From the time of development and throughout the life of an organism, cells of the body are constantly exposed to a variety of mechanical stimuli through the actions of muscle forces, gravity, blood flow, and other physical processes. The interactions between cells and mechanical factors are critical to the health and function of various tissues and organs of the body, and are also believed to play an important role in a variety of disease states such as atherosclerosis, osteoarthritis, and osteoporosis (Ingber, 2003
). Importantly, there is mounting evidence that mechanical factors can significantly influence the process of development, and may play critical roles in controlling stem cell fate and lineage determination.
As early as the last century, scientists recognized that the mechanical environment could influence development (reviewed in (Estes et al., 2004
)). For example, in a series of experiments in the 1930’s and 1940’s, Glücksmann demonstrated that cultured chick rudiments under static compression following displacement of the periosteum and perichondrium resulted in cartilaginous tissue formation whereas tensile stresses promoted bone formation (Glücksmann, 1942
). In similar fashion, paralysis of chick embryos resulted in a loss or significant inhibition of cartilage formation, while a mechanical environment in the form of membranous bone articulation resulted in secondary cartilage formation (Fang and Hall, 1995
; Hall and Herring, 1990
; Murray and Drachman, 1969
). The common thread in all of these early studies was an emphasis on the undeniable influence of the mechanical environment during development.
Despite such in vivo
evidence, little was known regarding the biomechanical and biochemical mechanisms by which such mechanical factors could affect gene expression and the determination of stem cell fate. One major difficulty in studying such interactions has been the complexity in determining the precise nature of mechanical “signals” perceived by stem cells in vivo
. For example, simple mechanical loading of tissues results in complex physical environments that consist of time-varying stress, strain, fluid flow and pressure, and potentially, other biophysical changes such as osmotic pressure or electric fields that are generated by the ubiquitous presence of fixed and mobile electric charge on biological molecules (Guilak et al., 1997
). In principle, these changes in the microenvironment may significantly alter the structure of ECM proteins and the activity of soluble growth factors and cytokines. As such, it is difficult to isolate the effects of mechanical force in vivo
from indirect effects associated with mechanically-driven changes in adhesive cues and/or paracrine signaling, and as described earlier, subsequent changes in cell shape. Nonetheless, mechanical forces can directly affect cellular function, and the transduction processes by which cells “sense” applied physical stimuli are only recently being uncovered (Liedtke and Kim, 2005
Importantly, cells are not simply passive biomaterials with constant mechanical properties, but rather use signals from the ECM to “tune” their mechanical properties by dynamically remodeling their cytoskeletal networks. Thus cellular responses to mechanical perturbations are not only a function of the input stimuli, but are also determined by the coupling of these stimuli to mechanosensitive changes in the cytoskeletal organization, interaction with the ECM, and cellular force production. In some experimental systems, these cell-generated forces may be necessary or sufficient to influence stem cell differentiation (Engler et al., 2006
; McBeath et al., 2004
). In this context, the field of “mechanobiology” has exploded in the past few years in characterizing the response of stem cells to more highly controlled mechanical (and other physical) loading, and in determining the biophysical mechanisms and biochemical signal transduction pathways that regulate lineage commitment (Wang and Thampatty, 2008
). As the biophysical signals to which stem cells respond may involve a variety of secondary factors that are engendered in the ECM subsequent to initial mechanical loading, several studies have attempted to isolate the influence of specific physical stimuli such as cell stretch (i.e., tension), compression, or fluid shear stress on stem cell behavior.
For example, the influence of cyclic strain on MSC phenotype has been studied extensively in vitro
for vascular tissue engineering. MSCs cultured on various protein coated flexible membranes and subjected to 5% or 10% cyclic uniaxial stretch demonstrated commitment toward a myogenic phenotype as noted by the expression of smooth muscle actin (SMA) among other factors (Gong and Niklason, 2008
; Hamilton et al., 2004
; Park et al., 2004
; Yang et al., 2000
). However, strains of 1% or 15% failed to either induce commitment to the myogenic lineage or caused a decrease in smooth muscle cell markers, pointing to the importance of the magnitude of strain during differentiation (Yang et al., 2000
). The strain-induced myogenic phenotype was dependent on the protein to which the cells were attached; not coating the substrate resulted in loss of strain-induced myogenesis (Gong and Niklason, 2008
). The effect of the mechanical environment is also dependent on cell type, as adipose-derived stem cells subjected to a similar uniaxial strain protocol (10% uniaxial cyclic strain at 1Hz for 7 days) showed decreased expression of myogenic markers (Lee et al., 2007
Uniform biaxial strain has also been applied in vitro
to MSCs to enhance osteogenic differentiation as noted by increases in specific osteogenic markers, namely Runx2, osterix, alkaline phosphatase, and calcium deposition (Sen et al., 2008
; Simmons et al., 2003
; Thomas and el Haj, 1996
; Yoshikawa et al., 1997
). Also, for bone tissue engineering, pulsatile fluid flow has been shown to upregulate osteogenic markers in adipose-derived stem cells, but only after some degree of osteogenesis had occurred (Knippenberg et al., 2005
) further alluding to the potential differential effects the mechanical environment may have dependent on both the cell type and differentiated phenotype of the cell.
In other studies, cyclic mechanical strain has been shown to induce mouse embryonic stem cell differentiation into vascular smooth muscle cells (Shimizu et al., 2008
). Flk-1-positive (Flk-1+) embryonic stem cells subjected to cyclic strain (4–12% strain, 1 Hz, 24 h) showed significant increases in proliferation and reoriented perpendicular to the direction of strain, exhibiting dose-dependent increases in smooth muscle alpha-actin and smooth muscle-myosin heavy chain at both the protein and gene level. Interestingly, inhibition of platelet-derived growth factor receptor beta completely blocked the mechanically-induced differentiation of embryonic stem cells, suggest that activation of the receptor by cyclic strain plays a critical role in vascular smooth muscle cell differentiation from Flk-1+ ES cells.
Mechanical strain has also been shown to inhibit the differentiation of human embryonic stem cells, while promoting self-renewal without selecting against survival of differentiated or undifferentiated cells (Saha et al., 2006
). Interestingly, stem cells cultured while being cyclically strained retained pluripotency, evidenced by their ability to differentiate to cell lineages in all three germ layers. The influence of mechanical strain appeared to involve the TGF-b/activin/nodal pathway, as strain was shown to upregulate TGF-β1, Activin A, Nodal, and SMAD2/3 phosphorylation in undifferentiated embryonic stem cells (Saha et al., 2008b
), whereas inhibition of the TGFβ/Activin/Nodal receptor stimulated differentiation.
Cyclic unconfined compression has also been shown to alter the phenotype of mesenchyme-derived stem cells. For example, the chondrogenic induction of stage 23/24 chick limb bud cells embedded in an agarose matrix was significantly enhanced by dynamic mechanical unconfined compression as a function of applied frequency, specifically noting an approximate doubling in the cartilage nodule density and a greater than 2-fold increase in proteoglycan synthesis rates compared to static compression (Elder et al., 2001
; Elder et al., 2000
). MSCs encapsulated in a 2% agarose matrix and subjected to unconfined dynamic compression exhibited increased aggrecan and collagen II transcript levels over non-loaded controls, which also translated into enhanced protein deposition in the matrix (Huang et al., 2004
; Mauck et al., 2007
). Additionally, intermittent hydrostatic pressure positively affected chondrogenically induced MSC aggregates resulting in significantly higher levels of collagen and proteoglycan content compared to unloaded controls (Angele et al., 2003
As might be expected, the influence of mechanical loading on stem cell response appears to depend on the type of stem cell as well as the state of (pre-)differentiation. For example, dynamic mechanical compression can significantly increase the chondrocytic expression (e.g., Sox-9, type II collagen, and aggrecan) of bone marrow-derived MSCs encapsulated in a hydrogel, irrespective of the presence of chondrogenic growth factors (Terraciano et al., 2007
). Under the same conditions, embryonic stem cell-derived embryoid bodies exhibit significant downregulation of cartilage-specific genes in response to mechanical compression. Following chondrogenic differentiation with TGFβ-1, however, these cells showed significant increases in the expression of cartilage-specific genes when exposed to mechanical compression (Terraciano et al., 2007
), suggesting that the mechanosensitivity of different types of stem cells is highly dependent on their state of differentiation. Taken together, it is that clear physical signals, in part, regulate differentiation but also that the ensuing phenotype is dependent on a myriad of factors including, but not limited to, the biochemical environment, biomaterials that are being employed, the differentiated state of the cell, and the precise mechanical loading protocol.