Nano-fibrous scaffolding promotes osteoblast differentiation and biomineralization

Nano-fibrous poly(l-lactic acid) (PLLA) scaffolds with interconnected pores were developed under the hypothesis that nano-fibrous scaffolding would mimic a morphological function of collagen fibrils to create a more favorable microenvironment for cells versus solid-walled scaffolds. In this study, an in vitro system was used to examine biological properties of the nano-fibrous scaffolds compared with those of solid-walled scaffolds for their potential use in bone tissue engineering. Biomineralization was enhanced substantially on the nano-fibous scaffolds compared to solid-walled scaffolds, and this was confirmed by von Kossa staining, measurement of calcium contents, and transmission electron microscopy. In support of this finding, osteoblasts cultured on the nano-fibrous scaffolds exhibited higher alkaline phosphatase activity and an earlier and enhanced expression of the osteoblast phenotype versus solid-walled scaffolds. Most notable were the increases in runx2 protein and in bone sialoprotein mRNA in cells cultured on nano-fibrous scaffolds versus solid-walled scaffolds. At the day 1 of culture, α2 and β1 integrins as well as αv and β3 integrins were highly expressed on the surface of cells seeded on nano-fibrous scaffolds, and linked to this were higher levels of phospho-Paxillin and phospho-FAK in cell lysates. In contrast, cells seeded on solid-walled scaffolds expressed significantly lower levels of these integrins, phospho-Paxillin, and phospho-FAK. To further examine the role of nano-fibrous architecture, we inhibited the formation of collagen fibrils by adding 3,4-dehydroproline to cultures and then assayed cells for expression of α2 integrin. Cells seeded on nano-fibrous scaffolds sustained expression of α2 integrin in the presence of dehydroproline, while suppression of α2 integrin was evident in cells seeded on solid-walled scaffolds. These results provide initial evidence that synthetic nano fibers may exhibit certain properties that are comparable to natural collagen fibers, and thus, the nano-fibrous architecture may serve as a superior scaffolding versus solid-walled architecture for promoting osteoblast differentiation and biomineralization.