Integration of refractory periods in the administration of low-magnitude mechanical signals increases mesenchymal stem cell numbers in the bone marrow

Abstract

High-frequency, low-magnitude mechanical signals (LMMS) applied exogenously as low-intensity vibrations (LIV) have been shown to bias mesenchymal stem cell (MSC) differentiation towards osteoblastogenesis at the expense of adipogenesis. Recent in-vitro work has indicated that refractory periods allow additional benefit from these mechanical signals when scheduled into the loading routine. Work is presented here from three distinct 6-week experiments conducted to investigate the role of refractory periods allowed in the administration of LMMS in an in-vivo animal model. Results from the first experiment using 7-week old male C57BL/6J mice suggested that multiple animal handling had an adverse effect on bone morphology, with more loading periods, and thus more handling, suppressing the relative anabolic response. A second experiment also using 7-week old male C57BL/6J mice employed a timer-controlled semi-automated vibration platform that minimized animal handling by allowing in-cage administration of LMMS. Data from this experiment showed no obvious phenotypic changes in bone morphology between LMMS-treated animals and untreated controls. However, animals that received either 30 minutes or 10 minutes three times daily bouts of LMMS showed a significant increase in MSC numbers (p<0.05) compared those that were not vibrated or only vibrated for 10 minutes daily. Importantly, animals were observed to have remained in a recumbent position when vibrated in their cages; unlike their standing, alert, position when moved to and vibrated in ventilated boxes, raising the issue that the automated system allowed the animals to sleep through the day portion of the diurnal cycle. In a third experiment that attempted to build on observations made from the previous two, 8-week old female BALB/cyJ mice that were vibrated twice daily for 15 minutes, separated by 6 hours, had 1.7 times the number of cells that stained positively for MSC antibodies compared to animals vibrated once daily for 30 minutes (p=0.004). Separating LMMS into discrete loading bouts separated by 6 hours therefore appeared to improve the ability of LMMS to proliferate MSCs. These results provide insight not only into the importance of recognizing the complications that may arise with handling animals, but also into optimizing a potential clinical intervention for increasing MSCs in conditions like aging, obesity, and osteoporosis characterized by a compromised bone marrow cell population.