OBJECTIVETo evaluate the effect of co-culture system of bone marrow mesenchymal stem cells (BMSC) and vascular endothelial cells (EC) on osteogenesis.

METHODSBMSC were isolated by whole bone marrow centrifugal adherent method. Then BMSC were induced into EC with induced medium. Co-culture system in different proportions of BMSC and EC (10:0, 10:1, 8:2, 7:3, 5:5, 3:7, 2:8, 1:10, 0:10) were further evaluated. The cell growth level of BMSC was examined. The CD44 expression of BMSC and von willebrand factor (vWF) expression of vascular EC were examined by immunofluorescence. Furthermore, calcium nodules exhibited by alizarin red staining, alkaline phosphatase activity, and the expression of osteogenic genes by reverse transcription-quantitative PCR (RT-qPCR) were observed to validate the osteogenesis of co-culture system.

RESULTSThe growth curve of P3 passage of BMSC demonstrated the doubling time of BMSC was 39.9 h. The positive specific markers of BMSC and EC showed efficient induction. Although the calcium nodules ratio of the co-culture [group 7:3 (19.0 ± 3.0) and group 5:5 (20.8 ± 2.9)] was not significantly different (P > 0.05), but higher than that of other co-culture groups with a significant difference (P < 0.01). Alkaline phosphatase activity was increased with prolonged induction of osteogenic medium. While alkaline phosphatase activity of group 10:0 (16.84 ± 0.82), group 10:1 (15.86 ± 3.10), group 8:2 (16.37 ± 1.33), group 7:3 (17.99 ± 1.98), and group 5:5 (17.49 ± 0.87) did not show significant difference after osteogenic induction for 7 days (P > 0.05), but significantly higher than that of other co-culture groups (P < 0.05). The co-culture ratio of 7:3 (33.74 ± 0.99) was slightly higher than that of 5:5 (31.09 ± 0.87), but significantly higher than that of other groups (P < 0.01). Moreover, the osteocalcin (OCN) and runt-related transcription factor 2 (RUNX2) expression of group 7:3 was significantly higher than that of other groups.

CONCLUSIONSThe EC that derived from BMSC can promote the BMSC differentiate into osteoblasts. The co-culture system of BMSC and EC with the ratio of 7:3 increases the alkaline phosphatase activity and facilitates the expression of osteogenic genes.

Alkaline Phosphatase ; metabolism ; Bone Marrow Cells ; cytology ; physiology ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Coculture Techniques ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Endothelial Cells ; cytology ; physiology ; Humans ; Hyaluronan Receptors ; metabolism ; Mesenchymal Stromal Cells ; cytology ; physiology ; Osteoblasts ; cytology ; Osteocalcin ; metabolism ; Osteogenesis ; physiology ; Time Factors ; von Willebrand Factor ; metabolism