Effect of Purmorphamine on Osteogenic Differentiation of Human Mesenchymal Stem Cells in a Three-Dimensional Dynamic Culture System

Abstract

We have previously demonstrated that purmorphamine exerts an osteoinductive effect on human bone marrow mesenchymal stem cells (hBM-MSCs) in a two-dimensional (2D) static culture system. In the present study, we have evaluated the effect of purmorphamine on osteogenic differentiation of human mesenchymal stem cells (hMSCs) in a three-dimensional (3D) dynamic culture system vs. a 2D static culture condition. In this study, hMSCs were seeded in either 3D collagen scaffolds using a perfusion bioreactor or on 2D culture plates. One day later, the medium was replaced with osteogenic medium supplemented with dexamethasone, l‐ascorbic acid and β-glycerophosphate in the presence or absence of purmorphamine. Histological staining, flow cytometry, and real-time PCR were conducted for evaluation of osteogenesis in the study groups. The expressions of RUNX-2, alkaline phosphatase (ALP), osteocalcin (OC), collagen I (Col I), and bone sialoprotein (BSP) were compared between the 3D and 2D culture systems at 14 and 21 days post-induction of differentiation. Based on our results, alizarin red staining showed deposition of the mineralized matrix in the 2D static and 3D dynamic cultures. Our flow cytometric analysis indicated that the number of ALP+/OC− cells increased in the presence of purmorphamine in the 2D culture rather than the 3D system at day14. One week later, the numbers of OC+/ALP− and OC+/Stro-1− cells increased significantly in the 3D dynamic model compared with the 2D cultures. Expression of RUNX-2 was upregulated in the presence of purmorphamine in both cultures at day 14. The purmorphamine response gene, Gli-1, was upregulated during both the early and late culture periods in the 3D dynamic system, while similar up-regulation was observed only during the early culture period in 2D culture. No difference was observed in expressions of collagen type I and BSP between groups. The present study indicates that purmorphamine as agonist of Shh signaling pathway affects on osteogenic differentiation program of hMSCs in static and dynamic culture systems.