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Current approaches for the treatment of skeletal defects are suboptimal, principally because the ability of bone to repair and regenerate is poor. Although the promise of effective cellular therapies for skeletal repair is encouraging, these approaches are limited by the risks of infection, cellular contamination, and tumorigenicity. Development of a pharmacological approach would therefore help avoid some of these potential risks. This study identifies transforming growth factor beta (TGFβ) signaling as a potential pathway for pharmacological modulation in vivo. We demonstrate that inhibition of TGFβ signaling by the small molecule SB431542 potentiates calvarial skeletal repair through activation of bone morphogenetic protein (BMP) signaling on osteoblasts and dura mater cells participating in healing of calvarial defects. Cells respond to inhibition of TGFβ signaling by producing higher levels of BMP2 that upregulates inhibitory Smad6 expression, thus providing a negative feedback loop to contain excessive BMP signaling. Importantly, study on human osteoblasts indicates that molecular mechanism(s) triggered by SB431542 are conserved. Collectively, these data provide insights into the use of small molecules to modulate key signaling pathways for repairing skeletal defects.

Bone regeneration is a complex event that requires the interaction of numerous growth factors. Fibroblast growth factor (Fgf)-ligands have been previously described for their importance in osteogenesis during development. In the current study, we investigated the role of Fgf-18 during bone regeneration. By utilizing a unicortical tibial defect model, we revealed that mice haploinsufficient for Fgf-18 have a markedly reduced healing capacity as compared with wild-type mice. Reduced levels of Runx2 and Osteocalcin but not Vegfa accompanied the impaired bone regeneration. Interestingly, our data indicated that upon injury angiogenesis was not impaired in Fgf-18(+/-) mice. Moreover, other Fgf-ligands and Bmp-2 could not compensate for the loss of Fgf-18. Finally, application of FGF-18 protein was able to rescue the impaired healing in Fgf-18(+/-) mice. Thus, we identified Fgf-18 as an important mediator of bone regeneration, which is required during later stages of bone regeneration. This study provides hints on how to engineering efficiently programmed bony tissue for long bone repair.

To delineate the competence window in which canonical wingless (Wnt)-signaling can either inhibit or promote osteogenic differentiation, we have analyzed cells with different status, specifically undifferentiated mesenchymal cells, such as adipose-derived stem cells and embryonic calvarial mesenchymal cells, and differentiated mesenchymal cells such as juvenile immature calvarial osteoblasts and adult calvarial osteoblasts. Our analysis indicated that undifferentiated mesenchymal cells and juvenile calvarial osteoblasts are endowed with higher levels of endogenous canonical Wnt signaling compared to fully differentiated adult calvarial osteoblasts, and that different levels of activation inversely correlated with expression levels of several Wnt antagonists. We have observed that activation of canonical Wnt signaling may elicit opposite biological activity in the context of osteogenic differentiation depending on the status of cell, the threshold levels of its activation, and Wnt ligands concentration. The results presented in this study indicate that treatment with Wnt3 and/or expression of constitutively activated#x03B2;-catenin inhibits osteogenic differentiation of undifferentiated mesenchymal cells, whereas expression of dominant negative transcription factor 4 (Tcf4) and/or secreted frizzled related protein 1 treatment enhances their osteogenic differentiation. Wnt3a treatment also inhibits osteogenesis in juvenile calvarial osteoblasts in a dose-dependent fashion. Conversely, Wnt3a treatment strongly induces osteogenesis in mature calvarial osteoblasts in a dose-dependent manner. Importantly, in vitro data correlated with in vivo results showing that Wnt3a treatment of calvarial defects, created in juvenile mice, promotes calvarial healing and bone regeneration only at low doses, whereas high doses of Wnt3a impairs tissue regeneration. In contrast, high doses of Wnt3a enhance bony tissue regeneration and calvarial healing in adult mice. Therefore, the knowledge of both endogenous activity of canonical Wnt signaling and appropriate concentrations of Wnt3a treatment may lead to significant improvement for bony tissue engineering, as well as for the efficient implement of adipose-derived stem cells in bone regeneration. Indeed, this study has important potential implications for tissue engineering, specifically for repair of juvenile bone defects.