Figure 4: Noni leaf extract increases mineralized matrix formation in MC3T3-E1 pre-osteoblasts. Matrix mineralization was assayed by Alizarin Red staining after 6 weeks in culture. (A) A confluent monolayer of MC3T3-E1 cells was observed in the untreated group without any cellular nodule formation. (B and C) Formation of multilayered MC3T3-E1 cells and nodules occurred after 3 weeks with 2.5% (g/ml) noni leaf extract or β-GP with ascorbic acid, respectively. (D) Untreated cells remained unstained by Alizarin Red. (E and F) MC3T3-E1 cells treated with noni leaf extract or β-GP with ascorbic acid, respectively, produced nodules that stained positively by Alizarin Red. (E) The strongest staining was found in the noni leaf extract group at the end of the culture period. Scale bars indicate 300 μm.