Metabolic bone disease affects hundreds of millions of people worldwide, and as a result, in vitro models of bone tissue have become essential tools to help analyze bone pathogenesis, develop drug screening, and test potential therapeutic strategies. Drugs that either promote or impair bone formation are in high demand for the treatment of metabolic bone diseases. These drugs work by targeting numerous signaling pathways responsible for regulating osteogenesis such as Hedgehog, Wnt/β-catenin, and PI3K-AKT. In this study, differentiated bone marrow-derived mesenchymal stem cell (BM-MSC) scaffold-free 3D bioprinted constructs and 2D monolayer cultures were utilized to screen four drugs predicted to either promote (Icariin and Purmorphamine) or impair osteogenesis (PD98059 and U0126). Osteogenic differentiation capacity was analyzed over a four week culture period by evaluating mineralization, alkaline phosphatase (ALP) activity, and osteogenesis related gene expression. Responses to drug treatment were observed in both 3D differentiated constructs and 2D monolayer cultures. After four weeks in culture, 3D differentiated constructs and 2D monolayer cultures treated with Icariin or Purmorphamine showed increased mineralization, ALP activity, and the gene expression of bone formation markers (BGLAP, SSP1, and COL1A1), signaling molecules (MAPK1, WNT1, and AKT1), and transcription factors (RUNX2 and GLI1) that regulate osteogenic differentiation relative to untreated. 3D differentiated constructs and 2D monolayer cultures treated with PD98059 or U0126 showed decreased mineralization, ALP activity, and the expression of the aforementioned genes BGLAP, SPP1, COL1A1, MAPK1, AKT1, RUNX2, and GLI1 relative to untreated. Differences in ALP activity and osteogenesis related gene expression relative to untreated cells cultured in a 2D monolayer were greater in 3D constructs compared to 2D monolayer cultures. These findings suggest that our bioprinted bone model system offers a more sensitive, biologically relevant drug screening platform than traditional 2D monolayer in vitro testing platforms.

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