Effect of substance P on co-cultured osteoblasts and vascular endothelial cells induced from bone marrow stromal cells in rabbits

Objective To study the effect of substance P (SP) on the co-cultured osteoblasts (OB) and endothelial cells (EC) induced from bone marrow stromal cells (BMSCs) in vitro in rabbits.Methods Four culture systems (OB,EC,indirect and direct co-culture of OB and EC at the ratio of 2∶ 1) were respectively established.Of each system,the experimental group was simultaneously added with 1 × 10-8mol/L SP (n =6) while the control was not.After culture for 48 hours,the cell cycles were analyzed by flow cytometry to detect the cell proliferation and activity; the expression of ALP (Alkaline phosphatase) was measured by ELISA; the mRNA expression of OC (osteocalcin) gene in osteoblasts was detected by real-time quantitative PCR.The changing of cell function was compared between the 2 groups in each cell culture system.Results In systems of mere OB culture and indirect co-culture,the cells were significantly decreased at phase G1 and significantly increased at phase G2/M in the experimental groups compared with the controls (P ＜ 0.05).In systems of mere OB culture and indirect and direct co-cuhures,the experimental groups had significantly higher ALP expression (2.877 ±0.188,3.053 ±0.179 and 4.345 ±0.305) and mRNA expression of OC (1.682 ±0.155,3.188 ± 0.213 and 3.720 ± 0.194) than the controls respectively (2.600±0.233,2.842 ±0.120 and 3.220 ±0.191; 1.360 ±0.141,2.272 ±0.143 and 2.507 ±0.176) (P ＜ 0.05).In the system of mere EC culture,there were no significant differences in ALP expression or mRNA expression of OC between the 2 groups (P ＞ 0.05).In the experimental groups,indirect and direct co-cultures produced significantly higher ALP expression and mRNA expression of OC than mere OB culture,and so did the direct co-culture than the indirect co-culture (P ＜ 0.05).Conclusions There is good cellular compatibility between the endothelial cells and osteoblasts induced from BMSCs when the 2 kinds of cells are co-cultured.SP can significantly promote the activity and proliferation of OB in a co-culture system. Key words: Bone marrow cells;  Substance P;  Osteoblasts;  Endothelial cells;  Coculture techniques