A biphasic multilayer computational model of human skin

The present study investigates the layer-specific mechanical behavior of human skin. Motivated by skin’s histology, a biphasic model is proposed which differentiates between epidermis, papillary and reticular dermis, and hypodermis. Inverse analysis of ex vivo tensile and in vivo suction experiments yields mechanical parameters for each layer and predicts a stiff reticular dermis and successively softer papillary dermis, epidermis and hypodermis. Layer-specific analysis of simulations underlines the dominating role of the reticular dermis in tensile loading. Furthermore, it shows that the observed out-of-plane deflection in ex vivo tensile tests is a direct consequence of the layered structure of skin. In in vivo suction experiments, the softer upper layers strongly influence the mechanical response, whose dissipative part is determined by interstitial fluid redistribution within the tissue. Magnetic resonance imaging-based visualization of skin deformation in suction experiments confirms the deformation pattern predicted by the multilayer model, showing a consistent decrease in dermal thickness for large probe opening diameters.

     

Softenin,a Novel Protein That Softens the Connective Tissue of Sea Cucumbers through Inhibiting Interaction between Collagen Fibrils

The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between extracellular materials.     

De novo synthesis of human dermis in vitro in the absence of a three-dimensional scaffold

Neonatal human dermal fibroblasts cultured in vitro synthesize an organized and physically substantial three-dimensional extracellular matrix, without the addition of exogenous matrix components or synthetic scaffolds. De novo matrix synthesis proceeds in an orderly manner over a 21-d culture period and beyond. Analysis of the fibroblast phenotype, i.e., matrix synthesis by the fibroblasts, suggests that both serum and serum-free conditions are conducive to the production of a human tissue-engineered “dermal equivalent”. We report that given the appropriate permissive environment, the fibroblasts establish and grow a tissue in vitro, which bears striking biochemical and physical resemblance to normal human dermis.     

Fibroblast state switching orchestrates dermal maturation and wound healing

Murine dermis contains functionally and spatially distinct fibroblast lineages that cease to proliferate in early postnatal life. Here, we propose a model in which a negative feedback loop between extracellular matrix (ECM) deposition and fibroblast proliferation determines dermal architecture. Virtual‐tissue simulations of our model faithfully recapitulate dermal maturation, predicting a loss of spatial segregation of fibroblast lineages and dictating that fibroblast migration is only required for wound healing. To test this, we performed in vivo live imaging of dermal fibroblasts, which revealed that homeostatic tissue architecture is achieved without active cell migration. In contrast, both fibroblast proliferation and migration are key determinants of tissue repair following wounding. The results show that tissue‐scale coordination is driven by the interdependence of cell proliferation and ECM deposition, paving the way for identifying new therapeutic strategies to enhance skin regeneration.     

Development of the Mouse Dermal Adipose Layer Occurs Independently of Subcutaneous Adipose Tissue and Is Marked by Restricted Early Expression of FABP4

The laboratory mouse is a key animal model for studies of adipose biology, metabolism and disease, yet the developmental changes that occur in tissues and cells that become the adipose layer in mouse skin have received little attention. Moreover, the terminology around this adipose body is often confusing, as frequently no distinction is made between adipose tissue within the skin, and so called subcutaneous fat. Here adipocyte development in mouse dorsal skin was investigated from before birth to the end of the first hair follicle growth cycle. Using Oil Red O staining, immunohistochemistry, quantitative RT-PCR and TUNEL staining we confirmed previous observations of a close spatio-temporal link between hair follicle development and the process of adipogenesis. However, unlike previous studies, we observed that the skin adipose layer was created from cells within the lower dermis. By day 16 of embryonic development (e16) the lower dermis was demarcated from the upper dermal layer, and commitment to adipogenesis in the lower dermis was signalled by expression of FABP4, a marker of adipocyte differentiation. In mature mice the skin adipose layer is separated from underlying subcutaneous adipose tissue by the panniculus carnosus. We observed that the skin adipose tissue did not combine or intermix with subcutaneous adipose tissue at any developmental time point. By transplanting skin isolated from e14.5 mice (prior to the start of adipogenesis), under the kidney capsule of adult mice, we showed that skin adipose tissue develops independently and without influence from subcutaneous depots. This study has reinforced the developmental link between hair follicles and skin adipocyte biology. We argue that because skin adipocytes develop from cells within the dermis and independently from subcutaneous adipose tissue, that it is accurately termed dermal adipose tissue and that, in laboratory mice at least, it represents a separate adipose depot.     

Adiponectin and leptin up-regulate extracellular matrix production by dermal fibroblasts

Adipocytes were recently shown to secrete adipocytokines, such as adiponectin and leptin, which may have an endocrine role. Subcutaneous adipose tissue lies just beneath the dermis, and dermal condition is correlated with body mass index (BMI). However, it is not clear whether adipocytokines released by adipocytes in subcutaneous adipose tissue influence the adjacent dermis. We found that human dermal fibroblasts express genes encoding receptors for adiponectin and leptin, and that those cytokines both significantly increase production of hyaluronic acid (HA), a major extracellular matrix component (ECM) of dermis, by dermal fibroblasts. This effect is accompanied with up-regulation of HA synthase 2 gene expression. Moreover, adiponectin significantly increases production of collagen, the most abundant component of ECM in dermis, by dermal fibroblasts. These results suggest that subcutaneous adipocytes influence dermal condition by up-regulating collagen and HA production by dermal fibroblasts via secretion of adiponectin and leptin.     

Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin

Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced by transgenic epidermal activation of β-catenin (EF skin). We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of β-catenin signalling. By contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal β-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis.     

Development of tendon structure and function: regulation of collagen fibrillogenesis

In the tendon, the development of mature mechanical properties is dependent on the assembly of a tendon-specific extracellular matrix. This matrix is synthesized by the tendon fibroblasts and composed of collagen fibrils organized as fibers, as well as fibril-associated collagenous and non-collagenous proteins. All of these components are integrated, during development and growth, to form a functional tissue. During tendon development, collagen fibrillogenesis and matrix assembly progress through multiple steps where each step is regulated independently, culminating in a structurally and functionally mature tissue. Collagen fibrillogenesis occurs in a series of extracellular compartments where fibril intermediates are assembled and mature fibrils grow through a process of post-depositional fusion of the intermediates. Linear and lateral fibril growth occurs after the immature fibril intermediates are incorporated into fibers. The processes are regulated by interactions of extracellular macromolecules with the fibrils. Interactions with quantitatively minor fibrillar collagens, fibril-associated collagens and proteoglycans influence different steps in fibrillogenesis and the extracellular microdomains provide a mechanism for the tendon fibroblasts to regulate these extracellular interactions.     

Matricellular hevin regulates decorin production and collagen assembly

Matricellular proteins such as SPARC, thrombospondin 1 and 2, and tenascin C and X subserve important functions in extracellular matrix synthesis and cellular adhesion to extracellular matrix. By virtue of its reported interaction with collagen I and deadhesive activity on cells, we hypothesized that hevin, a member of the SPARC gene family, regulates dermal extracellular matrix and collagen fibril formation. We present evidence for an altered collagen matrix and levels of the proteoglycan decorin in the normal dermis and dermal wound bed of hevin-null mice. The dermal elastic modulus was also enhanced in hevin-null animals. The levels of decorin protein secreted by hevin-null dermal fibroblasts were increased by exogenous hevin in vitro, data indicating that hevin might regulate both decorin and collagen fibrillogenesis. We also report a decorin-independent function for hevin in collagen fibrillogenesis. In vitro fibrillogenesis assays indicated that hevin enhanced fibril formation kinetics. Furthermore, cell adhesion assays indicated that cells adhered differently to collagen fibrils formed in the presence of hevin. Our observations support the capacity of hevin to modulate the structure of dermal extracellular matrix, specifically by its regulation of decorin levels and collagen fibril assembly.     

Hyaluronan concentration within a 3D collagen matrix modulates matrix viscoelasticity, but not fibroblast response

The use of 3D extracellular matrix (ECM) microenvironments to deliver growth-inductive signals for tissue repair and regeneration requires an understanding of the mechanisms of cell–ECM signaling. Recently, hyaluronic acid (HA) has been incorporated in collagen matrices in an attempt to recreate tissue specific microenvironments. However, it is not clear how HA alters biophysical properties (e.g. fibril microstructure and mechanical behavior) of collagen matrices or what impact these properties have on cell behavior. The present study determined the effects of varying high molecular weight HA concentration on 1) the assembly kinetics, fibril microstructure, and viscoelastic properties of 3D type I collagen matrices and 2) the response of human dermal fibroblasts, in terms of morphology, F-actin organization, contraction, and proliferation within the matrices. Results showed increasing HA concentration up to 1 mg/ml (HA:collagen ratio of 1:2) did not significantly alter fibril microstructure, but did significantly alter viscoelastic properties, specifically decreasing shear storage modulus and increasing compressive resistance. Interestingly, varied HA concentration did not significantly affect any of the measured fibroblast behaviors. These results show that HA-induced effects on collagen matrix viscoelastic properties result primarily from modulation of the interstitial fluid with no significant change to the fibril microstructure. Furthermore, the resulting biophysical changes to the matrix are not sufficient to modulate the cell–ECM mechanical force balance or proliferation of resident fibroblasts. These results provide new insight into the mechanisms by which cells sense and respond to microenvironmental cues and the use of HA in collagen-based biomaterials for tissue engineering.     

Marine algae as attractive source to skin care

As the largest organ in the human body, the skin has multiple functions of which one of the most important is the protection against various harmful stressors. The keratinised stratified epidermis and an underlying thick layer of collagen-rich dermal connective tissues are important components of the skin. The environmental stressors such as ultraviolet radiation (UVR) and pollution increase the levels of reactive oxygen species (ROS), contributing to clinical manifestations such as wrinkle formation and skin aging. Skin aging is related to the reduction of collagen production and decrease of several enzymatic activities including matrix metalloproteinases (MMPs), which degrade collagen structure in the dermis; and tissue inhibitor of metalloproteinases (TIMPs), which inhibit the action of MMPs. In addition to alterations of DNA, signal transduction pathways, immunology, UVR, and pollution activate cell surface receptors of keratinocytes and fibroblasts in the skin. This action leads to a breakdown of collagen in the extracellular matrix and a shutdown of new collagen synthesis. Therefore, an efficient antioxidants strategy is of major importance in dermis and epidermis layers. Marine resources have been recognised for their biologically active substances. Among these, marine algae are rich-sources of metabolites, which can be used to fight against oxidative stress and hence skin aging. These metabolites include, among others, mycosporine-like amino acids (MAAs), polysaccharides, sulphated polysaccharides, glucosyl glycerols, pigments, and polyphenols. This paper reviews the role of oxidative processes in skin damage and the action of the compounds from algae on the physiological processes to maintain skin health.     

Quantitative Analysis of Amyloid-Integrated Biofilms Formed by Uropathogenic Escherichia coli at the Air-Liquid Interface

Bacterial biofilms are complex multicellular assemblies, characterized by a heterogeneous extracellular polymeric matrix, that have emerged as hallmarks of persistent infectious diseases. New approaches and quantitative data are needed to elucidate the composition and architecture of biofilms, and such data need to be correlated with mechanical and physicochemical properties that relate to function. We performed a panel of interfacial rheological measurements during biofilm formation at the air-liquid interface by the Escherichia coli strain UTI89, which is noted for its importance in studies of urinary tract infection and for its assembly of functional amyloid fibers termed curli. Brewster-angle microscopy and measurements of the surface elasticity (G_s′) and stress-strain response provided sensitive and quantitative parameters that revealed distinct stages during bacterial colonization, aggregation, and eventual formation of a pellicle at the air-liquid interface. Pellicles that formed under conditions that upregulate curli production exhibited an increase in strength and viscoelastic properties as well as a greater ability to recover from stress-strain perturbation. The results suggest that curli, as hydrophobic extracellular amyloid fibers, enhance the strength, viscoelasticity, and resistance to strain of E. coli biofilms formed at the air-liquid interface.     

Region-specific deposition of dermal proteins between dermis and epidermis during induction of chick feather and scale rudiments

To begin to study the role of particular proteins in inductive tissue interactions, we have used density labelling techniques to determine whether any dermal proteins are found between embryonic chick dermis and epidermis at a stage when the dermis plays an important inductive role in epidermal differentiation. Epidermis will form feathers or scales depending on whether it interacts with dorsal or foot dermis, respectively, and the dermis can still influence epidermal differentiation when direct cell contact between the tissues is blocked by a membrane filter during culturing (Peterson & Grainger, 1985). In transfilter experiments, we detect a subset of dermal proteins within the filter between the tissues. Several of these dermal proteins are deposited in a region-specific manner, that is, they are only found associated with filters from either dorsal or foot dermis. We have previously shown that the expression of some of these proteins is specific to particular regions of dermis and is also associated with the inductive potential of the dermis (Peterson & Grainger, 1986). We detect only 17 dermal proteins which are transferred across the filter in these cultures and found in direct association with epidermis; of these 14 are common to both dorsal and foot dermis, and 3 are deposited in a region-specific manner. Our results lead us to hypothesize a significant function for certain dermal proteins in this inductive interaction either as part of the extracellular matrix or in direct association with epidermis.     

An in vitro model for the pathological degradation of articular cartilage in osteoarthritis

The objective of this study was to develop an in vitro cartilage degradation model that emulates the damage seen in early-stage osteoarthritis. To this end, cartilage explants were collagenase-treated to induce enzymatic degradation of collagen fibers and proteoglycans at the articular surface. To assess changes in mechanical properties, intact and degraded cartilage explants were subjected to a series of confined compression creep tests. Changes in extracellular matrix structure and composition were determined using biochemical and histological approaches. Our results show that collagenase-induced degradation increased the amount of deformation experienced by the cartilage explants under compression. An increase in apparent permeability as well as a decrease in instantaneous and aggregate moduli was measured following collagenase treatment. Histological analysis of degraded explants revealed the presence of surface fibrillation, proteoglycan depletion in the superficial and intermediate zones and loss of the lamina splendens. Collagen cleavage was confirmed by the Col II–3/4C_short antibody. Degraded specimens experienced a significant decrease in proteoglycan content but maintained total collagen content. Repetitive testing of degraded samples resulted in the gradual collapse of the articular surface and the compaction of the superficial zone. Taken together, our data demonstrates that enzymatic degradation with collagenase can be used to emulate changes seen in early-stage osteoarthritis. Further, our in vitro model provides information on cartilage mechanics and insights on how matrix changes can affect cartilage's functional properties. More importantly, our model can be applied to develop and test treatment options for tissue repair.     

Hyaluronan Disrupts Cardiomyocyte Organization within 3D Fibrin-Based Hydrogels

The extracellular matrix in vivo contains variable but often large amounts of glycosaminoglycans that influence cell and tissue function. Hyaluronan (HA) is an abundant glycosaminoglycan within the extracellular matrix of the myocardium during early development and in the aftermath of a myocardial infarction. Its flexible anionic structure has a strong influence on mechanical response and interstitial fluid flow within the matrix. Additionally, HA has a direct, biochemical effect on cells through an array of cell-surface receptors, including CD44, RHAMM/CD168, and other surface-exposed structures. Recent studies have shown that HA modulates the response of cardiomyocytes and other cell types to two-dimensional substrates of varying elastic moduli. This study investigates the force response to HA of cardiomyocytes and cardiac fibroblasts within three-dimensional matrices of variable composition and mechanical properties in vitro. HA significantly decreased the force exerted by the cell-matrix constructs in a tensiometer testing platform and within microfabricated tissue gauges. However, its effect was no different from that of alginate, an anionic polysaccharide with the same charge density but no specific transmembrane receptors. Therefore, these results establish that HA exerts a generic physical-chemical effect within three-dimensional hydrogels that must be accounted for when interrogating cell-matrix interactions.     

Collagen fibril assembly and deposition in the developing dermis: segmental deposition in extracellular compartments

The hierarchy of extracytoplasmic compartmentalization and fibrillar organization as well as the assembly and deposition of collagen fibrils was characterized in the 15-day chick embryo dermis using transmission electron microscopy. At least two levels of extracellular compartmentalization are recognizable at this stage of dermal development. The first compartment consists of a series of narrow channels containing single or small groups (less than 5) of collagen fibrils. These channels course deep within the cell and are open to the extracellular space. The second extracellular compartment consists of fibrils grouped as small bundles in close association with the cell surface and is most often defined by a single fibroblast. A third level of fibril organization and compartmentalization is sometimes apparent at this stage of dermal development consisting of laterally associated bundles, more characteristic of the mature dermis. This compartment is associated with the fibroblast surface, but is less well defined than the fibril channels or bundle-forming compartments. Dermal collagen fibrils within bundles are discontinuous. Numerous fibrils ends are identified from serial sections and the ends gradually taper. These data indicate that the dermal fibroblast compartmentalizes the extracellular space and deposits collagen fibril segments during dermal morphogenesis. A model for the genesis of the extracellular compartments and their role in collagen fibrillogenesis and development of regularly arranged connective tissues, tendon, and cornea has been proposed. Dermal development conforms to this model and we suggest that extracytoplasmic compartmentalization of the steps in matrix assembly and segmental deposition of collagen fibrils are important mechanisms in the development of a wide variety of connective tissues.     

Dermal connective tissue development in mice: an essential role for tenascin-X

Deficiency of the extracellular matrix protein tenascin-X (TNX) causes a recessive form of Ehlers‐Danlos syndrome (EDS) characterized by hyperextensible skin and hypermobile joints. It is not known whether the observed alterations of dermal collagen fibrils and elastic fibers in these patients are caused by disturbed assembly and deposition or by altered stability and turnover. We used biophysical measurements and immunofluorescence to study connective tissue properties in TNX knockout and wild-type mice. We found that TNX knockout mice, even at a young age, have greatly disturbed biomechanical properties of the skin. No joint abnormalities were noted at any age. The spatio-temporal expression of TNX during normal mouse skin development, during embryonic days 13–19 (E13–E19), was distinct from tropoelastin and the dermal fibrillar collagens type I, III, and V. Our data show that TNX is not involved in the earliest phase (E10–E14) of the deposition of collagen fibrils and elastic fibers during fetal development. From E15 to E19, TNX starts partially to colocalize with the dermal collagens and elastin, and in adult mice, TNX is present in the entire dermis. In adult TNX knockout mice, we observed an apparent increase of elastin. We conclude that TNX knockout mice only partially recapitulate the phenotype of TNX-deficient EDS patients, and that TNX could potentially be involved in maturation and/or maintenance of the dermal collagen and elastin network.     

Quantitative alterations of hyaluronan and dermatan sulfate in the hairless mouse dorsal skin exposed to chronic UV irradiation.

The quantitative alterations of hyaluronan and dermatan sulfate in the upper dermis (fibrous tissue) and the lower dermis (adipose tissue) of the hairless mouse skin chronically exposed to the UV irradiation as solar-simulating irradiation (lambda(max) 352 nm, UV distribution: 300-310 nm, 0.9%; 310-320 nm, 2.0%; 320-420 nm, 97.1%) were evaluated. Hyaluronan and dermatan sulfate contents in each part of dermis were determined as follows: skin sections on a glass slide prepared by histological technique were processed into the upper dermis and the lower dermis with a small surgical knife, and treated with chondroitinase ABC and ACII in the presence of bacterial collagenase. The resulting unsaturated disaccharides were determined by HPLC method. By applying this method to the UV-irradiated hairless mouse skin, it was found that the chronic UV irradiation increased dermatan sulfate in the upper dermis, whereas an increase of hyaluronan content was not statistically significant. In the lower dermis, on the contrary, both hyaluronan and dermatan sulfate contents remarkably increased as compared with the control mice. Furthermore, the histological study showed the accumulation of the collagen fibers in the lower dermis of the UV-irradiated hairless mouse skin following the disappearance of adipocytes. These findings indicate that the increases of glycosaminoglycan contents in the UV-irradiated skin are related to the accumulation of the extracellular matrix components in the lower dermis.