electrospun fibers but increased their polymer crystallinity and mechanical properties. The mRNA expression of RUNX2 was upregulated while the mRNA expression of scleraxis was downregulated in response to an increase in substrate stiffness, suggesting that increased stiffness favorably drives hMSCs into the osteogenic lineage. With subsequent induction of osteogenic differentiation, osteogenesis of hMSCs on stiffer substrates was increased compared to that of the cells on control substrates. Cells on stiffer substrates increasingly activated AKT and YAP and upregulated transcript expression of YAP target genes compared to those on control substrates, and inhibition of AKT led to decreased expression of YAP and RUNX2. Furthermore, macrophage migration inhibitory factor (MIF) was increasingly produced by the cell on stiffer substrates, and knocking down MIF by siRNA resulted in decreased AKT phosphorylation. Taken together, we hereby demonstrate that simply using the annealing approach can manipulate stiffness of an aligned fibrous substrate without altering the material chemistry, and substrate stiffness dictates hMSC differentiation through the MIF-mediated AKT/YAP/RUNX2 pathway. STATEMENT OF SIGNIFICANCE: Stiffness of biomaterial substrates plays a critical role in regulation of cell behavior. Although the effect of substrate stiffness on cell behavior has been extensively studied, molecular mechanisms of regulation rather than those involving cytoskeletal activities still remain elusive. In this manuscript, we report our new findings that simply using the annealing approach can manipulate stiffness of an aligned fibrous substrate without altering the material chemistry, and substrate stiffness dictates human mesenchymal stem cell (hMSC) differentiation through the macrophage migration inhibitory factor-mediated AKT/YAP/RUNX2 pathway. The findings are novel and interesting because we have identified a new mechanism rather than those involving cytoskeleton activity, by which substrate stiffness regulates hMSC behavior.