[Dose-dependence of bone morphogenetic protein 2-derived peptide on osteogenic induction in marrow mesenchymal stem cells in vitro].

OBJECTIVE

To investigate the effect of the synthetic bone morphogenetic protein 2 (BMP-2)-derived peptide on the osteogenic induction in the marrow mesenchymal stem cells (MSCs) and to evaluate the osteoinductivity and dose-dependence of the BMP-2-derived peptide in vitro.

METHODS

MSCs of 4-week old Wistar rats were separated and cultured. In the 3rd passage, the conditional culture medium was changed, in which the BMP-2-derived peptide in the following doses was added: 300,200, 100, 50, and 0 microg/ml, respectively (Groups A-E). The activity of alkaline phosphatase (ALP)and the amount of calcium deposition were meassured at 5, 10, 15 and 20 days during the culture with the conditional culture medium. The real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was performed to measure the mRNA expressions of collagen type I, osteopontin (OPN), and osteocalcin (OCN) and to measure the osteoinductivity of the BMP-2-derived peptide in the different concentrations.

RESULTS

Under the inverted phase contrast microscope, MSCs cultured in the conditional culture medium for 3-4 days were changed in shape, from long fusiform to short fusiform or polygon. As the concentration of the BMP-2-derived peptide increased, the time for MSCs to change into the osteoblasts decreased. There was a significantly greater level of the ALP activity and amount of the calcium deposition in Groups A and B than in the other groups (P < 0.05). However, there was no significant difference between Group A and Group B (P > 0.05). The result of FQ-PCR showed that after MSCs were cultured in the different doses of the conditional culture medium for 14 days, the mRNA expressions of collagen type I, OPN and OCN were at higher levels. An increasing order in the level of the cycle threshold (Ct) was found in the following groups: A > B > C > D). Almost no expression was found in Group E. The Ct levels were significantly greater in Groups A and B than in Groups C and D (P < 0. 05). However, there was no significant difference between Group A and Group B (P > 0.05).

CONCLUSION

The BMP-2-derived peptide can greatly promote differentiation of MSCs into the osteoblasts, the promotion of osteogenesis has a dose-dependent pattern, and the best inducing dosage is 200 microg/ml.