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Mechanical Stimulation

Most of the occurring bone fractures heal on their own. However about 10 percent of them end up forming a delayed-union or non-union bone. The performance of the invasive procedures is a way to deal with the bone pathologies, which often does not provide a satisfactory life quality to the patients. Low-Intensity Pulsed Ultrasound (LIPUS) is an alternative treatment of bone fractures, which was approved by FDA and represents a non-invasive and non-ionizing technique.

Numerous in-vitro and in-vivo studies confirmed the beneficial effects of ultrasound at all stages of the bone healing process (ref), with many of them remaining controversial (summarized in Puts et al., 2014). This happens due to inadequate quantification of the acoustic dose, used for the stimulation-experiments, hindering the understanding of the complex physical phenomena, introduced by ultrasound to the stimulated specimen and, this in turn, the biological mechanism.

In our laboratory we use a Focused Low-Intensity Pulsed Ultrasound set-up, which provides controlled sound-field geometry and direct deposition of well-defined acoustic dose, which is provided by stimulation of cells with far-field of the focused transducer. The set-up is depicted in Figure.

Schematic representation of SAM500 cell stimulation set-up used for in-vitro FLIPUS experiments in 24- well cell-culture plates.

As a result of our studies we showed that acoustic dose of 20.9 mW/cm2 enhances proliferation of rat mesenchymal stem cells (rMSCs) and murine pre-osteoblastic MC3T3-E1 cells, which were placed in the reduced-serum medium. The former cells also expressed more osteogenesis-associated genes on day 5, when FLIPUS was applied, which was quantified by RT-PCR. The up-regulated expression of early response genes c-jun and cyclooxygenase 2 (cox-2) was observed 30 min after the FLIPUS-stimulation in MC3T3-E1s.

In order to understand these changes upon the FLIPUS-exposure, we also look at the signaling pathways, possibly mediating these effects. One of them is BMP-signaling pathway. In our experiments we observed that combinational treatment of MC3T3-E1s with FLIPUS and BMP-2 results in more enhanced phosphorylation/activation of SMAD1/5/8 transcription factors, regulating the expression of the genes, associated with bone-morphogenesis. These results were reported on International Ultrasonics Symposium 2014 in Chicago, USA.

Using the FLIPUS system we also were able to show that osteogenic differentiation of rMCSs is dependent on the age of the rats they were isolated from. These results await publication.