Nanoscale mechanical properties of chitosan hydrogels as revealed by AFM.

In the context of tissue engineering, chitosan hydrogels are attractive biomaterials because they represent a family of natural polymers exhibiting several suitable features (cytocompatibility, bioresorbability, wound healing, bacteriostatic and fungistatic properties, structural similarity with glycosaminoglycans), and tunable mechanical properties. Optimizing the design of these biomaterials requires fine knowledge of its physical characteristics prior to assessment of the cell-biomaterial interactions. In this work, using atomic force microscopy (AFM), we report a characterization of mechanical and topographical properties at the submicron range of chitosan hydrogels, depending on physico-chemical parameters such as their polymer concentration (1.5%, 2.5% and 3.5%), their degree of acetylation (4% and 38.5%), and the conditions of the gelation process. Well-known polyacrylamide gels were used to validate the methodology approach for the determination and analysis of elastic modulus (i.e., Young's modulus) distribution at the gel surface. We present elastic modulus distribution and topographical and stiffness maps for different chitosan hydrogels. For each chitosan hydrogel formulation, AFM analyses reveal a specific asymmetric elastic modulus distribution that constitutes a useful hallmark for chitosan hydrogel characterization. Our results regarding the local mechanical properties and the topography of chitosan hydrogels initiate new possibilities for an interpretation of the behavior of cells in contact with such soft materials.

Prestress and adhesion site dynamics control cell sensitivity to extracellular stiffness.

This study aims at improving the understanding of mechanisms responsible for cell sensitivity to extracellular environment. We explain how substrate mechanical properties can modulate the force regulation of cell sensitive elements primarily adhesion sites. We present a theoretical and experimental comparison between two radically different approaches of the force regulation of adhesion sites that depends on their either stationary or dynamic behavior. The most classical stationary model fails to predict cell sensitivity to substrate stiffness whereas the dynamic model predicts extracellular stiffness dependence. This is due to a time dependent reaction force in response to actomyosin traction force exerted on cell sensitive elements. We purposely used two cellular models, i.e., alveolar epithelial cells and alveolar macrophages exhibiting respectively stationary and dynamic adhesion sites, and compared their sensitivity to theoretical predictions. Mechanical and structural results show that alveolar epithelial cells exhibit significant prestress supported by evident stress fibers and lacks sensitivity to substrate stiffness. On the other hand, alveolar macrophages exhibit low prestress and exhibit sensitivity to substrate stiffness. Altogether, theory and experiments consistently show that adhesion site dynamics and cytoskeleton prestress control cell sensitivity to extracellular environment with an optimal sensitivity expected in the intermediate range.

Mechanical and structural assessment of cortical and deep cytoskeleton reveals substrate-dependent alveolar macrophage remodeling.

The sensitivity of alveolar macrophages to substrate properties has been described in a recent paper (Féréol et al., Cell Motil. Cytoskel. 63 (2006), 321-340). It is presently re-analyzed in terms of F-actin structure (assessed from 3D-reconstructions in fixed cells) and mechanical properties (assessed by Magnetic Twisting Cytometry experiments in living cells) of cortical and deep cytoskeleton structures for rigid plastic (Young Modulus: 3 MPa) or glass (70 MPa) substrates and a soft (approximately 0.1 kPa) confluent monolayer of alveolar epithelial cells. The cortical cytoskeleton component (lowest F-actin density) is represented by the rapid and softer viscoelastic compartment while the deep cytoskeleton component (intermediate F-actin density) is represented by the slow and stiffer compartment. Stiffness of both cortical and deep cytoskeleton is significantly decreased when soft confluent monolayer of alveolar epithelial cells replace the rigid plastic substrate while F-actin reconstructions reveal a consistent actin cytoskeleton remodeling observable on both cytoskeleton components.

Abdominal muscle activity in sleep apnea during continuous positive airway pressure titration.

STUDY OBJECTIVES: The aim of this study was to investigate whether presence of expiratory abdominal muscle activity (EAMA) in obstructive sleep apnea syndrome (OSAS) patients during nasal continuous positive airway pressure (nCPAP) is due to either nCPAP overprescription or nCPAP underprescription. DESIGN: Airflow, esophageal pressure (Pes), and gastric pressure (Pga) were routinely measured during polysomnography aimed at determining the optimal nCPAP level, and the magnitude of EAMA was evaluated in relation to the nCPAP level and to the conventional indexes of upper-airway obstruction used during nCPAP titration. PATIENTS: The study was performed 12 patients with OSAS. RESULTS: Six patients displayed sustained EAMA, ie, EAMA lasting > 3 min, and characterized by a decrease in abdominal diameter and a paradoxical rise in Pga during expiration. In all six patients, EAMA decreased gradually as nCPAP neared optimal levels, and then disappeared when the optimal nCPAP level was achieved. The decrease in EAMA as nCPAP increased was associated with an increase in minute ventilation, decreases in both inspiratory and expiratory resistance, a decrease in Pes swing, and the normalization of the inspiratory flow contour. CONCLUSIONS: We conclude that the EAMA observed in some OSAS patients might be an indirect marker of upper-airway obstruction, and that the presence of EAMA during nCPAP titration might indicate a suboptimal nCPAP level rather than a deleterious effect of nCPAP.

Dual assessment of airway area profile and respiratory input impedance from a single transient wave.

This report concerns the inference of geometric and mechanical airway characteristics based on information derived from a single transient planar wave recorded at the airway opening. We describe a new method to simultaneously measure upper airway area and respiratory input impedance by performing dual analysis of a single pressure wave. The algorithms required to reconstruct airway dimensions and mechanical characteristics were developed, implemented, and tested with reference to known physical models. Our method appears suitable to estimate, even under severe intensive care unit conditions, the respiratory system frequency response (above 10 Hz) in intubated patients and the patency of the endotracheal tube used to connect the patients to the ventilator.

Segmental analysis of nasal cavity compliance by acoustic rhinometry.

To explore the determinants of possible collapse of the nasal valve region, a common cause of nasal obstruction, we evaluated the mechanical properties of the nasal wall. In this study, we determined the nasal cross-sectional area-to-negative pressure ratio (nasal wall compliance) in the anterior part of the nose in six healthy subjects by measuring nasal area by acoustic rhinometry at pressures ranging from atmospheric pressure to a negative pressure of -10 cmH(2)O. Measurements were performed at baseline and after nasal mucosal decongestion (oxymetazoline). At baseline, nasal wall compliance increased progressively from the nasal valve (0.031 +/- 0.016 cm2/cmH(2)O, mean +/- SD) to the anterior and medial part of the inferior turbinate (0.045 +/- 0.024 cm2/cmH(2)O) and to the middle meatus region (0.056 +/- 0.029 cm2/cmH(2)O). After decongestant, compliances decreased and became similar in the three regions. On the basis of these results, we hypothesize that compliance of the nasal wall is partly related to mucosal blood volume and quantity of vascular tissue, which differ in the three regions, increasing from the nasal valve to the middle meatus.

Gravity effects on upper airway area and lung volumes during parabolic flight.

We measured upper airway caliber and lung volumes in six normal subjects in the sitting and supine positions during 20-s periods in normogravity, hypergravity [1.8 + head-to-foot acceleration (Gz)], and microgravity ( approximately 0 Gz) induced by parabolic flights. Airway caliber and lung volumes were inferred by the acoustic reflection method and inductance plethysmography, respectively. In subjects in the sitting position, an increase in gravity from 0 to 1. 8 +Gz was associated with increases in the calibers of the retrobasitongue and palatopharyngeal regions (+20 and +30%, respectively) and with a concomitant 0.5-liter increase in end-expiratory lung volume (functional residual capacity, FRC). In subjects in the supine position, no changes in the areas of these regions were observed, despite significant decreases in FRC from microgravity to normogravity (-0.6 liter) and from microgravity to hypergravity (-0.5 liter). Laryngeal narrowing also occurred in both positions (about -15%) when gravity increased from 0 to 1.8 +Gz. We concluded that variation in lung volume is insufficient to explain all upper airway caliber variation but that direct gravity effects on tissues surrounding the upper airway should be taken into account.

Heavy snoring with upper airway resistance syndrome may induce intrinsic positive end-expiratory pressure.

We studied eight heavy snorers with upper airway resistance syndrome to investigate potential effects of sleep on expiratory airway and lung resistance, intrinsic positive end-expiratory pressure, hyperinflation, and elastic inspiratory work of breathing (WOB). Wakefulness and non-rapid-eye-movement sleep with high- and with low-resistance inspiratory effort (H-RIE and L-RIE, respectively) were compared. No differences in breathing pattern were seen across the three conditions. In contrast, we found increases in expiratory airway and lung resistance during H-RIE compared with L-RIE and wakefulness (56 +/- 24, 16 +/- 4, and 11 +/- 4 cmH2O . 1(-1) . s, respectively), with attendant increases in intrinsic positive end-expiratory pressure (5.4 +/- 1.8, 1.4 +/- 0.5, and 1.3 +/- 1.3 cmH2O, respectively) and elastic WOB (6.1 +/- 2.2, 3.7 +/- 1.2, and 3.4 +/- 0.7 J/min, respectively). The increase in WOB during H-RIE is partly caused by the effects of dynamic pulmonary hyperinflation produced by the increased expiratory resistance. Contrary to the Starling model, a multiple-element compliance model that takes into account the heterogeneity of the pharynx may explain flow limitation during expiration.

Interaction between steady flow and individualised compliant segments: application to upper airways.

To describe upper airway obstruction in patients with sleep apnea syndrome, the steady state solutions of a simple model of local pharyngeal obstruction were studied. Importantly, the present model embodies a series of two individualised elements, each having its own compliance, which enables the consideration, from a conceptual point of view, of local differences in anatomical and physiological properties between pharyngeal regions. The evolution of inspiratory flow and area variations were predicted using the transmural pressure at the downstream element as the controlled variable. Derivation and normalisation of fundamental governing equations, written for non-viscous and viscous fluids, reveal very different kinds of behaviour, depending on values of the speed index defined from the local distensibility, or its modulation by a friction factor for viscous fluid. The two-element model is able to describe a considerably rich mechanical behaviour, including the occurrence of critical conditions when area becomes very high or small, i.e. when the distensibility-dependent speed index at the upstream element tends toward unity. In spite of its simplicity, not only does the present model describe steady state behaviours that resemble the well-known phenomenon of choking in an elastic tube, but the viscous fluid conditions also reveal (i) an area evolution following a typical doubly folded shape, (ii) the occurrence of a close succession maximum and minimum flows which can be seen as a physiological 'flow plateau'. It is concluded that the concept of interaction behind the two-element model must be considered as soon as flow interacts in a compliant structure characterised by anatomical and/or functional singularities.

Force distribution on multiple bonds controls the kinetics of adhesion in stretched cells.

We show herein how mechanical forces at macro or micro scales may affect the biological response at the nanoscale. The reason resides in the intimate link between chemistry and mechanics at the molecular level. These interactions occur under dynamic conditions such as the shear stress induced by flowing blood or the intracellular tension. Thus, resisting removal by mechanical forces, e.g., shear stresses, is a general property of cells provided by cellular adhesion. Using classical models issued from theoretical physics, we review the force regulation phenomena of the single bond. However, to understand the force regulation of cellular adhesion sites, we need to consider the collective behavior of receptor-ligand bonds. We discuss the applicability of single bond theories to describe collective bond behavior. Depending on bond configuration, e.g., presently "parallel" and "zipper", the number of bonds and dissociation forces variably affect the kinetics of multiple bonds. We reveal a marked efficiency of the collective organization to stabilize multiple bonds by sharply increasing bond lifetime compared to single bond. These theoretical predictions are then compared to experimental results of the literature concerning the kinetic parameters of bonds measured by atomic force microscopy and by shear flow. These comparisons reveal that the force-control of bonds strongly depends on whether the force distribution on multiple bonds is homogeneous, e.g., in AFM experiments, or heterogeneous, e.g., in shear flow experiments. This reinforces the need of calculating the stress/strain fields exerted on living tissues or cells at various scales and certainly down to the molecular scale.

Micropatterned ECM substrates reveal complementary contribution of low and high affinity ligands to neurite outgrowth.

Growth and guidance of developing or regenerating axons require sensing of environmental cues (EC) by the growth cone. To explore the role of a spatially defined distribution of ligands on guidance, extension, and branching, we used a microcontact-printing technique allowing to deposit ligands as discrete spots of a size smaller than a cell body. Micropatterned substrates (MS) were created with varying distance between spots and two different ligands (laminin (LN) and fibronectin (FN)). Dissociated dorsal root ganglion neurons were seeded on either monocomponent MS made from LN or FN alone, or multicomponent MS made from alternating lines of LN and FN spots. On monocomponent MS the high-affinity ligand LN not only stimulated neurite extension, but also provided guidance and branching control, associated with marked cytoskeleton remodeling. The latter was assessed by evaluating the increase in rigidity of the distal neurite segment by Atomic Force Microscopy. In contrast, FN alone acts as a low-affinity ligand which dramatically limits neurite outgrowth. Surprisingly, observation of growth cone dynamics on multicomponent MS revealed that FN constitute a transient support for neurite progression, facilitating exploration of other EC present within a certain distance. Such a mutual contribution of high and low affinity ligands to neurite outgrowth is consistent with a recent theory of force regulation of dynamic adhesion sites showing cell's sensitivity to EC properties would actually depend on the rate of change of the reacting force, the latter controlling the otherwise instantaneous chemical binding process.

Comparison of patient-ventilator interfaces based on their computerized effective dead space.

PURPOSE: Non-invasive ventilation is largely used to treat acute and chronic respiratory failure. This ventilation encounters a non-negligible rate of failure related to the used interface/mask, but the reasons for this failure remain unclear. In order to shed light on this issue and to better understand the effects of the geometrical design of interfaces, we aimed to quantify flow, pressure and gas composition in terms of CO(2) and O(2) at the passage through different types of interface (oronasal mask, integral mask and helmet). In particular, we postulated that due to specific gas flow passing throughout the interface, the effective dead space added by the interface is not always related to the whole gas volume included in the interface. METHODS: Numerical simulations, using computational fluid dynamics, were used to describe pressure, flow and gas composition during ventilation with the different interfaces. RESULTS: Between the different interfaces the effective dead spaces differed only modestly (110-370 ml), whereas their internal volumes were markedly different (110-10,000 ml). Effective dead space was limited to half the tidal volume for the most voluminous interface, whereas it was close to the interface gas volume for the less voluminous interfaces. Pressure variations induced by the flow ventilation throughout the interface were negligible. CONCLUSIONS: Effective dead space is not related to the internal gas volume included in the interface, suggesting that this internal volume should not be considered as a limiting factor for their efficacy during non-invasive ventilation. Patient's comfort and synchrony have also to be taken into account.

Inaccuracy of tidal volume delivered by home mechanical ventilators.

Ideally, the inspired (tidal) volume (V(T)) provided by a volume-controlled ventilation device should not change when the pressure imposed on the ventilator varies. A bench study evaluation of V(T) versus pressure was performed on 10 commercially available devices. The difference between the desired V(T) and the observed V(T) reached 100 mL for some devices when inspiratory resistance was at its lowest, rising to 150 mL when inspiratory resistance was increased to obtain peak airway pressure of 60 cmH2O. The present data indicate that some home ventilators are inaccurate in delivering the preset tidal volume when the pressure imposed on the ventilator is increased to simulate high airway resistance.

Helium-oxygen in the postextubation period decreases inspiratory effort.

After tracheal extubation, upper and total airway resistances may frequently be increased resulting in an increase in inspiratory effort to breathe. We tested whether breathing a helium-oxygen mixture (HeO(2)) would reduce inspiratory effort in the period after extubation. Eighteen consecutive patients with no chronic obstructive pulmonary disease who had received mechanical ventilation (> 48 h) were successively studied immediately after extubation (N(2)O(2)), 15 min after breathing HeO(2), and after return to N(2)O(2). Effort to breathe, assessed by the transdiaphragmatic pressure swings (DeltaPdi) and the pressure-time index of the diaphragm (PTI), comfort, and gas exchange, were the main end points. The mean reduction of the transdiaphragmatic pressure under HeO(2) was 19 +/- 5%. All but three patients presented a decrease in transdiaphragmatic pressure under HeO(2), ranging from - 4 to - 55%, and a significant reduction in DeltaPdi was observed between HeO(2) and N(2)O(2) (10.2 +/- 0.7 versus 8.6 +/- 1.1 versus 10.0 +/- 0.8 cm H(2)O for the three consecutive periods; p < 0.05). PTI also differed significantly between HeO(2) and N(2)O(2) (197 +/- 19 versus 166 +/- 22 versus 201 +/- 23 cm H(2)O/s/min for the three periods; p < 0.05). Breathing HeO(2) significantly improved comfort, whereas gas exchange was not modified. We conclude that the use of HeO(2) in the immediate postextubation period decreases inspiratory effort and improves comfort.

Noninvasive ventilation with helium-oxygen in acute exacerbations of chronic obstructive pulmonary disease.

The use of helium-oxygen (HeO(2)) was tested in combination with noninvasive ventilation (NIV) in 10 patients with acute exacerbation of chronic obstructive pulmonary disease (COPD). Effort to breathe as assessed by the respiratory muscle pressure-time index (PTI), work of breathing (WOB), and gas exchange were the main endpoints. Results of NIV-HeO(2) were compared with those obtained with standard NIV (AirO(2)), at two levels of pressure-support ventilation (PSV), 9 +/- 2 cm H(2)O and 18 +/- 3 cm H(2)O. Significant reductions in PTI were observed between HeO(2) and AirO(2) at both the low PSV level (n = 9; 160 +/- 58 versus 198 +/- 78 cm H(2)O/s/ min; p < 0.05) and the high PSV level (n = 10; 100 +/- 45 versus 150 +/- 82 cm H(2)O/s/min; p < 0.01). WOB also differed significantly between HeO(2) and AirO(2) (7.8 +/- 4.1 versus 10.9 +/- 6.1 J/min at the low PSV level, p < 0.05; and 5.7 +/- 3.3 versus 9.2 +/- 5. J/min, p < 0.01 at the high PSV level). HeO(2) reduced Pa(CO(2)) at both the low PSV level (61 +/- 13 versus 64 +/- 15 mm Hg; p < 0.05) and the high PSV level (56 +/- 13 versus 58 +/- 14 mm Hg; p < 0.05), without significantly changing breathing pattern or oxygenation. We conclude that use of HeO(2) during NIV markedly enhances the ability of NIV to reduce patient effort and to improve gas exchange.