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Published data on the mechanical strength and elasticity of lung tissue is widely variable, primarily due to differences in how testing was conducted across individual studies. This makes it extremely difficult to find a benchmark modulus of lung tissue when designing synthetic extracellular matrices (ECMs). To address this issue, we tested tissues from various areas of the lung using multiple characterization techniques, including micro-indentation, small amplitude oscillatory shear (SAOS), uniaxial tension, and cavitation rheology. We report the sample preparation required and data obtainable across these unique but complimentary methods to quantify the modulus of lung tissue. We highlight cavitation rheology as a new method, which can measure the modulus of intact tissue with precise spatial control, and reports a modulus on the length scale of typical tissue heterogeneities. Shear rheology, uniaxial, and indentation testing require heavy sample manipulation and destruction; however, cavitation rheology can be performed in situ across nearly all areas of the lung with minimal preparation. The Young's modulus of bulk lung tissue using micro-indentation (1.4±0.4 kPa), SAOS (3.3±0.5 kPa), uniaxial testing (3.4±0.4 kPa), and cavitation rheology (6.1±1.6 kPa) were within the same order of magnitude, with higher values consistently reported from cavitation, likely due to our ability to keep the tissue intact. Although cavitation rheology does not capture the non-linear strains revealed by uniaxial testing and SAOS, it provides an opportunity to measure mechanical characteristics of lung tissue on a microscale level on intact tissues. Overall, our study demonstrates that each technique has independent benefits, and each technique revealed unique mechanical features of lung tissue that can contribute to a deeper understanding of lung tissue mechanics.

For an airway or a blood vessel to narrow, there must be a connected path that links the smooth muscle (SM) cells with each other, and transmits forces around the organ, causing it to constrict. Currently, we know very little about the mechanisms that regulate force transmission pathways in a multicellular SM ensemble. Here, we used extracellular matrix (ECM) micropatterning to study force transmission in a two-cell ensemble of SM cells. Using the two-SM cell ensemble, we demonstrate (a) that ECM stiffness acts as a switch that regulates whether SM force is transmitted through the ECM or through cell-cell connections. (b) Fluorescent imaging for adherens junctions and focal adhesions show the progressive loss of cell-cell borders and the appearance of focal adhesions with the increase in ECM stiffness (confirming our mechanical measurements). (c) At the same ECM stiffness, we show that the presence of a cell-cell border substantially decreases the overall contractility of the SM cell ensemble. Our results demonstrate that connectivity among SM cells is a critical factor to consider in the development of diseases such as asthma and hypertension.

Knowledge of cell mechanical properties, such as elastic modulus, is essential to understanding the mechanisms by which cells carry out many integrated functions in health and disease. Cellular stiffness is regulated by the composition, structural organization, and indigenous mechanical stress (or prestress) borne by the cytoskeleton. Current methods for measuring stiffness and cytoskeletal prestress of living cells necessitate either limited spatial resolution but with high speed, or spatial maps of the entire cell at the expense of long imaging times. We have developed a novel technique, called biomechanical imaging, for generating maps of both cellular stiffness and prestress that requires less than 30 s of interrogation time, but which provides subcellular spatial resolution. The technique is based on the ability to measure tractions applied to the cell while simultaneously observing cell deformation, combined with capability to solve an elastic inverse problem to find cell stiffness and prestress distributions. We demonstrated the application of this technique by carrying out detailed mapping of the shear modulus and cytoskeletal prestress distributions of 3T3 fibroblasts, making no assumptions regarding those distributions or the correlation between them. We also showed that on the whole cell level, the average shear modulus is closely associated with the average prestress, which is consistent with the data from the literature. Data collection is a straightforward procedure that lends itself to other biochemical/biomechanical interventions. Biomechanical imaging thus offers a new tool that can be used in studies of cell biomechanics and mechanobiology where fast imaging of cell properties and prestress is desired at subcellular resolution.

For an airway or a blood vessel to narrow, there must be a connected path that links the smooth muscle (SM) cells with each other, and transmits forces around the organ, causing it to constrict. Currently, we know very little about the mechanisms that regulate force transmission pathways in a multicellular SM ensemble. Here, we used extracellular matrix (ECM) micropatterning to study force transmission in a two-cell ensemble of SM cells. Using the two-SM cell ensemble, we demonstrate (a) that ECM stiffness acts as a switch that regulates whether SM force is transmitted through the ECM or through cell-cell connections. (b) Fluorescent imaging for adherens junctions and focal adhesions show the progressive loss of cell-cell borders and the appearance of focal adhesions with the increase in ECM stiffness (confirming our mechanical measurements). (c) At the same ECM stiffness, we show that the presence of a cell-cell border substantially decreases the overall contractility of the SM cell ensemble. Our results demonstrate that connectivity among SM cells is a critical factor to consider in the development of diseases such as asthma and hypertension. Footnotes * The manuscript has been revised based on comments received.

Cells respond to a number of cues that affect how they interact with their surrounding environment, such as topology, the presentation of adhesive ligands, and stiffness. Recent advancements in the field of mechanobiology have revealed that one of the main ways in which cells sense these cues is through contractile forces. Mechanobiology research seeks to understand how environmental cues affect the forces that cells exert on their surrounding environment and how these mechanical forces are communicated to the cell and transformed into biochemical signals. Therefore, quantitative methods have been developed to determine cell contractility on soft, optically transparent, deformable surfaces by quantifying substrate deformation in terms of cellular traction forces. However, the currently available tools that are used to study cell interactions are limited in their applicability due to the need for specialized technical expertise that is not amenable to the widespread adaptation of these techniques. Therefore, we have sought to develop a novel traction force microscopy technique known as micropattem traction microscopy. With this technique, we hope to greatly simplify the current traction force microscopy techniques and provide a method which will be able to be adopted by a wide range of laboratories. This dissertation describes the process of the development and application of this novel traction force technique to probe questions in mechanobiology that have not been previously broached due to the lack of appropriate tools. The technique itself uses indirect microcontact printing to create a regularized array of fluorescent protein onto a glass substrate, which is then transferred to an optically transparent, soft, elastic polyacrylamide hydrogel. Cells, limited by their ability to adhere only to patterned regions, will deform the pattern at these defined points. Thus, with knowledge of the bulk elastic properties of the substrate and a priori knowledge of the pattern, we are able to quantify the force a cell is exerting without its removal. We also developed and released a robust, automated MATLAB program that will aid users in the calculation of traction forces so that people with limited experience with programming can utilize the program without significant investments into training. This indirect approach allows for not only individual proteins, but also for multiple, spatially distinct, fluorescent proteins such as fibronectin and gelatin to be simultaneously patterned onto this surface as well. The ability to pattern multiple proteins in a spatially defined region significantly aids in giving users control over as many parameters as possible. Finally, we will explore the current and future potential that this technique has to offer to researchers in the field of mechanobiology.

Published data on the mechanical strength and elasticity of lung tissue is widely variable, primarily due to differences in how testing was conducted across individual studies. This makes it extremely difficult to find a benchmark modulus of lung tissue when designing synthetic extracellular matrices (ECMs). To address this issue, we tested tissues from various areas of the lung using multiple characterization techniques, including micro-indentation, small amplitude oscillatory shear (SAOS), uniaxial tension, and cavitation rheology. We report the sample preparation required and data obtainable across these unique but complimentary methods to quantify the modulus of lung tissue. We highlight cavitation rheology as a new method, which can measure the modulus of intact tissue with precise spatial control, and reports a modulus on the length scale of typical tissue heterogeneities. Shear rheology, uniaxial, and indentation testing require heavy sample manipulation and destruction; however, cavitation rheology can be performed in situ across nearly all areas of the lung with minimal preparation. The Young's modulus of bulk lung tissue using micro-indentation (1.9 0.5 kPa), SAOS (3.2 0.6 kPa), uniaxial testing (3.4 0.4 kPa), and cavitation rheology (6.1 1.6 kPa) were within the same order of magnitude, with higher values consistently reported from cavitation, likely due to our ability to keep the tissue intact. Although cavitation rheology does not capture the non-linear strains revealed by uniaxial testing and SAOS, it provides an opportunity to measure mechanical characteristics of lung tissue on a microscale level on intact tissues. Overall, our study demonstrates that each technique has independent benefits, and each technique revealed unique mechanical features of lung tissue that can contribute to a deeper understanding of lung tissue mechanics. Footnotes * Added additional figures on a comparative polymeric material (Figure 5b, Suppl. Figure 3, and Suppl. Figure 4).

The current therapeutic approach to asthma focuses exclusively on targeting inflammation and reducing airway smooth muscle force in an effort to prevent the recurrence of symptoms. However, the treatment is not a cure and has little beneficial effect on the progression of asthma. This suggests that there are mechanisms at play that are likely triggered by inflammation and eventually become self-sustaining so that even when airway inflammation is brought back under control, these alternative mechanisms continue to drive airway hyperreactivity in asthmatics. In this study, we hypothesized that the stiffening of the airway extracellular matrix is a core pathological change sufficient to support excessive bronchoconstriction in asthmatics even when in the absence of inflammation. To test this hypothesis, we increased the stiffness of airway ECM by photocrosslinking collagen fibers within the airway wall using riboflavin (vitamin B2) and Ultraviolet-A radiation. In our experiments, collagen crosslinking led to a three-fold increase in stiffness of the airway extracellular matrix. This change was sufficient to cause airways to constrict to a greater degree, at a faster rate when exposed to a low dose of contractile agonist. Our results highlight the need for therapeutic approaches that target matrix remodeling to develop a lasting cure for this disease. Competing Interest Statement The authors have declared no competing interest.