Title: Bacterial flagella as an osteogenic differentiation nano-promoter

Abstract: Flagella as protein nanofibers (∼14 nm wide) on the surface of swimming bacteria are molecular machines for assisting bacteria to swim in the liquid. They are mainly assembled from protein subunits (FliC) that can be genetically engineered to display peptides. However, so far, no study has been made to show whether flagella with or without displaying peptides could direct stem cell fate. Here we show that flagella detached from bacteria could promote the osteogenic differentiation of bone marrow derived mesenchymal stem cells (BMSCs), and the display of a functional peptide and mineralization of bone mineral (hydroxylapatite, HAP) on the flagella further collectively enhance the promotion effect. The functional peptide is made of two fused amino acid sequences, RGD and E8, which are responsible for promoting cell adhesion onto flagella-bearing substrates and inducing HAP mineralization on flagella from an HAP-supersaturated solution, respectively. Our work shows that the unique nanotopography and surface chemistry of both mineralized and non-mineralized flagella enable them to present physical and chemical cues favoring the osteogenic differentiation of stem cells. Thus flagella are nanofibrous osteogenic differentiation promoters that can be used to build extracellular matrix-like materials.