Characterization of the elastic properties of extracellular matrix models by atomic force microscopy.

Tissue elasticity is a critical regulator of cell behavior in normal and diseased conditions like fibrosis and cancer. Since the extracellular matrix (ECM) is a major regulator of tissue elasticity and function, several ECM-based models have emerged in the last decades, including in vitro endogenous ECM, decellularized tissue ECM and ECM hydrogels. The development of such models has urged the need to quantify their elastic properties particularly at the nanometer scale, which is the relevant length scale for cell-ECM interactions. For this purpose, the versatility of atomic force microscopy (AFM) to quantify the nanomechanical properties of soft biomaterials like ECM models has emerged as a very suitable technique. In this chapter we provide a detailed protocol on how to assess the Young's elastic modulus of ECM models by AFM, discuss some of the critical issues, and provide troubleshooting guidelines as well as illustrative examples of AFM measurements, particularly in the context of cancer.

Finite element simulation for the mechanical characterization of soft biological materials by atomic force microscopy.

The characterization of the mechanical properties of soft materials has been traditionally performed through uniaxial tensile tests. Nevertheless, this method cannot be applied to certain extremely soft materials, such as biological tissues or cells that cannot be properly subjected to these tests. Alternative non-destructive tests have been designed in recent years to determine the mechanical properties of soft biological tissues. One of these techniques is based on the use of atomic force microscopy (AFM) to perform nanoindentation tests. In this work, we investigated the mechanical response of soft biological materials to nanoindentation with spherical indenters using finite element simulations. We studied the responses of three different material constitutive laws (elastic, isotropic hyperelastic and anisotropic hyperelastic) under the same process and analyzed the differences thereof. Whereas linear elastic and isotropic hyperelastic materials can be studied using an axisymmetric simplification, anisotropic hyperelastic materials require three-dimensional analyses. Moreover, we established the limiting sample size required to determine the mechanical properties of soft materials while avoiding boundary effects. Finally, we compared the results obtained by simulation with an estimate obtained from Hertz theory. Hertz theory does not distinguish between the different material constitutive laws, and thus, we proposed corrections to improve the quantitative measurement of specific material properties by nanoindentation experiments.

Local micromechanical properties of decellularized lung scaffolds measured with atomic force microscopy.

Bioartificial lungs re-engineered from decellularized organ scaffolds are a promising alternative to lung transplantation. Critical features for improving scaffold repopulation depend on the mechanical properties of the cell microenvironment. However, the mechanics of the lung extracellular matrix (ECM) is poorly defined. The local mechanical properties of the ECM were measured in different regions of decellularized rat lung scaffolds with atomic force microscopy. Lungs excised from rats (n=11) were decellularized with sodium dodecyl sulfate (SDS) and cut into ~7µm thick slices. The complex elastic modulus (G(∗)) of lung ECM was measured over a frequency band ranging from 0.1 to 11.45Hz. Measurements were taken in alveolar wall segments, alveolar wall junctions and pleural regions. The storage modulus (G', real part of G(∗)) of alveolar ECM was ~6kPa, showing small changes between wall segments and junctions. Pleural regions were threefold stiffer than alveolar walls. G' of alveolar walls and pleura increased with frequency as a weak power law with exponent 0.05. The loss modulus (G″, imaginary part of G(∗)) was 10-fold lower and showed a frequency dependence similar to that of G' at low frequencies (0.1-1Hz), but increased more markedly at higher frequencies. Local differences in mechanical properties and topology of the parenchymal site could be relevant mechanical cues for regulating the spatial distribution, differentiation and function of lung cells.

Respiratory impedance during weaning from mechanical ventilation in a mixed population of critically ill patients.

BACKGROUND: Worsening of respiratory mechanics during a spontaneous breathing trial (SBT) has been traditionally associated with weaning failure, although this finding is based on studies with chronic obstructive pulmonary disease patients only. The aim of our study was to assess the course of respiratory impedance non-invasively measured by forced oscillation technique (FOT) during a successful and failed SBT in a mixed population. METHODS: Thirty-four weaning trials were reported in 29 consecutive mechanically ventilated patients with different causes of initiation of ventilation. During the SBT, the patient was breathing through a conventional T-piece connected to the tracheal tube. FOT (5 Hz, +/- 1 cm H(2)O, 30 s) was applied at 5, 10, 15, 20, 25, and 30 min. Respiratory resistance (Rrs) and reactance (Xrs) were computed from pressure and flow measurements. The frequency to tidal volume ratio f/V(t) was obtained from the flow signal. At the end of the trial, patients were divided into two groups: SBT success and failure. RESULTS: Mixed model analysis showed no significant differences in Rrs and Xrs over the course of the SBT, or between the success (n=16) and the failure (n=18) groups. In contrast, f/V(t) was significantly (P<0.001) higher in the failure group. CONCLUSIONS: Worsening of respiratory impedance measured by FOT is not a common finding during a failed SBT in a typically heterogeneous intensive care unit population of mechanically ventilated patients.

The temperature dependence of cell mechanics measured by atomic force microscopy.

The cytoskeleton is a complex polymer network that regulates the structural stability of living cells. Although the cytoskeleton plays a key role in many important cell functions, the mechanisms that regulate its mechanical behaviour are poorly understood. Potential mechanisms include the entropic elasticity of cytoskeletal filaments, glassy-like inelastic rearrangements of cross-linking proteins and the activity of contractile molecular motors that sets the tensional stress (prestress) borne by the cytoskeleton filaments. The contribution of these mechanisms can be assessed by studying how cell mechanics depends on temperature. The aim of this work was to elucidate the effect of temperature on cell mechanics using atomic force microscopy. We measured the complex shear modulus (G*) of human alveolar epithelial cells over a wide frequency range (0.1-25.6 Hz) at different temperatures (13-37 degrees C). In addition, we probed cell prestress by mapping the contractile forces that cells exert on the substrate by means of traction microscopy. To assess the role of actomyosin contraction in the temperature-induced changes in G* and cell prestress, we inhibited the Rho kinase pathway of the myosin light chain phosphorylation with Y-27632. Our results show that with increasing temperature, cells become stiffer and more solid-like. Cell prestress also increases with temperature. Inhibiting actomyosin contraction attenuated the temperature dependence of G* and prestress. We conclude that the dependence of cell mechanics with temperature is dominated by the contractile activity of molecular motors.

Thermal activation and ATP dependence of the cytoskeleton remodeling dynamics.

The cytoskeleton (CSK) is a nonequilibrium polymer network that uses hydrolyzable sources of free energy such as adenosine triphosphate (ATP) to remodel its internal structure. As in inert nonequilibrium soft materials, CSK remodeling has been associated with structural rearrangements driven by energy-activated processes. We carry out particle tracking and traction microscopy measurements of alveolar epithelial cells at various temperatures and ATP concentrations. We provide the first experimental evidence that the remodeling dynamics of the CSK is driven by structural rearrangements over free-energy barriers induced by thermally activated forces mediated by ATP. The measured activation energy of these forces is approximately 40k_{B}T_{r} ( k_{B} being the Boltzmann constant and T_{r} being the room temperature). Our experiments provide clues to understand the analogy between the dynamics of the living CSK and that of inert nonequilibrium soft materials.

Upper airway collapse and reopening induce inflammation in a sleep apnoea model.

The upper airway of obstructive sleep apnoea patients is subjected to recurrent negative pressure swings promoting its collapse and reopening. The aim of the present study was to ascertain whether this mechanical stress induces upper airway inflammation in a rat model. The upper airway of Sprague-Dawley rats was subjected to a periodic pattern of recurrent negative (-40 cmH2O, 1 s) and positive (4 cmH2O, 2 s) pressures inducing collapse and reopening for 5 h. Rats that were instrumented but not subjected to negative pressure swings were used as controls. The gene expression of the pro-inflammatory biomarkers macrophage inflammatory protein (MIP)-2, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and P-selectin in the soft palate and larynx tissues was assessed by real-time PCR. A marked overexpression of MIP-2, TNF-alpha, IL-1beta and P-selectin (approximately 40-, 24-, 47- and 7-fold greater than controls, respectively) was observed in the larynx tissue; similar results were found in the soft palate tissue (approximately 14-, 7-, 35- and 11-fold greater than controls, respectively). Recurrent upper airway collapse and reopening mimicking those experienced by obstructive sleep apnoea patients triggered an early local inflammatory process. These results could explain the inflammation observed in the upper airway of obstructive sleep apnoea patients.

Estrategias de interpretación de las pruebas de función pulmonar / No disponible

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Consideraciones generales acerca de las pruebas de función pulmonar / Tittle

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Estandarización de la espirometría / Tittle

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Performance of mechanical ventilators at the patient's home: a multicentre quality control study.

BACKGROUND: Quality control procedures vary considerably among the providers of equipment for home mechanical ventilation (HMV). METHODS: A multicentre quality control survey of HMV was performed at the home of 300 patients included in the HMV programmes of four hospitals in Barcelona. It consisted of three steps: (1) the prescribed ventilation settings, the actual settings in the ventilator control panel, and the actual performance of the ventilator measured at home were compared; (2) the different ventilator alarms were tested; and (3) the effect of differences between the prescribed settings and the actual performance of the ventilator on non-programmed readmissions of the patient was determined. RESULTS: Considerable differences were found between actual, set, and prescribed values of ventilator variables; these differences were similar in volume and pressure preset ventilators. The percentage of patients with a discrepancy between the prescribed and actual measured main ventilator variable (minute ventilation or inspiratory pressure) of more than 20% and 30% was 13% and 4%, respectively. The number of ventilators with built in alarms for power off, disconnection, or obstruction was 225, 280 and 157, respectively. These alarms did not work in two (0.9%), 52 (18.6%) and eight (5.1%) ventilators, respectively. The number of non-programmed hospital readmissions in the year before the study did not correlate with the index of ventilator error. CONCLUSIONS: This study illustrates the current limitations of the quality control of HMV and suggests that improvements should be made to ensure adequate ventilator settings and correct ventilator performance and ventilator alarm operation.

Noninvasive detection of expiratory flow limitation in COPD patients during nasal CPAP.

The difference between mean inspiratory and expiratory respiratory reactance (delta(rs)) measured with forced oscillation technique (FOT) at 5 Hz allows the detection of expiratory flow limitation (EFL) in chronic obstructive pulmonary disease (COPD) patients breathing spontaneously. This aim of this study was to evaluate whether this approach can be applied to COPD patients during noninvasive pressure support. Delta(rs) was measured in seven COPD patients subjected to nasal continuous positive airway pressure (CPAP) at 0, 4, 8 and 12 cmH2O in sitting and supine positions. Simultaneous recording of oesophageal pressure and the Mead and Whittenberger (M-W) method provided a reference for scoring each breath as flow-limited (FL), non-flow-limited (NFL) or indeterminate (I). For each patient, six consecutive breaths were analysed for each posture and CPAP level. According to M-W scoring, 47 breaths were FL, 166 NFL and 51 I. EFL scoring using FOT coincided with M-W in 94.8% of the breaths. In the four patients who were FL in at least one condition, delta(rs) was reduced with increasing CPAP. These data suggest that the forced oscillation technique may be useful in chronic obstructive pulmonary disease patients on nasal pressure support by identifying continuous positive airway pressure levels that support breathing without increasing lung volume, which in turn increase the work of breathing and reduce muscle effectiveness and efficiency.