Mechanical signals provide regulatory information to prokaryotic and eukaryotic cells, catalyzing adaptive changes in morphology that accommodate new loading challenges. In humans, mechanical signals – most often considered in the context of exercise – affect a range of physiologic systems and are essential in achieving a robust musculoskeletal system, and central to a goal of slowing formation of adipose tissue. Despite general agreement that mechanical signals play a central role in defining fat and bone/muscle phenotypes (Engler et al., 2006
; Lee et al., 2010
), the ideal characteristics of an effective loading regimen, or the molecular cues which control the adaptive responses are poorly understood. Here we provide insights into the characteristics by which physical input controls MSC lineage allocation, and ultimately the formation of both fat and bone.
Extremely small bone strains (<0.003%), induced at a sufficiently high frequency (10–100Hz), are anabolic to bone (Rubin et al., 2001a
). Importantly, mechanically mediated bone remodeling exhibits a strong interdependence of strain magnitude and cycle number, such that bone mass can be enhanced either with a few large strain events or 100,000’s of extremely low magnitude strain signals (Qin et al., 1998
). This leads to the hypothesis that bone structure depends as much on extremely low magnitude, high frequency strains arising during predominant activities (i.e., standing), as it does on rarer, large strain events generated during strenuous activity (Fritton et al., 2000
). Recent evidence confirms that extremely small mechanical signals are both anabolic to bone (Rubin et al., 2001a
; Slatkovska et al., 2010
), and suppress the isocaloric formation of adipose tissue: growing mice subject to a daily LIV signal for 12 weeks acquired about 25% less fat than controls (Rubin et al., 2007b). The work presented here, with the suppression of adipogenesis by the LIV signal as achieved by negligible strain yet significant accelerations, suggests that bone matrix deformation may not be the exclusive agent of mechanotransduction in musculoskeletal tissues (Garman et al., 2007b
The ability of both high and low intensity mechanical challenges to promote bone and decrease fat has been demonstrated in vivo, but difficulty in delivering physiologically relevant mechanical signals to cells in vitro has created hurdles in identifying molecular events that control these adaptive responses. With the loading systems described here it becomes possible to compare the biologic responsiveness to spectral extremes of mechanical signals, and through controlled changes in loading schedules, to begin to optimize influential loading parameters. While prior studies have demonstrated the importance of cell or substrate distortion (Song et al., 2007
), a similar anti-adipogenic response was here obtained with both high and low magnitude strain. Recent in vivo evidence suggests that some mechanical responses even occur in the absence of matrix strain, through acceleration rather than tissue loading (Judex and Rubin, 2010
). As such the physical acceleration of a cell may represent a generic signal that transmits information via cytoskeletal or cell/matrix interrelationships, rather than requiring substrate deviation.
Independent of the magnitude of the regimen, mechanical control of MSC lineage allocation was markedly enhanced when the stimulus was separated into multiple applications separated by refractory periods of at least one hour, indicating that scheduling of events was at least as important as input duration. However, under extreme adipogenic constraints, only the HMS regimen effectively inhibited adipogenesis, suggesting that there is, ultimately, a critical combination of signal parameters necessary to combat environmental cues. Nevertheless, application of LIV three times daily, while adhering to a 3 hour refractory period between bouts, markedly increased the ability of this extremely low intensity mechanical challenge to inhibit fat formation. Interestingly, incorporation of a rest cycle between individual loading cycles has also been shown to enhance cell response, and represents another parameter which can be optimized to maximize mechanical response (Srinivasan et al., 2002
; Batra et al., 2005
). In sum, within a 24 hour period, brief but repetitive loading challenges can develop an adaptive response greater than that achieved in a single extended daily bout.
Although the LIV signal is several orders of magnitude smaller than the mechanical challenge of HMS, both inputs are transduced through the GSK3β locus. Both HMS (Sen et al., 2009
), and LIV as shown here, suppressed adipogenesis through processes involving β-catenin: when β-catenin was deficient, LIV was unable to restrain adipogenesis. As β-catenin activation by LIV treatment paralleled effects of pharmacological GSK3β inhibition it was not surprising that LIV also activated NFATc1 and increased COX2. Although LIV induced signaling events are not as great in magnitude as those resulting from HMS, LIV appears to bias MSC from the adipocyte pathway using similar signaling pathways.
While mechanical induction of osteogenic genes in MSC has been reported (Li et al., 2004
; David et al., 2007
), mechanical inhibition of MSC adipogenesis per se does not reciprocally promote osteogenesis: neither HMS (Sen et al., 2008
) nor LIV induced typical osteogenic genes (Runx2, osterix or osteocalcin). However, BMP2 induction of PTH receptor and osteocalcin was enhanced after pretreatment with LIV, suggesting that biochemical and biophysical factors combine to enhance osteogenesis. As both HMS and LIV stimulate bone formation in animal models (Rubin and Lanyon 1987
; Rubin et al., 2001a
; Garman et al., 2007
; de Oliveira et al., 2010
), it is not surprising that mechanical signals enhance the effect of other environmental factors.
In sum, both high-magnitude/low-frequency and low-magnitude/high-frequency challenges influence the fate of MSC, repressing adipogenesis and protecting their capacity to respond to osteogenic stimuli. Our data suggest that magnitude is not the dominant determinant of efficacy. Further, that the MSC response to mechanical signals is markedly augmented by incorporating a refractory period of at least 1 hour between loading events, indicates that the scheduling of mechanical signals is perhaps as important as the signals themselves. Additional bouts within 24 hours further build upon the response. This ability of the MSC pool to “reset” its sensitivity to a loading challenge suggests that the impact of mechanical regimens, whether strenuous or subtle, might be effectively delivered with several brief periods daily, rather than a single extended session. As such, multiple daily bouts of mechanical signals could be leveraged in cases of rehabilitation and recreation, particularly in the context of the degradation of the musculoskeletal system that accompanies both aging and functional compromise.