Biomechanical activation of keloid fibroblasts promotes lysosomal remodelling and exocytosis.

Keloids are a severe form of scarring for which the underlying mechanisms are poorly understood, and treatment options are limited or inconsistent. While biomechanical forces are potential drivers of keloid scarring, the direct cellular responses to mechanical cues have yet to be defined. The aim of this study was to examine the distinct responses of normal dermal fibroblasts (NDFs) and keloid-derived fibroblasts (KDFs) to changes in extracellular matrix (ECM) stiffness. When cultured on hydrogels mimicking the elasticity of normal or scarred skin, KDFs displayed greater stiffness-dependent increases in cell spreading, F-actin stress fibre formation, and focal adhesion assembly. Elevated acto-myosin contractility in KDFs disrupted the normal mechanical regulation of ECM deposition and conferred resistance myosin inhibitors. Transcriptional profiling identified mechanically-regulated pathways in NDFs and KDFs, including the actin cytoskeleton, Hippo signalling, and autophagy. Further analysis of the autophagy pathway revealed that autophagic flux was intact in both fibroblast populations and depended on acto-myosin contractility. However, KDFs displayed marked changes in lysosome organisation and an increase in lysosomal exocytosis, which was mediated by acto-myosin contractility. Together, these findings demonstrate that KDFs possess an intrinsic increase in cytoskeletal tension, which heightens the response to ECM mechanics and promotes lysosomal exocytosis.

Design of an Integrated Microvascularized Human Skin-on-a-Chip Tissue Equivalent Model.

Tissue-engineered skin constructs have been under development since the 1980s as a replacement for human skin tissues and animal models for therapeutics and cosmetic testing. These have evolved from simple single-cell assays to increasingly complex models with integrated dermal equivalents and multiple cell types including a dermis, epidermis, and vasculature. The development of micro-engineered platforms and biomaterials has enabled scientists to better recreate and capture the tissue microenvironment in vitro, including the vascularization of tissue models and their integration into microfluidic chips. However, to date, microvascularized human skin equivalents in a microfluidic context have not been reported. Here, we present the design of a novel skin-on-a-chip model integrating human-derived primary and immortalized cells in a full-thickness skin equivalent. The model is housed in a microfluidic device, in which a microvasculature was previously established. We characterize the impact of our chip design on the quality of the microvascular networks formed and evidence that this enables the formation of more homogenous networks. We developed a methodology to harvest tissues from embedded chips, after 14 days of culture, and characterize the impact of culture conditions and vascularization (including with pericyte co-cultures) on the stratification of the epidermis in the resulting skin equivalents. Our results indicate that vascularization enhances stratification and differentiation (thickness, architecture, and expression of terminal differentiation markers such as involucrin and transglutaminase 1), allowing the formation of more mature skin equivalents in microfluidic chips. The skin-on-a-chip tissue equivalents developed, because of their realistic microvasculature, may find applications for testing efficacy and safety of therapeutics delivered systemically, in a human context.

Multi-Scale Analysis of the Composition, Structure, and Function of Decellularized Extracellular Matrix for Human Skin and Wound Healing Models.

The extracellular matrix (ECM) is a complex mixture of structural proteins, proteoglycans, and signaling molecules that are essential for tissue integrity and homeostasis. While a number of recent studies have explored the use of decellularized ECM (dECM) as a biomaterial for tissue engineering, the complete composition, structure, and mechanics of these materials remain incompletely understood. In this study, we performed an in-depth characterization of skin-derived dECM biomaterials for human skin equivalent (HSE) models. The dECM materials were purified from porcine skin, and through mass spectrometry profiling, we quantified the presence of major ECM molecules, including types I, III, and VI collagen, fibrillin, and lumican. Rheological analysis demonstrated the sol-gel and shear-thinning properties of dECM materials, indicating their physical suitability as a tissue scaffold, while electron microscopy revealed a complex, hierarchical structure of nanofibers in dECM hydrogels. The dECM materials were compatible with advanced biofabrication techniques, including 3D printing within a gelatin microparticle support bath, printing with a sacrificial material, or blending with other ECM molecules to achieve more complex compositions and structures. As a proof of concept, we also demonstrate how dECM materials can be fabricated into a 3D skin wound healing model using 3D printing. Skin-derived dECM therefore represents a complex and versatile biomaterial with advantageous properties for the fabrication of next-generation HSEs.

Fabrication of Human Skin Equivalents Using Decellularized Extracellular Matrix.

There is a growing demand for in vitro models of human tissues that recapitulate the complex structures and functions found in vivo, and the biomaterials that support these physiologically relevant models are essential underpinning technologies. Here, we present an optimized protocol for generating human skin equivalents (HSEs) using a dermal matrix isolated from decellularized porcine skin. The decellularized extracellular matrix (dECM) contains a complex mixture of fibrillar collagens and matrisomal proteins that mimic native skin and can be produced in large quantities. The procedure for decellularization, digestion, and solubilization of the dECM is described in detail. In addition, we provide instructions for how to construct a three-dimensional HSE model using the dECM as the dermal support matrix for human keratinocytes and dermal fibroblasts. Recent studies from our laboratory have shown that HSEs generated using porcine dECM display improved epidermal differentiation and stratification compared to existing protocols using type I collagen gels. Thus, dECM-based biomaterials are a useful tool for replicating human skin physiology in vitro and developing advanced human skin models for therapeutic discovery and testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of decellularized extracellular matrix from porcine skin Basic Protocol 2: Generation of human skin equivalents.

Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function.

Adhesive cues from the extracellular matrix (ECM) specify the size and shape of the nucleus via mechanical forces transmitted through the cytoskeleton. However, the effects of these biophysical stimuli on internal nuclear architecture and cellular responses remain poorly understood. This study investigates the direct impact of ECM adhesion on nucleolar remodeling in human keratinocytes using micropatterned substrates. Limited adhesion on small micropatterns promotes fusion of nucleoli, alongside a reduction in nuclear volume and condensation of heterochromatin. These changes in nucleolar architecture are mediated by altered chromatin biomechanics and depend on integration of the nucleus with the actin cytoskeleton. Functionally, nucleolar remodeling regulates ribogenesis and protein synthesis in keratinocytes and is associated with specific transcriptional changes in ribogenesis genes. Together, these findings demonstrate that cell shape and nuclear morphology control nucleolar structure and function and implicate the nucleolus as a key mechano-sensing element within the cell.

Antibody Screening Results for Anti-Nucleocapsid Antibodies Toward the Development of a Lateral Flow Assay to Detect SARS-CoV-2 Nucleocapsid Protein.

The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably rapid and inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance, they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow (immuno)assays (LFAs) in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.

Measurement Biases Distort Cell-Free DNA Fragmentation Profiles and Define the Sensitivity of Metagenomic Cell-Free DNA Sequencing Assays.

BACKGROUND: Metagenomic sequencing of microbial cell-free DNA (cfDNA) in blood and urine is increasingly used as a tool for unbiased infection screening. The sensitivity of metagenomic cfDNA sequencing assays is determined by the efficiency by which the assay recovers microbial cfDNA vs host-specific cfDNA. We hypothesized that the choice of methods used for DNA isolation, DNA sequencing library preparation, and sequencing would affect the sensitivity of metagenomic cfDNA sequencing. METHODS: We characterized the fragment length biases inherent to select DNA isolation and library preparation procedures and developed a model to correct for these biases. We analyzed 305 cfDNA sequencing data sets, including publicly available data sets and 124 newly generated data sets, to evaluate the dependence of the sensitivity of metagenomic cfDNA sequencing on pre-analytical variables. RESULTS: Length bias correction of fragment length distributions measured from different experimental procedures revealed the ultrashort (<100 bp) nature of microbial-, mitochondrial-, and host-specific urinary cfDNA. The sensitivity of metagenomic sequencing assays to detect the clinically reported microorganism differed by more than 5-fold depending on the combination of DNA isolation and library preparation used. CONCLUSIONS: Substantial gains in the sensitivity of microbial and other short fragment recovery can be achieved by easy-to-implement changes in the sample preparation protocol, which highlights the need for standardization in the liquid biopsy field.

Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Research Techniques Made Simple: Analysis of Skin Cell and Tissue Mechanics Using Atomic Force Microscopy.

Atomic force microscopy (AFM) is a powerful technique for nanoscale imaging and mechanical analysis of biological specimens. It is based on the highly sensitive detection of forces and displacement of a sharp-tipped cantilever as it scans the surface of an object. Because it requires minimal sample processing and preparation, AFM is particularly advantageous for the analysis of cells and tissues in their near-native state. Moreover, recent advances in Bio-AFM systems and the combination with light microscopy imaging have greatly enhanced the application of AFM in biological research. In the field of dermatology, the method has led to important insights into our understanding of the biomechanics of normal healthy skin and the pathogenesis of a variety of skin diseases. In this Research Techniques Made Simple article, we review the fundamental principles of AFM, how AFM can be applied to the analysis of cell and tissue mechanics, and recent applications of AFM in skin science and dermatology.

Field evaluation of a prototype tuberculosis lipoarabinomannan lateral flow assay on HIV-positive and HIV-negative patients.

Detection of tuberculosis at the point-of-care (POC) is limited by the low sensitivity of current commercially available tests. We describe a diagnostic accuracy field evaluation of a prototype urine Tuberculosis Lipoarabinomannan Lateral Flow Assay (TB-LAM LFA) in both HIV-positive and HIV-negative patients using fresh samples with sensitivity and specificity as the measures of accuracy. This prototype combines a proprietary concentration system with a sensitive LFA. In a prospective study of 292 patients with suspected pulmonary tuberculosis in Uganda, the clinical sensitivity and specificity was compared against a microbiological reference standard including sputum Xpert MTB/RIF Ultra and solid and liquid culture. TB-LAM LFA had an overall sensitivity of 60% (95%CI 51-69%) and specificity of 80% (95%CI 73-85%). When comparing HIV-positive (N = 86) and HIV-negative (N = 206) patients, there was no significant difference in sensitivity (sensitivity difference 8%, 95%CI -11% to +24%, p = 0.4351) or specificity (specificity difference -9%, 95%CI -24% to +4%, p = 0.2051). Compared to the commercially available Alere Determine TB-LAM Ag test, the TB-LAM LFA prototype had improved sensitivity in both HIV-negative (difference 49%, 95%CI 37% to 59%, p<0.0001) and HIV-positive patients with CD4+ T-cell counts >200cells/µL (difference 59%, 95%CI 32% to 75%, p = 0.0009). This report is the first to show improved performance of a urine TB LAM test for HIV-negative patients in a high TB burden setting. We also offer potential assay refinement solutions that may further improve sensitivity and specificity.

Regulation of collective cell polarity and migration using dynamically adhesive micropatterned substrates.

Collective cell migration is a fundamental biological process in which groups of cells move together in a coordinated manner, and it is essential for tissue development and wound repair. However, the underlying mechanisms that orchestrate directionality in collectively migrating cells remain poorly understood. In this study, we employed dynamically adhesive micropatterned substrates to investigate the role of adhesive cues in directing epithelial migration. Our findings demonstrate that epithelial cells collectively polarize in response to asymmetric patterns of extracellular matrix (ECM), and the degree of polarization depends on the degree of asymmetry and requires calcium-dependent cell-cell adhesion. When released from the micropatterns, epithelial cells collectively migrate according to the direction of pre-established polarity, and cohesive migration specifically requires E-cadherin-containing adherens junctions. Finally, disruption of the microtubule network blocks collective polarization and functionally inhibits directed migration. Together, these results indicate that adhesive cues from the ECM guide collective epithelial polarity and migration, and this response depends on adherens junctions and microtubules. STATEMENT OF SIGNIFICANCE: This study employs a dynamically adhesive micropatterning platform to investigate the role of adhesive cues in directing the polarity and directional migration of epithelial cells. The findings demonstrate how asymmetric tissue geometry influences the collective directionality in simple epithelia and that this response is mediated by adherens junctions and the microtubule network. This work provides new insight into fundamental cellular processes involved in wound healing and has important implications for biomaterial and scaffold design.

Multiplexed and Extraction-Free Amplification for Simplified SARS-CoV-2 RT-PCR Tests.

The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/µL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.

The keratin network of intermediate filaments regulates keratinocyte rigidity sensing and nuclear mechanotransduction.

The keratin network of intermediate filaments provides keratinocytes with essential mechanical strength and resilience, but the contribution to mechanosensing remains poorly understood. Here, we investigated the role of the keratin cytoskeleton in the response to altered matrix rigidity. We found that keratinocytes adapted to increasing matrix stiffness by forming a rigid, interconnected network of keratin bundles, in conjunction with F-actin stress fiber formation and increased cell stiffness. Disruption of keratin stability by overexpression of the dominant keratin 14 mutation R416P inhibited the normal mechanical response to substrate rigidity, reducing F-actin stress fibers and cell stiffness. The R416P mutation also impaired mechanotransduction to the nuclear lamina, which mediated stiffness-dependent chromatin remodeling. By contrast, depletion of the cytolinker plectin had the opposite effect and promoted increased mechanoresponsiveness and up-regulation of lamin A/C. Together, these results demonstrate that the keratin cytoskeleton plays a key role in matrix rigidity sensing and downstream signal transduction.

Contractile myosin rings and cofilin-mediated actin disassembly orchestrate ECM nanotopography sensing.

The nanotopography and nanoscale geometry of the extra-cellular matrix (ECM) are important regulators of cell adhesion, motility and fate decision. However, unlike the sensing of matrix mechanics and ECM density, the molecular processes regulating the direct sensing of the ECM nanotopography and nanoscale geometry are not well understood. Here, we use nanotopographical patterns generated via electrospun nanofibre lithography (ENL) to investigate the mechanisms of nanotopography sensing by cells. We observe the dysregulation of actin dynamics, resulting in the surprising formation of actin foci. This alteration of actin organisation is regulated by myosin contractility but independent of adapter proteins such as vinculin. This process is highly dependent on differential integrin expression as ß3 integrin expressing cells, more sensitive to nanopattern dimensions than ß1 integrin expressing cells, also display increased perturbation of actin assembly and actin foci formation. We propose that, in ß3 integrin expressing cells, contractility results in the destabilisation of nanopatterned actin networks, collapsing into foci and sequestering regulators of actin dynamics such as cofilin that orchestrate disassembly. Therefore, in contrast to the sensing of substrate mechanics and ECM ligand density, which are directly orchestrated by focal adhesion assembly, we propose that nanotopography sensing is regulated by a long-range sensing mechanism, remote from focal adhesions and mediated by the actin architecture.

Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces.

Desmoglein 3 (Dsg3) plays a crucial role in cell-cell adhesion and tissue integrity. Increasing evidence suggests that Dsg3 acts as a regulator of cellular mechanotransduction, but little is known about its direct role in mechanical force transmission. The present study investigated the impact of cyclic strain and substrate stiffness on Dsg3 expression and its role in mechanotransduction in keratinocytes. A direct comparison was made with E-cadherin, a well-characterized mechanosensor. Exposure of oral and skin keratinocytes to equiaxial cyclic strain promoted changes in the expression and localization of junction assembly proteins. The knockdown of Dsg3 by siRNA blocked strain-induced junctional remodeling of E-cadherin and Myosin IIa. Importantly, the study demonstrated that Dsg3 regulates the expression and localization of yes-associated protein (YAP), a mechanosensory, and an effector of the Hippo pathway. Furthermore, we showed that Dsg3 formed a complex with phospho-YAP and sequestered it to the plasma membrane, while Dsg3 depletion had an impact on both YAP and phospho-YAP in their response to mechanical forces, increasing the sensitivity of keratinocytes to the strain or substrate rigidity-induced nuclear relocation of YAP and phospho-YAP. Plakophilin 1 (PKP1) seemed to be crucial in recruiting the complex containing Dsg3/phospho-YAP to the cell surface since its silencing affected Dsg3 junctional assembly with concomitant loss of phospho-YAP at the cell periphery. Finally, we demonstrated that this Dsg3/YAP pathway has an influence on the expression of YAP1 target genes and cell proliferation. Together, these findings provide evidence of a novel role for Dsg3 in keratinocyte mechanotransduction.

Minor collagens of the skin with not so minor functions.

The structure and function of the skin relies on the complex expression pattern and organisation of extracellular matrix macromolecules, of which collagens are a principal component. The fibrillar collagens, types I and III, constitute over 90% of the collagen content within the skin and are the major determinants of the strength and stiffness of the tissue. However, the minor collagens also play a crucial regulatory role in a variety of processes, including cell anchorage, matrix assembly, and growth factor signalling. In this article, we review the expression patterns, key functions and involvement in disease pathogenesis of the minor collagens found in the skin. While it is clear that the minor collagens are important mediators of normal tissue function, homeostasis and repair, further insight into the molecular level structure and activity of these proteins is required for translation into clinical therapies.

Ultrasensitive detection of lipoarabinomannan with plasmonic grating biosensors in clinical samples of HIV negative patients with tuberculosis.

BACKGROUND: Timely diagnosis of tuberculosis disease is critical for positive patient outcomes, yet potentially millions go undiagnosed or unreported each year. Sputum is widely used as the testing input, but limited by its complexity, heterogeneity, and sourcing problems. Finding methods to interrogate noninvasive, non-sputum clinical specimens is indispensable to improving access to tuberculosis diagnosis and care. In this work, economical plasmonic gratings were used to analyze tuberculosis biomarker lipoarabinomannan (LAM) from clinical urine samples by single molecule fluorescence assay (FLISA) and compared with gold standard sputum GeneXpert MTB/ RIF, culture, and reference ELISA testing results. METHODS AND FINDINGS: In this study, twenty sputum and urine sample sets were selected retrospectively from a repository of HIV-negative patient samples collected before initiation of anti-tuberculosis therapy. GeneXpert MTB/RIF and culture testing of patient sputum confirmed the presence or absence of pulmonary tuberculosis while all patient urines were reference ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA demonstrated good qualitative agreement with gold standard tests on 19 of 20 patients, including accurately predicting the gold-standard-negative patients, while one gold-standard-positive patient produced no observable LAM in urine. CONCLUSIONS: Single molecule FLISA by plasmonic grating demonstrated the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from the complex media itself. Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively.

High-Content Analysis of Cell Migration Dynamics within a Micropatterned Screening Platform.

Cell migration is a fundamental biological process that is dynamically regulated by complex interactions between the microenvironment and intrinsic gene expression programs. Here, a high-throughput cell migration assay is developed using micropatterned and dynamically adhesive polymer brush substrates, which support highly precise and consistent control over cell-matrix interactions within a 96-well cell culture plate format. This system is combined with automated imaging and quantitation of both cell motility and organization of the F-actin cytoskeleton for high-content analysis of cell migration phenotypes. Using this platform to screen a library of 147 epigenetic inhibitors identifies a set of EZH2-specific compounds that promote cytoskeletal remodeling and accelerates keratinocyte migration through derepression of an epithelial to mesenchymal transition-like gene expression program. Together, these studies establish the high-throughput, micropatterned assay as a powerful tool for discovery of novel therapeutic targets and for dissecting complex gene-environment interactions involved in wound repair.

Tissue stiffening promotes keratinocyte proliferation through activation of epidermal growth factor signaling.

Tissue biomechanics regulate a wide range of cellular functions, but the influences on epidermal homeostasis and repair remain unclear. Here, we examined the role of extracellular matrix stiffness on human keratinocyte behavior using elastomeric substrates with defined mechanical properties. Increased matrix stiffness beyond normal physiologic levels promoted keratinocyte proliferation but did not alter the ability to self-renew or terminally differentiate. Activation of epidermal growth factor (EGF) signaling mediated the proliferative response to matrix stiffness and depended on focal adhesion assembly and cytoskeletal tension. Comparison of normal skin with keloid scar tissue further revealed an upregulation of EGF signaling within the epidermis of stiffened scar tissue. We conclude that matrix stiffness regulates keratinocyte proliferation independently of changes in cell fate and is mediated by EGF signaling. These findings provide mechanistic insights into how keratinocytes sense and respond to their mechanical environment, and suggest that matrix biomechanics may play a role in the pathogenesis keloid scar formation.