Figure 3.

Mesenchymal stem cells differentiating into the osteoblast lineage. (A) Alkaline phosphatase (AP) quantification at 5 days after the differentiation cascade is initiated. (B) Alizarin red (AR) staining and quantification for calcium deposits 14 days after mesenchymal stem cells have been in culture under different conditions as indicated. Both AP and AR signals have been represented as fold increase or decrease of the Control +L-AA/β-GP case. A single factor ANOVA test allowed rejection of the null hypothesis for all assays; and a Tukey test between all groups was performed (s.d., n=9, ** p < 0.01; * p < 0.05; ns = not significant all others are p < 0.001). Temporal expression patterns of osteogenic markers in mesenchymal stem cells. Quantification of osteogenic markers (C) Osteocalcin, (D) visualized (green) from differentiating cells by fluorescence over the course of the study. (E) Osteoprotonegrin and (F) Osteopontin expression follows a similar trend to osteocalcin. In all the assays, the synergistic effect of having an osteoconductive and an osteoinductive layer maximizes AP and AR signals. Expression of osteogenic markers may be accelerated and amplified by the introduction of the osteophilic multilayers leading to an environment conducive for bone formation. Peak and cumulative expression levels of the combination osteoconductive and an osteoinductive multilayers are statistically significant compared to other groups (p<0.001, Fig S9–S11).