AFM and Microrheology in the Zebrafish Embryo Yolk Cell.

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples.

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

Polarized cortical tension drives zebrafish epiboly movements.

The principles underlying the biomechanics of morphogenesis are largely unknown. Epiboly is an essential embryonic event in which three tissues coordinate to direct the expansion of the blastoderm. How and where forces are generated during epiboly, and how these are globally coupled remains elusive. Here we developed a method, hydrodynamic regression (HR), to infer 3D pressure fields, mechanical power, and cortical surface tension profiles. HR is based on velocity measurements retrieved from 2D+T microscopy and their hydrodynamic modeling. We applied HR to identify biomechanically active structures and changes in cortex local tension during epiboly in zebrafish. Based on our results, we propose a novel physical description for epiboly, where tissue movements are directed by a polarized gradient of cortical tension. We found that this gradient relies on local contractile forces at the cortex, differences in elastic properties between cortex components and the passive transmission of forces within the yolk cell. All in all, our work identifies a novel way to physically regulate concerted cellular movements that might be instrumental for the mechanical control of many morphogenetic processes.

Microelastic mapping of the rat dentate gyrus.

The lineage commitment of many cultured stem cells, including adult neural stem cells (NSCs), is strongly sensitive to the stiffness of the underlying extracellular matrix. However, it remains unclear how well the stiffness ranges explored in culture align with the microscale stiffness values stem cells actually encounter within their endogenous tissue niches. To address this question in the context of hippocampal NSCs, we used atomic force microscopy to spatially map the microscale elastic modulus (E) of specific anatomical substructures within living slices of rat dentate gyrus in which NSCs reside during lineage commitment in vivo. We measured depth-dependent apparent E-values at locations across the hilus (H), subgranular zone (SGZ) and granule cell layer (GCL) and found a two- to threefold increase in stiffness at 500 nm indentation from the H (49 ± 7 Pa) and SGZ (58 ± 8 Pa) to the GCL (115 ± 18 Pa), a fold change in stiffness we have previously found functionally relevant in culture. Additionally, E exhibits nonlinearity with depth, increasing significantly for indentations larger than 1 µm and most pronounced in the GCL. The methodological advances implemented for these measurements allow the quantification of the elastic properties of hippocampal NSC niche at unprecedented spatial resolution.

Snail1-expressing fibroblasts in the tumor microenvironment display mechanical properties that support metastasis.

Crosstalk between tumor and stromal cells in the tumor microenvironment alter its properties in ways that facilitate the invasive behavior of tumor cells. Here, we demonstrate that cancer-associated fibroblasts (CAF) increase the stiffness of the extracellular matrix (ECM) and promote anisotropic fiber orientation, two mechanical signals generated through a Snail1/RhoA/αSMA-dependent mechanism that sustains oriented tumor cell migration and invasiveness. Snail1-depleted CAF failed to acquire myofibroblastic traits in response to TGFß, including RhoA activation, αSMA-positive stress fibers, increased fibronectin fibrillogenesis, and production of a stiff ECM with oriented fibers. Snail1 expression in human tumor-derived CAF was associated with an ability to organize the ECM. In coculture, a relatively smaller number of Snail1-expressing CAF were capable of imposing an anisotropic ECM architecture, compared with nonactivated fibroblasts. Pathologically, human breast cancers with Snail1(+) CAF tended to exhibit desmoplastic areas with anisotropic fibers, lymph node involvement, and poorer outcomes. Snail1 involvement in driving an ordered ECM was further confirmed in wound-healing experiments in mice, with Snail1 depletion preventing the anisotropic organization of granulation tissue and delaying wound healing. Overall, our results showed that inhibiting Snail1 function in CAF could prevent tumor-driven ECM reorganization and cancer invasion.

Inhomogeneity of local stiffness in the extracellular matrix scaffold of fibrotic mouse lungs.

Lung disease models are useful to study how cell engraftment, proliferation and differentiation are modulated in lung bioengineering. The aim of this work was to characterize the local stiffness of decellularized lungs in aged and fibrotic mice. Mice (2- and 24-month old; 14 of each) with lung fibrosis (N=20) and healthy controls (N=8) were euthanized after 11 days of intratracheal bleomycin (fibrosis) or saline (controls) infusion. The lungs were excised, decellularized by a conventional detergent-based (sodium-dodecyl sulfate) procedure and slices of the acellular lungs were prepared to measure the local stiffness by means of atomic force microscopy. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous within the lung and increased according to the degree of the structural fibrotic lesion. Local stiffness of the acellular lungs did not show statistically significant differences caused by age. The group of mice most affected by fibrosis exhibited local stiffness that were ~2-fold higher than in the control mice: from 27.2±1.64 to 64.8±7.1kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8kPa in the tunica intima. Since acellular lungs from mice with bleomycin-induced fibrosis present considerable micromechanical inhomogeneity, this model can be a useful tool to better investigate how different degrees of extracellular matrix lesion modulate cell fate in the process of organ bioengineering from decellularized lungs.

Heterogeneous micromechanical properties of the extracellular matrix in healthy and infarcted hearts.

Infarcted hearts are macroscopically stiffer than healthy organs. Nevertheless, although cell behavior is mediated by the physical features of the cell niche, the intrinsic micromechanical properties of healthy and infarcted heart extracellular matrix (ECM) remain poorly characterized. Using atomic force microscopy, we studied ECM micromechanics of different histological regions of the left ventricle wall of healthy and infarcted mice. Hearts excised from healthy (n=8) and infarcted mice (n=8) were decellularized with sodium dodecyl sulfate and cut into 12 µm thick slices. Healthy ventricular ECM revealed marked mechanical heterogeneity across histological regions of the ventricular wall with the effective Young's modulus ranging from 30.2 ± 2.8 to 74.5 ± 8.7 kPa in collagen- and elastin-rich regions of the myocardium, respectively. Infarcted ECM showed a predominant collagen composition and was 3-fold stiffer than collagen-rich regions of the healthy myocardium. ECM of both healthy and infarcted hearts exhibited a solid-like viscoelastic behavior that conforms to two power-law rheology. Knowledge of intrinsic micromechanical properties of the ECM at the length scale at which cells sense their environment will provide further insight into the cell-scaffold interplay in healthy and infarcted hearts.

Effects of the decellularization method on the local stiffness of acellular lungs.

Lung bioengineering, a novel approach to obtain organs potentially available for transplantation, is based on decellularizing donor lungs and seeding natural scaffolds with stem cells. Various physicochemical protocols have been used to decellularize lungs, and their performance has been evaluated in terms of efficient decellularization and matrix preservation. No data are available, however, on the effect of different decellularization procedures on the local stiffness of the acellular lung. This information is important since stem cells directly sense the rigidity of the local site they are engrafting to during recellularization, and it has been shown that substrate stiffness modulates cell fate into different phenotypes. The aim of this study was to assess the effects of the decellularization procedure on the inhomogeneous local stiffness of the acellular lung on five different sites: alveolar septa, alveolar junctions, pleura, and vessels' tunica intima and tunica adventitia. Local matrix stiffness was measured by computing Young's modulus with atomic force microscopy after decellularizing the lungs of 36 healthy rats (Sprague-Dawley, male, 250-300 g) with four different protocols with/without perfusion through the lung circulatory system and using two different detergents (sodium dodecyl sulfate [SDS] and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate [CHAPS]). The local stiffness of the acellular lung matrix significantly depended on the site within the matrix (p<0.001), ranging from ∼ 15 kPa at the alveolar septum to ∼ 60 kPa at the tunica intima. Acellular lung stiffness (p=0.003) depended significantly, albeit modestly, on the decellularization process. Whereas perfusion did not induce any significant differences in stiffness, the use of CHAPS resulted in a ∼ 35% reduction compared with SDS, the influence of the detergent being more important in the tunica intima. In conclusion, lung matrix stiffness is considerably inhomogeneous, and conventional decellularization procedures do not result in substantially different local stiffness in the acellular lung.

Development of a three-dimensional bone-like construct in a soft self-assembling peptide matrix.

This work describes the development of a three-dimensional (3D) model of osteogenesis using mouse preosteoblastic MC3T3-E1 cells and a soft synthetic matrix made out of self-assembling peptide nanofibers. By adjusting the matrix stiffness to very low values (around 120 Pa), cells were found to migrate within the matrix, interact forming a cell-cell network, and create a contracted and stiffer structure. Interestingly, during this process, cells spontaneously upregulate the expression of bone-related proteins such as collagen type I, bone sialoprotein, and osteocalcin, indicating that the 3D environment enhances their osteogenic potential. However, unlike MC3T3-E1 cultures in 2D, the addition of dexamethasone is required to acquire a final mature phenotype characterized by features such as matrix mineralization. Moreover, a slight increase in the hydrogel stiffness (threefold) or the addition of a cell contractility inhibitor (Rho kinase inhibitor) abrogates cell elongation, migration, and 3D culture contraction. However, this mechanical inhibition does not seem to noticeably affect the osteogenic process, at least at early culture times. This 3D bone model intends to emphasize cell-cell interactions, which have a critical role during tissue formation, by using a compliant unrestricted synthetic matrix.

Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells.

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm(2) cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca(2+) signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

Pre-treatment with mesenchymal stem cells reduces ventilator-induced lung injury.

Bone marrow-derived mesenchymal stem cells (MSCs) reduce acute lung injury in animals challenged by bleomycin or bacterial lipopolysaccaride. It is not known, however, whether MSCs protect from ventilator-induced lung injury (VILI). This study investigated whether MSCs have a potential role in preventing or modulating VILI in healthy rats subjected to high-volume ventilation. 24 Sprague-Dawley rats (250-300 g) were subjected to high-volume mechanical ventilation (25 mL·kg(-1)). MSCs (5 × 10(6)) were intravenously or intratracheally administered (n=8 each) 30 min before starting over-ventilation and eight rats were MSC-untreated. Spontaneously breathing anesthetised rats (n=8) served as controls. After 3 h of over-ventilation or control the animals were sacrificed and lung tissue and bronchoalveolar lavage fluid (BALF) were sampled for further analysis. When compared with controls, MSC-untreated over-ventilated rats exhibited typical VILI features. Lung oedema, histological lung injury index, concentrations of total protein, interleukin-1ß, macrophage inflammatory protein-2 and number of neutrophils in BALF and vascular cell adhesion protein-1 in lung tissue significantly increased in over-ventilated rats. All these indices of VILI moved significantly towards normalisation in the rats treated with MSCs, whether intravenously or intratracheally. Both local and systemic pre-treatment with MSCs reduced VILI in a rat model.