High frequency vibration enhances the expression of osteogenic genes and extracellular matrix deposition in human Bone Marrow Stromal Cells (hBMSCs

INTRODUCTION Human bone marrow stromal cells have the potentiality to differentiate into ligament, tendon, muscle, nerve, endothelium and bone [1]. Previous studies have demonstrated the efficacy of high frequency vibration in accelerating the in vitro differentiation of SAOS-2 cells [2] and human Adipose-Derived Stem Cells (hASCs) toward bone tissue [3]. So, we decided to treat BMSC cells with high frequency vibration (HFV) in order to determine whether the promising results obtained with SAOS-2 and hASCs could be extended to hBMSCs. The cells were stimulated with 30 Hz vibrations for 45 minutes a day, for 21 and 40 days as in previous experiments with other cell lines. EXPERIMENTAL METHODS To stimulate the cells, a previously described custom made “bioreactor” was used [2]. We cultured hBMSCs in osteogenic medium (15% Osteogenic Stimulatory supplement™, 10-8M Dexamethasone, 50 µg/mL Ascorbic Acid and 3.5mM β-Glycerophosphate). hBMSCs were divided into two groups of samples: one subjected to mechanical treatment (T) and one as control, (C). We measured the expression of osteogenic genes with (q) Real-Time PCR: OP, RUNX2, ALP and BOSP. In addition we evaluated the levels of the more important osteogenic proteins (collagen I, collagen III, osteocalcin, human decorin, osteopontin, alkaline phosphatase, osteonectin and bone sialoprotein), commonly used to test the level of bone differentiation [4]. RESULTS AND DISCUSSION The results of the (q)Real-time PCR are presented in Fig.1. At 40 days, the expression of BOSP and OP was higher in treated cells with respect to control ones (Fig. 1C and 1D, p<0,001). Also RUNX-2, that is an important transcription factor associated with osteoblasts differentiation, was higher in treated cells with respect to controls (Fig.1B). Also the effects at 21 days was evident: all the osteogenic genes were higher in treated hBMSCs with respect to control cells (Fig.1). In order to evaluate the amount of the extracellular matrix constituents produced by the cells, an ELISA assay was performed. In Table 1, the protein content results are presented for treated and control samples, as fg/(cells x dish). At 21 days and at 40 days the deposition of bone proteins in HFV stimulated samples was considerably enhanced (p