Abstract
The discovery and application of small molecules is one of the practical strategies of safe osteogenic drugs. The small molecule CHIR99021 (C91) is a highly specific, safe, and most effective GSK-3β Inhibitor. This study found that it efficiently activates the canonical Wnt signaling of bone marrow stromal cell ST2 and promotes osteoblast differentiation and mineralization. C91 increases the production and biochemical activity of osteoblast marker alkaline phosphatase, the expression of osteoblast marker genes Alpl, Bglap, Runx2, and Sp7, and the formation of bone nodules. Triptonide is a transcription inhibitor of Wnt target gene, which diminishes C91-induced osteoblast differentiation in a dose-dependent manner. Meanwhile, C91 also induces autophagy through autophagosome formation and conversion of autophagy biomarker LC-3I into LC-3II. Autophagy inhibitor 3MA partially reduces C91-induced osteoblast differentiation and mineralization; autophagy inducer Rapamycin increases the expression of β-catenin to promote osteogenic differentiation, but cannot alleviate the inhibition of Triptonide on C91-induced osteogenic differentiation, indicating the crosstalk of canonical Wnt signaling and autophagy regulates C91-induced osteoblast differentiation. Furthermore, in order to simulate the in vivo detection of C91 in osteogenesis process, we made a C91 slow-release hydrogel with our newly established polycaprolactone and cell-integrated 3D printing system (PCCI3D module). The sustained release C91 promotes the differentiation and mineralization of ST2 cells. C91 can also enhance the proliferative activity of ST2 cells. The release rate of C91 from hydrogel gradually decreases within 7 days. During this period, the C91 is released by 83.0% and the cell viability maintained at 96.4%. Therefore, the small molecule Wnt agonist C91 promotes osteogenesis through caonical and autophagy-mediated Wnt signaling pathway with an option for translational application.