Effects of Low Amplitude, High Frequency Vibrations on Proliferation and Differentiation of SAOS-2 Human Osteogenic cell line.

The aim of the work is to understand the consequences of low amplitude, high frequency vibrations on proliferation and differentiation of SAOS-2 cells (sarcoma osteogenetic), an osteoblastic and tumorigenic cell line. We realized a bioreactor composed by an eccentric motor which produces a displacement of 11 mm at frequencies between 1 and 120 Hz on a plate connected to the motor. The cultures of SAOS-2 cells were fixed on the plate and the linear acceleration provoked by the motor to the cultures was measured. We used 30 Hz as stimulating frequency after a preliminary test on the effect of different frequencies on differentiation of cells. Afterwards SAOS-2 cells were stimulated with 30 Hz for different durations, every day for 4 days. The expression of some genes involved in the differentiation process was analyzed firstly with a rt-PCR and afterwards with a real time PCR on the most expressed genes. Moreover the proliferation of cells was evaluated. The results suggest a strong increase in the expression of the genes involved in tissue differentiation in the treated groups with respect to the controls. On the other hand, the proliferation seems to be slowed down, so probably the acceleration perceived by the mechanosensors of the cells changes the cellular cycle by blocking the duplication in order to early differentiate toward bone tissue

Low-amplitude high frequency vibration down-regulates myostatin and atrogin-1 expression, two components of the atrophy pathway in muscle cells

Whole body vibration (WBV) is a very widespread mechanical stimulus used in physical therapy, rehabilitation and fitness centres. It has been demonstrated that vibration induces improvements in muscular strength and performance and increases bone density. We investigated the effects of low-amplitude, high frequency vibration (HFV) at the cellular and tissue levels in muscle. We developed a system to produce vibrations adapted to test several parameters in vitro and in vivo. For in vivo experiments, we used newborn CD1 wild-type mice, for in vitro experiments, we isolated satellite cells from 6-day-old CD1 mice, while for proliferation studies, we used murine cell lines. Animals and cells were treated with high frequency vibration at 30 Hz. We analyzed the effects of mechanical stimulation on muscle hypertrophy/atrophy pathways, fusion enhancement of myoblast cells and modifications in the proliferation rate of cells. Results demonstrated that mechanical vibration strongly down-regulates atrophy genes both in vivo and in vitro. The in vitro experiments indicated that mechanical stimulation promotes fusion of satellite cells treated directly in culture compared to controls. Finally, proliferation experiments indicated that stimulated cells had a decreased growth rate compared to controls. We concluded that vibration treatment at 30 Hz is effective in suppressing the atrophy pathway both in vivo and in vitro and enhances fusion of satellite muscle cells. © 2012 John Wiley & Sons, Lt

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<0.05) in comparison with the control samples. CONCLUSION Although these encouraging findings indicate that high frequency vibration treatment accelerates the differentiation of BMSCs toward bone, other tests should be carried out on BMSCs plated on scaffolds or on specific biomaterials in order to translate this information into clinical application