Cellular Microbiaxial Stretching to Measure a Single-Cell Strain Energy Density Function.

The stress in a cell due to extracellular mechanical stimulus is determined by its mechanical properties, and the structural organization of many adherent cells suggests that their properties are anisotropic. This anisotropy may significantly influence the cells' mechanotransductive response to complex loads, and has important implications for development of accurate models of tissue biomechanics. Standard methods for measuring cellular mechanics report linear moduli that cannot capture large-deformation anisotropic properties, which in a continuum mechanics framework are best described by a strain energy density function (SED). In tissues, the SED is most robustly measured using biaxial testing. Here, we describe a cellular microbiaxial stretching (CµBS) method that modifies this tissue-scale approach to measure the anisotropic elastic behavior of individual vascular smooth muscle cells (VSMCs) with nativelike cytoarchitecture. Using CµBS, we reveal that VSMCs are highly anisotropic under large deformations. We then characterize a Holzapfel-Gasser-Ogden type SED for individual VSMCs and find that architecture-dependent properties of the cells can be robustly described using a formulation solely based on the organization of their actin cytoskeleton. These results suggest that cellular anisotropy should be considered when developing biomechanical models, and could play an important role in cellular mechano-adaptation.

Empirically Determined Vascular Smooth Muscle Cell Mechano-Adaptation Law.

Cardiovascular disease can alter the mechanical environment of the vascular system, leading to mechano-adaptive growth and remodeling. Predictive models of arterial mechano-adaptation could improve patient treatments and outcomes in cardiovascular disease. Vessel-scale mechano-adaptation includes remodeling of both the cells and extracellular matrix. Here, we aimed to experimentally measure and characterize a phenomenological mechano-adaptation law for vascular smooth muscle cells (VSMCs) within an artery. To do this, we developed a highly controlled and reproducible system for applying a chronic step-change in strain to individual VSMCs with in vivo like architecture and tracked the temporal cellular stress evolution. We found that a simple linear growth law was able to capture the dynamic stress evolution of VSMCs in response to this mechanical perturbation. These results provide an initial framework for development of clinically relevant models of vascular remodeling that include VSMC adaptation.

Vascular smooth muscle cell functional contractility depends on extracellular mechanical properties.

Vascular smooth muscle cells' primary function is to maintain vascular homeostasis through active contraction and relaxation. In diseases such as hypertension and atherosclerosis, this function is inhibited concurrent to changes in the mechanical environment surrounding vascular smooth muscle cells. It is well established that cell function and extracellular mechanics are interconnected; variations in substrate modulus affect cell migration, proliferation, and differentiation. To date, it is unknown how the evolving extracellular mechanical environment of vascular smooth muscle cells affects their contractile function. Here, we have built upon previous vascular muscular thin film technology to develop a variable-modulus vascular muscular thin film that measures vascular tissue functional contractility on substrates with a range of pathological and physiological moduli. Using this modified vascular muscular thin film, we found that vascular smooth muscle cells generated greater stress on substrates with higher moduli compared to substrates with lower moduli. We then measured protein markers typically thought to indicate a contractile phenotype in vascular smooth muscle cells and found that phenotype is unaffected by substrate modulus. These data suggest that mechanical properties of vascular smooth muscle cells' extracellular environment directly influence their functional behavior and do so without inducing phenotype switching.

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films.

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues. Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

Guidelines for the isolation and characterization of murine vascular smooth muscle cells. A report from the International Society of Cardiovascular Translational Research.

Vascular smooth muscle cells (VSMCs) play important roles in cardiovascular disorders and biology. Outlined in this paper is a step-by-step procedure for isolating aortic VSMCs from adult C57BL6J male mice by enzymatic digestion of the aorta using collagenase. The plating, culturing, and subculturing of the isolated cells are discussed in detail along with techniques to characterize VSMC phenotype by gene expression and immunofluorescence. Traction force microscopy was used to characterize contractility of single subcultured VSMCs at baseline.

Smooth muscle architecture within cell-dense vascular tissues influences functional contractility.

The role of vascular smooth muscle architecture in the function of healthy and dysfunctional vessels is poorly understood. We aimed at determining the relationship between vascular smooth muscle architecture and contractile output using engineered vascular tissues. We utilized microcontact printing and a microfluidic cell seeding technique to provide three different initial seeding conditions, with the aim of influencing the cellular architecture within the tissue. Cells seeded in each condition formed confluent and aligned tissues but within the tissues, the cellular architecture varied. Tissues with a more elongated cellular architecture had significantly elevated basal stress and produced more contractile stress in response to endothelin-1 stimulation. We also found a correlation between the contractile phenotype marker expression and the cellular architecture, contrary to our previous findings in non-confluent tissues. Taken with previous results, these data suggest that within cell-dense vascular tissues, smooth muscle contractility is strongly influenced by cell and tissue architectures.

Long-term vascular contractility assay using genipin-modified muscular thin films.

Vascular disease is a leading cause of death globally and typically manifests chronically due to long-term maladaptive arterial growth and remodeling. To date, there is no in vitro technique for studying vascular function over relevant disease time courses that both mimics in vivo-like tissue structure and provides a simple readout of tissue stress. We aimed to extend tissue viability in our muscular thin film contractility assay by modifying the polydimethylsiloxane (PDMS) substrate with micropatterned genipin, allowing extracellular matrix turnover without cell loss. To achieve this, we developed a microfluidic delivery system to pattern genipin and extracellular matrix proteins on PDMS prior to cell seeding. Tissues constructed using this method showed improved viability and maintenance of in vivo-like lamellar structure. Functional contractility of tissues fabricated on genipin-modified substrates remained consistent throughout two weeks in culture. These results suggest that muscular thin films with genipin-modified PDMS substrates are a viable method for conducting functional studies of arterial growth and remodeling in vascular diseases.