Nonmulberry Silk Braids Direct Terminal Osteocytic Differentiation through Activation of Wnt-Signaling

Silk polymers can regulate osteogenesis by mimicking some features of the extracellular matrix of bone and facilitate mineralized deposition on their surface by cultured osteoprogenitors. However, terminal differentiation of these mineralizing osteoblasts into osteocytic phenotypes has not yet been demonstrated on silk. Therefore, in this study we test the hypothesis that flat braids of natively (nonregenerated) spun nonmulberry silk A. mylitta, possessing mechanical stiffness in the range of trabecular bone, can regulate osteocyte differentiation within their 3D microenvironment. We seeded human preosteoblasts onto these braids and cultured them under varied temperatures (33.5 and 39 degrees C), soluble factors (dexamethasone, ascorbic acid, and beta-glycerophosphate), and cytokine (TGF-beta 1). After 1 week, cell dendrites were conspicuously evident, confirming osteocyte differentiation, especially, in the presence of osteogenic factors and TGF-beta 1 expressing all characteristic osteocyte markers (podoplanin, DMP-1, and sclerostin). A. mylitta silk braids alone were sufficient to induce this differentiation, albeit only transiently. Therefore, we believe that the combinatorial effect of A. mylitta silk (surface chemistry, braid rigidity, and topography), osteogenic differentiation factors, and TGF-beta 1 were critical in stabilizing the mature osteocytic phenotype. Interestingly, Wnt signaling promoted osteocytic differentiation as evidenced by the upregulated expression of beta-catenin in the presence of osteogenic factors and growth factor. This study highlights the role of nonmulberry silk braids in regulating stable osteocytic differentiation. Future studies could benefit from this understanding of the signaling mechanisms associated with silk-based matrices in order to develop 3D in vitro bone model systems.