Even after several decades of research, the most optimal source of silk for promoting osteogenesis in situ is still a subject of debate. A major gap in existing knowledge is role of underlying signaling mechanisms in both the mulberry and nonmulberry silk species that leads to the development of differential levels of osteogenesis. In our previous study, we elucidated the role of Wnt/β-catenin signaling for promoting superior osteogenic differentiation in nonmulberry silk braids in the presence of TGF-β and pro-osteogenic supplements. Here, we provide a comparative osteogenic analysis of the two most popular silk species (mulberry and nonmulberry silk), in the form of silk braids prepared from natively spun fibers, by conducting detailed gene expression profiling using 25 different osteogenic markers, followed by further validation by immunohistochemistry. Our study provides novel insights into the direct regulatory role of nonmulberry silk fibroin braids on hedgehog and parathyroid signaling pathways in controlling osteogenic differentiation of cultured human fetal osteoblasts (hFOBs), a phenomenon not very evident in the mulberry silk textile braids. Although both silk braids enabled adequate cellular attachment, proliferation, and extracellular collagen matrix formation, superior expression of osteogenic markers (ALP, VDR, Runx2), matrix proteins (Col1A2, OPN), and signaling molecules (GLI1, GLI2, Shh) with characteristic terminal osteocytic phenotype could only be observed in nonmulberry silk. Therefore, our study provided detailed insights into the development of engineered bone to be a prospective tissue equivalent with potential to provide the essential instructive elements for activating physiological pathways of bone differentiation. Such engineered constructs have potential for use as an in vitro model for drug testing and as scaffolds for bone regeneration strategies.
Keywords: hedgehog/parathyroid signaling; in vitro model; mulberry and nonmulberry silk; osteogenesis.