Effect of sphingosine and inoculum concentrations on Staphylococcus aureus and Staphylococcus epidermidis biofilms.

Atopic dermatitis (AD) is a chronic inflammatory skin disease where patients are more susceptible to infection and inflammation. The most salient symptoms of atopic dermatitis (AD) are skin dysbiosis and ceramide deficiency. Here, the effect of AD conditions on S. aureus resilience was investigated. S. aureus and S. epidermidis biofilms were co-inoculated at healthy and AD bacterial ratios and exposed to various sphingosine dosing regimens. In both healthy (S. epidermidis dominant) and AD (S. aureus dominant) conditions the viability of the non-dominant bacterial species was affected. Quorum sensing (QS)-impaired S. aureus was overall more susceptible to sphingosine. Despite the general resilience of QS-intact S. aureus against sphingosine, modulation of S. epidermidis (healthy ratio) and sphingosine (healthy Sph) led to a lack of recovery from its initial killing. Overall, it was found that when in biofilms, S. epidermidis increases S. aureus resilience to sphingosine, possibly enhancing the pathogen's recalcitrance in AD skin.

Biomechanical fracture mechanics of composite layered skin-like materials.

Most protective biological tissues are structurally comprised of a stiff and thin outer layer on top of a soft underlying substrate. Examples include mammalian skin, fish scales, crustacean shells, and nut and seed shells. While these composite skin-like tissues are ubiquitous in nature, their mechanics of failure and what potential mechanical advantages their composite structures offer remains unclear. In this work, changes in the puncture mechanics of composite hyperelastic elastomers with differing non-dimensional layer thicknesses are explored. Puncture behavior of these membranes is measured for dull and sharp conical indenters. Membranes with a stiff outer layer of only 1% of the overall composite thickness exhibit a puncture energy comparable to membranes with a stiff outer layer approximately 20 times thicker. This puncture energy, scaled by its flexural capacity, achieves a local maximum when the top layer is approximately 1% of the total membrane, similar to the structure of numerous mammalian species. The mode of failure for these regimes is also investigated. In contrast with puncture directly beneath sharp tips caused by high stress concentrations, a new type of 'coring' type fracture emerges at large indentation depths, resulting from accumulated tensile strain energy along the sides of the divot as the membrane is deformed with a blunt indenter. These results could enhance the durability and robustness of stretchable materials used for products such as surgical gloves, packaging, and flexible electronics.

Patterning droplets with durotaxis.

Numerous cell types have shown a remarkable ability to detect and move along gradients in stiffness of an underlying substrate--a process known as durotaxis. The mechanisms underlying durotaxis are still unresolved, but generally believed to involve active sensing and locomotion. Here, we show that simple liquid droplets also undergo durotaxis. By modulating substrate stiffness, we obtain fine control of droplet position on soft, flat substrates. Unlike other control mechanisms, droplet durotaxis works without imposing chemical, thermal, electrical, or topographical gradients. We show that droplet durotaxis can be used to create large-scale droplet patterns and is potentially useful for many applications, such as microfluidics, thermal control, and microfabrication.

Traction force microscopy in physics and biology.

Adherent cells, crawling slugs, peeling paint, sessile liquid drops, bearings and many other living and non-living systems apply forces to solid substrates. Traction force microscopy (TFM) provides spatially-resolved measurements of interfacial forces through the quantification and analysis of the deformation of an elastic substrate. Although originally developed for adherent cells, TFM has no inherent size or force scale, and can be applied to a much broader range of mechanical systems across physics and biology. In this paper, we showcase the wide range of applicability of TFM, describe the theory, and provide experimental details and code so that experimentalists can rapidly adopt this powerful technique.

Ultraviolet light induces mechanical and structural changes in full thickness human skin.

While the detrimental health effects of prolonged ultraviolet (UV) irradiation on skin health have been widely accepted, the biomechanical process by which photoaging occurs and the relative effects of irradiation with different UV ranges on skin biomechanics have remained relatively unexplored. In this study, the effects of UV-induced photoageing are explored by quantifying the changes in the mechanical properties of full-thickness human skin irradiated with UVA and UVB light for incident dosages up to 1600 J/cm2. Mechanical testing of skin samples excised parallel and perpendicular to the predominant collagen fiber orientation show a rise in the fractional relative difference of elastic modulus, fracture stress, and toughness with increased UV irradiation. These changes become significant with UVA incident dosages of 1200 J/cm2 for samples excised both parallel and perpendicular to the dominant collagen fiber orientation. However, while mechanical changes occur in samples aligned with the collagen orientation at UVB dosages of 1200 J/cm2, statistical differences in samples perpendicular to the collagen orientation emerge only for UVB dosages of 1600 J/cm2. No notable or consistent trend is observed for the fracture strain. Analyses of toughness changes with maximum absorbed dosage reveals that no one UV range is more impactful in inducing mechanical property changes, but rather these changes scale with maximum absorbed energy. Evaluation of the structural characteristics of collagen further reveals an increase in collagen fiber bundle density with UV irradiation, but not collagen tortuosity, potentially linking mechanical changes to altered microstructure.

Scaling of traction forces with the size of cohesive cell colonies.

To understand how the mechanical properties of tissues emerge from interactions of multiple cells, we measure traction stresses of cohesive colonies of 1-27 cells adherent to soft substrates. We find that traction stresses are generally localized at the periphery of the colony and the total traction force scales with the colony radius. For large colony sizes, the scaling appears to approach linear, suggesting the emergence of an apparent surface tension of the order of 10(-3) N/m. A simple model of the cell colony as a contractile elastic medium coupled to the substrate captures the spatial distribution of traction forces and the scaling of traction forces with the colony size.

Mechanisms and Implications of Bacterial Invasion across the Human Skin Barrier.

Atopic dermatitis (AD) is associated with a deficiency of skin lipids, increased populations of Staphylococcus aureus in the microbiome, and structural defects in the stratum corneum (SC), the outermost layer of human skin. However, the pathogenesis of AD is ambiguous, as it is unclear whether observed changes are the result of AD or contribute to the pathogenesis of the disease. Previous studies have shown that S. aureus is capable of permeating across isolated human SC tissue when lipids are depleted to levels consistent with AD conditions. In this study, we expand upon this discovery to determine the mechanisms and implications of bacterial penetration into the SC barrier. Specifically, we establish if bacteria are permeating intercellularly or employing a combination of both inter- and intracellular travel. The mechanical implications of bacterial invasion, lipid depletion, and media immersion are also evaluated using a newly developed, physiologically relevant, temperature-controlled drip chamber. Results reveal for the first time that S. aureus can be internalized by corneocytes, indicating transcellular movement through the tissue during permeation, consistent with previous theoretical models. S. aureus also degrades the mechanical integrity of human SC, particularly when the tissue is partially depleted of lipids. These observed mechanical changes are likely the cause of broken or ruptured tissue seen as exudative lesions in AD flares. This work further highlights the necessity of lipids in skin microbial barrier function. IMPORTANCE Millions of people suffer from the chronic inflammatory skin disease atopic dermatitis (AD), whose symptoms are associated with a deficiency of skin lipids that exhibit antimicrobial functions and increased populations of the opportunistic pathogen Staphylococcus aureus. However, the pathogenesis of AD is ambiguous, and it remains unclear if these observed changes are merely the result of AD or contribute to the pathogenesis of the disease. In this article, we demonstrate the necessity of skin lipids in preventing S. aureus from penetrating the outermost barrier of human skin, thereby causing a degradation in tissue integrity. This bacterial permeation into the viable epidermis could act as an inflammatory trigger of the disease. When coupled with delipidated AD tissue conditions, bacterial permeation can also explain increased tissue fragility, potentially causing lesion formation in AD patients that results in further enhancing bacterial permeability across the stratum corneum and the development of chronic conditions.

Systematic evaluating and modeling of SARS-CoV-2 UVC disinfection.

The ongoing COVID-19 global pandemic has necessitated evaluating various disinfection technologies for reducing viral transmission in public settings. Ultraviolet (UV) radiation can inactivate pathogens and viruses but more insight is needed into the performance of different UV wavelengths and their applications. We observed greater than a 3-log reduction of SARS-CoV-2 infectivity with a dose of 12.5 mJ/cm2 of 254 nm UV light when the viruses were suspended in PBS, while a dose of 25 mJ/cm2 was necessary to achieve a similar reduction when they were in an EMEM culture medium containing 2%(v/v) FBS, highlighting the critical effect of media in which the virus is suspended, given that SARS-CoV-2 is always aerosolized when airborne or deposited on a surface. It was found that SARS-CoV-2 susceptibility (a measure of the effectiveness of the UV light) in a buffer such as PBS was 4.4-fold greater than that in a cell culture medium. Furthermore, we discovered the attenuation of UVC disinfection by amino acids, vitamins, and niacinamide, highlighting the importance of determining UVC dosages under a condition close to aerosols that wrap the viruses. We developed a disinfection model to determine the effect of the environment on UVC effectiveness with three different wavelengths, 222 nm, 254 nm, and 265 nm. An inverse correlation between the liquid absorbance and the viral susceptibility was observed. We found that 222 nm light was most effective at reducing viral infectivity in low absorbing liquids such as PBS, whereas 265 nm light was most effective in high absorbing liquids such as cell culture medium. Viral susceptibility was further decreased in N95 masks with 222 nm light being the most effective. The safety of 222 nm was also studied. We detected changes to the mechanical properties of the stratum corneum of human skins when the 222 nm accumulative exposure exceeded 50 J/cm2.The findings highlight the need to evaluate each UV for a given application, as well as limiting the dose to the lowest dose necessary to avoid unnecessary exposure to the public.

The precision of macroscale mechanical measurements is limited by the inherent structural heterogeneity of human stratum corneum.

Biological tissues are structurally heterogenous mosaics at cellular and sub-cellular length scales. Some tissues, like the outermost layer of human skin, or stratum corneum (SC), also exhibit a rich topography of microchannels at larger mesoscopic length scales. Although this is well understood, modern studies continue to characterize the mechanical properties of biological tissues, including the SC, using macroscale techniques that assume these materials are homogenous in structure, thickness, and composition. Macroscale failure testing of SC is commonly associated with large sample to sample variability. We anticipate that microscale heterogeneities play an important role in defining the global mechanical response of the tissue. To evaluate the validity of the prevailing paradigm that macroscopic testing techniques can provide meaningful information about failure in soft heterogenous tissues, the macroscale work of fracture in isolated human SC samples is measured using conventional macroscale testing techniques and compared with the energy cost of creating new crack interfaces at the microscale, measured using a modified traction force microscopy technique. Results show that measured micro- and macroscale energy costs per unit crack path length are highly consistent. However, crack propagation is found to be guided by microscale topographical features in the tissue. This correlation reveals that macroscale mechanical sample to sample variability is caused by notable differences in crack propagation pathways. STATEMENT OF SIGNIFICANCE: Although designed to test homogeneous materials, macroscopic uniaxial tensometry is currently the gold standard for measuring the mechanical properties of biological tissues. All tissues, including human stratum corneum are structurally heterogeneous at the microscale and mechanical measurements are commonly highly variable, even for specimens from the same source. This study explores the fundamental causes of this disparity and evaluates the prevailing paradigm that macroscopic testing techniques can provide meaningful information about failure in soft heterogeneous tissues. Results conclude that the cause of large variability in mechanical work of fracture is due to inherent structural heterogeneities governing crack propagation pathways and altering the total crack length. Structural heterogeneities in tissue therefore limits the precision of macroscale biomechanical testing.

Effect of collagen degradation on the mechanical behavior and wrinkling of skin.

Chronological skin aging is a complex process that is controlled by numerous intrinsic and extrinsic factors. One major factor is the gradual degradation of the dermal collagen fiber network. As a step toward understanding the mechanistic importance of dermal tissue in the process of aging, this study employs analytical and multiscale computational models to elucidate the effect of collagen fiber bundle disintegration on the mechanical properties and topography of skin. Here, human skin is modeled as a soft composite with an anisotropic dermal layer. The anisotropy of the tissue is governed by collagen fiber bundles with varying densities, average fiber alignments, and normalized alignment distributions. In all finite element models examined, collagen fiber bundle degradation results in progressive decreases in dermal and full-thickness composite stiffness. This reduction is more profound when collagen bundles align with the compression axis. Aged skin models with low collagen fiber bundle densities under compression exhibit notably smaller critical wrinkling strains and larger critical wavelengths than younger skin models, in agreement with in vivo wrinkling behavior with age. The propensity for skin wrinkling can be directly attributable to the degradation of collagen fiber bundles, a relationship that has previously been assumed but unsubstantiated. While linear-elastic analytical models fail to capture the postbuckling behavior in skin, nonlinear finite element models can predict the complex bifurcations of the compressed skin with different densities of collagen bundles.

Lipid depletion enables permeation of Staphylococcus aureus bacteria through human stratum corneum.

Atopic dermatitis (AD) is a chronic inflammatory disease that affects approximately 2-5% of adults worldwide. The pathogenesis of AD continues to be a well-debated point of conjecture, with numerous hypotheses having been proposed. AD conditions are associated with increased populations of Staphylococcus aureus and reduced skin lipids. In this study, we evaluate the ability of S. aureus to permeate across human stratum corneum (SC) exhibiting both normal and depleted lipid conditions consistent with AD. This permeation would enable bacteria to interact with underlying viable epidermal cells, which could serve as a trigger for inflammation and disease onset. Our results indicate that permeation of S. aureus through SC exhibiting normal lipid conditions is not statistically significant. However, bacteria can readily permeate through lipid depleted tissue over a 9-d period. These findings suggest that S. aureus may potentially act as the mechanistic cause, rather than merely the result of AD. ABBREVIATIONS: AD: Atopic dermatitis; SC: Stratum Corneum; AMP: Antimicrobial peptide; DIW: Deionized water; PDMS: Polydimethylsiloxane; GFP: Green fluorescent protein; BHI: Brain heart infusion medium.

Ultraviolet light degrades the mechanical and structural properties of human stratum corneum.

Prolonged exposure of human skin to sunlight causes photodamage, which induces the early onset of wrinkles and increased tissue fragility. While solar ultraviolet (UV) light is considered to have the most damaging effect, the UV range that is most harmful remains a topic of significant debate. In this study, we take a first step towards elucidating biomechanical photoageing effects by quantifying how exposure to different UV ranges and dosages impacts the mechanical and structural properties of human stratum corneum (SC), the most superficial skin layer. Mechanical testing reveals that irradiation of isolated human SC to UVA (365 nm), UVB (302 nm), or UVC (265 nm) light with dosages of up to 4000 J/cm2 notably alters the elastic modulus, fracture stress, fracture strain, and work of fracture. For equivalent incident dosages, UVC degrades SC the greatest. However, upon discounting reflected and transmitted components of the incident light, a generalized scaling law relating the photonic energy absorbed by the SC to the energy cost of tissue fracture emerges. This relationship indicates that no one UV range is more damaging than another. Rather, the magnitude of absorbed UV energy governs the degradation of tissue mechanical integrity. Subsequent structural studies are performed to elucidate the cause of this mechanical degradation. UV absorption scales with the spatial dispersion of desmoglein 1 (Dsg 1), a component of corneocyte cell-cell junctions, away from intercellular sites. Combining both scaling laws, we establish a mechanical-structural model capable of predicting UV induced tissue mechanical integrity from Dsg 1 dispersion.

Embedding topography enables fracture guidance in soft solids.

The natural topographical microchannels in human skin have recently been shown to be capable of guiding propagating cracks. In this article we examine the ability to guide fracture by incorporating similar topographical features into both single, and dual layer elastomer membranes that exhibit uniform thickness. In single layer membranes, crack guidance is achieved by minimizing the nadir thickness of incorporated v-shaped channels, maximizing the release of localized strain energy. In dual layer membranes, crack guidance along embedded channels is achieved via interfacial delamination, which requires less energy to create a new surface than molecular debonding. In both membrane types, guided crack growth is only temporary. However, utilizing multiple embedded channels, non-contiguous crack control can be maintained at angles up to 45° from the mode I fracture condition. The ability to control and deflect fracture holds great potential for improving the robustness and lifespan of flexible electronics and stretchable sensors.

Viscoelastic properties of Pseudomonas aeruginosa variant biofilms.

Pseudomonas aeruginosa evolves during chronic pulmonary infections of cystic fibrosis (CF) patients, forming pathoadapted variants that are persistent. Mucoid and rugose small-colony variants (RSCVs) are typically isolated from sputum of CF patients. These variants overproduce exopolysaccharides in the biofilm extracellular polymeric substance (EPS). Currently, changes to the biophysical properties of RSCV and mucoid biofilms due to variations in EPS are not well understood. This knowledge may reveal how lung infections resist host clearance mechanisms. Here, we used mechanical indentation and shear rheometry to analyse the viscoelasticity of RSCV and mucoid colony-biofilms compared to their isogenic parent at 2-, 4-, and 6-d. While the viscoelasticity of parental colony-biofilms underwent fluctuating temporal changes, in contrast, RSCV and mucoid colony-biofilms showed a gradual progression to more elastic-solid behaviour. Theoretical indices of mucociliary and cough clearance predict that mature 6-d parental and RSCV biofilms may show reduced cough clearance from the lung, while early mucoid biofilms may show reduced clearance by both mechanisms. We propose that viscoelasticity be considered a virulence property of biofilms.

Heterogeneous ceramide distributions alter spatially resolved growth of Staphylococcus aureus on human stratum corneum.

Contemporary studies have revealed dramatic changes in the diversity of bacterial microbiota between healthy and diseased skin. However, the prevailing use of swabs to extract the microorganisms has meant that only population 'snapshots' are obtained, and all spatially resolved information of bacterial growth is lost. Here we report on the temporospatial growth of Staphylococcus aureus on the surface of the human stratum corneum (SC); the outermost layer of skin. This bacterial species dominates bacterial populations on skin with atopic dermatitis (AD). We first establish that the distribution of ceramides naturally present in the SC is heterogeneous, and correlates with the tissue's structural topography. This distribution subsequently impacts the growth of bacterial biofilms. In the SC retaining healthy ceramide concentrations, biofilms exhibit no spatial preference for growth. By contrast, a depletion of ceramides consistent with reductions known to occur with AD enables S. aureus to use the patterned network of topographical canyons as a conduit for growth. The ability of ceramides to govern bacterial growth is confirmed using a topographical skin canyon analogue coated with the ceramide subcomponent d-sphingosine. Our work appears to explain the causal link between ceramide depletion and increased S. aureus populations that is observed in AD. It may also provide insight into disease transmission as well as improving pre-operative skin cleansing techniques.

Measuring and Modeling Contractile Drying in Human Stratum Corneum.

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.

Non-ionising UV light increases the optical density of hygroscopic self assembled DNA crystal films.

We report on ultraviolet (UV) light induced increases in the UV optical density of thin and optically transparent crystalline DNA films formed through self assembly. The films are comprised of closely packed, multi-faceted and sub micron sized crystals. UV-Vis spectrophotometry reveals that DNA films with surface densities up to 0.031 mg/mm2 can reduce the transmittance of incident UVC and UVB light by up to 90%, and UVA transmittance by up to 20%. Subsequent and independent film irradiation with either UVA or UVB dosages upwards of 80 J/cm2 both reduce UV transmittance, with reductions scaling monotonically with UV dosage. To date the induction of a hyperchromic effect has been demonstrated using heat, pH, high salt mediums, and high energy ionising radiation. Both hyperchromicity and increased light scattering could account for the increased film optical density after UV irradiation. Additional characterisation of the films reveal they are highly absorbent and hygroscopic. When coated on human skin, they are capable of slowing water evaporation and keeping the tissue hydrated for extended periods of time.

Nanoparticle size-specific actin rearrangement and barrier dysfunction of endothelial cells.

In this work, we evaluated the impact of gold nanoparticles on endothelial cell behavior and function beyond the influence on cell viability. Five types of gold nanoparticles were studied: 5 nm and 20 nm bare gold nanoparticles, 5 nm and 20 nm gold nanoparticles with biocompatible polyethylene glycol (PEG) coating and 60 nm bare gold nanoparticles. We found that all tested gold nanoparticles did not affect cell viability significantly and reduced the reactive oxygen species (ROS) level in endothelial cells. Only 20 nm bare gold nanoparticles caused an over 50% increase in endothelial barrier permeability and slow recovery of barrier function was observed after the gold nanoparticles were removed. This impairment in endothelial barrier function was caused by unbalanced forces between intracellular tensions and paracellular forces, actin microfilament rearrangement, which occurred through a Rho/ROCK kinase-dependent pathway and broke the force balance between intracellular tensions and paracellular forces. The size-specific effect of gold nanoparticles on endothelial cells may have important implications regarding the behavior of nanoparticles in the biological system and provide valuable guidance in nanomaterial design and biomedical applications.