4D Force Detection of Cell Adhesion and Contractility.

Mechanical signals establish two-way communication between mammalian cells and their environment. Cells contacting a surface exert forces via contractility and transmit them at the areas of focal adhesions. External stimuli, such as compressive and pulling forces, typically affect the adhesion-free cell surface. Here, we demonstrate the collaborative employment of Fluidic Force Microscopy and confocal Traction Force Microscopy supported by the Cellogram solver to enable a powerful integrated force probing approach, where controlled vertical forces are applied to the free surface of individual cells, while the concomitant deformations are used to map their transmission to the substrate. Force transmission across human cells is measured with unprecedented temporal and spatial resolution, enabling the investigation of the cellular mechanisms involved in the adaptation, or maladaptation, to external mechanical stimuli. Altogether, the system enables facile and precise force interrogation of individual cells, with the capacity to perform population-based analysis.

Systems of conductive skin for power transfer in clinical applications.

The primary aim of this article is to review the clinical challenges related to the supply of power in implanted left ventricular assist devices (LVADs) by means of transcutaneous drivelines. In effect of that, we present the preventive measures and post-operative protocols that are regularly employed to address the leading problem of driveline infections. Due to the lack of reliable wireless solutions for power transfer in LVADs, the development of new driveline configurations remains at the forefront of different strategies that aim to power LVADs in a less destructive manner. To this end, skin damage and breach formation around transcutaneous LVAD drivelines represent key challenges before improving the current standard of care. For this reason, we assess recent strategies on the surface functionalization of LVAD drivelines, which aim to limit the incidence of driveline infection by directing the responses of the skin tissue. Moreover, we propose a class of power transfer systems that could leverage the ability of skin tissue to effectively heal short diameter wounds. In this direction, we employed a novel method to generate thin conductive wires of controllable surface topography with the potential to minimize skin disruption and eliminate the problem of driveline infections. Our initial results suggest the viability of the small diameter wires for the investigation of new power transfer systems for LVADs. Overall, this review uniquely compiles a diverse number of topics with the aim to instigate new research ventures on the design of power transfer systems for IMDs, and specifically LVADs.

Mechanical Fingerprint of Senescence in Endothelial Cells.

Endothelial senescence entails alterations of the healthy cell phenotype, which accumulate over time and contribute to cardiovascular disease. Mechanical aspects regulating cell adhesion, force generation, and the response to flow contribute to the senescence-associated drift; however, they remain largely unexplored. Here, we exploit force microscopy to resolve variations of the cell anchoring to the substrate and the tractions generated upon aging in the nanonewton (nN) range. Senescent endothelial cells display a multifold increase in the levels of basal adhesion and force generation supported by mature and strong focal adhesions. The enhanced mechanical interaction with the substrate yields static endothelial monolayers that polarize in response to flow but fail the process of coordinated cell shape remodeling and reorientation. The emerging picture indicates that senescence reinforces the local cell interaction with the substrate and may therefore prevent endothelial denudation; however, it compromises the ability to functionally adapt to the local hemodynamic conditions.

Mean Airway Pressure As a Predictor of 90-Day Mortality in Mechanically Ventilated Patients.

OBJECTIVES: To determine the association between mean airway pressure and 90-day mortality in patients with acute respiratory failure requiring mechanical ventilation and to compare the predictive ability of mean airway pressure compared with inspiratory plateau pressure and driving pressure. DESIGN: Prospective observational cohort. SETTING: Five ICUs in Lima, Peru. SUBJECTS: Adults requiring invasive mechanical ventilation via endotracheal tube for acute respiratory failure. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of potentially eligible participants (n = 1,500), 65 (4%) were missing baseline mean airway pressure, while 352 (23.5%) were missing baseline plateau pressure and driving pressure. Ultimately, 1,429 participants were included in the analysis with an average age of 59 ± 19 years, 45% female, and a mean PaO2/FIO2 ratio of 248 ± 147 mm Hg at baseline. Overall, 90-day mortality was 50.4%. Median baseline mean airway pressure was 13 cm H2O (interquartile range, 10-16 cm H2O) in participants who died compared to a median mean airway pressure of 12 cm H2O (interquartile range, 10-14 cm H2O) in participants who survived greater than 90 days (p < 0.001). Mean airway pressure was independently associated with 90-day mortality (odds ratio, 1.38 for difference comparing the 75th to the 25th percentile for mean airway pressure; 95% CI, 1.10-1.74) after adjusting for age, sex, baseline Acute Physiology and Chronic Health Evaluation III, baseline PaO2/FIO2 (modeled with restricted cubic spline), baseline positive end-expiratory pressure, baseline tidal volume, and hospital site. In predicting 90-day mortality, baseline mean airway pressure demonstrated similar discriminative ability (adjusted area under the curve = 0.69) and calibration characteristics as baseline plateau pressure and driving pressure. CONCLUSIONS: In a multicenter prospective cohort, baseline mean airway pressure was independently associated with 90-day mortality in mechanically ventilated participants and predicts mortality similarly to plateau pressure and driving pressure. Because mean airway pressure is readily available on all mechanically ventilated patients and all ventilator modes, it is a potentially more useful predictor of mortality in acute respiratory failure.

Cellogram: On-the-Fly Traction Force Microscopy.

Traction force microscopy (TFM) derives maps of cell-generated forces, typically in the nanonewton range, transmitted to the extracellular environment upon actuation of complex biological processes. In traditional approaches, force rendering requires a terminal, time-consuming step of cell deadhesion to obtain a reference image. A conceptually opposite approach is provided by reference-free methods, opening to the on-the-fly generation of force maps from an ongoing experiment. This requires an image processing algorithm keeping the pace of the biological phenomena under investigation. Here, we introduce an integrated software pipeline rendering force maps from single reference-free TFM images seconds to minutes after their acquisition. The algorithm tackles image processing, reference image estimation, and finite element analysis as a single problem, yielding a robust and fully automatic solution. The method's capabilities are demonstrated in two applications. First, the mechanical annihilation of cancer cells is monitored as a function of rising environmental temperature, setting a population threshold at 45 °C. Second, the fast temporal correlation of forces produced across individual cells is used to map physically connected adhesion points, yielding typical lengths that vary as a function of the cell cycle phase.

Sedation practices and clinical outcomes in mechanically ventilated patients in a prospective multicenter cohort.

OBJECTIVES: We sought to study the association between sedation status, medications (benzodiazepines, opioids, and antipsychotics), and clinical outcomes in a resource-limited setting. DESIGN: A longitudinal study of critically ill participants on mechanical ventilation. SETTING: Five intensive care units (ICUs) in four public hospitals in Lima, Peru. PATIENTS: One thousand six hundred fifty-seven critically ill participants were assessed daily for sedation status during 28 days and vital status by day 90. RESULTS: After excluding data of participants without a Richmond Agitation Sedation Scale score and without sedation, we followed 1338 (81%) participants longitudinally for 18,645 ICU days. Deep sedation was present in 98% of participants at some point of the study and in 12,942 ICU days. Deep sedation was associated with higher mortality (interquartile odds ratio (OR) = 5.42, 4.23-6.95; p < 0.001) and a significant decrease in ventilator (- 7.27; p < 0.001), ICU (- 4.38; p < 0.001), and hospital (- 7.00; p < 0.001) free days. Agitation was also associated with higher mortality (OR = 39.9, 6.53-243, p < 0.001). The most commonly used sedatives were opioids and benzodiazepines (9259 and 8453 patient days respectively), and the latter were associated with a 41% higher mortality in participants with a higher cumulative dose (75th vs 25th percentile, interquartile OR = 1.41, 1.12-1.77; p < 0.01). The overall cumulative dose of benzodiazepines and opioids was high, 774.5 mg and 16.8 g, respectively, by day 7 and by day 28; these doses approximately doubled. Haloperidol was only used in 3% of ICU days; however, the use of it was associated with a 70% lower mortality (interquartile OR = 0.3, 0.22-0.44, p < 0.001). CONCLUSIONS: Deep sedation, agitation, and cumulative dose of benzodiazepines were all independently associated with higher 90-day mortality. Additionally, deep sedation was associated with less ventilator-, ICU-, and hospital-free days. In contrast, haloperidol was associated with lower mortality in our study.

Force and Collective Epithelial Activities.

Cells apply forces to their surroundings to perform basic biological activities, including division, adhesion, and migration. Similarly, cell populations in epithelial tissues coordinate forces in physiological processes of morphogenesis and repair. These activities are highly regulated to yield the correct development and function of the body. The modification of this order is at the onset of pathological events and malfunctions. Mechanical forces and their translation into biological signals are the focus of an emerging field of research, shaping as a central discipline in the study of life and gathering knowledge at the interface of engineering, physics, biology and medicine. Novel engineering methods are needed to complement the classic instruments developed by molecular biology, physics and medicine. These should enable the measurement of forces at the cellular and multicellular level, and at a temporal and spatial resolution which is fully compatible with the ranges experienced by cells in vivo.

Pore Shape Defines Paths of Metastatic Cell Migration.

Invasion of dense tissues by cancer cells involves the interplay between the penetration resistance offered by interstitial pores and the deformability of cells. Metastatic cancer cells find optimal paths of minimal resistance through an adaptive path-finding process, which leads to successful dissemination. The physical limit of nuclear deformation is related to the minimal cross section of pores that can be successfully penetrated. However, this single biophysical parameter does not fully describe the architectural complexity of tissues featuring pores of variable area and shape. Here, employing laser nanolithography, we fabricate pore microenvironment models with well-controlled pore shapes, through which human breast cells (MCF10A) and their metastatic offspring (MCF10CA1a.cl1) could pervade. In these experimental settings, we demonstrate that the actual pore shape, and not only the cross section, is a major and independent determinant of cancer penetration efficiency. In complex architectures containing pores demanding large deformations from invading cells, tall and narrow rectangular openings facilitate cancer migration. In addition, we highlight the characteristic traits of the explorative behavior enabling metastatic cells to identify and select such pore shapes in a complex multishape pore environment, pinpointing paths of least resistance to invasion.

Endocytic reawakening of motility in jammed epithelia.

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination.

Endothelialization of Rationally Microtextured Surfaces with Minimal Cell Seeding Under Flow.

The generation of a confluent and functional endothelium at the luminal surface of cardiovascular devices represents the ideal solution to avoid contact between blood and synthetic materials thus allowing the long-term body integration of the implants. Due to the foreseen paucity of source cells in cardiovascular patients, surface engineering strategies to achieve full endothelialization, while minimizing the amount of endothelial cells required to seed the surface leading to prompt and full coverage with an endothelium are necessary. A stable endothelialization is the result of the interplay between endothelial cells, the flow-generated walls shear stress and the substrate topography. Here a novel strategy is designed and validated based on the use of engineered surface textures combined with confined islands of seeded endothelial cells. Upon release of the confinement, the cell island populations are able to migrate on the texture and merge under physiological flow conditions to promptly generate a fully connected endothelium. The interaction between endothelial cells and surface textures supports the process of endothelialization through the stabilization of cell-to-substrate adhesions and cell-to-cell junctions. It is shown that with this approach, when ≈50% of a textured surface is initially covered with cell seeding, the time to full endothelialization compared to an untextured surface is almost halved, underpinning the viability and effectiveness of the method for the quick and stable coverage of cardiovascular implants.

Acoustophoretic contactless transport and handling of matter in air.

Levitation and controlled motion of matter in air have a wealth of potential applications ranging from materials processing to biochemistry and pharmaceuticals. We present a unique acoustophoretic concept for the contactless transport and handling of matter in air. Spatiotemporal modulation of the levitation acoustic field allows continuous planar transport and processing of multiple objects, from near-spherical (volume of 0.1-10 µL) to wire-like, without being limited by the acoustic wavelength. The independence of the handling principle from special material properties (magnetic, optical, or electrical) is illustrated with a wide palette of application experiments, such as contactless droplet coalescence and mixing, solid-liquid encapsulation, absorption, dissolution, and DNA transfection. More than a century after the pioneering work of Lord Rayleigh on acoustic radiation pressure, a path-breaking concept is proposed to harvest the significant benefits of acoustic levitation in air.

Toward a rational design of surface textures promoting endothelialization.

The safe integration of cardiovascular devices requires the sustainable coverage of their luminal surface by endothelial cells (ECs). The engineering of active surface textures has the potential to coordinate cellular adhesion and migration under the action of hemodynamic forces. We define a paradigm to rationally design textures maximizing EC activities as a function of the applied stresses. This is based on harnessing the adhesions established by ECs through fine-tuning of the vertical extend of the underlying surface nanotopography.

Lack of association between chronic exposure to biomass fuel smoke and markers of right ventricular pressure overload at high altitude.

BACKGROUND: Chronic exposure to biomass fuel smoke has been implicated in the development of pulmonary hypertension and right ventricular pressure/volume overload through activation of inflammation, increase in vascular resistance, and endothelial dysfunction. We sought to compare N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) and echocardiography-derived pulmonary artery systolic pressure (PASP) levels in a high-altitude population-based study in Peru with and without chronic exposure to biomass fuel smoke. METHODS: NT-pro-BNP levels were measured in 519 adults (275 with and 244 without chronic exposure to biomass fuel smoke). Participants answered sociodemographics and clinical history questionnaires, underwent a clinical examination and blood testing for cardiopulmonary biomarkers. PASP was measured in a subgroup of 153 (31%) subjects. RESULTS: The study group consisted of 280 men (54%) and 239 women (46%). Average age was 56 years and average body mass index was 27 kg/m(2). In multivariable analysis, there was no association between chronic exposure to biomass fuel smoke and NT-pro-BNP (P = .31) or PASP (P = .31). In the subgroup in which both NT-pro-BNP levels and PASP were measured, there was strong evidence of an association between these two variables (ρ = 0.24, 95% CI 0.09-0.39; P = .003). We found that age, high sensitivity C-reactive protein, being male, and systolic blood pressure were positively associated with NT-pro-BNP levels whereas body mass index, low-density/high-density lipoprotein ratio, and Homeostasis Model of Assessment-Insulin Resistance were negatively associated (all P ≤ .02). CONCLUSIONS: In this population-based study in a high-altitude setting, neither NT-pro-BNP levels nor echocardiography-derived PASP were associated with chronic exposure to biomass fuel smoke.

Biophysical Reviews' Topical issue: The 7th Nanoengineering for Mechanobiology Symposium 2024 Camogli, Genoa, Italy.

This Commentary describes an open call for submissions to the upcoming Biophysical Reviews' Issue Focus: The 7th Nanoengineering for Mechanobiology (Genova, Italy). The submission deadline is August 1st of 2024. Interested parties are requested to make contact with the Issue Focus editors prior to submission.

N2FXm, a method for joint nuclear and cytoplasmic volume measurements, unravels the osmo-mechanical regulation of nuclear volume in mammalian cells.

In eukaryotes, cytoplasmic and nuclear volumes are tightly regulated to ensure proper cell homeostasis. However, current methods to measure cytoplasmic and nuclear volumes, including confocal 3D reconstruction, have limitations, such as relying on two-dimensional projections or poor vertical resolution. Here, to overcome these limitations, we describe a method, N2FXm, to jointly measure cytoplasmic and nuclear volumes in single cultured adhering human cells, in real time, and across cell cycles. We find that this method accurately provides joint size over dynamic measurements and at different time resolutions. Moreover, by combining several experimental perturbations and analyzing a mathematical model including osmotic effects and tension, we show that N2FXm can give relevant insights on how mechanical forces exerted by the cytoskeleton on the nuclear envelope can affect the growth of nucleus volume by biasing nuclear import. Our method, by allowing for accurate joint nuclear and cytoplasmic volume dynamic measurements at different time resolutions, highlights the non-constancy of the nucleus/cytoplasm ratio along the cell cycle.

Transdermal wires for improved integration in vivo.

Alternative engineering approaches have led the design of implants with controlled physical features to minimize adverse effects in biological tissues. Similar efforts have focused on optimizing the design features of percutaneous VAD drivelines with the aim to prevent infection, omitting however a thorough look on the implant-skin interactions that govern local tissue reactions. Here, we utilized an integrated approach for the biophysical modification of transdermal implants and their evaluation by chronic sheep implantation in comparison to the standard of care VAD drivelines. We developed a novel method for the transfer of breath topographical features on thin wires with modular size. We examined the impact of implant's diameter, surface topography, and chemistry on macroscopic, histological, and physical markers of inflammation, fibrosis, and mechanical adhesion. All implants demonstrated infection-free performance. The fibrotic response was enhanced by the increasing diameter of implants but not influenced by their surface properties. The implants of small diameter promoted mild inflammatory responses with improved mechanical adhesion and restricted epidermal downgrowth, in both silicone and polyurethane coated transdermal wires. On the contrary, the VAD drivelines with larger diameter triggered severe inflammatory reactions with frequent epidermal downgrowth. We validated these effects by quantifying the infiltration of macrophages and the level of vascularization in the fibrotic zone, highlighting the critical role of size reduction for the benign integration of transdermal implants with skin. This insight on how the biophysical properties of implants impact local tissue reactions could enable new solutions on the transdermal transmission of power, signal, and mass in a broad range of medical devices.

In-vitro investigation of endothelial monolayer retention on an inflow VAD cannula inside a beating heart phantom.

Ventricular assist devices (VADs) provide an alternative solution to heart transplantation for patients with end-stage heart failure. Insufficient hemocompatibility of VAD components can result in severe adverse events, such as thromboembolic stroke, and readmissions. To enhance VAD hemocompatibility, and avoid thrombus formation, surface modification techniques and endothelialization strategies are employed. In this work, a free form patterning topography is selected to facilitate the endothelialization of the outer surface of the inflow cannula (IC) of a commercial VAD. An endothelialization protocol for convoluted surfaces such as the IC is produced, and the retainment of the endothelial cell (EC) monolayer is evaluated. To allow this evaluation, a dedicated experimental setup is developed to simulate realistic flow phenomena inside an artificial, beating heart phantom with a VAD implanted on its apex. The procedural steps of mounting the system result to the impairment of the EC monolayer, which is further compromised by the developed flow and pressure conditions, as well as by the contact with the moving inner structures of the heart phantom. Importantly, the EC monolayer is better maintained in the lower part of the IC, which is more susceptible to thrombus formation and may therefore aid in minimizing the hemocompatibility related adverse events after the VAD implantation.

Accelerated epithelial layer healing induced by tactile anisotropy in surface topography.

Mammalian cells respond to tactile cues from topographic elements presented by the substrate. Among these, anisotropic features distributed in an ordered manner give directionality. In the extracellular matrix, this ordering is embedded in a noisy environment altering the contact guidance effect. To date, it is unclear how cells respond to topographical signals in a noisy environment. Here, using rationally designed substrates, we report morphotaxis, a guidance mechanism enabling fibroblasts and epithelial cells to move along gradients of topographic order distortion. Isolated cells and cell ensembles perform morphotaxis in response to gradients of different strength and directionality, with mature epithelia integrating variations of topographic order over hundreds of micrometers. The level of topographic order controls cell cycle progression, locally delaying or promoting cell proliferation. In mature epithelia, the combination of morphotaxis and noise-dependent distributed proliferation provides a strategy to enhance wound healing as confirmed by a mathematical model capturing key elements of the process.

PIP4K2B is mechanoresponsive and controls heterochromatin-driven nuclear softening through UHRF1.

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4,5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localisation. Here, we unveil the role of PIP4K2B as a mechanoresponsive enzyme. PIP4K2B protein level strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to softness, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.