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.

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.

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.

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.

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.

Importance of the pulse oximeter averaging time when measuring oxygen desaturation in sleep apnea.

The accuracy of pulse oximeters in measuring transient changes in oxygen saturation (SaO2) may be affected by the oximeter time response. The aim of this study was to assess the effect of modifying the pulse oximeter averaging time (T) on the measurement of SaO2 in patients with the sleep apnea-hypopnea syndrome (SAHS). Twelve patients with severe SAHS were studied during a nap with conventional oximeters: Ohmeda 3740 and Criticare 501. We compared the readings of each patient's oxygen desaturation measured simultaneously with two identical pulse oximeters. One oximeter was the control (T = 3 seconds), and in the other T was set from 3 seconds to 21 seconds. No significant differences in SaO2 were found when both oximeters were set to the same T (3 seconds). In contrast, increasing T to 12 seconds and 21 seconds in one of the oximeters resulted in considerable and significant differences in the measured SaO2: oxygen desaturation was underestimated by up to 60% when compared with the control. The misestimation of SaO2 induced by settings of T which are within the range selectable in conventional oximeters may be of epidemiological significance when pulse oximetry is used as a complementary diagnostic tool to classify sleep events in SAHS.

Performance of nasal prongs in sleep studies : spectrum of flow-related events.

OBJECTIVES: The use of nasal prongs connected to a pressure transducer is a noninvasive, sensitive method to detect respiratory events, and can be easily implemented in routine sleep studies. Moreover, its good time response allows the detection of several flow-related phenomena of high interest, in addition to apnea and hypopnea. The aims of the study were to examine the quality and performance of the nasal prong flow signal, and to describe other flow-related events during full-night polysomnography studies. METHODS: Twenty-seven subjects were studied (16 male subjects; mean +/- SD age, 49 +/- 14 years; mean body mass index, 27 +/- 4 kg/m(2)): 15 subjects recruited from the general population and 12 consecutive patients with suspected sleep apnea/hypopnea syndrome (SAHS). RESULTS: A blind analysis of the respiratory events detected both by nasal prongs and thermistor was done. The quality of the nasal prong signal recordings was considered optimal for scoring purposes in 78% of cases, and no recording was considered uninterpretable. The nasal prong signal detected additional flow-related events not observed by the thermistor: (1) short and long (> 2 min) periods of inspiratory flow limitation morphology without decrease in the amplitude of the signal; (2) periods of mouth expiration; and (3) snoring. The apnea/hypopnea index was significantly higher with the nasal prong scoring (18 vs 11 [p < 0.05] in the general population and 37 vs 27 [p < 0.001] in the group with suspected SAHS). CONCLUSIONS: The incorporation of nasal prongs in routine full-night studies is an attainable technical option that provides adequate recordings in most cases. Additionally, relevant information not scored by thermistors is obtained on flow-related respiratory events, thus increasing diagnostic accuracy.

Optimized estimation of respiratory impedance by signal averaging in the time domain.

The spontaneous breathing of a subject during measurements of respiratory impedance (Zrs) by the forced oscillation technique (FOT) induces errors that result in biased impedance estimates, especially at low frequencies. Although in standard measurements this bias may be avoided by using special impedance estimators, there are two applications of FOT for which such estimators are not useful: when a head generator is used and when measurements are made during intubation. In this paper we describe a data-processing procedure for unbiased impedance estimation for all FOT setups. The proposed estimator (Z) was devised for pseudorandom excitation and is based on time-domain signal averaging before frequency analysis. The performance of estimator Z was first analyzed by computer simulation of a head generator setup and a setup including an endotracheal tube to measure (2-32 Hz) a resistance-inertance-elastance model mimicking Zrs of a healthy subject. Second, Z was assessed during real measurements in 16 healthy subjects. The results obtained in the simulation (e.g., error in elastance was reduced from 15.6% with most conventional estimators to 3.3% with Z in simulation of head generator setup) and in the measurements in subjects (differences of less than 1.6% between Z and a reference) confirmed the theoretical lack of bias of Z and its practical suitability for the different FOT setups. In addition to its applicability in the situations in which no other unbiased estimators are available, estimator Z is also advantageous in most conventional applications of FOT, since it requires much less computing time and thus allows on-line Zrs measurements.

Respiratory input impedance up to 256 Hz in healthy humans breathing foreign gases.

Currently available data concerning respiratory input impedance (Zrs) at frequencies up to 300 Hz indicate that Zrs is determined mainly by the airways and, in particular, the gas compressibility in the airways and the airway wall compliance. Hence, measurements of Zrs when breathing gases with different physical properties would be useful in investigating airway mechanics and the role of acoustic propagation. Zrs measured with a standard generator (Zst) and corrected for the upper airway shunt (Zrs*) were measured in nine healthy subjects breathing air or a gas mixture consisting of 20% O2 and 80% He or SF6. The frequency band was extended up to 256 Hz for air and He-O2 and up to 128 Hz for SF6-O2. Zrs exhibited a similar pattern for the three gases, with a shift toward low frequencies as the gas density increased. Moreover, the resonance peaks tended to be narrower and higher as the gas density increased. The second frequency of resonance for He-O2, air, and SF6-O2 were 220, 180, and 50 Hz, respectively, for Zrs* and were systematically higher for Zst. Zrs* and Zst data were interpreted in terms of a tricompartmental model that partitioned the airways into two segments: a central one featuring the acoustic propagation in the airways and a peripheral one that included bronchial wall elasticity (Farré et al. J. Appl. Physiol. 67: 1973-1981, 1989). The model was able to interpret the gas dependence of Zrs* but not that of Zst. The influence of the gas physical properties on both Zrs* and Zst confirms that total Zrs at high frequencies is basically that of the airways and that the second resonance is related mainly to the gas compressibility in the airways.

Human lung impedance from spontaneous breathing frequencies to 32 Hz.

Lung impedance (ZL) was measured from 0.1875 to 32 Hz in spontaneously breathing healthy subjects by spectral analysis of the pressure and flow signals generated simultaneously by the muscular generator of breathing and by a forced oscillation system. This method did not require cooperation from the subject to perform panting or special ventilatory maneuvers and therefore allowed us to analyze the frequency dependence of lung resistance, reactance, and elastance (-2 pi.frequency.reactance) at the physiological conditions of normal breathing. Resistance and elastance parameters were also computed by multiple linear regression of the time-domain pressure and flow data on a simple resistance-elastance model. Resistances and elastances computed at the breathing frequency by spectral analysis and by multiple linear regression were similar (nonsignificant differences < 4 and 10%, respectively). The results obtained when comparing ZL from the breathing component (0.1875-0.75 Hz) of the recorded signals and from the forced oscillation component (2-32 Hz) were fairly consistent. ZL (0.1875-10 Hz) was interpreted in terms of a model consisting of an airway compartment, including a resistance and an inertance, in series with a viscoelastic tissue compartment (J. Hildebrandt. J. Appl. Physiol. 28: 365-372, 1970) characterized by two parameters. The model analysis provided parameter values (resistance 2.49 +/- 0.58 hPa.l-1.s, inertance 1.70 +/- 0.29 Pa.l-1.s2, Hildebrandt parameters 4.87 +/- 2.28 and 0.73 +/- 0.99 hPa/l) consistent with the hypothesis that lung tissue in healthy humans during spontaneous breathing behaves as a viscoelastic structure with a hysteresivity of approximately 0.10.

Assessment of respiratory pressure-volume nonlinearity in rabbits during mechanical ventilation.

The volume dependence of respiratory elastance makes it difficult to recognize actual changes in lung and chest wall elastic properties in artificially ventilated subjects. We have assessed in six anesthetized, tracheotomized, and paralyzed rabbits whether reliable information on the static pressure-volume (PV) curve could be obtained from recordings performed during step variations of the end-expiratory pressure without interrupting mechanical ventilation. Pressure and flow data recorded during 5- and 10-hPa positive-pressure steps were analyzed in the time domain with a nonlinear model featuring a sigmoid PV curve and with a model that, in addition, accounted for tissue viscoelastic properties. The latter fitted the data substantially better. Both models provided reasonably reproducible coefficients, but the PV curves obtained from the 5- and 10-hPa steps were systematically different. When the PV curves were used to predict respiratory effective elastance, the best predictor was the curve derived from the 10-hPa step with the viscoelastic model: unsigned differences averaged 8.6 +/- 11.1, 26.9 +/- 36.4, and 5.5 +/- 5.8% at end-expiratory pressures of 0, 5, and 10 hPa, respectively. This approach provides potentially useful, although not highly accurate, estimates of respiratory effective elastance-volume dependence.

Effect of expiratory flow limitation on respiratory mechanical impedance: a model study.

Large phasic variations of respiratory mechanical impedance (Zrs) have been observed during induced expiratory flow limitation (EFL) (M. Vassiliou, R. Peslin, C. Saunier, and C. Duvivier. Eur. Respir. J. 9: 779-786, 1996). To clarify the meaning of Zrs during EFL, we have measured from 5 to 30 Hz the input impedance (Zin) of mechanical analogues of the respiratory system, including flow-limiting elements (FLE) made of easily collapsible rubber tubing. The pressures upstream (Pus) and downstream (Pds) from the FLE were controlled and systematically varied. Maximal flow (Vmax) increased linearly with Pus, was close to the value predicted from wave-speed theory, and was obtained for Pus-Pds of 4-6 hPa. The real part of Zin started increasing abruptly with flow (V) > 85% Vmax and either further increased or suddenly decreased in the vicinity of Vmax. The imaginary part of Zin decreased markedly and suddenly above 95% Vmax. Similar variations of Zin during EFL were seen with an analogue that mimicked the changes of airway transmural pressure during breathing. After pressure and V measurements upstream and downstream from the FLE were combined, the latter was analyzed in terms of a serial (Zs) and a shunt (Zp) compartment. Zs was consistent with a large resistance and inertance, and Zp with a mainly elastic element having an elastance close to that of the tube walls. We conclude that Zrs data during EFL mainly reflect the properties of the FLE.

Dynamic viscoelastic nonlinearity of lung parenchymal tissue.

To investigate the contribution of nonlinear tissue viscoelasticity to the dynamic behavior of lung, time and frequency responses of isolated parenchymal strips of degassed dog lungs were investigated. The strips were subjected to loading and unloading stretch steps for 60 s and to sinusoidal oscillations (0.03-3 Hz) of different stretch amplitudes (delta lambda = 0.05, 0.1, and 0.2) and at different operating stresses (T(o) = 0.5, 1, and 2 kPa). Elastance (E) increased linearly with the logarithm of frequency (approximately 10% per frequency decade), and resistance (R) decreased hyperbolically with frequency. Both E and R varied little with delta lambda but they increased proportionally with T(o). Hysteresivity (eta = R x 2 pi x frequency/E) ranged from 0.07 to 0.10. In agreement with the frequency response, the magnitude of the unit step response increased with T(o) and was higher when loading than when unloading, and the stress relaxation ratio (approximately 0.10) did not vary greatly with T(o) or with delta lambda. The time and frequency behavior of the strips were interpreted in terms of the quasilinear viscoelastic model of Navajas et al. (J. Appl. Physiol. 73:2681-2692, 1992). The model explains most of the dependencies of step and oscillatory responses on the measurement conditions, in particular the proportional dependence of E and R on T(o). According to the model, about two-thirds of energy dissipated during oscillation arises from tissue viscoelasticity. The remaining dissipated energy could be accounted for by plasticity. Thus the effect of nonlinear elasticity on the dynamic behavior of lung tissue can be empirically described by a simple quasilinear model characterized by only two parameters.

Lung and respiratory impedance at low frequency during mechanical ventilation in rabbits.

We have tested in eight rabbits the feasibility of measuring respiratory (Zrs) and lung (ZL) impedances in the low-frequency domain, including below the breathing frequency (fb), during conventional mechanical ventilation (CMV). The animals were tracheotomized and ventilated with a tidal volume (VT) of 20 ml at a fb of 1 Hz. The excitation signal was provided by a flow generator connected in parallel with the ventilator; it included six components ranging from 0.45 to 14.8 Hz, which met the neither-sum-nor-difference criterion of B. Suki and K. Lutchen (IEEE Trans. Biomed. Eng. 39: 1142-1151, 1992) to minimize the influence of nonlinearities. Zrs and ZL were also measured at the same mean lung volume and with the same excitation signal both during apnea and when the ventilator signal was replaced by a sine wave with the same VT and fb (SMV). The real parts (Re) of both Zrs and ZL, as well as the effective elastances, were significantly larger during apnea than during CMV and SMV over the whole frequency range. Re(Zrs) and Re(ZL) were similar during CMV and SMV above fb but they were lower during CMV at 0.45 Hz. The latter difference seems to be related to the presence of harmonics of fb and of additional frequency components due to pulse amplitude modulation. We conclude that, because of nonlinearities, it is feasible to measure Zrs and ZL during CMV only at and above fb.

T model partition of lung and respiratory system impedances.

The aim of this work was to demonstrate that the three compartments of the lung T network and the chest wall impedance (Zcw) can be identified from input and transfer impedances of the respiratory system if the pleural pressure is recorded during the measurements. The method was tested in six healthy volunteers in the range of 8-32 Hz. The impedances resulting from the decomposition confirm the adequacy of the monoalveolar structure commonly used in healthy subjects. Indeed, the T shunt impedance is well modeled by a purely compliant element, the mean compliance [0.038 +/- 0.081 (SD) l/kPa], which coincides within 9.5 +/- 6.3% of the alveolar gas compressibility derived from thoracic gas volume (0.036 +/- 0.011 l/kPa). The results obtained provide experimental evidence that the alveolar gas compression is predominantly isothermal and that lung tissue impedance is negligible throughout the whole frequency range. The shape of Zcw is consistent with a low compliance-low inertance pathway in parallel with a high compliance-high inertance pathway. We conclude that the proposed method is able to reliably identify the T network featuring the lung and Zcw.

Validity of the esophageal balloon technique at high frequencies.

The reliability of the esophageal balloon technique in measuring high-frequency changes in pleural pressure (Ppl) was investigated in six normal subjects by studying the amplitude ratio (A) and phase angle (phi) of esophageal (Pes) and mouth (Pm) pressures during airway occlusion and while pseudorandom pressure variations (2-32 Hz) were applied to the chest. The measurements were made with a common esophageal balloon-catheter system connected to a high-impedance piezoresistive transducer. When the cheeks were firmly supported, A averaged 1.08 +/- 0.063 at 2 Hz and 1.06 +/- 0.11 at 32 Hz. Pes increasingly led Pm with increasing frequency, and phi averaged 20.8 +/- 4.0 degrees at 32 Hz. Washing the airways with 80% He-20% O2 reduced phi by 50%. When the cheeks were not supported, A exhibited a strong positive frequency dependence, averaging 1.71 +/- 0.34 at 32 Hz, whereas phi increased much faster below 20 Hz and tended to decrease afterward. Because the esophageal transfer function Pes/Ppl = (Pes/Pm)/(Ppl/Pm), we could estimate Pes/Ppl by computing for individual subjects the pressure difference between the pleura and the mouth based on the lung and upper airway wall properties that were measured separately. The results suggest that the ratio of Pes and Ppl remains close to unity from 2 to 32 Hz, but Pes lags slightly behind Ppl (phi equals about -7 degrees at 32 Hz).

Density dependence of respiratory input and transfer impedances in humans.

Total respiratory input (Zin) and transfer (Ztr) impedances were obtained from 4 to 30 Hz in 10 healthy subjects breathing air and He-O2. Zin was measured by applying pressure oscillations around the head to minimize the upper airway shunt and Ztr by applying pressure oscillations around the chest. Ztr was analyzed with a six-coefficient model featuring airways resistance (Raw) and inertance (Iaw), alveolar gas compressibility, and tissue resistance, inertance, and compliance. Breathing He-O2 significantly decreased Raw (1.35 +/- 0.32 vs. 1.74 +/- 0.49 cmH2O.l-1.s in air, P less than 0.01) and Iaw (0.59 +/- 0.33 vs. 1.90 +/- 0.44 x 10(-2) cmH2O.l-1.s2), but, as expected, it did not change the tissue coefficients significantly. Airways impedance was also separately computed by combining Zin and Ztr data. This approach demonstrated similar variations in Raw and Iaw with the lighter gas mixture. With both analyses, however, the changes in Iaw were more than what was expected from the change in density. This indicates that factors other than gas inertance are included in Iaw and reveals the short-comings of the six-coefficient model to interpret impedance data.