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.

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.

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.

Role of lipopolysaccharides and lipoteichoic acids on C-Chrysophsin-1 interactions with model Gram-positive and Gram-negative bacterial membranes.

Antimicrobial peptides (AMPs) are attractive as biomaterial coatings because they have broad spectrum activity against different microbes, with a low likelihood of incurring antimicrobial resistance. Direct action against the bacterial membrane is the most common mechanism of action (MOA) of AMPs, with specific MOAs dependent on membrane composition, peptide concentration, and environmental factors that include temperature. Chrysophsin-1 (CHY1) is a broad spectrum salt-tolerant AMP that is derived from a marine fish. A cysteine modification was made to the peptide to facilitate attachment to a surface, such as a biomedical device. The authors used quartz crystal microbalance with dissipation monitoring to study how temperature (23 and 37 °C) and lipid composition influence the MOA of cysteine-modified peptide (C-CHY1) with model membranes comprised of supported lipid bilayers (SLBs). These two temperatures were used so that the authors could better understand the differences in behavior between typical lab temperatures and physiologic conditions. The authors created model membranes that mimicked properties of Gram-negative and Gram-positive bacteria in order to understand how the mechanisms might differ for different types of bacterial systems. SLB models of Gram-positive bacterial membranes were formed using combinations of phosphatidylcholine, phosphatidylglycerol (PG), and S. aureus-derived lipoteichoic acid (LTA). SLB models of Gram-negative bacterial membranes were formed using combinations of phosphatidylethanolamine (PE), PG, and E. coli-derived lipopolysaccharides (LPS). The molecules that distinguish Gram-positive and Gram-negative membranes (LTA and LPS) have the potential to alter the MOA of C-CHY1 with the SLBs. The authors' results showed that the MOA for the Gram-positive SLBs was not sensitive to temperature, but the LTA addition did have an effect. Specifically, similar trends in frequency and dissipation changes across all overtones were observed, and the same mechanistic trends were observed in the polar plots at 23 and 37 °C. However, when LTA was added, polar plots showed an association between C-CHY1 and LTA, leading to SLB saturation. This was demonstrated by significant changes in dissipation, while the frequency (mass) was not increasing after the saturation point. For the Gram-negative SLBs, the composition did not have a significant effect on MOA, but the authors saw more differences between the two temperatures studied. The authors believe this is due to the fact that the gel-liquid crystal transition temperature of PE is 25 °C, which means that the bilayer is more rigid at 23 °C, compared to temperatures above the transition point. At 23 °C, a significant energetic shift would be required to allow for additional AMP insertion. This could be seen in the polar plots, where there was a steep slope but there was very little mass addition. At 37 °C, the membrane is more fluid and there is less of an energetic requirement for insertion. Therefore, the authors observed greater mass addition and fewer changes in dissipation. A better understanding of C-CHY1 MOA using different SLB models will allow for the more rational design of future therapeutic solutions that make use of antimicrobial peptides, including those involving biomaterial coatings.

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.

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.