Fig. 4.

Bone mimetic ECM engineered by cocultured cells facilitated cell proliferation, adhesion and osteogenic differentiation. (a) Schematic diagram of cellular guiding effects of ECM on cell proliferation, adhesion and osteogenic differentiation. M-ECM, MN-ECM and N-ECM were prepared, following with seeding of BMSCs and osteoblasts to assess the effects. (b–c) MN-ECM promoted cell proliferation of BMSCs (b) and MC3T3-E1 (c), especially at the ratio of 9:1 (MC:NIH) (n = 6). (d–i) Cell spreading morphology of BMSCs (d–f) and MC3T3-E1 (g–i) on MN-ECM (9:1) was unique. F-actin of BMSCs (d) and MC3T3-E1 (g) was stained by FITC-phalloidin. Representative images were shown. Scale bars represented 50 μm. Cell area (e&h) and cell perimeter (f&i) were calculated based on images by ImageJ software. Twenty cells (n = 20) were measured for each individual samples and four individual experiments (n = 4) were performed for each group. (j–m) MN-ECM (9:1) promoted osteogenic mineralization of BMSCs (j&k) (n = 3) and MC3T3-E1 (l&m) (n = 4). BMSCs were cultured in osteogenic induction medium (100 nM dexamethazone, 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate). Mineralization was stained by Alizarin red S and captured by a camera (j&l). The stained dye was extracted with cetylpyridinium chloride and measured at 560 nm (k&m).