H&E indicated the presence of calcified tissue in DE-DM constructs after 1 wk in vivo culture (A), but not in DE (B), or DM cell only (C) constructs. Greater numbers of calcified nodules were observed in both DE-DM cell constructs (D) and DE cell only constructs (E) after 4 wk in vivo culture. DM cell alone constructs (F) exhibited no evidence of calcified tissue formation. G) High magnification view of the black boxed area in Panel E viewed under light (G) and polarized light (H), and von Kossa stained. Abbreviations: de, DE cells with Matrigel; dm, DM cells with Collagen gel; m, Matrigel without DE cells; c, Collagen gel without DM cells. Scale bar=100 μm (A–F), and 20 μm (G–I).