Approaches to Manipulating the Dimensionality and Physicochemical Properties of Common Cellular Scaffolds

A major hurdle in studying biological systems and administering effective tissue engineered therapies is the lack of suitable cell culture models that replicate the dynamic nature of cell-microenvironment interactions. Advances in the field of surface chemistry and polymer science have allowed researchers to develop novel methodologies to manipulate materials to be extrinsically tunable. Usage of such materials in modeling tissues in vitro has offered valuable insights into numerous cellular processes including motility, invasion, and alterations in cell morphology. Here, we discuss novel techniques devised to more closely mimic cell-tissue interactions and to study cell response to distinct physico-chemical changes in biomaterials, with an emphasis on the manipulation of collagen scaffolds. The benefits and pitfalls associated with using collagen are discussed in the context of strategies proposed to control the engineered microenvironment. Tunable systems such as these offer the ability to alter individual features of the microenvironment in vitro, with the promise that the molecular basis of mechanotransduction in vivo may be laid out in future

Towards Tuning the Mechanical Properties of Three-Dimensional Collagen Scaffolds Using a Coupled Fiber-Matrix Model

Scaffold mechanical properties are essential in regulating the microenvironment of three-dimensional cell culture. A coupled fiber-matrix numerical model was developed in this work for predicting the mechanical response of collagen scaffolds subjected to various levels of non-enzymatic glycation and collagen concentrations. The scaffold was simulated by a Voronoi network embedded in a matrix. The computational model was validated using published experimental data. Results indicate that both non-enzymatic glycation-induced matrix stiffening and fiber network density, as regulated by collagen concentration, influence scaffold behavior. The heterogeneous stress patterns of the scaffold were induced by the interfacial mechanics between the collagen fiber network and the matrix. The knowledge obtained in this work could help to fine-tune the mechanical properties of collagen scaffolds for improved tissue regeneration applications

Age-related vascular stiffening: causes and consequences

Arterial stiffening occurs with age and is closely associated with the progression of cardiovascular disease. Stiffening is most often studied at the level of the whole vessel because increased stiffness of the large arteries can impose increased strain on the heart leading to heart failure. Interestingly, however, recent evidence suggests that the impact of increased vessel stiffening extends beyond the tissue scale and can also have deleterious microscale effects on cellular function. Altered extracellular matrix (ECM) architecture has been recognized as a key component of the pre-atherogenic state. Here, the underlying causes of age-related vessel stiffening are discussed, focusing on age-related crosslinking of the ECM proteins as well as through increased matrix deposition. Methods to measure vessel stiffening at both the macroscale and microscale are described, spanning from the pulse wave velocity measurements performed clinically to microscale measurements performed largely in research laboratories. Additionally, recent work investigating how arterial stiffness and the changes in the ECM associated with stiffening contributed to endothelial dysfunction will be reviewed. We will highlight how changes in ECM protein composition contribute to atherosclerosis in the vessel wall. Lastly, we will discuss very recent work that demonstrates endothelial cells are mechano-sensitive to arterial stiffening, where changes in stiffness can directly impact endothelial cell health. Overall, recent studies suggest that stiffening is an important clinical target not only because of potential deleterious effects on the heart but also because it promotes cellular level dysfunction in the vessel wall, contributing to a pathological atherosclerotic state

Microfabricated Physical Spatial Gradients for Investigating Cell Migration and Invasion Dynamics

We devise a novel assay that introduces micro-architectures into highly confining microchannels to probe the decision making processes of migrating cells. The conditions are meant to mimic the tight spaces in the physiological environment that cancer cells encounter during metastasis within the matrix dense stroma and during intravasation and extravasation through the vascular wall. In this study we use the assay to investigate the relative probabilities of a cell 1) permeating and 2) repolarizing (turning around) when it migrates into a spatially confining region. We observe the existence of both states even within a single cell line, indicating phenotypic heterogeneity in cell migration invasiveness and persistence. We also show that varying the spatial gradient of the taper can induce behavioral changes in cells, and different cell types respond differently to spatial changes. Particularly, for bovine aortic endothelial cells (BAECs), higher spatial gradients induce more cells to permeate (60%) than lower gradients (12%). Furthermore, highly metastatic breast cancer cells (MDA-MB-231) demonstrate a more invasive and permeative nature (87%) than non-metastatic breast epithelial cells (MCF-10A) (25%). We examine the migration dynamics of cells in the tapered region and derive characteristic constants that quantify this transition process. Our data indicate that cell response to physical spatial gradients is both cell-type specific and heterogeneous within a cell population, analogous to the behaviors reported to occur during tumor progression. Incorporation of micro-architectures in confined channels enables the probing of migration behaviors specific to defined geometries that mimic in vivo microenvironments

Traction forces exerted by endothelial cells on deformable substrates

Endothelial cells comprise the nearly impermeable single cell barrier that lines the lumen of all blood vessels. During angiogenesis, endothelial cells migrate from an existing capillary into the surrounding extracellular matrix to ultimately form a new capillary. Physiologically, blood vessel formation is important for wound healing; however, aberrant endothelial cell behavior can lead to tumor formation and numerous vascular diseases. This thesis extends the current knowledge of endothelial cell adhesion and migration by investigating the traction forces endothelial cells exert on their substrate. We are the first to apply Traction Force Microscopy (TFM), a technique that quantifies the magnitude, direction and spatial location of the traction forces exerted by a cell on its substrate, to the study of endothelial cell adhesion. We have created well-characterized substrates to investigate changes in endothelial cell traction resulting from changes in ligand density and mechanical compliance. Using TFM, we have determined the relationship between cell force and area over a wide range of ligand concentrations, and have found fundamental differences in cell behavior. Our results indicate the total magnitude of force that a cell exerts is related to its area. Moreover, we characterized the effects of ligand density on the ability of endothelial cells to adhere and exert forces immediately after plating and throughout spreading. Ligand density, in part, dictates the rate of spreading, the extent of spreading and the shape changes that the cells undergo during spreading. To further probe the relationship between traction and cell area, we also investigated the cellular adhesion and contractile mechanisms of focal adhesion and stress fiber formation during spreading. Interestingly, we found that focal adhesion, as marked by vinculin clustering, and actin stress fibers, as marked by phalloidin staining, are not necessary for traction force generation. In a first step towards understanding tissue formation, we explored endothelial cell-cell cohesion by investigating the changes cellular traction during cell-cell contact. Interestingly, we found that cell contact results in both global and local changes in traction generation. Even more notably, we identified a previously undescribed form of cell-cell communication where cells sense and respond to adjacent cells through traction-driven tension created in the substrate. This data coincides with recent studies of durotaxis and demonstrate a novel mechanism of cell-cell communication. These insights into the mechanics of endothelial cell behavior will ultimately benefit the rational design of biomaterials and tissue-engineered therapeutics

Traction forces exerted by endothelial cells on deformable substrates

Endothelial cells comprise the nearly impermeable single cell barrier that lines the lumen of all blood vessels. During angiogenesis, endothelial cells migrate from an existing capillary into the surrounding extracellular matrix to ultimately form a new capillary. Physiologically, blood vessel formation is important for wound healing; however, aberrant endothelial cell behavior can lead to tumor formation and numerous vascular diseases. This thesis extends the current knowledge of endothelial cell adhesion and migration by investigating the traction forces endothelial cells exert on their substrate. We are the first to apply Traction Force Microscopy (TFM), a technique that quantifies the magnitude, direction and spatial location of the traction forces exerted by a cell on its substrate, to the study of endothelial cell adhesion. We have created well-characterized substrates to investigate changes in endothelial cell traction resulting from changes in ligand density and mechanical compliance. Using TFM, we have determined the relationship between cell force and area over a wide range of ligand concentrations, and have found fundamental differences in cell behavior. Our results indicate the total magnitude of force that a cell exerts is related to its area. Moreover, we characterized the effects of ligand density on the ability of endothelial cells to adhere and exert forces immediately after plating and throughout spreading. Ligand density, in part, dictates the rate of spreading, the extent of spreading and the shape changes that the cells undergo during spreading. To further probe the relationship between traction and cell area, we also investigated the cellular adhesion and contractile mechanisms of focal adhesion and stress fiber formation during spreading. Interestingly, we found that focal adhesion, as marked by vinculin clustering, and actin stress fibers, as marked by phalloidin staining, are not necessary for traction force generation. In a first step towards understanding tissue formation, we explored endothelial cell-cell cohesion by investigating the changes cellular traction during cell-cell contact. Interestingly, we found that cell contact results in both global and local changes in traction generation. Even more notably, we identified a previously undescribed form of cell-cell communication where cells sense and respond to adjacent cells through traction-driven tension created in the substrate. This data coincides with recent studies of durotaxis and demonstrate a novel mechanism of cell-cell communication. These insights into the mechanics of endothelial cell behavior will ultimately benefit the rational design of biomaterials and tissue-engineered therapeutics

Tuning cell migration: contractility as an integrator of intracellular signals from multiple cues [version 1; referees: 2 approved]

There has been immense progress in our understanding of the factors driving cell migration in both two-dimensional and three-dimensional microenvironments over the years. However, it is becoming increasingly evident that even though most cells share many of the same signaling molecules, they rarely respond in the same way to migration cues. To add to the complexity, cells are generally exposed to multiple cues simultaneously, in the form of growth factors and/or physical cues from the matrix. Understanding the mechanisms that modulate the intracellular signals triggered by multiple cues remains a challenge. Here, we will focus on the molecular mechanism involved in modulating cell migration, with a specific focus on how cell contractility can mediate the crosstalk between signaling initiated at cell-matrix adhesions and growth factor receptors

A balance of substrate mechanics and matrix chemistry regulates endothelial cell network assembly. Cellular and Molecular Bioengineering

Abstract-Driven by specific extracellular matrix cues, endothelial cells can spontaneously assemble into networks. Cell network assembly is, in part, dictated by both substrate stiffness and extracellular matrix chemistry; however, the balance between substrate mechanics and matrix chemistry in promoting cell network assembly is not well understood. Because both mechanics and chemistry can alter cell-substrate and cell-cell adhesion, we hypothesized that cell network assembly can be promoted on substrates that minimize cell-substrate adhesivity while promoting cell-cell connections. To investigate these hypotheses, bovine aortic endothelial cells (BAEC) were seeded on variably compliant polyacrylamide (PA) substrates derivatized with type I collagen and observed over time. Our results indicate that cell network assembly can be induced on substrates that are sufficiently compliant (Young's modulus, E = 200 Pa) and present significant amounts of substrate-bound ligand, and on substrates that are stiffer (E = 10,000 Pa) but which present less adhesive ligand. In both of these cases, cellsubstrate adhesivity is decreased, which may enhance cellcell adhesivity. Moreover, our data indicate that fibronectin polymerization stabilizes cell-cell contacts and is necessary for network formation to occur regardless of substrate compliance or the density of substrate-bound ligand. These data demonstrate the balance between substrate mechanics and chemistry in directing cell network assembly