Inhibition of Wnt Signaling Antagonists Induces Osteogenesis in Vitro

Objectives: The Wnt signaling pathway plays a critical role in craniofacial development. Our previous work in animal models of cleft palate demonstrated the safety and efficacy of targeted inhibition of Wnt signaling antagonists (Dkk1 and Sclerostin). These studies tested the delivery of a small-molecule (IIIc3a) inhibitor of Dkk1, and, a monoclonal antibody (Romosozumab, mAb-Scl), that is an inhibitor of Sclerostin. The primary aim of this study is to analyze the effects of these Wnt agonist therapeutics on a human osteogenic progenitor cell line.
Methods: Human pediatric bone-derived osteoblast-like cells (HBO) were isolated by collagenase digestion of fibular bones from three pediatric patients (HBO19, HBO23, HBO24). Cells were grown in basal media (DMEM + 10% FBS + 1X Antibiotics) or osteogenic media supplemented with or without mAb-Scl, 50ng/ml and/or 25ng/ml and or IIIc3a, 6.5µg/ml and/or 1.5µg/ml and/or BMP-2 (100nM). Alkaline phosphatase (ALP) activity was assessed at day 6, normalized to crystal violet staining, as well as Alizarin Red (AR) staining at day 21. Cell proliferation was quantified by way of the Methyl Green staining assay. Analysis of variance (ANOVA) was conducted against controls after all treatment periods.
Results: HBO cells treated with either low or high dose of mAb-Scl and/or IIIc3a demonstrated a trend toward elevated ALP activity and in vitro mineralization compared to controls. Furthermore, HBO cells treated with combination doses of mAb-Scl and IIIc3a demonstrated exponential trends toward increase in ALP activity.
Conclusions: Targeted inhibition of Wnt signaling antagonists, Sclerostin and Dkk1, resulted in activation of osteoblastic activity and in vitro mineralization in HBO cells. These data provide a molecular insight into our in vivo demonstration that Wnt agonist therapies correct the failure of palatal shelf fusion.