Shelly R. Peyton

The effects of matrix stiffness and RhoA on the phenotypic plasticity of smooth muscle cells in a 3-D biosynthetic hydrogel system

Studies using 2-D cultures have shown that the mechanical properties of the extracellular matrix (ECM) influence cell migration, spreading, proliferation, and differentiation; however, cellular mechanosensing in 3-D remains under-explored. To investigate this topic, a unique biomaterial system based on poly(ethylene glycol)-conjugated fibrinogen was adapted to study phenotypic plasticity in smooth muscle cells (SMCs) as a function of ECM mechanics in 3-D. Tuning the compressive modulus between 448 and 5804 Pa modestly regulated SMC cytoskeletal assembly in 3-D, with spread cells in stiff matrices having a slightly higher degree of F-actin bundling after prolonged culture. However, vinculin expression in all 3-D conditions was qualitatively low and was not assembled into the classic focal adhesions typically seen in 2-D cultures. Given the evidence that RhoA-mediated cytoskeletal contractility represents a critical node in mechanosensing, we molecularly upregulated contractility by inducing SMCs to express constitutively active RhoA. In these cells, F-actin bundling and total vinculin expression increased, and focal adhesion-like structures began to emerge, consistent with RhoAs mechanism of action in cells cultured on 2-D substrates. Furthermore, SMC proliferation in 3-D did not depend significantly on matrix stiffness, and was reduced by constitutive activation of RhoA irrespective of ECM mechanical properties. Conversely, the expression of contractile markers globally increased with constitutive RhoA activation and depended on 3-D matrix stiffness only in cells with heightened RhoA activity. Combined, these data suggest that the synergistic effects of ECM mechanics and RhoA activity on SMC phenotype in 3-D are distinct from those in 2-D, and highlight the importance of studying the mechanical role of cell-matrix interactions in tunable 3-D environments.

The emergence of ECM mechanics and cytoskeletal tension as important regulators of cell function

The ability to harvest and maintain viable cells from mammalian tissues represented a critical advance in biomedical research, enabling individual cells to be cultured and studied in molecular detail. However, in these traditional cultures, cells are grown on rigid glass or polystyrene substrates, the mechanical properties of which often do not match those of the in vivo tissue from which the cells were originally derived. This mechanical mismatch likely contributes to abrupt changes in cellular phenotype. In fact, it has been proposed that mechanical changes in the cellular microenvironment may alone be responsible for driving specific cellular behaviors. Recent multidisciplinary efforts from basic scientists and engineers have begun to address this hypothesis more explicitly by probing the effects of ECM mechanics on cell and tissue function. Understanding the consequences of such mechanical changes is physiologically relevant in the context of a number of tissues in which altered mechanics may either correlate with or play an important role in the onset of pathology. Examples include changes in the compliance of blood vessels associated with atherosclerosis and intimal hyperplasia, as well as changes in the mechanical properties of developing tumors. Compelling evidence from 2-D in vitro model systems has shown that substrate mechanical properties induce changes in cell shape, migration, proliferation, and differentiation, but it remains to be seen whether or not these same effects translate to 3-D systems or in vivo. Furthermore, the molecular “mechanotransduction” mechanisms by which cells respond to changes in ECM mechanics remain unclear. Here, we provide some historical context for this emerging area of research, and discuss recent evidence that regulation of cytoskeletal tension by changes in ECM mechanics (either directly or indirectly) may provide a critical switch that controls cell function.

ECM compliance regulates osteogenesis by influencing MAPK signaling downstream of RhoA and ROCK.

The compliance of the extracellular matrix (ECM) regulates osteogenic differentiation by modulating extracellular signal‐regulated kinase (ERK) activity. However, the molecular mechanism linking ECM compliance to the ERK‐mitogen‐activated protein kinase (MAPK) pathway remains unclear. Furthermore, RhoA has been widely implicated in integrin‐mediated signaling and mechanotransduction. We studied the relationship between RhoA and ERK‐MAPK signaling to determine their roles in the regulation of osteogenesis by ECM compliance. Inhibition of RhoA and ROCK in MC3T3‐E1 pre‐osteoblasts cultured on substrates of varying compliance reduced ERK activity, whereas constitutively active RhoA enhanced it. The expression of RUNX2, a potent osteogenic transcription factor, was increased on stiffer matrices and correlated with elevated ERK activity. Inhibition of RhoA, ROCK, or the MAPK pathway diminished RUNX2 activity and delayed the onset of osteogenesis as shown by altered osteocalcin (OCN) and bone sialoprotein (BSP) gene expression, alkaline phosphatase (ALP) activity, and matrix mineralization. These data establish that one possible mechanism by which ECM rigidity regulates osteogenic differentiation involves MAPK activation downstream of the RhoA‐ROCK signaling pathway.

The regulation of osteogenesis by ECM rigidity in MC3T3‐E1 cells requires MAPK activation

Once thought to provide only structural support to tissues by acting as a scaffold to which cells bind, it is now widely recognized that the extracellular matrix (ECM) provides instructive signals that dictate cell behavior. Recently we demonstrated that mechanical cues intrinsic to the ECM directly regulate the behavior of pre‐osteoblastic MC3T3‐E1 cells. We hypothesized that one possible mechanism by which ECM compliance exerts its influence on osteogenesis is by modulating the mitogen‐activated protein kinase (MAPK) pathway. To address this hypothesis, the differentiation of MC3T3‐E1 cells cultured on poly(ethylene glycol) (PEG)‐based model substrates with tunable mechanical properties was assessed. Alkaline phosphatase (ALP) levels at days 7 and 14 were found to be significantly higher in cells grown on stiffer substrates (423.9 kPa hydrogels and rigid tissue culture polystyrene (TCPS) control) than on a soft hydrogel (13.7 kPa). Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression levels followed a similar trend. In parallel, MAPK activity was significantly higher in cells cultured on stiffer substrates at both time points. Inhibiting this activation pharmacologically, using PD98059, resulted in significantly lower ALP levels, OCN, and BSP gene expression levels on the hydrogels. Interestingly, the effectiveness of PD98059 was itself dependent on substrate stiffness, with marked inhibition of MAPK phosphorylation in cells grown on compliant hydrogels but insignificant reduction in cells grown on TCPS. Together, these data confirm a role for MAPK in the regulation of osteogenic differentiation by ECM compliance. J. Cell. Physiol. 211: 661–672, 2007.

Marrow‐Derived stem cell motility in 3D synthetic scaffold is governed by geometry along with adhesivity and stiffness

Design of 3D scaffolds that can facilitate proper survival, proliferation, and differentiation of progenitor cells is a challenge for clinical applications involving large connective tissue defects. Cell migration within such scaffolds is a critical process governing tissue integration. Here, we examine effects of scaffold pore diameter, in concert with matrix stiffness and adhesivity, as independently tunable parameters that govern marrow‐derived stem cell motility. We adopted an “inverse opal” processing technique to create synthetic scaffolds by crosslinking poly(ethylene glycol) at different densities (controlling matrix elastic moduli or stiffness) and small doses of a heterobifunctional monomer (controlling matrix adhesivity) around templating beads of different radii. As pore diameter was varied from 7 to 17 µm (i.e., from significantly smaller than the spherical cell diameter to approximately cell diameter), it displayed a profound effect on migration of these stem cells—including the degree to which motility was sensitive to changes in matrix stiffness and adhesivity. Surprisingly, the highest probability for substantive cell movement through pores was observed for an intermediate pore diameter, rather than the largest pore diameter, which exceeded cell diameter. The relationships between migration speed, displacement, and total path length were found to depend strongly on pore diameter. We attribute this dependence to convolution of pore diameter and void chamber diameter, yielding different geometric environments experienced by the cells within. Bioeng. 2011; 108:1181–1193.

Mechanics of intact bone marrow.

The current knowledge of bone marrow mechanics is limited to its viscous properties, neglecting the elastic contribution of the extracellular matrix. To get a more complete view of the mechanics of marrow, we characterized intact yellow porcine bone marrow using three different, but complementary techniques: rheology, indentation, and cavitation. Our analysis shows that bone marrow is elastic, and has a large amount of intra- and inter-sample heterogeneity, with an effective Young׳s modulus ranging from 0.25 to 24.7 kPa at physiological temperature. Each testing method was consistent across matched tissue samples, and each provided unique benefits depending on user needs. We recommend bulk rheology to capture the effects of temperature on tissue elasticity and moduli, indentation for quantifying local tissue heterogeneity, and cavitation rheology for mitigating destructive sample preparation. We anticipate the knowledge of bone marrow elastic properties for building in vitro models will elucidate mechanisms involved in disease progression and regenerative medicine.

The influence of ascorbic acid, TGF-β1, and cell-mediated remodeling on the bulk mechanical properties of 3-D PEG-fibrinogen constructs

Two-dimensional cell culture studies have shown that matrix rigidity can influence cell function, but little is known about how matrix physical properties, and their changes with time, influence cell function in 3-D. Biosynthetic hydrogels based on PEGylated fibrinogen permit the initial decoupling of matrix chemical and mechanical properties, and are thus potentially attractive for addressing this question. However, the mechanical stability of these gels due to passive hydrolysis and cell-mediated remodeling has not previously been addressed. Here, we show that the bulk mechanical properties of acellular PEG-fibrinogen hydrogels significantly decrease over time in PBS regardless of matrix cross-linking density in 7 days. To compensate, smooth muscle cells (SMCs) were encapsulated and stimulated to produce their own matrix using ascorbic acid or TGF-beta1. Ascorbic acid treatment improved the mechanical properties of the constructs after 14 days in less cross-linked matrices, but TGF-beta1 did not. The increase in matrix modulus of the constructs was not due to an increase in type I collagen deposition, which remained low and pericellular regardless of cross-link density or the soluble factor applied. Instead, ascorbic acid, but not TGF-beta1, preferentially enhanced the contractile SMC phenotype in the less cross-linked gels. Inhibition of contractility reduced cell spreading and the expression of contractile markers, and eliminated any beneficial increase in matrix modulus induced by cell-generated contraction of the gels. Together, these data show that PEG-fibrinogen hydrogels are susceptible to both hydrolysis and proteolysis, and suggest that some soluble factors may stimulate matrix remodeling by modulating SMC phenotype instead of inducing ECM synthesis in a 3-D matrix.

A novel three-dimensional model to quantify metastatic melanoma invasion

Although attempts to develop any viable chemotherapeutic approaches to combat metastatic cancers have largely failed, potential genetic targets to halt metastatic progression continue to be identified. As drugs are developed to address these targets, there is a need for high-throughput systems that accurately reproduce in vivo microenvironments to gauge their efficacy. Accordingly, we have developed a three-dimensional in vitro culture system representative of the environment present upon secondary metastasis to quantitatively measure tumor cell invasion in this setting three-dimensionally. Culturing melanomas of different metastatic capacities within the system showed that each cell type invades the matrix in a manner commensurate to its known metastatic potential in vivo. Moreover, the developed quantitative schemes were put to use to characterize the effect of microenvironmental influences (i.e., matrix components, interstitial cell presence) on planar and vertical melanoma invasion. We propose this novel, quantitative system as a useful tool to assess the effects of pharmacologic and/or microenvironmental influences on tumor cell invasion at a metastatic site. [Mol Cancer Ther 2007;6(2):552–61]

Thermal-Responsive Behavior of a Cell Compatible Chitosan/Pectin Hydrogel

Biopolymer hydrogels are important materials for wound healing and cell culture applications. While current synthetic polymer hydrogels have excellent biocompatibility and are nontoxic, they typically function as a passive matrix that does not supply any additional bioactivity. Chitosan (CS) and pectin (Pec) are natural polymers with active properties that are desirable for wound healing. Unfortunately, the synthesis of CS/Pec materials have previously been limited by harsh acidic synthesis conditions, which further restricted their use in biomedical applications. In this study, a zero-acid hydrogel has been synthesized from a mixture of chitosan and pectin at biologically compatible conditions. For the first time, we demonstrated that salt could be used to suppress long-range electrostatic interactions to generate a thermoreversible biopolymer hydrogel that has temperature-sensitive gelation. Both the hydrogel and the solution phases are highly elastic, with a power law index of close to -1. When dried hydrogels were placed into phosphate buffered saline solution, they rapidly rehydrated and swelled to incorporate 2.7× their weight. As a proof of concept, we removed the salt from our CS/Pec hydrogels, thus, creating thick and easy to cast polyelectrolyte complex hydrogels, which proved to be compatible with human marrow-derived stem cells. We suggest that our development of an acid-free CS/Pec hydrogel system that has excellent exudate uptake, holds potential for wound healing bandages.

Fewer Bacteria Adhere to Softer Hydrogels

Clinically, biofilm-associated infections commonly form on intravascular catheters and other hydrogel surfaces. The overuse of antibiotics to treat these infections has led to the spread of antibiotic resistance and underscores the importance of developing alternative strategies that delay the onset of biofilm formation. Previously, it has been reported that during surface contact, bacteria can detect surfaces through subtle changes in the function of their motors. However, how the stiffness of a polymer hydrogel influences the initial attachment of bacteria is unknown. Systematically, we investigated poly(ethylene glycol) dimethacrylate (PEGDMA) and agar hydrogels that were 20 times thicker than the cumulative size of bacterial cell appendages, as a function of Youngs moduli. Soft (44.05-308.5 kPa), intermediate (1495-2877 kPa), and stiff (5152-6489 kPa) hydrogels were synthesized. Escherichia coli and Staphylococcus aureus attachment onto the hydrogels was analyzed using confocal microscopy after 2 and 24 h incubation periods. Independent of hydrogel chemistry and incubation time, E. coli and S. aureus attachment correlated positively to increasing hydrogel stiffness. For example, after a 24 h incubation period, there were 52 and 82% fewer E. coli adhered to soft PEGDMA hydrogels than to the intermediate and stiff PEGDMA hydrogels, respectively. A 62 and 79% reduction in the area coverage by the Gram-positive microbe S. aureus occurred after 24 h incubation on the soft versus intermediate and stiff PEGDMA hydrogels. We suggest that hydrogel stiffness is an easily tunable variable that could potentially be used synergistically with traditional antimicrobial strategies to reduce early bacterial adhesion and therefore the occurrence of biofilm-associated infections.