In order to verify whether differentiation of adult stem cells toward bone tissue is promoted by high-frequency vibration (HFV), bone marrow stromal cells (BMSCs) were mechanically stimulated with HFV (30？Hz) for 45 minutes a day for 21 or 40 days. Cells were seeded in osteogenic medium, which enhances differentiation towards bone tissue. The effects of the mechanical treatment on differentiation were measured by Alizarin Red test, (q) real-time PCR, and protein content of the extracellular matrix. In addition, we analyzed the proliferation rate and apoptosis of BMSC subjected to mechanical stimulation. A strong increase in all parameters characterizing differentiation was observed. Deposition of calcium was almost double in the treated samples; the expression of genes involved in later differentiation was significantly increased and protein content was higher for all osteogenic proteins. Lastly, proliferation results indicated that stimulated BMSCs have a decreased growth rate in comparison with controls, but both treated and untreated cells do not enter the apoptosis process. These findings could reduce the gap between research and clinical application for bone substitutes derived from patient cells by improving the differentiation protocol for autologous cells and a further implant of the bone graft into the patient. 1. Introduction Despite an increased interest in tissue regeneration, the availability of cells remains a serious limitation for clinical applications in regenerative medicine. Autologous adult stem cells must be expanded and induced along specific cell lines; this process implies long culture periods. Thus, a great challenge consists in finding methods to selectively improve differentiation toward tissue, which would consequently reduce the differentiation period. Several stimulation methods that use different sources of energy have been widely tested to improve the rate of differentiation of primary cell culture to osteoblasts, that is, ultrasound [1, 2] and electromagnetic fields [3–5]. In particular, following a biomimetic approach, mechanical stress has been demonstrated to be a good stimulus. It promotes early cell differentiation into bone [6, 7] and may be achieved by either flow perfusion bioreactors [6, 8] or physical mechanical stress [9, 10]. In fact, cells such as osteoblasts and fibroblasts respond to a variety of stresses, including compression, torsion, and tension. These facilitate matrix turnover and remodelling [11, 12] through mechanical signals which are translated by cell surface receptors via intracellular pathway
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