4 – SYNERGISTIC EFFECT OF RHBMP-7 AND OSTEOGENIC DIFFERENTIATION MEDIUM ON HUMAN BONE MARROW-DERIVED PRIMARY MESENCHYMAL STEM CELLS IN VITRO

Abstract

Purpose: The current clinical treatment protocol for bone healing applies super-physiological dose of rhBMP7. Unfortunately, it may result in adverse side effects. Some studies have demonstrated a dose-dependent osteogenic differentiation using rodent bone marrow derived stem cells (BMSCs). However, the dose effect of BMP7 on osteogenic differentiation of normal human BMSCs is largely unknown. In the present study, we investigated in vitro osteogenic differentiation of hBMSCs with a gradient concentration of rhBMP7. The interaction between rhBMP7 and osteogenic differentiation medium (ODM) was also examined.

Method: The primary BMSCs from human bone marrow were cultured and maintained in MSC growth medium (MGM). Six study groups were designed: MGM only, MGM with rhBMP7 of 0.1ug/ml, ODM only, and ODM with 3 concentration of rhBMP7 of 0.01μg/ml, 0.1μg/ml, and 1.0μg/ml, respectively. Alkaline phosphatase level (ALP) at day 17 and cumulative calcium deposit at both day 17 and day 35 were examined. mRNA expression level of osteogenic markers including osteocalcin (OC), osteopontin (OPN) and ALP were quantified using real-time RT-PCR at day 17.

Results: ALP activity at day17 did not increase in MGM with or without 0.1μg/ml of rhBMP7, ODM alone and ODM with 0.01μg/ml of rhBMP7. ALP activity was much higher with 0.1μg/ml of rhBMP7 plus ODM (0.22±0.02IU) than that in MGM with 0.1μg/ml of rhBMP7 (0.01±0.01IU, P< 0.05).

Conclusion: Our study demonstrated that rhBMP7 induced osteogenic differentiation of hBMSCs in a dose-dependent manner in the presence of ODM and the minimal dose for inducing in vitro osteoblastic differentiation was 0.1ug/ml of rhBMP7 under synergistic effect of ODM. The results of this study provide some insights into further investigation of synergy of rhBMP7 with other molecules. The types and amounts of simple molecules could significantly reduce therapeutic dose of rhBMP7 and achieve equivalent or better outcomes in clinical application warrant further investigation.