Fiber alignment in 3D collagen networks as a biophysical marker for cell contractility.

Cells cultured in 3D fibrous biopolymer matrices exert traction forces on their environment that induce deformations and remodeling of the fiber network. By measuring these deformations, the traction forces can be reconstructed if the mechanical properties of the matrix and the force-free matrix configuration are known. These requirements limit the applicability of traction force reconstruction in practice. In this study, we test whether force-induced matrix remodeling can instead be used as a proxy for cellular traction forces. We measure the traction forces of hepatic stellate cells and different glioblastoma cell lines and quantify matrix remodeling by measuring the fiber orientation and fiber density around these cells. In agreement with simulated fiber networks, we demonstrate that changes in local fiber orientation and density are directly related to cell forces. By resolving Rho-kinase (ROCK) inhibitor-induced changes of traction forces, fiber alignment, and fiber density in hepatic stellate cells, we show that the method is suitable for drug screening assays. We conclude that differences in local fiber orientation and density, which are easily measurable, can be used as a qualitative proxy for changes in traction forces. The method is available as an open-source Python package with a graphical user interface.

Long-term stability of aerophilic metallic surfaces underwater.

Aerophilic surfaces immersed underwater trap films of air known as plastrons. Plastrons have typically been considered impractical for underwater engineering applications due to their metastable performance. Here, we describe aerophilic titanium alloy (Ti) surfaces with extended plastron lifetimes that are conserved for months underwater. Long-term stability is achieved by the formation of highly rough hierarchically structured surfaces via electrochemical anodization combined with a low-surface-energy coating produced by a fluorinated surfactant. Aerophilic Ti surfaces drastically reduce blood adhesion and, when submerged in water, prevent adhesion of bacteria and marine organisms such as barnacles and mussels. Overall, we demonstrate a general strategy to achieve the long-term stability of plastrons on aerophilic surfaces for previously unattainable underwater applications.

Silicone-Based Lubricant-Infused Slippery Coating Covalently Bound to Aluminum Substrates for Underwater Applications.

Wetting of solid surfaces is crucial for biological and industrial processes but is also associated with several harmful phenomena such as biofouling and corrosion that limit the effectiveness of various technologies in aquatic environments. Despite extensive research, these challenges remain critical today. Recently, we have developed a facile UV-grafting technique to covalently attach silicone-based coatings to solid substrates. In this study, the grafting process was evaluated as a function of UV exposure time on aluminum substrates. While short-time exposure to UV light results in the formation of lubricant-infused slippery surfaces (LISS), a flat, nonporous variant of slippery liquid-infused porous surfaces, longer exposure leads to the formation of semi-rigid cross-linked polydimethylsiloxane (PDMS) coatings, both covalently bound to the substrate. These coatings were exposed to aquatic media to evaluate their resistance to corrosion and biofouling. While the UV-grafted cross-linked PDMS coating effectively inhibits aluminum corrosion in aquatic environments and allows organisms to grow on the surface, the LISS coating demonstrates improved corrosion resistance but inhibits biofilm adhesion. The synergy between facile and low-cost fabrication, rapid binding kinetics, eco-friendliness, and nontoxicity of the applied materials to aquatic life combined with excellent wetting-repellent characteristics make this technology applicable for implementation in aquatic environments.

Synthetic mucus for an ex vivo phonation setup: Creation, application, and effect on excised porcine larynges.

Laryngeal mucus hydrates and lubricates the deformable tissue of the vocal folds and acts as a boundary layer with the airflow from the lungs. However, the effects of the mucus' viscoelasticity on phonation remain widely unknown and mucus has not yet been established in experimental procedures of voice research. In this study, four synthetic mucus samples were created on the basis of xanthan with focus on physiological frequency-dependent viscoelastic properties, which cover viscosities and elasticities over 2 orders of magnitude. An established ex vivo experimental setup was expanded by a reproducible and controllable application method of synthetic mucus. The application method and the suitability of the synthetic mucus samples were successfully verified by fluorescence evidence on the vocal folds even after oscillation experiments. Subsequently, the impact of mucus viscoelasticity on the oscillatory dynamics of the vocal folds, the subglottal pressure, and acoustic signal was investigated with 24 porcine larynges (2304 datasets). Despite the large differences of viscoelasticity, the phonatory characteristics remained stable with only minor statistically significant differences. Overall, this study increased the level of realism in the experimental setup for replication of the phonatory process enabling further research on pathological mucus and exploration of therapeutic options.

Human Laryngeal Mucus from the Vocal Folds: Rheological Characterization by Particle Tracking Microrheology and Oscillatory Shear Rheology.

Mucus consistency affects voice physiology and is connected to voice disorders. Nevertheless, the rheological characteristics of human laryngeal mucus from the vocal folds remain unknown. Knowledge about mucus viscoelasticity enables fabrication of artificial mucus with natural properties, more realistic ex-vivo experiments and promotes a better understanding and improved treatment of dysphonia with regard to mucus consistency. We studied human laryngeal mucus samples from the vocal folds with two complementary approaches: 19 samples were successfully applied to particle tracking microrheology (PTM) and five additional samples to oscillatory shear rheology (OSR). Mucus was collected by experienced laryngologists from patients together with demographic data. The analysis of the viscoelasticity revealed diversity among the investigated mucus samples according to their rigidity (absolute G' and G″). Moreover some samples revealed throughout solid-like character (G' > G″), whereas some underwent a change from solid-like to liquid-like (G' < G″). This led to a subdivision into three groups. We assume that the reason for the differences is a variation in the hydration level of the mucus, which affects the mucin concentration and network formation factors of the mucin mesh. The demographic data could not be correlated to the differences, except for the smoking behavior. Mucus of predominant liquid-like character was associated with current smokers.

Collective forces of tumor spheroids in three-dimensional biopolymer networks.

We describe a method for quantifying the contractile forces that tumor spheroids collectively exert on highly nonlinear three-dimensional collagen networks. While three-dimensional traction force microscopy for single cells in a nonlinear matrix is computationally complex due to the variable cell shape, here we exploit the spherical symmetry of tumor spheroids to derive a scale-invariant relationship between spheroid contractility and the surrounding matrix deformations. This relationship allows us to directly translate the magnitude of matrix deformations to the total contractility of arbitrarily sized spheroids. We show that our method is accurate up to strains of 50% and remains valid even for irregularly shaped tissue samples when considering only the deformations in the far field. Finally, we demonstrate that collective forces of tumor spheroids reflect the contractility of individual cells for up to 1 hr after seeding, while collective forces on longer timescales are guided by mechanical feedback from the extracellular matrix.

Measurement of Skeletal Muscle Fiber Contractility with High-Speed Traction Microscopy.

We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels under auxotonic loading conditions. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Young's modulus of the matrix without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/mL Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 ± 0.15 µN and tetanic forces (100-Hz stimulation frequency) of 2.38 ± 0.71 µN, corresponding to a tension of 0.44 ± 0.25 kPa and 2.53 ± 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium stabilizer tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high-speed method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.