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Designing Tunable Artificial Matrices for Stem Cell Culture
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Designing Tunable Artificial Matrices for Stem Cell Culture Elizabeth F. Irwin,* Jacob F. Pollock,* David V. Schaffery, ** and Kevin E. Healy*, z Department of Bioengineering, yDepartment of Chemical Engineering, **The Helen Wills Neuroscience Institute, zDepartment of Materials Science and
*
Engineering, University of California at Berkeley, Berkeley, CA, USA
Chapter Outline Introduction The Extracellular Matrix Physical Properties of the Extracellular Matrix Cell Adhesion and Mechanotransduction Mechanics of the Natural ECM Developing Artificial Matrices with Tunable Moduli for Stem Cell Culture Physical Structure of the Matrix Choice of Material Natural Materials
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INTRODUCTION Embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells can generate a myriad of different cells types in the body and thus have enormous potential for use in regenerative medicine. In vivo, the fate of these stem cells, that is, to remain undifferentiated or to differentiate into a particular cell type, is determined in large part by their local microenvironment, where the regulatory signaling mechanisms include cellecell interactions; cellematrix interactions; growth factors; cytokines; and the physiochemical nature of the environment, including the oxygen tension, osmolarity, and pH. The local microenvironment, however, is highly dynamic, not only as organismal development progresses, but also within the fluctuating nature of adult tissue. Adult stem cells grow in niches and are often maintained in a dormant, multipotent state where they retain the ability to either self-renew or divide. These cells receive signals through a diverse population of neighboring differentiated cell types, which secrete growth factors and cytokines and organize the extracellular matrix (Fuchs et al., 2004). Niche cells thereby provide an environment that isolates stem cells from differentiation stimuli,
Synthetic Materials Synthetic Materials with Bioactive Ligands Creating Matrices with Tunable Moduli Characterization of Matrix Mechanics Atomic Force Microscopy Rheology Role of Matrix Mechanics in Stem Cell Behavior Conclusions and Future Directions References
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apoptotic stimuli, and excessive stem cell proliferation that could lead to cancer (Moore and Lemischka, 2006). However, with tissue injury and other processes associated with tissue turnover, the surrounding microenvironment actively signals to these adult stem cells to begin to proliferate, self-renew, and/or differentiate to form new tissues. The fate of the inner cell mass, the in vivo precursors of ES cells, is likewise determined by a complex sequence of signaling from the local environment, which in this case provides a more dynamic set of chemical and mechanical signals that orchestrate tissue formation and differentiation. During the earliest stages of this process, inner cell mass constituents interact with the matrix as it guides fundamental processes of development including migration in the early embryo, and the modulation of growth and differentiation programs of cells (Zagris, 2001). Subsequently, as groups of cells form tissues, they experience not only morphogen patterns but also tension, compression, and shear forces, and these mechanical forces can regulate the expression of various genes (Brouzes and Farge, 2004). In order to grow stem cells and tissues in vitro, it is necessary to understand and attempt to reproduce the
Handbook of Stem Cells, Two-Volume Set. DOI: http://dx.doi.org/10.1016/B978-0-12-385942-6.00076-7 Copyright Ó 2013 Elsevier Inc. All rights reserved.
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complex microenvironment presented to these cells in vivo; however, current culture technologies are not sufficient to mimic this dynamic and intricate natural environment. This chapter focuses on progress in the design and characterization of artificial matrices that attempt to recapitulate microenvironmental cues for in vitro stem cell culture and differentiation.
THE EXTRACELLULAR MATRIX Physical Properties of the Extracellular Matrix The natural extracellular matrix (ECM) provides a network of chemically and physically associated proteins and polysaccharides that allow cells to attach, migrate, and proliferate and also presents biochemical and physical signals affecting cell fate (Roskelley et al., 1995). A schematic of these interactions is shown in Figure 76.1. In addition, the ECM is not a static entity but instead provides a very dynamic environment whose components are locally secreted and restructured by cells. For example, as they move through the matrix, cells deposit new proteins as well as locally cleave proteins by releasing metalloproteinases (Streuli, 1999). In addition, the remodeling of the ECM is even more rapid in developing tissues, a process particularly relevant to embryonic and fetal stem cells (Zagris, 2001). Collagen scaffolds, which have been extensively studied, exemplify the basic architecture of the ECM. In tissues such as bone, cartilage, and tendons, collagen is arranged in fibrils to provide tensile strength. In contrast, in epithelial tissue, collagen forms a network of fibers as a basement membrane (Bosman and Stamenkovic, 2003),
FIGURE 76.1 Schematic of the mechanical interaction between a cell and the surrounding ECM. Integrin receptors engage binding sites on structural ECM proteins, bridging the cytoskeleton of the cell with the surrounding matrix. Integrin binding at the surface can influence structural rearrangements in both the cytoplasm and the nucleus.
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an open structure that allows rapid diffusion of nutrients, metabolites, and hormones between the blood and constituent cells. In addition to these two structures, there is huge variability in collagen structure from tissue to tissue, as their complex architectures are composed of more than 28 genetically distinct collagen molecules (Martin et al., 1985; Gordon and Hahn, 2010). In addition to the collagen network in the ECM, there exists long chain glycoaminoglycans (GAGs) and adhesive proteins, including fibronectin, tenascin, and laminin. GAGs are highly hydrated and provide some compressive strength to the network. Adhesive proteins present an immense number of physically immobilized and nonimmobilized signals to the cells. Fibronectin, for example, is an important protein in guiding cell attachment and migration during embryonic development, where its absence leads to defects in mesodermal, neural tube, and vascular development. Similarly, laminin has been shown to affect cell migration and differentiation in numerous systems (Kubota et al., 1988).
Cell Adhesion and Mechanotransduction Cell adhesion to the ECM is crucial for both development and tissue maintenance (de Arcangelis and Georges-Labouesse, 2000). Cell adhesion events mediate cell spreading, migration, neurite extension, muscle-cell contraction, cell-cycle progression, and differentiation (Giancotti and Ruoslahti, 1999). Cells adhere to distinct adhesion domains on the ECM (Ruoslahti and Pierschbacher, 1987) through cell-surface receptors, primarily from the integrin family (Hynes, 2002). Upon engagement, these receptors provide chemical and mechanical signals to the cell that can lead to altered gene expression and in some cases cell fate, including apoptosis, migration, differentiation, and proliferation. Integrins are a family of approximately 25 membranespanning heterodimeric proteins containing ligand-binding regions on the outer-membrane region and microfilamentsdocking domains within the ectodomain. They are composed of a (~120 kD) and b subunits (~180 kD), and each combination has a different binding affinity and signaling properties. Most integrins are expressed on a wide variety of cell types, and most cells express several integrin receptors. Integrins signal both from the outside-in (binding of the integrin with the ECM induces intracellular signaling events) and the inside-out (the binding activity and expression of integrins is regulated by the cell) (Giancotti and Ruoslahti, 1999). Following activation (via a binding event on the cytoplasmic domain), the focal adhesion complexes (FACs) bind actin-associated proteins such as talin, vinculin, zyxin, and paxillin and provide a direct physical link to the cytoskeleton, which also links to the nuclear scaffold. Integrin binding at the surface can
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therefore influence structural rearrangements in both the cytoplasm and the nucleus (Geiger et al., 2001). This is particularly relevant since mechanical signals can potentially travel faster than signals that are mediated via diffusion either across or through the cell. Once bound to the ECM, integrins enable cells to sense the physical and mechanical properties of the matrix. As a result, changes in physical or mechanical properties of the matrix can activate signaling pathways including MAPK and JNK that direct cell-cycle progression and differentiation, and this conversion of physical signals into biochemical signals is termed mechanotransduction.
Mechanics of the Natural ECM In vivo, tissues including bone, arteries, and brain naturally have distinct moduli (Black and Hastings, 1998), where the modulus of each is primarily defined by the properties of the ECM. Accordingly, ex situ measurements of natural tissues demonstrate the wide range of stiffnesses of different tissues in the body. Engler et al. (2006) observed that the osteoid matrix that surrounds osteoblasts in culture had a Young’s modulus of ~27 10 kPa, much stiffer than other tissues in the body. In addition, a few years prior, Engler et al. (2004) sectioned arteries ex situ and measured an intermediate stiffness with a measured Young’s modulus of 5e8 kPa. Finally, Elkin et al. (2007) and Lu et al. (2006) made measurements of native brain tissue, which had a much lower modulus of ~500 Pa. The different moduli of these tissues in vivo indicate there may be a significant role of the stiffness of the matrix in cell fate and cell behavior.
DEVELOPING ARTIFICIAL MATRICES WITH TUNABLE MODULI FOR STEM CELL CULTURE The physical and chemical properties of the ECM play a key role in stem cell fate; therefore, the field of tissue engineering has the difficult task of creating artificial matrices that impart the desired signals to the cells in direct contact with those matrices. In addition, since the natural microenvironment is dynamic in nature, it may be necessary to create tunable systems that the user is able to modify, for example to direct progressive processes such as cell fate specification or tissue organization. This section focuses on designing matrices for in vitro stem cell culture, both for maintaining stem cell self-renewal and differentiating stem cells into a variety of cell lineages.
Physical Structure of the Matrix The physical structure, or microarchitecture, of an artificial matrix must provide appropriate physical signals, present
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or allow access to biochemical cues, and permit nutrient and waste exchange. Synthetic matrices should mimic some aspects of the natural properties of the collagen scaffold and adjacent proteins of the ECM, which constitutes a highly hydrated and fibrous network that supports cell attachment, migration, and other functions. One approach to mimicking the physical structure of the ECM is the creation of matrices of nanofibers prepared with electrospun polymers. In 2003, Yoshimoto et al. grew mesenchymal stem cells (MSCs) on scaffolds created by electrospinning poly(ε-caprolactone) (PCL). They demonstrated increased osteogenic differentiation on the nanofiber matrices compared with standard tissue culture surfaces. In 2006, Nur-E-Kamal et al. cultured mouse ES (mES) cells on a synthetic polyamide matrix whose threedimensional (3D) nanofibrillar organization resembled the ECM/basement membrane and found that this surface greatly enhanced proliferation and self-renewal compared with propagation on tissue culture surfaces without nanofibers. This important work indicated that mimicking the nanofiber structure of the ECM can yield enhanced cell behavior, and subsequent work (discussed on p. 931) has built upon these efforts to incorporate material designs that allow tuning of a wide range of mechanical properties. Another approach to the design of artificial matrices for stem cells is to employ a hydrogel to mimic the physical properties of the natural ECM. Hydrogels emulate the high water content and porous nature of most natural soft tissues. In addition, scaffolds have been designed to enable cells to proteolytically cleave certain domains of the network as they move through it, allowing for the creation of pores (West and Hubbell, 1999; Schense et al., 2000; Kim et al., 2005; Raeber et al., 2005; Levesque and Shoichet, 2007). Hydrogels can thus provide the diverse physical properties of an artificial matrix, while also providing a system that can be chemically and mechanically tuned for a desired application.
Choice of Material For the desired microarchitecture, a variety of both natural and synthetic polymer chemistries can be employed to create the nanofiber or hydrogel systems discussed above that offer different material characteristics.
Natural Materials Naturally occurring polymer components from the ECM can be isolated and employed as artificial microenvironments for stem cell culture. For decades, natural materials e including alginate (Barralet et al., 2005), chitosan (Azab et al., 2006), hyaluronic acid (Masters et al., 2004), collagen (Butcher and Nerem, 2004), laminin, fibronectin, and fibrin (Eyrich et al., 2007) e have been used as matrices for
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a variety of primary cells and cell lines. As they are part of the natural ECM, these proteoglycan and protein molecules contain binding sequences to engage cell surface receptors and allow for cell attachment and migration. However, disadvantages of using natural, isolated materials include lot-to-lot variability in the signals the matrix presents to the cells, the potential transfer of immunogens to cells, and the potential for viral or bacterial contamination. Therefore, the primary value of most work using natural materials has been to elucidate the roles of natural ECM molecule(s) on cell fate. In 2005, Battista et al. employed collagen, fibronectin (FN), and laminin (LM) matrices for the culture of mEScell-derived embryoid bodies (EBs) in an attempt to direct stem cell behavior. They showed that the composition of the matrix plays an important role in EB development, where high collagen concentrations inhibited EB differentiation, FN constructs stimulated endothelial cell differentiation and vascularization, and LM constructs increased the percentage of cells that differentiated into beating cardiomyocytes. This work indicates that there is key regulatory “information,” i.e., ligands, and possibly physical cues within these ECM proteins that regulate mES cell fate decisions. In addition, several groups have utilized surface arrays for the high-throughput analysis of how different combinations of natural ECM molecules can impact stem cell fate. Flaim et al. (2005) designed a platform to study the effects of 32 different combinations of collagen I, collagen III, collagen IV, laminin, and fibronectin on the differentiation of mouse ES cells towards an early hepatic fate. They identified ECM combinations that impacted both hepatocyte function and ES cell differentiation. Soen et al. (2006) printed mixtures of ECM components and signaling factors on a glass surface to generate an array of immobilized “molecular microenvironments” and found the composition of the microenvironment affected the degree of differentiation of primary human neural precursor cells. These studies provide fundamental information that increases our understanding of the role of matrix composition on stem cell behavior, which can be harnessed to design synthetic hydrogels that can offer more precise control over matrix properties and signals presented to cells.
Synthetic Materials In contrast to natural materials, synthetic polymer hydrogels offer improved control, repeatability, safety, and scalability. However, it can be challenging to functionalize synthetic materials with the highly complex bioactivities of natural materials. Synthetic materials used commonly for tissue engineering of all cell types include poly(glycolic acid), poly(lactic acid), and their copolymers; polyethylene
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glycol (PEG) (Sawhney et al., 1993); polyvinyl (PVA) (Martens and Anseth, 2000); polyNIPAAm (Stile et al., 2004); polyacrylamides; and polyacrylates. Several reviews describe the use of these synthetic polymer matrices for the growth of anchorage-dependent, differentiated cells (Lutolf and Hubbell, 2005; Lin and Anseth, 2009; Tibbitt and Anseth, 2009), and these chemistries likewise have potential for use in scaffolds for stem cell culture. When selecting polymer chemistry for a particular application, design parameters include the toxicity of the material to the cells, hydrophilicity, swelling behavior, degradation properties, interactions the polymer chains have with neighboring cells, biofunctionalization, and crosslinking properties. Hydrogel matrices have been used to support the potency and differentiation of stem cells. Biodegradable polymer scaffolds were employed for the differentiation of hES cells into 3D structures with characteristics of developing neural tissues, cartilage, or liver (Levenberg et al., 2003). These synthetic matrices were superior to their natural counterparts in scalability, repeatability, and control over design parameters. In addition, advanced screening methods have been employed to identify hydrogel surfaces for the self-renewal of pluripotent stem cells (Yang et al., 2009; Derda et al., 2010; Hook et al., 2010). However, there is still a great deal of work to be done to evaluate various hydrogels and their influence on stem cell behavior, particularly engineering them with the biochemical and mechanical signals inherent in natural matrix.
Synthetic Materials with Bioactive Ligands Synthetic hydrogels can be modified with bioactive ligands to allow cells to attach, proliferate, and/or differentiate upon otherwise inert surfaces as shown schematically in Figure 76.2. Extensive work has been performed to identify binding sequences in natural ECM molecules (Derda et al., 2007) and then generate short peptides or small recombinant proteins encompassing these sequences for incorporation into artificial matrices (Orner et al., 2004; Derda et al., 2007). The resulting bioactive materials can support cell receptoreligand adhesion, which enables cells to sense and respond to the stiffness of the matrix. However, many key parameters must be tuned to control cell and stem cell behavior, including the ligand identity, presentation, and density (Nowakowski et al., 2004; Shin et al., 2004; Yim and Leong, 2005; Beckstead et al., 2006). Peptides are typically conjugated to hydrogels using either primary amines or sulfhydryl groups on the peptides themselves. The method of conjugation, as well as spacerarm length, can be varied to modulate the steric accessibility of the peptide sequence to the cell. In addition, it can be difficult to generate synthetic analogues of the complex motifs that natural ECM proteins present. In some cases,
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FIGURE 76.2 Schematic of a cell embedded in a 3D synthetic hydrogel. Integrin receptors bind to pendant cellbinding domains, and growth factor receptors bind to soluble ligands. Cell-secreted proteases enzymatically cleave substrates incorporated into the polymer network, locally degrading it.
the natural conformation of the binding site on the protein can be more closely approximated by cyclizing short peptide sequences that are otherwise linear (Schense et al., 2000). The most common molecules used to mediate cell attachment to synthetic matrices are short peptide sequences (usually between 6 and 15 amino acids) containing the consensus sequence arginine-glycine-aspartate (RGD), which is present in several ECM proteins. Many studies have tethered this peptide to hydrogels and demonstrated its ability to bind anchorage-dependent cells through a subset of RGD-binding integrins, such as avb3 (Massia and Hubbell, 1991; Hubbell, 1995). In another approach, Meng et al. (2010) physisorbed short peptide sequences to cell culture plates to bind specific integrins identified on hES cells to create a completely synthetic defined cell culture system for medium-term self-renewal of hES cells. Using synthetic matrices functionalized with bioactive ligands, Saha et al. (2008) demonstrated both long-term self-renewal and multipotent differentiation of neural stem cells on interpenetrating networks of polyacrylamide and polyethylene glycol functionalized with a 15-mer RGD sequence isolated from bone rat sialoprotein (bsp-RGD15). In another example, Li et al. (2006) were able to maintain hESC pluripotency employing synthetic semi-interpenetrating polymer networks (sIPNs) functionalized with the same peptide. In addition, Hwang et al. (2006) differentiated hES cells into mesenchymal stem cells and then evaluated their chondrogenic capacity upon encapsulation in poly(ethylene glycol)-diacrylate (PEGDA) hydrogels functionalized with an RGD peptide. This combination yielded neocartilage within 3 weeks of culture. In another
study, Ferreira et al. (2007) formed a 3D matrix from the natural polymer dextran, then functionalized the material with RGD peptides and microencapsulated VEGF. They were able to increase the fraction of cells displaying a vascular marker by 20-fold compared to spontaneously differentiated EBs and propose that this hydrogel enables the derivation of vascular cells in large quantities.
Creating Matrices with Tunable Moduli Mechanical design parameters for artificial matrices include elasticity, compressibility, viscoelastic behavior, and tensile strength. Controlling the mechanical properties of a material at the cellular level can help elicit a desired cell response, and, in addition, the bulk mechanical properties of the matrix must be controlled such that the matrix is able to withstand loads that may be involved in downstream applications. The mechanical properties of hydrogels can be varied and controlled via chemical synthesis and processing. Hydrogels are composed of long, hydrophilic polymer chains either physically entangled or chemically crosslinked to form a network, and their mechanical properties can be chemically altered by controlling crosslinking density (entanglements or chemical crosslinks). For the AAm gels described on page 933, the input crosslinker (bisacrylamide) concentration of the AAm gels was varied, and a linear relationship between input crosslinker density and gel modulus was found. Based on prior work (see p. 933) (Engler et al., 2006; Saha et al., 2007, 2008; Boonen et al., 2009), tuning the crosslink density of hydrogels may aid in designing systems to support stem cell self-renewal
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or differentiation, depending on the desired application. An increasing number of studies have illustrated a role of stiffness in regulating stem cell function in two dimensions, and initial evidence to date indicates that the mechanical properties of a material are likely to also influence stem cell behavior in 3D (Banerjee et al., 2009). Although a direct correlation with matrix stiffness and behavior of hES or iPS cells has yet to be demonstrated, Li et al. (2006) proposed that the soft mechanical properties of their hydrogels improve the self-renewal of hES cells on their defined, synthetic hydrogels. In this work, pNIPAAm hydrogels functionalized with bsp-RGD15 and with a complex shear modulus of ~50e100 Pa (depending on the frequency of the measurement) and were able to maintain pluripotency in the short-term. Future studies are very likely to focus on analyzing the effects of stiffness and other mechanical properties on the self-renewal, lineage commitment, and differentiation of numerous cell types, providing additional key design parameters to control cell function for downstream applications.
CHARACTERIZATION OF MATRIX MECHANICS The mechanical properties of synthetic and natural matrices are typically characterized by either atomic force microscopy or rheology, and each is addressed in further detail.
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the cantilever is measured as shown in Figure 76.3. To reduce strain at the point of contact, and ensure uniform curvature at the point of contact, a bead can be attached to the cantiliver tip as shown. The AFM collects data by reflecting a laser off a cantilever with a known spring constant. The laser is reflected into a photodiode (detector) as the tip (on the end of a cantilever) is indented into the surface and bends in response to the force between the tip and the sample. A constant force is maintained on the sample by the tip by using a feedback loop with piezoelectric translators that adjust the z-axis of the stage. The elastic response of the underlying material is analyzed by applying Hertzian mechanical models to the slope of the forceedisplacement curves to estimate the elastic modulus and other material properties or structural parameters (Dimitriadis et al., 2002). The indentation curves are then analyzed with a Hertzian mechanics model with the following relationship: F ¼ ð2=PÞ½E=ð1 v2 Þd2 tanðaÞ
(1)
where F is the applied force, E is the elastic modulus, n is the Poisson ratio of the material, d is the indentation depth, and a is the angle of the indenting cone. This model assumes infinite depth of the sample, and therefore the indentation of the tip into the material must be less than 10% of the film thickness or the stiffness of the underlying substrate may be sensed by the AFM tip.
Atomic Force Microscopy
Rheology
The mechanical stiffness of two-dimensional (2D) hydrogels can be characterized by force mode atomic force microscopy (AFM). AFMs have been used widely as microindenters to probe the physical properties of the materials (Burnham and Colton, 1989; Tao et al., 1992; Rotsch et al., 1997; Domke and Radmacher, 1998; Dimitriadis et al., 2002; Irwin et al., 2008). In force mode, the AFM tip is indented into the surface, and the deflection of
The mechanical properties of engineered 3D hydrogel systems, like natural biological tissues, are viscoelastic and are typically characterized using rheological techniques. The mechanical characteristics of such materials are intermediate between an ideal solid and an ideal liquid and are dependent on loading rate and history. Rheometry measures the flow and deformation behavior of materials under stress, for example by using rotational parallel-plate devices.
FIGURE 76.3 Schematic of the indentation of a gel sample with a rigid sphere using an AFM. Adapted from Dimitriadis et al. (2002).
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Oscillatory strain-controlled parallel-plate rheometers apply a sinusoidal shear strain to a hydrogel and measure the resulting stress (torque) response. The ratio of the amplitudes and phase difference of the stress and strain waves provide the storage (elastic), G0 , and loss (viscous), G00 , moduli. The phase angle, d ¼ arctanðG00 =G0 Þ;
(2)
indicates the degree to which a material is like an elastic solid or a viscous liquid, while the complex modulus, (3) G ¼ sqrt½ðG0 Þ2 þ ðG00 Þ2 ; indicates the overall resistance to shear deformation. These properties are measured over a range of frequencies to determine their dependence on loading rate. Strain sweeps are performed to define the linear viscoelastic regime and yield point of the material. Rheology is particularly applicable for the analysis of environmentally responsive and in situ-forming hydrogels for tissue engineering and cell biology. Kinetic changes in mechanical properties can be measured as the materials transition from liquid to solid. Liquids are indicated by low, frequency-dependent elastic moduli and high phase angles while solids have high, frequency-independent elastic moduli and low phase angles.
ROLE OF MATRIX MECHANICS IN STEM CELL BEHAVIOR The biochemistry, physical architecture, and modulus of the microenvironment are all important parameters in influencing cell behavior. Cells are traditionally cultured on tissue culture polystyrene (TCPS) and glass, which have Young’s moduli of ~108 and ~1010 Pa, respectively: values that are orders of magnitude higher than the moduli of most natural tissues, ~102e105 Pa. Given this mismatch in mechanical properties, and given that it has been demonstrated in multiple anchorage-dependent cell types that a material’s modulus impacts cell morphology, cytoskeletal formation, and gene expression, this key parameter must be considered in the design of cell culture systems. Pelham and Wang developed a system to evaluate the effect of material stiffness on cell behavior (Pelham and Wang, 1998). This system was composed of 2D, variable moduli polyacrylamide (pAAm) gels functionalized with collagen to allow for cell attachment, and has been since employed by multiple research laboratories to demonstrate modulus dependent behavior of a variety of different anchorage-dependent cell types. These gels, which greatly contrast with the current tissue culture polystyrene used for standard cell culture that is orders of magnitude more rigid (~ GPa), provided moduli that more accurately matched those of native tissue. In 1998, Pelham and Wang first
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demonstrated that fibroblast and epithelial cell behavior were regulated by the mechanical properties of the underlying synthetic matrix on which the cells were cultured. They found that both focal adhesion and cytoskeletal formation depended on the stiffness of the underlying pAAm gels. Thomas and Dimilla (2000) cultured human SNB-19 glioblastoma cells on poly(methylphenyl)siloxane (PDMS) films of variable moduli and showed that the average projected cell area decreases by over 60% with a two-orders-of-magnitude increase in compliance. Lo et al. (2000) cultured fibroblasts on pAAm gels with a spatial gradient in modulus and demonstrated that the fibroblasts preferentially migrated to the stiffer areas of the gel, a process termed durotaxis, indicating the cells were able to sample the stiffness of the underlying substrate. In addition, by applying mechanical strain to the substrate with a microneedle, they demonstrated that cell movement is also guided by strain in the substrate. In 2004, Engler et al. employed the same pAAm gel system and demonstrated that matrix stiffness affected the cell spreading, actin cytoskeletal formation, and focal adhesion organization of smooth muscle cells (SMCs), where stiffer gels cause an increase in all three. Several groups have been able to show that, in addition to varying a number of properties of differentiated cells, the stiffness of the matrix can regulate the lineage commitment processes of adult stem cells. In 2006, the lab of Engler et al. used the pAAm gel system to test the effect of matrix stiffness on the differentiation of adult stem cells. A variation in stiffness alone was able to control lineage commitment, where softer matrices resulted in neurogenic commitment, intermediate stiffnesses yielded myogenic commitment, and finally the stiffest matrices resulted in osteogenic commitment. This work suggests that mesenchymal stem cells differentiate according to the stiffness of the environment in which they were cultured. Reflecting this observation, Saha et al. (2008) demonstrated that adult neural stem cell differentiation also depended on the stiffness of the underlying matrix. In this work, an interpenetrating polymer network (IPN) of AAm and PEG functionalized with bspRGD(15) was employed to demonstrate that softer gels (100e500 Pa) greatly favored differentiation into neurons, whereas harder gels (1,000e10,000 Pa) promoted glial cultures. Recently, Boonen et al. cultured muscle progenitor cells (MPCs) onto pAAm gels of varying stiffness and found that proliferation and differentiation were influenced by elasticity (Boonen et al., 2009). An intermediate stiffness of 21 kPa was optimal for the proliferation of MPCs, where only gels with elasticities greater than 3 kPa led to maturation with cross-striations and contractions. Collectively, these studies demonstrate that the stiffness of the matrix is a crucial parameter in designing matrices for stem cells; stiffness collaborates with soluble cues to direct lineage commitment of the cells.
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CONCLUSIONS AND FUTURE DIRECTIONS In engineering matrices for stem cell culture, it is evident that ligand identity and presentation, as well as material architecture and mechanical properties, are key design parameters in controlling stem cell fate. Although there have been significant advances in the design and synthesis of artificial ECMs, there is still a great need for more sophisticated scaffolds that play an active role in guiding tissue regeneration and functional adaptation of the newly formed tissue. In particular, while it is recognized as a signal to the cells, the modulus of the material is still not often varied and optimized for the particular application. Future work is likely to increasingly tap into this and other opportunities that materials offer to afford greater control over cell fate and function and thereby enhance the potential of numerous downstream applications for stem cell engineering.
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de Arcangelis, A., Georges-Labouesse, E., 2000. Integrin and ECM functions e roles in vertebrate development. Trends Genet. 16, 389e395. Derda, R., Li, L.Y., Orner, B.P., Lewis, R.L., Thomson, J.A., Kiessling, L.L., 2007. Defined substrates for human embryonic stem cell growth identified from surface arrays. Acs Chem. Biol. 2, 347e355. Derda, R., Musah, S., Orner, B.P., Klim, J.R., Li, L.Y., Kiessling, L.L., 2010. High-throughput discovery of synthetic surfaces that support proliferation of pluripotent cells. J. Am. Chem. Soc. 132, 1289e1295. Dimitriadis, E.K., Horkay, F., Kachar, B., Chadwick, R.S., 2002. Measurement of the elastic modulus of polymer gels using the atomic force microscope. Abstr. Papers Am. Chem. Soc. 224 U417eU417. Domke, J., Radmacher, M., 1998. Measuring the elastic properties of thin polymer films with the atomic force microscope. Langmuir 14, 3320e3325. Elkin, B.S., Azeloglu, E.U., Costa, K.D., Morrison, B., 2007. Mechanical heterogeneity of the rat hippocampus measured by atomic force microscope indentation. J. Neurotrauma. 24, 812e822. Engler, A., Bacakova, L., Newman, C., Hategan, A., Griffin, M., Discher, D., 2004. Substrate compliance versus ligand density in cell on gel responses. Biophysical J. 86, 617e628. Engler, A.J., Sen, S., Sweeney, H.L., Discher, D.E., 2006. Matrix elasticity directs stem cell lineage specification. Cell 126, 677e689. Eyrich, D., Wiese, H., Mailer, G., Skodacek, D., Appel, B., Sarhan, H., et al., 2007. In vitro and in vivo cartilage engineering using a combination of chondrocyte-seeded long-term stable fibrin gels and polycaprolactonebased polyurethane scaffolds. Tissue Engin. 13, 2207e2218. Ferreira, L.S., Gerecht, S., Fuller, J., Shieh, H.F., Vunjak-Novakovic, G., Langer, R., 2007. Bioactive hydrogel scaffolds for controllable vascular differentiation of human embryonic stem cells. Biomaterials 28, 2706e2717. Flaim, C.J., Chien, S., Bhatia, S.N., 2005. An extracellular matrix microarray for probing cellular differentiation. Nat. Methods 2, 119e125. Fuchs, E., Tumbar, T., Guasch, G., 2004. Socializing with the neighbors: stem cells and their niche. Cell 116, 769e778. Geiger, B., Bershadsky, A., Pankov, R., Yamada, K.M., 2001. Transmembrane extracellular matrix-cytoskeleton crosstalk. Nat. Rev. Mol. Cell Biol. 2, 793e805. Giancotti, F.G., Ruoslahti, E., 1999. Transduction e integrin signaling. Science 285, 1028e1032. Gordon, M.K., Hahn, R.A., 2010. Collagens. Cell Tissue Res. 339, 247e257. Hook, A.L., Anderson, D.G., Langer, R., Williams, P., Davies, M.C., Alexander, M.R., 2010. High throughput methods applied in biomaterial development and discovery. Biomaterials 31, 187e198. Hubbell, J.A., 1995. Biomaterials in tissue engineering. Bio-Technology 13, 565e576. Hwang, N.S., Varghese, S., Zhang, Z., Elisseeff, J., 2006. Chondrogenic differentiation of human embryonic stem cell-derived cells in arginine-glycine-aspartate modified hydrogels. Tissue Eng. 12, 2695e2706. Hynes, R.O., 2002. Integrins: bidirectional, allosteric signaling machines. Cell 110, 673e687. Irwin, E.F., Saha, K., Rosenbluth, M., Gamble, L.J., Castner, D.G., Healy, K.E., 2008. Modulus-dependent macrophage adhesion and behavior. J. Biomater. Sci. Polymer Ed. 19, 1363e1382. Kim, S., Chung, E.H., Gilbert, M., Healy, K.E., 2005. Synthetic MMP-13 degradable ECMs based on poly(N-isopropylacrylamide-co-acrylic
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Designing Tunable Artificial Matrices for Stem Cell Culture Elizabeth F. Irwin,* Jacob F. Pollock,* David V. Schaffery, ** and Kevin E. Healy*, z Department of Bioengineering, yDepartment of Chemical Engineering, **The Helen Wills Neuroscience Institute, zDepartment of Materials Science and
*
Engineering, University of California at Berkeley, Berkeley, CA, USA
Chapter Outline Introduction The Extracellular Matrix Physical Properties of the Extracellular Matrix Cell Adhesion and Mechanotransduction Mechanics of the Natural ECM Developing Artificial Matrices with Tunable Moduli for Stem Cell Culture Physical Structure of the Matrix Choice of Material Natural Materials
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INTRODUCTION Embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells can generate a myriad of different cells types in the body and thus have enormous potential for use in regenerative medicine. In vivo, the fate of these stem cells, that is, to remain undifferentiated or to differentiate into a particular cell type, is determined in large part by their local microenvironment, where the regulatory signaling mechanisms include cellecell interactions; cellematrix interactions; growth factors; cytokines; and the physiochemical nature of the environment, including the oxygen tension, osmolarity, and pH. The local microenvironment, however, is highly dynamic, not only as organismal development progresses, but also within the fluctuating nature of adult tissue. Adult stem cells grow in niches and are often maintained in a dormant, multipotent state where they retain the ability to either self-renew or divide. These cells receive signals through a diverse population of neighboring differentiated cell types, which secrete growth factors and cytokines and organize the extracellular matrix (Fuchs et al., 2004). Niche cells thereby provide an environment that isolates stem cells from differentiation stimuli,
Synthetic Materials Synthetic Materials with Bioactive Ligands Creating Matrices with Tunable Moduli Characterization of Matrix Mechanics Atomic Force Microscopy Rheology Role of Matrix Mechanics in Stem Cell Behavior Conclusions and Future Directions References
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apoptotic stimuli, and excessive stem cell proliferation that could lead to cancer (Moore and Lemischka, 2006). However, with tissue injury and other processes associated with tissue turnover, the surrounding microenvironment actively signals to these adult stem cells to begin to proliferate, self-renew, and/or differentiate to form new tissues. The fate of the inner cell mass, the in vivo precursors of ES cells, is likewise determined by a complex sequence of signaling from the local environment, which in this case provides a more dynamic set of chemical and mechanical signals that orchestrate tissue formation and differentiation. During the earliest stages of this process, inner cell mass constituents interact with the matrix as it guides fundamental processes of development including migration in the early embryo, and the modulation of growth and differentiation programs of cells (Zagris, 2001). Subsequently, as groups of cells form tissues, they experience not only morphogen patterns but also tension, compression, and shear forces, and these mechanical forces can regulate the expression of various genes (Brouzes and Farge, 2004). In order to grow stem cells and tissues in vitro, it is necessary to understand and attempt to reproduce the
Handbook of Stem Cells, Two-Volume Set. DOI: http://dx.doi.org/10.1016/B978-0-12-385942-6.00076-7 Copyright Ó 2013 Elsevier Inc. All rights reserved.
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complex microenvironment presented to these cells in vivo; however, current culture technologies are not sufficient to mimic this dynamic and intricate natural environment. This chapter focuses on progress in the design and characterization of artificial matrices that attempt to recapitulate microenvironmental cues for in vitro stem cell culture and differentiation.
THE EXTRACELLULAR MATRIX Physical Properties of the Extracellular Matrix The natural extracellular matrix (ECM) provides a network of chemically and physically associated proteins and polysaccharides that allow cells to attach, migrate, and proliferate and also presents biochemical and physical signals affecting cell fate (Roskelley et al., 1995). A schematic of these interactions is shown in Figure 76.1. In addition, the ECM is not a static entity but instead provides a very dynamic environment whose components are locally secreted and restructured by cells. For example, as they move through the matrix, cells deposit new proteins as well as locally cleave proteins by releasing metalloproteinases (Streuli, 1999). In addition, the remodeling of the ECM is even more rapid in developing tissues, a process particularly relevant to embryonic and fetal stem cells (Zagris, 2001). Collagen scaffolds, which have been extensively studied, exemplify the basic architecture of the ECM. In tissues such as bone, cartilage, and tendons, collagen is arranged in fibrils to provide tensile strength. In contrast, in epithelial tissue, collagen forms a network of fibers as a basement membrane (Bosman and Stamenkovic, 2003),
FIGURE 76.1 Schematic of the mechanical interaction between a cell and the surrounding ECM. Integrin receptors engage binding sites on structural ECM proteins, bridging the cytoskeleton of the cell with the surrounding matrix. Integrin binding at the surface can influence structural rearrangements in both the cytoplasm and the nucleus.
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an open structure that allows rapid diffusion of nutrients, metabolites, and hormones between the blood and constituent cells. In addition to these two structures, there is huge variability in collagen structure from tissue to tissue, as their complex architectures are composed of more than 28 genetically distinct collagen molecules (Martin et al., 1985; Gordon and Hahn, 2010). In addition to the collagen network in the ECM, there exists long chain glycoaminoglycans (GAGs) and adhesive proteins, including fibronectin, tenascin, and laminin. GAGs are highly hydrated and provide some compressive strength to the network. Adhesive proteins present an immense number of physically immobilized and nonimmobilized signals to the cells. Fibronectin, for example, is an important protein in guiding cell attachment and migration during embryonic development, where its absence leads to defects in mesodermal, neural tube, and vascular development. Similarly, laminin has been shown to affect cell migration and differentiation in numerous systems (Kubota et al., 1988).
Cell Adhesion and Mechanotransduction Cell adhesion to the ECM is crucial for both development and tissue maintenance (de Arcangelis and Georges-Labouesse, 2000). Cell adhesion events mediate cell spreading, migration, neurite extension, muscle-cell contraction, cell-cycle progression, and differentiation (Giancotti and Ruoslahti, 1999). Cells adhere to distinct adhesion domains on the ECM (Ruoslahti and Pierschbacher, 1987) through cell-surface receptors, primarily from the integrin family (Hynes, 2002). Upon engagement, these receptors provide chemical and mechanical signals to the cell that can lead to altered gene expression and in some cases cell fate, including apoptosis, migration, differentiation, and proliferation. Integrins are a family of approximately 25 membranespanning heterodimeric proteins containing ligand-binding regions on the outer-membrane region and microfilamentsdocking domains within the ectodomain. They are composed of a (~120 kD) and b subunits (~180 kD), and each combination has a different binding affinity and signaling properties. Most integrins are expressed on a wide variety of cell types, and most cells express several integrin receptors. Integrins signal both from the outside-in (binding of the integrin with the ECM induces intracellular signaling events) and the inside-out (the binding activity and expression of integrins is regulated by the cell) (Giancotti and Ruoslahti, 1999). Following activation (via a binding event on the cytoplasmic domain), the focal adhesion complexes (FACs) bind actin-associated proteins such as talin, vinculin, zyxin, and paxillin and provide a direct physical link to the cytoskeleton, which also links to the nuclear scaffold. Integrin binding at the surface can
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therefore influence structural rearrangements in both the cytoplasm and the nucleus (Geiger et al., 2001). This is particularly relevant since mechanical signals can potentially travel faster than signals that are mediated via diffusion either across or through the cell. Once bound to the ECM, integrins enable cells to sense the physical and mechanical properties of the matrix. As a result, changes in physical or mechanical properties of the matrix can activate signaling pathways including MAPK and JNK that direct cell-cycle progression and differentiation, and this conversion of physical signals into biochemical signals is termed mechanotransduction.
Mechanics of the Natural ECM In vivo, tissues including bone, arteries, and brain naturally have distinct moduli (Black and Hastings, 1998), where the modulus of each is primarily defined by the properties of the ECM. Accordingly, ex situ measurements of natural tissues demonstrate the wide range of stiffnesses of different tissues in the body. Engler et al. (2006) observed that the osteoid matrix that surrounds osteoblasts in culture had a Young’s modulus of ~27 10 kPa, much stiffer than other tissues in the body. In addition, a few years prior, Engler et al. (2004) sectioned arteries ex situ and measured an intermediate stiffness with a measured Young’s modulus of 5e8 kPa. Finally, Elkin et al. (2007) and Lu et al. (2006) made measurements of native brain tissue, which had a much lower modulus of ~500 Pa. The different moduli of these tissues in vivo indicate there may be a significant role of the stiffness of the matrix in cell fate and cell behavior.
DEVELOPING ARTIFICIAL MATRICES WITH TUNABLE MODULI FOR STEM CELL CULTURE The physical and chemical properties of the ECM play a key role in stem cell fate; therefore, the field of tissue engineering has the difficult task of creating artificial matrices that impart the desired signals to the cells in direct contact with those matrices. In addition, since the natural microenvironment is dynamic in nature, it may be necessary to create tunable systems that the user is able to modify, for example to direct progressive processes such as cell fate specification or tissue organization. This section focuses on designing matrices for in vitro stem cell culture, both for maintaining stem cell self-renewal and differentiating stem cells into a variety of cell lineages.
Physical Structure of the Matrix The physical structure, or microarchitecture, of an artificial matrix must provide appropriate physical signals, present
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or allow access to biochemical cues, and permit nutrient and waste exchange. Synthetic matrices should mimic some aspects of the natural properties of the collagen scaffold and adjacent proteins of the ECM, which constitutes a highly hydrated and fibrous network that supports cell attachment, migration, and other functions. One approach to mimicking the physical structure of the ECM is the creation of matrices of nanofibers prepared with electrospun polymers. In 2003, Yoshimoto et al. grew mesenchymal stem cells (MSCs) on scaffolds created by electrospinning poly(ε-caprolactone) (PCL). They demonstrated increased osteogenic differentiation on the nanofiber matrices compared with standard tissue culture surfaces. In 2006, Nur-E-Kamal et al. cultured mouse ES (mES) cells on a synthetic polyamide matrix whose threedimensional (3D) nanofibrillar organization resembled the ECM/basement membrane and found that this surface greatly enhanced proliferation and self-renewal compared with propagation on tissue culture surfaces without nanofibers. This important work indicated that mimicking the nanofiber structure of the ECM can yield enhanced cell behavior, and subsequent work (discussed on p. 931) has built upon these efforts to incorporate material designs that allow tuning of a wide range of mechanical properties. Another approach to the design of artificial matrices for stem cells is to employ a hydrogel to mimic the physical properties of the natural ECM. Hydrogels emulate the high water content and porous nature of most natural soft tissues. In addition, scaffolds have been designed to enable cells to proteolytically cleave certain domains of the network as they move through it, allowing for the creation of pores (West and Hubbell, 1999; Schense et al., 2000; Kim et al., 2005; Raeber et al., 2005; Levesque and Shoichet, 2007). Hydrogels can thus provide the diverse physical properties of an artificial matrix, while also providing a system that can be chemically and mechanically tuned for a desired application.
Choice of Material For the desired microarchitecture, a variety of both natural and synthetic polymer chemistries can be employed to create the nanofiber or hydrogel systems discussed above that offer different material characteristics.
Natural Materials Naturally occurring polymer components from the ECM can be isolated and employed as artificial microenvironments for stem cell culture. For decades, natural materials e including alginate (Barralet et al., 2005), chitosan (Azab et al., 2006), hyaluronic acid (Masters et al., 2004), collagen (Butcher and Nerem, 2004), laminin, fibronectin, and fibrin (Eyrich et al., 2007) e have been used as matrices for
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a variety of primary cells and cell lines. As they are part of the natural ECM, these proteoglycan and protein molecules contain binding sequences to engage cell surface receptors and allow for cell attachment and migration. However, disadvantages of using natural, isolated materials include lot-to-lot variability in the signals the matrix presents to the cells, the potential transfer of immunogens to cells, and the potential for viral or bacterial contamination. Therefore, the primary value of most work using natural materials has been to elucidate the roles of natural ECM molecule(s) on cell fate. In 2005, Battista et al. employed collagen, fibronectin (FN), and laminin (LM) matrices for the culture of mEScell-derived embryoid bodies (EBs) in an attempt to direct stem cell behavior. They showed that the composition of the matrix plays an important role in EB development, where high collagen concentrations inhibited EB differentiation, FN constructs stimulated endothelial cell differentiation and vascularization, and LM constructs increased the percentage of cells that differentiated into beating cardiomyocytes. This work indicates that there is key regulatory “information,” i.e., ligands, and possibly physical cues within these ECM proteins that regulate mES cell fate decisions. In addition, several groups have utilized surface arrays for the high-throughput analysis of how different combinations of natural ECM molecules can impact stem cell fate. Flaim et al. (2005) designed a platform to study the effects of 32 different combinations of collagen I, collagen III, collagen IV, laminin, and fibronectin on the differentiation of mouse ES cells towards an early hepatic fate. They identified ECM combinations that impacted both hepatocyte function and ES cell differentiation. Soen et al. (2006) printed mixtures of ECM components and signaling factors on a glass surface to generate an array of immobilized “molecular microenvironments” and found the composition of the microenvironment affected the degree of differentiation of primary human neural precursor cells. These studies provide fundamental information that increases our understanding of the role of matrix composition on stem cell behavior, which can be harnessed to design synthetic hydrogels that can offer more precise control over matrix properties and signals presented to cells.
Synthetic Materials In contrast to natural materials, synthetic polymer hydrogels offer improved control, repeatability, safety, and scalability. However, it can be challenging to functionalize synthetic materials with the highly complex bioactivities of natural materials. Synthetic materials used commonly for tissue engineering of all cell types include poly(glycolic acid), poly(lactic acid), and their copolymers; polyethylene
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glycol (PEG) (Sawhney et al., 1993); polyvinyl (PVA) (Martens and Anseth, 2000); polyNIPAAm (Stile et al., 2004); polyacrylamides; and polyacrylates. Several reviews describe the use of these synthetic polymer matrices for the growth of anchorage-dependent, differentiated cells (Lutolf and Hubbell, 2005; Lin and Anseth, 2009; Tibbitt and Anseth, 2009), and these chemistries likewise have potential for use in scaffolds for stem cell culture. When selecting polymer chemistry for a particular application, design parameters include the toxicity of the material to the cells, hydrophilicity, swelling behavior, degradation properties, interactions the polymer chains have with neighboring cells, biofunctionalization, and crosslinking properties. Hydrogel matrices have been used to support the potency and differentiation of stem cells. Biodegradable polymer scaffolds were employed for the differentiation of hES cells into 3D structures with characteristics of developing neural tissues, cartilage, or liver (Levenberg et al., 2003). These synthetic matrices were superior to their natural counterparts in scalability, repeatability, and control over design parameters. In addition, advanced screening methods have been employed to identify hydrogel surfaces for the self-renewal of pluripotent stem cells (Yang et al., 2009; Derda et al., 2010; Hook et al., 2010). However, there is still a great deal of work to be done to evaluate various hydrogels and their influence on stem cell behavior, particularly engineering them with the biochemical and mechanical signals inherent in natural matrix.
Synthetic Materials with Bioactive Ligands Synthetic hydrogels can be modified with bioactive ligands to allow cells to attach, proliferate, and/or differentiate upon otherwise inert surfaces as shown schematically in Figure 76.2. Extensive work has been performed to identify binding sequences in natural ECM molecules (Derda et al., 2007) and then generate short peptides or small recombinant proteins encompassing these sequences for incorporation into artificial matrices (Orner et al., 2004; Derda et al., 2007). The resulting bioactive materials can support cell receptoreligand adhesion, which enables cells to sense and respond to the stiffness of the matrix. However, many key parameters must be tuned to control cell and stem cell behavior, including the ligand identity, presentation, and density (Nowakowski et al., 2004; Shin et al., 2004; Yim and Leong, 2005; Beckstead et al., 2006). Peptides are typically conjugated to hydrogels using either primary amines or sulfhydryl groups on the peptides themselves. The method of conjugation, as well as spacerarm length, can be varied to modulate the steric accessibility of the peptide sequence to the cell. In addition, it can be difficult to generate synthetic analogues of the complex motifs that natural ECM proteins present. In some cases,
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FIGURE 76.2 Schematic of a cell embedded in a 3D synthetic hydrogel. Integrin receptors bind to pendant cellbinding domains, and growth factor receptors bind to soluble ligands. Cell-secreted proteases enzymatically cleave substrates incorporated into the polymer network, locally degrading it.
the natural conformation of the binding site on the protein can be more closely approximated by cyclizing short peptide sequences that are otherwise linear (Schense et al., 2000). The most common molecules used to mediate cell attachment to synthetic matrices are short peptide sequences (usually between 6 and 15 amino acids) containing the consensus sequence arginine-glycine-aspartate (RGD), which is present in several ECM proteins. Many studies have tethered this peptide to hydrogels and demonstrated its ability to bind anchorage-dependent cells through a subset of RGD-binding integrins, such as avb3 (Massia and Hubbell, 1991; Hubbell, 1995). In another approach, Meng et al. (2010) physisorbed short peptide sequences to cell culture plates to bind specific integrins identified on hES cells to create a completely synthetic defined cell culture system for medium-term self-renewal of hES cells. Using synthetic matrices functionalized with bioactive ligands, Saha et al. (2008) demonstrated both long-term self-renewal and multipotent differentiation of neural stem cells on interpenetrating networks of polyacrylamide and polyethylene glycol functionalized with a 15-mer RGD sequence isolated from bone rat sialoprotein (bsp-RGD15). In another example, Li et al. (2006) were able to maintain hESC pluripotency employing synthetic semi-interpenetrating polymer networks (sIPNs) functionalized with the same peptide. In addition, Hwang et al. (2006) differentiated hES cells into mesenchymal stem cells and then evaluated their chondrogenic capacity upon encapsulation in poly(ethylene glycol)-diacrylate (PEGDA) hydrogels functionalized with an RGD peptide. This combination yielded neocartilage within 3 weeks of culture. In another
study, Ferreira et al. (2007) formed a 3D matrix from the natural polymer dextran, then functionalized the material with RGD peptides and microencapsulated VEGF. They were able to increase the fraction of cells displaying a vascular marker by 20-fold compared to spontaneously differentiated EBs and propose that this hydrogel enables the derivation of vascular cells in large quantities.
Creating Matrices with Tunable Moduli Mechanical design parameters for artificial matrices include elasticity, compressibility, viscoelastic behavior, and tensile strength. Controlling the mechanical properties of a material at the cellular level can help elicit a desired cell response, and, in addition, the bulk mechanical properties of the matrix must be controlled such that the matrix is able to withstand loads that may be involved in downstream applications. The mechanical properties of hydrogels can be varied and controlled via chemical synthesis and processing. Hydrogels are composed of long, hydrophilic polymer chains either physically entangled or chemically crosslinked to form a network, and their mechanical properties can be chemically altered by controlling crosslinking density (entanglements or chemical crosslinks). For the AAm gels described on page 933, the input crosslinker (bisacrylamide) concentration of the AAm gels was varied, and a linear relationship between input crosslinker density and gel modulus was found. Based on prior work (see p. 933) (Engler et al., 2006; Saha et al., 2007, 2008; Boonen et al., 2009), tuning the crosslink density of hydrogels may aid in designing systems to support stem cell self-renewal
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or differentiation, depending on the desired application. An increasing number of studies have illustrated a role of stiffness in regulating stem cell function in two dimensions, and initial evidence to date indicates that the mechanical properties of a material are likely to also influence stem cell behavior in 3D (Banerjee et al., 2009). Although a direct correlation with matrix stiffness and behavior of hES or iPS cells has yet to be demonstrated, Li et al. (2006) proposed that the soft mechanical properties of their hydrogels improve the self-renewal of hES cells on their defined, synthetic hydrogels. In this work, pNIPAAm hydrogels functionalized with bsp-RGD15 and with a complex shear modulus of ~50e100 Pa (depending on the frequency of the measurement) and were able to maintain pluripotency in the short-term. Future studies are very likely to focus on analyzing the effects of stiffness and other mechanical properties on the self-renewal, lineage commitment, and differentiation of numerous cell types, providing additional key design parameters to control cell function for downstream applications.
CHARACTERIZATION OF MATRIX MECHANICS The mechanical properties of synthetic and natural matrices are typically characterized by either atomic force microscopy or rheology, and each is addressed in further detail.
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the cantilever is measured as shown in Figure 76.3. To reduce strain at the point of contact, and ensure uniform curvature at the point of contact, a bead can be attached to the cantiliver tip as shown. The AFM collects data by reflecting a laser off a cantilever with a known spring constant. The laser is reflected into a photodiode (detector) as the tip (on the end of a cantilever) is indented into the surface and bends in response to the force between the tip and the sample. A constant force is maintained on the sample by the tip by using a feedback loop with piezoelectric translators that adjust the z-axis of the stage. The elastic response of the underlying material is analyzed by applying Hertzian mechanical models to the slope of the forceedisplacement curves to estimate the elastic modulus and other material properties or structural parameters (Dimitriadis et al., 2002). The indentation curves are then analyzed with a Hertzian mechanics model with the following relationship: F ¼ ð2=PÞ½E=ð1 v2 Þd2 tanðaÞ
(1)
where F is the applied force, E is the elastic modulus, n is the Poisson ratio of the material, d is the indentation depth, and a is the angle of the indenting cone. This model assumes infinite depth of the sample, and therefore the indentation of the tip into the material must be less than 10% of the film thickness or the stiffness of the underlying substrate may be sensed by the AFM tip.
Atomic Force Microscopy
Rheology
The mechanical stiffness of two-dimensional (2D) hydrogels can be characterized by force mode atomic force microscopy (AFM). AFMs have been used widely as microindenters to probe the physical properties of the materials (Burnham and Colton, 1989; Tao et al., 1992; Rotsch et al., 1997; Domke and Radmacher, 1998; Dimitriadis et al., 2002; Irwin et al., 2008). In force mode, the AFM tip is indented into the surface, and the deflection of
The mechanical properties of engineered 3D hydrogel systems, like natural biological tissues, are viscoelastic and are typically characterized using rheological techniques. The mechanical characteristics of such materials are intermediate between an ideal solid and an ideal liquid and are dependent on loading rate and history. Rheometry measures the flow and deformation behavior of materials under stress, for example by using rotational parallel-plate devices.
FIGURE 76.3 Schematic of the indentation of a gel sample with a rigid sphere using an AFM. Adapted from Dimitriadis et al. (2002).
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Oscillatory strain-controlled parallel-plate rheometers apply a sinusoidal shear strain to a hydrogel and measure the resulting stress (torque) response. The ratio of the amplitudes and phase difference of the stress and strain waves provide the storage (elastic), G0 , and loss (viscous), G00 , moduli. The phase angle, d ¼ arctanðG00 =G0 Þ;
(2)
indicates the degree to which a material is like an elastic solid or a viscous liquid, while the complex modulus, (3) G ¼ sqrt½ðG0 Þ2 þ ðG00 Þ2 ; indicates the overall resistance to shear deformation. These properties are measured over a range of frequencies to determine their dependence on loading rate. Strain sweeps are performed to define the linear viscoelastic regime and yield point of the material. Rheology is particularly applicable for the analysis of environmentally responsive and in situ-forming hydrogels for tissue engineering and cell biology. Kinetic changes in mechanical properties can be measured as the materials transition from liquid to solid. Liquids are indicated by low, frequency-dependent elastic moduli and high phase angles while solids have high, frequency-independent elastic moduli and low phase angles.
ROLE OF MATRIX MECHANICS IN STEM CELL BEHAVIOR The biochemistry, physical architecture, and modulus of the microenvironment are all important parameters in influencing cell behavior. Cells are traditionally cultured on tissue culture polystyrene (TCPS) and glass, which have Young’s moduli of ~108 and ~1010 Pa, respectively: values that are orders of magnitude higher than the moduli of most natural tissues, ~102e105 Pa. Given this mismatch in mechanical properties, and given that it has been demonstrated in multiple anchorage-dependent cell types that a material’s modulus impacts cell morphology, cytoskeletal formation, and gene expression, this key parameter must be considered in the design of cell culture systems. Pelham and Wang developed a system to evaluate the effect of material stiffness on cell behavior (Pelham and Wang, 1998). This system was composed of 2D, variable moduli polyacrylamide (pAAm) gels functionalized with collagen to allow for cell attachment, and has been since employed by multiple research laboratories to demonstrate modulus dependent behavior of a variety of different anchorage-dependent cell types. These gels, which greatly contrast with the current tissue culture polystyrene used for standard cell culture that is orders of magnitude more rigid (~ GPa), provided moduli that more accurately matched those of native tissue. In 1998, Pelham and Wang first
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demonstrated that fibroblast and epithelial cell behavior were regulated by the mechanical properties of the underlying synthetic matrix on which the cells were cultured. They found that both focal adhesion and cytoskeletal formation depended on the stiffness of the underlying pAAm gels. Thomas and Dimilla (2000) cultured human SNB-19 glioblastoma cells on poly(methylphenyl)siloxane (PDMS) films of variable moduli and showed that the average projected cell area decreases by over 60% with a two-orders-of-magnitude increase in compliance. Lo et al. (2000) cultured fibroblasts on pAAm gels with a spatial gradient in modulus and demonstrated that the fibroblasts preferentially migrated to the stiffer areas of the gel, a process termed durotaxis, indicating the cells were able to sample the stiffness of the underlying substrate. In addition, by applying mechanical strain to the substrate with a microneedle, they demonstrated that cell movement is also guided by strain in the substrate. In 2004, Engler et al. employed the same pAAm gel system and demonstrated that matrix stiffness affected the cell spreading, actin cytoskeletal formation, and focal adhesion organization of smooth muscle cells (SMCs), where stiffer gels cause an increase in all three. Several groups have been able to show that, in addition to varying a number of properties of differentiated cells, the stiffness of the matrix can regulate the lineage commitment processes of adult stem cells. In 2006, the lab of Engler et al. used the pAAm gel system to test the effect of matrix stiffness on the differentiation of adult stem cells. A variation in stiffness alone was able to control lineage commitment, where softer matrices resulted in neurogenic commitment, intermediate stiffnesses yielded myogenic commitment, and finally the stiffest matrices resulted in osteogenic commitment. This work suggests that mesenchymal stem cells differentiate according to the stiffness of the environment in which they were cultured. Reflecting this observation, Saha et al. (2008) demonstrated that adult neural stem cell differentiation also depended on the stiffness of the underlying matrix. In this work, an interpenetrating polymer network (IPN) of AAm and PEG functionalized with bspRGD(15) was employed to demonstrate that softer gels (100e500 Pa) greatly favored differentiation into neurons, whereas harder gels (1,000e10,000 Pa) promoted glial cultures. Recently, Boonen et al. cultured muscle progenitor cells (MPCs) onto pAAm gels of varying stiffness and found that proliferation and differentiation were influenced by elasticity (Boonen et al., 2009). An intermediate stiffness of 21 kPa was optimal for the proliferation of MPCs, where only gels with elasticities greater than 3 kPa led to maturation with cross-striations and contractions. Collectively, these studies demonstrate that the stiffness of the matrix is a crucial parameter in designing matrices for stem cells; stiffness collaborates with soluble cues to direct lineage commitment of the cells.
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CONCLUSIONS AND FUTURE DIRECTIONS In engineering matrices for stem cell culture, it is evident that ligand identity and presentation, as well as material architecture and mechanical properties, are key design parameters in controlling stem cell fate. Although there have been significant advances in the design and synthesis of artificial ECMs, there is still a great need for more sophisticated scaffolds that play an active role in guiding tissue regeneration and functional adaptation of the newly formed tissue. In particular, while it is recognized as a signal to the cells, the modulus of the material is still not often varied and optimized for the particular application. Future work is likely to increasingly tap into this and other opportunities that materials offer to afford greater control over cell fate and function and thereby enhance the potential of numerous downstream applications for stem cell engineering.
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