Silk fibroins are biomaterials that have been applied to surgical sutures, drug delivery systems, food supplements, and tissue engineering. Studies have shown the antiadipogenic effects of silk proteins in 3T3-L1 cells and obese mice. Furthermore, other studies have shown that silk proteins increase osteogenic marker expression in osteoblast-like cells. Because osteogenic and adipogenic differentiation from common mesenchymal progenitor cells are often regulated reciprocally, we hypothesized that silk proteins would stimulate osteoblast differentiation. The objective of this study was to evaluate the effects of silk proteins on promoting osteoblast differentiation and identify the underlying mechanism. We showed that silk proteins dose dependently stimulated alkaline phosphatase (ALP) activity, osteoblast differentiation, and induced expression of osteoblast markers in C3H10T1/2 and M2-10B4 multipotent cells. In addition, silk proteins also induced the expression of osteoblast markers in primary rat bone marrow cells isolated from tibiae. Molecular studies showed that silk proteins suppressed the expression of Notch-activated genes and blocked activation of the Notch-specific reporter. Similarly, inhibiting Notch signaling with pharmacologic inhibitors and by small interfering RNA-mediated Notch1 silencing also induced ALP activity and messenger RNA expression. Finally, induction of ALP activity and messenger RNA expression by silk proteins was blunted in Notch1 knock-downed cells, further demonstrating Notch signaling as an important mediator for the pro-osteogenic effects of silk proteins. Taken together, our data suggest that silk proteins may serve as functional foods to promote bone healing and therapeutic interventions for bone fractures and osteoporosis.
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