Figure 3: Noni leaf extract increases mineralized matrix formation in hPDL cells. Matrix mineralization was assayed by Alizarin Red staining after 6 weeks in culture. (A-C) Phase-contrast micrographs showed the monolayer of primary hPDL cells with spindle-like morphology after 7 days in culture with complete medium, 2.5% (g/ml) noni leaf extract, or β-GP together with ascorbic acid, respectively. (D) Formation of a cell cluster associated with a small nodule (indicated by arrow) was observed in the experimental group treated with 2.5% (g/ml) noni leaf extract after 3 weeks. (E and F) Representative nodules gradually increased in size after 4-6 weeks of the noni leaf extract treatment. (G) hPDL cells cultured in complete medium stained negative for Alizarin Red, indicating that untreated cells did not form mineralized matrix (Note that bright red spots found in the untreated group were an artifact of Alizarin Red precipitation). (H and I) Mineralized nodules formed by noni-treated hPDL cells were stained strongly positive by Alizarin Red. (J) The β-GP with ascorbic acid treated group revealed cell aggregation with only weakly positive Alizarin Red staining after 6 weeks. Scale bars indicate 300 μm (D), and 200 μm (A-C and E-J).