Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part ...because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an
system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, we present a novel hydrophobic hydrogel system
chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, we show that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, we reveal that stiffness mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. Our material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function.
Physical features of the extracellular matrix (ECM) heavily influence cell migration strategies and efficiency. Migration in and on fibrous ECMs is of significant physiologic importance, but ...limitations in the ability to experimentally define the diameter, density, and alignment of native ECMs in vitro have hampered our understanding of how these properties affect this basic cell function. Here, we designed a high-throughput in vitro platform that models fibrous ECM as collections of lines of cell-adhesive fibronectin on a flat surface to eliminate effects of dimensionality and topography. Using a microcontact printing approach to orthogonally vary line alignment, density, and size, we determined each factor's individual influence on NIH3T3 fibroblast migration. High content imaging and statistical analyses revealed that ECM alignment is the most critical parameter in influencing cell morphology, polarization, and migratory behavior. Specifically, increasing ECM alignment led cells to adopt an elongated uniaxial morphology and migrate with enhanced speed and persistence. Intriguingly, migration speeds were tightly correlated with the organization of focal adhesions, where cells with the most aligned adhesions migrated fastest. Highly organized focal adhesions and associated actin stress fibers appeared to define the number and location of protrusive fronts, suggesting that ECM alignment influences active Rac1 localization. Utilizing a novel microcontact-printing approach that lacks confounding influences of substrate dimensionality, mechanics, or differences in the adhesive area, this work highlights the effect of ECM alignment on orchestrating the cytoskeletal machinery that governs directed uniaxial cell migration.
Cellular phenotype is heavily influenced by the extracellular matrix (ECM), a complex and tissue-specific three-dimensional structure with distinct biophysical and biochemical properties. As ...naturally derived cell culture platforms are difficult to controllably modulate, engineered synthetic ECMs have facilitated our understanding of how specific matrix properties direct cell behavior. However, synthetic approaches typically lack fibrous topography, a hallmark of stromal and interstitial ECMs in vivo. To construct tunable biomimetic models with physiologic microstructure, we developed a versatile approach to generate modular fibrous architectures in 3D. Photo-cross-linkable polymers were electrospun, photopatterned into desired lengths, and coencapsulated alongside cells within natural biopolymer, semisynthetic, and synthetic hydrogels. Cells encapsulated within fiber-reinforced hydrogel composites (FHCs) demonstrated accelerated spreading rates compared to in gels lacking such fibrous topography. Furthermore, increases in fiber density at constant bulk hydrogel elastic modulus produced morphologically distinct cell populations and modulated cellular mechanosensing in 3D, as evidenced by increased nuclear localization of the mechanosensitive transcription factor, Yes-associated protein (YAP). This work documents the impact of physical guidance cues in 3D and establishes a novel approach to generating more physiologic tissue- and disease-specific biomimetic models.
Fibrocartilaginous tissues such as the meniscus serve critical load-bearing roles, relying on arrays of collagen fibers to resist tensile loads experienced with normal activity. As these structures ...are frequently injured and possess limited healing capacity, there exists great demand for tissue-engineered replacements. Toward recreating the structural features of these anisotropic tissues in vitro, we employ scaffolds composed of co-aligned nanofibers that direct mesenchymal stem cell (MSC) orientation and the formation of organized extracellular matrix (ECM). Concomitant with ECM synthesis, the mechanical properties of constructs increase with free-swelling culture, but ultimately failed to achieve equivalence with meniscal fibrocartilage. As mechanical forces are essential to the development and maintenance of musculoskeletal tissues, this work examined the effect of cyclic tensile loading on MSC-laden nanofibrous constructs. We hypothesized that loading would modulate the transcriptional behavior of MSCs, spur the deposition of ECM, and lead to enhancements in construct mechanical properties compared to free-swelling controls. Fiber-aligned scaffolds were seeded with MSCs and dynamically loaded daily in tension or maintained as nonloaded controls for 4 weeks. With mechanical stimulation, fibrous gene expression increased, collagen deposition increased, and the tensile modulus increased by 16% relative to controls. These results show that dynamic tensile loading enhances the maturation of MSC-laden aligned nanofibrous constructs, suggesting that recapitulation of the structural and mechanical environment of load-bearing tissues results in increases in functional properties that can be exploited for tissue engineering applications.
The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochemical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues ...influence transformed epithelial cells, prompting a diversity of migration modes spanning single cell and multicellular phenotypes. Purported consequences of these variations in tumor escape strategies include differential metastatic capability and therapy resistance. Therefore, understanding how cues from the peritumoral stromal microenvironment regulate migration mode has both prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel mechanics can be orthogonally tuned to investigate the contribution of these two key matrix attributes on MCF10A migration mode phenotypes, epithelial-mesenchymal transition (EMT), and invasive potential. We develop an automated computational image analysis framework to extract migratory phenotypes from fluorescent images and determine 3D migration metrics relevant to metastatic spread. Using this analysis, we find that matrix fiber density and bulk hydrogel mechanics distinctly contribute to a variety of MCF10A migration modes including amoeboid, single mesenchymal, clusters, and strands. We identify combinations of physical and soluble cues that induce a variety of migration modes originating from the same MCF10A spheroid and use these settings to examine a functional consequence of migration mode –resistance to apoptosis. We find that cells migrating as strands are more resistant to staurosporine-induced apoptosis than either disconnected clusters or individual invading cells. Improved models of the peritumoral stromal microenvironment and understanding of the relationships between matrix attributes and cell migration mode can aid ongoing efforts to identify effective cancer therapeutics that address cell plasticity-based therapy resistances.
Stromal extracellular matrix structure dictates both cell homeostasis and activation towards migratory phenotypes. However decoupling the effects of myriad biophysical cues has been difficult to achieve. Here, we encapsulate electrospun fiber segments within an amorphous hydrogel to create a fiber-reinforced hydrogel composite in which fiber density and hydrogel stiffness can be orthogonally tuned. Quantification of 3D cell migration reveal these two parameters uniquely contribute to a diversity of migration phenotypes spanning amoeboid, single mesenchymal, multicellular cluster, and collective strand. By tuning biophysical and biochemical cues to elicit heterogeneous migration phenotypes, we find that collective strands best resist apoptosis. This work establishes a composite approach to modulate fibrous topography and bulk hydrogel mechanics and identified biomaterial parameters to direct distinct 3D cell migration phenotypes.
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Hydrogels are often used to study the impact of biomechanical and topographical cues on cell behavior. Conventional hydrogels are designed a priori, with characteristics that cannot be dynamically ...changed in an externally controlled, user-defined manner. We developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), that enables non-invasive, spatiotemporally controlled modulation of mechanical and morphological properties using focused ultrasound. An ARS consists of a phase-shift emulsion distributed in a fibrin matrix. Ultrasound non-thermally vaporizes the emulsion into bubbles, which induces localized, radial compaction and stiffening of the fibrin matrix. In this in vitro study, we investigate how this mechanism can control the differentiation of fibroblasts into myofibroblasts, a transition correlated with substrate stiffness on 2D substrates. Matrix compaction and stiffening was shown to be highly localized using confocal and atomic force microscopies, respectively. Myofibroblast phenotype, evaluated by α-smooth muscle actin (α-SMA) immunocytochemistry, significantly increased in matrix regions proximal to bubbles compared to distal regions, irrespective of the addition of exogenous transforming growth factor-β1 (TGF-β1). Introduction of the TGF-β1 receptor inhibitor SB431542 abrogated the proximal enhancement. This approach providing spatiotemporal control over biophysical signals and resulting cell behavior could aid in better understanding fibrotic disease progression and the development of therapeutic interventions for chronic wounds.
Hydrogels are used in cell culture to recapitulate both biochemical and biophysical aspects of the native extracellular matrix. Biophysical cues like stiffness can impact cell behavior. However, with conventional hydrogels, there is a limited ability to actively modulate stiffness after polymerization. We have developed an ultrasound-based method of spatiotemporally-controlling mechanical and morphological properties within a composite hydrogel, termed an acoustically-responsive scaffold (ARS). Upon exposure to ultrasound, bubbles are non-thermally generated within the fibrin matrix of an ARS, thereby locally compacting and stiffening the matrix. We demonstrate how ARSs control the differentiation of fibroblasts into myofibroblasts in 2D. This approach could assist with the study of fibrosis and the development of therapies for chronic wounds.
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Mechanical interactions between fibroblasts and their surrounding extracellular matrix (ECM) guide fundamental behaviors such as spreading, migration, and proliferation that underlie disease ...pathogenesis. The challenges of studying ECM mechanics in vivo have motivated the development of in vitro models of the fibrous ECM in which fibroblasts reside. Natural materials such as collagen hydrogels bear structural and biochemical resemblance to stromal ECM, but mechanistic studies in these settings are often confounded by cell-mediated material degradation and the lack of structural and mechanical tunability. Here, we established a new material system composed of electrospun dextran vinyl sulfone (DexVS) polymeric fibers. These fibrous matrices exhibit mechanical tunability at both the single fiber (80–340 MPa) and bulk matrix (0.77–11.03 kPa) level, as well as long-term stability in mechanical properties over a two-week period. Cell adhesion to these matrices can be either user-defined by functionalizing synthetic fibers with thiolated adhesive peptides or methacrylated heparin to sequester cell-derived ECM proteins. We utilized DexVS fibrous matrices to investigate the role of matrix mechanics on the activation of fibroblasts into myofibroblasts, a key step of the fibrotic progression. In contrast to previous findings with non-fibrous hydrogel substrates, we find that fibroblasts in soft and deformable matrices exhibit increased spreading, focal adhesion formation, proliferation, and myofibroblast activation as compared to cells on stiffer matrices with equivalent starting architecture.
Cellular mechanosensing of fibrillar extracellular matrices plays a critical role in homeostasis and disease progression in stromal connective tissue. Here, we established a new material system composed of electrospun dextran vinyl sulfone polymeric fibers. These matrices exhibit architectural, mechanical, and biochemical tunability to accurately model diverse tissue microenvironments found in the body. In contrast to previous observations with non-fibrous hydrogels, we find that fibroblasts in soft and deformable fibrous matrices exhibit increased spreading and focal adhesion formation as compared to those in stiffer matrices with equivalent architecture. We also investigated the role of matrix stiffness on myofibroblast activation, a critical step in the fibrotic cascade, and find that low stiffness matrices promote increased myofibroblast activation.
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While matrix stiffness regulates cell behavior on 2D substrates, recent studies using synthetic hydrogels have suggested that in 3D environments, cell behavior is primarily impacted by matrix ...degradability, independent of stiffness. However, these studies did not consider the potential impact of other confounding matrix parameters that typically covary with changes in stiffness, particularly, hydrogel swelling and hydrolytic stability, which may explain the previously observed distinctions in cell response in 2D versus 3D settings. To investigate how cells sense matrix stiffness in 3D environments, a nonswelling, hydrolytically stable, linearly elastic synthetic hydrogel model is developed in which matrix stiffness and degradability can be tuned independently. It is found that matrix degradability regulates cell spreading kinetics, while matrix stiffness dictates the final spread area once cells achieve equilibrium spreading. Importantly, the differentiation of human mesenchymal stromal cells toward adipocytes or osteoblasts is regulated by the spread state of progenitor cells upon initiating differentiation. These studies uncover matrix stiffness as a major regulator of cell function not just in 2D, but also in 3D environments, and identify matrix degradability as a critical microenvironmental feature in 3D that in conjunction with matrix stiffness dictates cell spreading, cytoskeletal state, and stem cell differentiation outcomes.
This study establishes a linearly elastic, 3D hydrogel cell culture model, in which matrix stiffness, adhesiveness, and degradability can be independently tuned without concurrent changes in other hydrogel properties, in particular swelling and hydrolytic stability. Using this model, the authors determine that matrix degradability regulates cell spreading kinetics, while matrix stiffness dictates the final spread state once cells achieve equilibrium spreading.
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