Session GB: Cellular Mechanics and Biomechanics

Mechanical Property measurements of Single Nanofibers

Mechanical properties of biological materials play an important role in physiology. Specifically, mechanical properties of nanofibers are important to the extracellular matrix as well as in blood coagulation. Previous studies measured mechanical and structural properties such as creep, storage modulus, G', and loss modulus, G'' of nanofiber mats or bundles however, individual fiber properties were not measured due to measurement limitations because of the size of the fibers. However, advances in technologies, instrumentations and techniques now allow us to probe the properties of individual nanofibers. The mechanical properties of individual fibers help us to understand and model the properties of the bulk fiber network. Our research focuses on the mechanical properties of fibrin fibers and electrospun fibrinogen. Studies on electrospun fibers have determined their bending modulus and extensibility and elastic limit. We have worked in concert with these efforts to expand the knowledge of single fiber mechanical properties using a combined atomic force microscope (AFM) and inverted optical microscope. We found fibrin fiber have a modulus of 4 $+$/- 3 MPa when uncrosslinked and 15 $+$/- 7 MPa when crosslinked. When we measured a variant fibrinogen molecule, which eliminates gamma-gamma crosslinking, the modulus was 10 $+$/- 12 MPa. We also measured the extensibility of the fibers, the extensibilities were 221 $+$/- 44 {\%}, 177 $+$/- 58 {\%} and 236 $+$/- 96 {\%} for uncrosslinked, crosslinked and variant fibrin, respectively. In addition we measured the modulus (17.6 $+$/- 1.5 MPa) and extensibility (130 $+$/- 10 {\%}) for electrospun fibrinogen fibers. These studies provide insight into the similarities and differences between native and electrospun fibrin/fibrinogen fibers as well as, provide insight in to the role of crosslinking on the mechanical properties of fibrin fibers.   

How a Surface Bound Polymer Matrix Can Regulate Surface Accessibility and Function of Cells

The external interface of many cell types is not the plasma membrane. Rather, the gateway to a sub-class of cells is a sizable surface bound polymer brush like structure. This so-called pericellular matrix (PCM) extends a few hundred nanometers to tens of microns from the cell surface. The PCM affects filtration and transport of molecules to and from the cell surface. It also influences interactions of the plasma membrane with surrounding cells and extracellular matrix. Studies suggest that the PCM plays key physicochemical roles in processes as diverse as molecular sequestration, mediation of cell adhesion in proliferating and migrating cells, the formation of neuronal synapses, embryogenesis, and cancer metastasis. This talk will present insights into the macromolecular structure and the mechanics of the PCM gained from a spectrum of original biophysical assays, ranging from optical force probe microscopy to quantitative particle exclusion assays to fluorescent single molecule tracking. Accessibility of the cell surface and its screening by the PCM will be addressed. Preliminary experiments investigating (model) growth factor sequestration via electrostatic interaction with PCM molecules and its possible relation to the influence of PCM on mesenchymal stem cell differentiation will be discussed.   

Matrices and Mechanics to Direct hASC Fate

Functional tissue engineering uses physical stimulation to direct cell populations to produce tissue with anatomically and physiologically correct structures and with material properties similar to native tissue. Adipose-derived stem cells (ASC) are a particularly promising cell source for functional tissue engineering applications due to their multilineage differentiation potential and their abundance and ease of harvest relative to many other cell types. However, mechanobiological understanding of human ASC (hASC) is still emerging and many questions remain to be answered. Approaches and mechanisms associated with physical stimuli-induced hASC lineage specification and functional tissue formation comprise an increasingly active area of investigation and much remains to be learned. A primary objective of the Loboa lab is to understand and elucidate the role of physical stimuli on the mechanobiology of hASC and attempt to optimize these effects for functional tissue engineering using hASC. Both computational and empirical approaches are utilized in our investigations of hASC mechanobiology for tissue regeneration. Methods include: 1) application of external physical stimuli via use of custom bioreactor systems that mimic in vivo physical stimuli; 2) finite element analyses of cell-seeded constructs exposed to mechanical load to determine local stresses and strains associated with global strain applications; 3) investigations of mechanotransduction mechanisms associated with hASC response to physical stimuli; and 4) creation of biomimetic 3D scaffolds to induce hASC proliferation and controlled differentiation. Studies performed with hASC on novel biomaterials developed in our lab have recently led to our further development of biomimetic engineered nanofibrous scaffolds for wound healing applications. Fibrous materials are constructed from biocompatible, biodegradable materials that possess structural and physical similarities to the native extracellular matrix. In addition to mimicking the \textit{in vivo} topographical environment, fibrous materials provide an ideal substrate for bioactive molecule delivery based on their superior surface area to volume ratio (yielding maximum interaction with a surrounding medium), and the ability to generate controlled release kinetics based on biomolecule placement within the fibrous scaffold. Specifically, bioactive dopants can be homogenized within the polymeric matrix of fibrous assemblies, be preferentially located in either the core or shell of a fiber, and unique fiber architectures can also be generated with porous morphologies along the length of the fiber. We have found distinct differences in release profiles as a function of fiber morphology and initial drug concentration that significantly affect human stem cell and human skin cell fate. Using these approaches, we have created novel scaffolds that successfully inhibit and kill multiple bacteria of critical concern in wound healing while also maintaining viability, or in some cases promoting proliferation, of human skin cells.   

TNF-alpha mediated modulation of cell biophysical properties enhances cell adhesion and transendothelial migration

Tumor necrosis factor alpha (TNF-alpha) is a widely studied inflammatory cytokine involved in apoptosis, cell survival, inflammation and tumor angiogenesis. It has also been shown to promote cell migration and cytoskeletal rearrangement. We hypothesized that TNF-alpha promotes prometastatic changes in normal mammary epithelial cells by altering their biophysical properties. These changes likely facilitate adhesive interactions with the extracellular matrix (ECM) and promote cell migration and metastasis. We carried out atomic force microscope cell stiffness measurements of the mammary epithelial cell lines. The obtained force-indentation curves were fitted to the Young's modulus. In addition, AFM single cell force spectroscopy (SCFS) was used to measure cell adhesion to collagen type I. Further, adhesion receptor distribution and cell to cell adhesion measurements were conducted to reveal if inflammatory conditions promoted cell transendothelial migration. We found that TNF-alpha treatment decreased the measured cell stiffness of mammary epithelial cells. These changes facilitated enhanced mammary epithelial cell adhesion to collagen following TNF-alpha stimulation, which was largely mediated by increased tether formation. Further, we determined that combined treatment with TNF-alpha and INF-gamma enhanced cell transendothelial migration. AFM adhesion mapping revealed greater adhesion receptor clustering near HUVEC junctions. This facilitated enhanced cell adhesion as measured by SCFS. Taken together, our results implicate that pro-inflammatory mediators, such as TNF-alpha, play an important role in modulating epithelial cell biophysical properties toward a more metastatic cell phenotype.   

Mechanisms of Cell Adhesion and Migration on Simple and Complex Surfaces

Coordinated motion is a hallmark of animal behavior at diverse length scales, ranging over about 9 orders of magnitude for individuals and about 3 orders of magnitude more for populations. My laboratory is studying selected biophysical and biochemical aspects of cell adhesion and migration with a view towards novel materials applications in biotechnology and medicine. The cells of this talk are normal human dermal fibroblasts, skin cells. The materials are non-woven electrospun fibers made of synthetic polypeptides. We have characterized physical properties of these materials. We have analyzed cell interactions with fibers by microscopy. Specific protein constituents of focal adhesions (FAs) have been stained with fluorescent antibodies, and cell migration in real time has been monitored by phase-contrast microscopy and confocal laser-scanning microscopy. Analysis of samples stained for specific focal adhesion proteins showed that the surface density of FAs on fibers, 6 x 10$^{-3}\mu $m$^{2}$, was about 2-fold higher than on glass, essentially a planar substrate. Further analysis showed that the average angle between the major axis of focal adhesions and the fiber trajectory was 22 deg., roughly half of the expected value for random orientation. The alignment data have been interpreted in terms of beam statistical mechanics, yielding a flexural rigidity of 8.3 x 10$^{-26}$ N-m$^{2}$. This stiffness is about 10$^{3}$-fold smaller than for microtubules and 25{\%} greater than for actin filaments. Modeling based on this result is consistent with the experimental result that FA alignment increases as fiber diameter decreases. We have also utilized near-UV circular dichroism spectroscopy and intrinsic fluorescence emission spectroscopy to obtain Langmuir isotherms for cytoplasmic tails of integrin $\beta $ subunits associating with intracellular focal adhesion constituents in vitro. Dissociation constants obtained by fitting a single-site model to the experimental data range from 8.3 $\mu $M to 50 nM at ambient temperature. Taken together, the results suggest that the coupling between FAs and stress fibers is tight and highly specific, and there is a quantifiable thermal limit to the energy cost a cell will pay to form an adhesion site. The results further suggest a limit to the benefits that can be derived from cellular interaction with disorganized nanostructured materials.   

Visualizing Molecular Forces Across Specific Proteins in Living Cells

In vivo, cells adhere to a deformable extracellular matrix that is both a source of applied forces and a means of mechanical support. Cells detect and interpret mechanical signals, such as force and rigidity, from the extracellular environment through mechanotransduction. This process is central to cell migration, tissue organization, and many disease states. Progress has largely been limited by an inability to measure dynamic forces across proteins in living cells. Recently we developed an experimentally calibrated Forster resonance energy transfer (FRET)-based biosensor that measures forces across specific proteins in cells with pico-Newton sensitivity. While appropriate for focal adhesion proteins, such as vinculin, the previous sensor was only sensitive to 1-6 pN forces. As this sensor may not be optimal for studying vinculin in fibroblasts or appropriate for other contexts, such as use in highly contractile cells, we have designed and constructed a new class of tension sensors. These are based on unstructured polypeptides. We have developed a model, based on simple theories from polymer physics, which suggests these sensors should have force sensitivities ranging from 0.5-25 pN. This range is expected to be sufficient for many studies of mechanotransduction and mechanotransmission. Current work focuses on determining if these new linkers are well-described by this simple model, which is unlike the existing flagelliform-based sensors, and assessing the function of the new sensors. These efforts should enable the rational design of a new generation of FRET-based tension sensors appropriate for a wide range of studies in mechanobiology in many novel systems.   

Mechanics and Malignancy: Biophysical Approaches for Investigating the Tumor Microenvironment

Despite huge advances in the molecular regulators of cancer growth and metastasis, patient survival rates have largely stagnated, with over 90{\%} of cancer-related deaths due to metastasis. The majority of cancer drugs target cancer cells in the primary tumor, which doesn't prevent the development of metastatic tumors from cells dormant in the tissues. Bone marrow derived mesenchymal stem cells (MSCs) that accumulate in the primary tumor due to their natural tropism for inflammatory tissues may also enhance the metastatic potential of tumor cells through direct interactions or paracrine signaling. A series of recent studies have highlighted that in addition to molecular changes, cancer cells also undergo biophysical changes.~ Though emerging work highlights the importance of tumor stromal cells and microenvironment in cancer progression, the interplay of these factors has not been fully investigated. My research combines molecular and gene expression analysis with quantitative biophysical analysis using sensitive mechanical tools (such as time-lapsed cell tracking, traction force microscopy, and particle tracking microrheology) to provide genetic and mechanical profiles of tumor and stromal cells in conditions that more closely mimic the tumor microenvironment. This approach has recently been used to demonstrate that ovarian cancer cells, which metastasize to the soft omentum fat pad, preferentially engraft on adipose-mimetic substrates or MSCs differentiated into soft adipocytes. Moreover, after engrafting they display a gene expression signature characteristic of epithelial-mesenchymal transition with corresponding increases in motility, proliferation, and chemoresistance. ~Though this preference for soft matrices is in contrast to what has been documented in breast and other cancers, our studies have confirmed that an increased malignant phenotype is still associated with higher traction forces. Work from my lab has also shown that both murine and human MSCs undergo dramatic cytoskeletal stiffening in response to pro-migratory molecules in the tumor microenvironment, including a cocktail of molecules released by tumor cells in culture and individual molecules like TGF-$\beta $1 and PDGF. The degree of stiffening is a key differentiating factor between MSCs and their less migratory fibroblast counterparts and even predictive of decreased MSC function with extended culture.   

Biophysical Modeling and Mechanobiology of Cartilaginous Tissues

Cartilaginous tissues (e.g., knee cartilage and intervertebral disc) are subjected to a wide range of mechanical loading associated with the daily physical activities. The development and maintenance of tissue structure and mechanical characteristics are tied directly to the effect of mechanical loading on the biology of cartilage cells and the extracellular matrix (ECM). Mechanical loadings produce complex physical-chemical environment changes around cartilage cells, including spatial-temporal variations of stress, strain, fluid flow, fluid pressure, osmotic pressure, fixed charge density, pH, electrical field, and solute transport within the ECM. These physicochemical signals across the ECM, which are difficult to be measured, can be quantitatively determined by using appropriate theoretical models for the mechano-electrochemical behaviors of cartilage. These models provide an essential framework for correlating the spatial-temporal distributions of physical stimuli surrounding cells with external loading at the tissue level. We introduce a multiphasic mechano-electrochemical model for cartilaginous tissues based on the general mixture theory. Correspondingly, the techniques are discussed for establishing constitutive relationships in the model for mechanical, electrical, and transport properties. In addition, the effect of physicochemical signals on cartilage cell homeostasis is demonstrated. The presented studies are important to the understanding of cartilage mechanobiology and shed light on how cartilage cells convert physical stimuli into cellular responses which can ultimately guide the maintenance of healthy cartilage and treatment of related disease.