Putrescine Promotes Human Marrow Mesenchymal Stem Cells to Differentiate along Osteogenic Pathway

Abstract

To investigate the effects of putrescine on the growth and differentiation of human bone marrow mesenchymal stem cells (MSC) to develop a new inductive medium mixture for their osteogenic differentiation. Human bone marrow MSC were collected from three healthy donors and were used to observe the growth-promoting activity of putrescine with MTT test. Experiments were divided into 3 groups: (1) putrescine group, (2) positive control group (presence of dexamethasone, ascorbate, and glycerol phosphate) and negative group (d-alpha with 5% FCS). The cellular expression level of Runx-2 was detected by PCR assay after the culture was maintained for 1 week. After 2 weeks, the intracellular activity of alkaline phosphatase was revealed by histochemistry staining, the phosphatase activity, and the protein concentration in the cell lysates were also detected. Furthermore, MSC were cultured in the presence of putrescine for 2 weeks and Oil-red O staining was performed to reveal the differentiated adipocytes; the cells induced by the standard agent cocktail were used as the positive control. Putrescine promoted the proliferation of human marrow MSC in a dose-dependent manner. MSC exposed to putrescine at a concentration of 100 µmol/L for 1 week expressed greatly higher level of Runx-2, compared with the negative control. Alkaline phosphatase activity was evidently observed after MSC were maintained in the presence of putrescine for 2 weeks. The phosphatase activity contrasted to the protein content in putrescine-treated MSC was significantly higher than that of the control cells (0.87±0.012 vs 0.52±0.010) (P<0.01), and also greatly higher than that of the positive control (0.83±0.029) (P=0.02). Oil red O staining showed that MSC treated by putrescine did not differentiate into adipoblasts. Putrescine can promote the proliferation and osteogenic differentiation of MSC, suggesting the potential application of putrescine as a novel inductive agent for in vitro osteogenesis of MSC.