The pericellular environment regulates cytoskeletal development and the differentiation of mesenchymal stem cells and determines their response to hydrostatic pressure

Abstract

The objective of this study was to examine the interplay between matrix stiffness and hydrostatic pressure (HP) in regulating chondrogenesis of mesenchymal stem cells (MSCs) and to further elucidate the mechanotransductive roles of integrins and the cytoskeleton. MSCs were seeded into 1 %, 2 % or 4 % agarose hydrogels and exposed to cyclic hydrostatic pressure. In a permissive media, the stiffer hydrogels supported an osteogenic phenotype, with little evidence of chondrogenesis observed regardless of the matrix stiffness. In a chondrogenic media, the stiffer gels suppressed cartilage matrix production and gene expression, with the addition of RGDS (an integrin blocker) found to return matrix synthesis to similar levels as in the softer gels. Vinculin, actin and vimentin organisation all adapted within stiffer hydrogels, with the addition of RGDS again preventing these changes. While the stiffer gels inhibited chondrogenesis, they enhanced mechanotransduction of HP. RGDS suppressed the mechanotransduction of HP, suggesting a role for integrin binding as a regulator of both matrix stiffness and HP. Intermediate filaments also appear to play a role in the mechanotransduction of HP, as only vimentin organisation adapted in response to this mechanical stimulus. To conclude, the results of this study demonstrate that matrix density and/or stiffness modulates the development of the pericellular matrix and consequently integrin binding and cytoskeletal structure. The study further suggests that physiological cues such as HP enhance chondrogenesis of MSCs as the pericellular environment matures and the cytoskeleton adapts, and points to a novel role for vimentin in the transduction of HP.

Cyclic hydrostatic pressure promotes a stable cartilage phenotype and enhances the functional development of cartilaginous grafts engineered using multipotent stromal cells isolated from bone marrow and infrapatellar fat pad

“…Since the pericellular environment will become stiffer as ECM content increases with time in culture, this may contribute to the development of an endochondral phenotype in BMSC constructs. However, it has also been shown that HP can override the influence of a stiffening microenvironment on chondrogenesis of MSCs (Steward et al, 2013), possibly by modulating changes to the cytoskeleton. Taken together, these results raise the possibility that HP may act to suppress a substrate stiffness mediated progression of hypertrophy in BMSCs, although further work is clearly required to directly test this hypothesis.…”

“…Mechanical signals, such as hydrostatic pressure (HP), are a key component of the in vivo joint environment and have been shown to play a significant role in regulating the chondrogenic differentiation of mesenchymal stem cells. Previous studies have shown varying and occasionally conflicting results (Parkkinen et al, 1993;Carver and Heath, 1999;Suh et al, 1999;Carver and Heath, 2000;Jortikka et al, 2000;Smith et al, 2000Smith et al, , 2005Hu and Athanasiou, 2006;Finger et al, 2007;Ogawa et al, 2009;Meyer et al, 2011;Liu et al, 2012;Maxson and Burg, 2012;Puetzer et al, 2012;Safshekan et al, 2012;Steward et al, 2012;Vinardell et al, 2012a;Steward et al, 2013), but it would appear that the application of physiological levels (3-10 MPa) of intermittent HP can enhance proteoglygan and collagen synthesis and upregulate aggrecan and collagen II mRNA expression in chondrocytes and stem/progenitor cells isolated from bone marrow, synovial tissues and adipose tissues. There is also evidence to suggest that dynamic HP can act to stabilise the phenotype of chondrogenically primed joint tissue derived MSCs (Vinardell et al, 2012a).…”

Exploring the roles of integrin binding and cytoskeletal reorganization during mesenchymal stem cell mechanotransduction in soft and stiff hydrogels subjected to dynamic compression

“…Denser and stiffer hydrogels were found to alter PCM organization, cytoskeletal organization, and integrin binding relative to less dense and softer hydrogels (Steward et al, 2013). These changes led to a more robust response to the application of extrinsic mechanical signals in the stiffer hydrogels, demonstrating the importance of the PCM for determining the response of MSCs to such cues (Steward et al, 2013). These changes to the PCM occurred in parallel with changes to the internal cytoskeleton, raising the possibility that the temporal response of MSCs to extrinsic mechanical cues could be related to changes in either the PCM or to the cytoskeleton during chondrogenesis.…”

“…Recently, studies have begun investigating the interplay between extrinsic and intrinsic mechanical cues and their subsequent effects on cellular differentiation. Denser and stiffer hydrogels were found to alter PCM organization, cytoskeletal organization, and integrin binding relative to less dense and softer hydrogels (Steward et al, 2013). These changes led to a more robust response to the application of extrinsic mechanical signals in the stiffer hydrogels, demonstrating the importance of the PCM for determining the response of MSCs to such cues (Steward et al, 2013).…”

“…those generated outside the cell and transmitted inwards via the pericellular matrix (PCM) and those generated within the cell in response to its substrate) have been well characterized as key regulators of mesenchymal stem/stromal cell (MSC) differentiation (Engler et al, 2006;Kelly and Jacobs, 2010;Reilly and Engler, 2010;Steward et al, 2011Steward et al, , 2012Steward et al, , 2013Thorpe et al, 2012). Dynamic compression (DC) has been shown to enhance chondrogenesis of MSCs depending on the timing and duration of its application (Campbell et al, 2006;Haugh et al, 2011;Huang et al, 2004Huang et al, , 2005Huang et al, , 2010Mouw et al, 2007;Thorpe et al, 2010); however, the underlying mechanisms responsible for this temporal mechanosensitivity are not well understood.…”