Objective      To investigate the effect of EphB6 on osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs).       Methods      The BMSCs seeded on decalcified bone matrix by biphasic seeding were divided into an osteogenic differentiation 5-day group (group A)

an osteogenic differentiation 14-day group (group B)

a non-induced 5-day group (group C) and a non-induced 14-day group (group D). Alizarin red staining was performed to detect calcium nodules. The activity of alkaline phosphatase (ALP) and mRNA expression of Runx2

Osterix and EphB6 were determined by ALP kit and quantitative real-time PCR

respectively. The indicators above were examined again after the BMSCs were treated with soluble ephrinB1-Fc to activate EphB6 in all the groups.       Results      ALP activity and mRNA expression of Runx2 and Osterix increased in group A and group B

and ALP activity (7.32±2.02 King’s unit/100 ml) and mRNA expression of Runx2 (3.442 6±0.258 5) and Osterix (2.482 3±0.329 2) reached the peak levels in group B. EphB6 mRNA expression was downregulated in group A and group B

and reached the bottom level in group B (0.754 3±0.023 5). After the treatment of soluble ephrinB1-Fc for EphB6 activation

a significant reduction of calcium nodules was observed and ALP activity significantly decreased (P0.05). High-dose ephrinB1-Fc showed stronger ALP activity decrease effects than low-dose ephrinB1-Fc (12.20±1.30 vs 15.40±1.03 King’s unit/100 ml

P=0.008). Similarly

the mRNA expression of Runx2 and Osterix was downregulated under ephrinB1-Fc treatment in a dose-dependent manner.       Conclusion      EphB6 inhibits the osteogenic differentiation of BMSCs via mRNA expression regulation of Runx2 and Osterix.