Multiple roles for elastic fibers in the skin.

Dermal elastic fibers are believed to have a primary role in providing elastic stretch and recoil to the skin. Here we compare the structural arrangement of dermal elastic fibers of chick skin and different animal species. Most elastic fibers in chick skin are derived from cells that line the feather follicle and/or smooth muscle that connects the pterial and apterial muscle bundles to feather follicles. Elastic fibers in the dermis of animals with single, primary hair follicles are derived from cells lining the hair follicle or from the ends of the pili muscle, which anchors the muscle to the matrix or to the hair follicle. Each follicle is interconnected with elastic fibers. Follicles of animals with primary and secondary (wool) hair follicles are also interconnected by elastic fibers, yet only the elastic fibers derived from the primary follicle are connected to each primary follicle. Only the primary hair follicles are connected to the pili muscle. Human skin, but not the skin of other primates, is significantly different from other animals with respect to elastic fiber organization and probably cell of origin. The data suggest that the primary role for elastic fibers in animals, with the possible exception of humans, is movement and/or placement of feathers or hair.     

Quantitative Determination of Murine Dermal Elastic Fibers by Color Image Analysis: Comparison of Three Staining Methods

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Klig-man's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.     

Homotypic Versican G1 Domain Interactions Enhance Hyaluronan Incorporation into Fibrillin Microfibrils

Versican G1 domain-containing fragments (VG1Fs) have been identified in extracts from the dermis in which hyaluronan (HA)-versican-fibrillin complexes are found. However, the molecular assembly of VG1Fs in the HA-versican-microfibril macrocomplex has not yet been elucidated. Here, we clarify the role of VG1Fs in the extracellular macrocomplex, specifically in mediating the recruitment of HA to microfibrils. Sequential extraction studies suggested that the VG1Fs were not associated with dermal elements through HA binding properties alone. Overlay analyses of dermal tissue sections using the recombinant versican G1 domain, rVN, showed that rVN deposited onto the elastic fiber network. In solid-phase binding assays, rVN bound to isolated nondegraded microfibrils. rVN specifically bound to authentic versican core protein produced by dermal fibroblasts. Furthermore, rVN bound to VG1Fs extracted from the dermis and to nondenatured versican but not to fibrillin-1. Homotypic binding of rVN was also seen. Consistent with these binding properties, macroaggregates containing VG1Fs were detected in high molecular weight fractions of sieved dermal extracts and visualized by electron microscopy, which revealed localization to microfibrils at the microscopic level. Importantly, exogenous rVN enhanced HA recruitment both to isolated microfibrils and to microfibrils in tissue sections in a dose-dependent manner. From these data, we propose that cleaved VG1Fs can be recaptured by microfibrils through VG1F homotypical interactions to enhance HA recruitment to microfibrils.     

The effect of composition and microstructure on the viscoelastic properties of dermis

Dermis is a heterogeneous tissue in which extracellular matrix components change in relative amount and spatial assembly across the tissue thickness. The effect of the microstructural and compositional heterogeneities on the overall mechanical response of dermis is, however, largely ignored. In this work, we aimed at gaining a better insight into the effect of extracellular matrix microstructure and composition on the mechanical behaviour of different dermal strata by combining mechanical analysis and selective enzymatic digestion of extracellular matrix components. The dynamical–mechanical tests we performed on bovine dermal splits show that the upper dermal stratum, which is richer in papillary dermis, is characterized by higher mechanical properties than the lower one, which is almost composed of reticular dermis. Moreover, the depletion of interfibrillar proteins, proteoglycans and glycosamminoglycans decreases the dynamic moduli of dermis, especially at small frequencies. Of the two dermal layers tested, the upper dermal layer is more sensitive to the enzymatic treatment than the lower layer. Interestingly, the disruption of the elastic network greatly influenced the viscoelastic properties of upper dermis, inducing a dramatic decrease of both storage (G′) and loss (G″) moduli, suggesting that the spatial assembly of the elastin and collagen networks as well as their mutual interactions dominate the dynamical mechanical response of the tissue.     

Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro

The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.     

Comparison of long-term survival of pigmented epidermal reconstructs cultured in vitro vs. xenografted on nude mice

Epidermal reconstructs incorporating pigment cells have been used in vitro over the last decade to study the physiology of the epidermal melanin unit. However, the major limitation of this technology is the duration of the assays, which need to be completed within 2-3 weeks to obviate the problem of epidermal senescence and excessive terminal differentiation. This becomes a major problem for studying long-term biological phenomena in photoprotection and epidermal skin cancers. We report here a simplified surgical technique in immunotolerant mice allowing long-term studies. The creation of a vascularized mouse skin flap is the key point of the surgical procedure. Long-term pigmentation of the xenografts seemed macroscopically successful, but surprisingly microscopy at 11 and 16 weeks postgrafting showed mostly dermal pigment aggregates and rare Melan-A positive dermal and epidermal pigment cells. In the same reconstructs maintained in vitro, dermal pigment and dermal pigment cells were never noted. It could be speculated that in our model, the colonization of the xenografted dead human dermis by murine cells influences melanocyte survival.     

Versican is expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis

The expression of the large chondroitin sulfate proteoglycan versican was studied in human adult skin. For this purpose, bacterial fusion proteins containing unique portions of the versican core protein were prepared. Polyclonal antibodies against the fusion proteins specifically reacted with versican from a proteoglycan fraction of MG63 osteosarcoma cells. In immunohistochemical experiments, the affinity- purified antibodies localized versican in the stratum basale of the epidermis, as well as in the papillary and reticular layers of the dermis. An apparent codistribution of versican with the various fiber forms of the elastic network of the dermis suggested an association of versican with microfibrils. Both dermal fibroblasts and keratinocytes expressed versican in culture during active cell proliferation. In line with the observation that versican is absent in the suprabasal layers of the epidermis where keratinocytes terminally differentiate, culture conditions promoting keratinocyte differentiation induced a down- regulation of versican synthesis. In Northern blots versican mRNA could be detected in extracts from proliferating keratinocytes and dermal fibroblasts. Comparison of RNA preparations from semi-confluent and confluent fibroblast cultures demonstrated decreasing amounts of versican mRNA at higher cell densities. This inverse correlation of versican expression and cell density was confirmed by indirect immunofluorescence staining of cultured fibroblasts and keratinocytes. The localization of versican in the basal zone of the epidermis as well as the density dependence of versican in cell cultures suggest a general function of versican in cell proliferation processes that may not solely be confined to the skin.     

Dermal melanocytosis in Japanese monkeys (Macaca fuscata)

The skin of Japanese monkeys (Macaca fuscata) shows diffuse discolorations resembling human dermal melanocytosis. Very few laboratory animals have melanocytes in the dermis. The purpose of this study was to clarify the dermatologic characteristics of Japanese monkeys in terms of gross appearance, skin color, and histopathologic findings. A colorimeter was used to record the skin colors of pigmented and nonpigmented sites. Tissue specimens obtained from both types of sites were examined histopathologically. All animals examined had pigmented sites on their bodies, and the discolorations extended over 25% to 33% of the body surface. The colorimeter could detect differences in skin color due to dermal melanocytosis. All parameters of the colorimetric systems used (Yxy, L*a*b*, and L*C*h* systems) demonstrated significant differences between pigmented and nonpigmented sites. In pigmented sites, the epidermis lacked melanocytes, but the dermis had numerous melanocytes with abundant melanin. Activated melanocytes with well-developed dendrites were distributed throughout the upper part of the dermal layer. Melanocytes were not arranged in clusters, and elastic and collagen fibers in the dermis showed no histological abnormalities. Nonpigmented sites lacked melanin granules in both the epidermis and dermis. This study revealed that gross dermal melanocytosis correlated well with colorimetric results and histopathologic findings. These findings suggest that the pigmentation of Japanese monkeys is equivalent to dermal melanocytosis in humans, to the end that Japanese monkeys may be a useful animal model for investigating dermal melanogenesis.     

"De-epithelization" and dermal pedicles.

Biopsy specimens from 38 "de-epithelized" dermal pedicles were examined microscopically. There was considerable variation in the depth of the plane among surgeons, procedures, and even between specimens from two sides of a bilateral procedure done by the same surgeon. Usually, the "de-epithelization" removed all the epidermis plus the upper layer of dermis containing the pilosebaceous apparatus. The significance of this finding as related to the future development of epidermal inclusion cysts is uncertain. Also, it brings into question the importance of "the dermal plexus circulation," which many have thought to be critical for viability of the nipple.     

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.     

Roles for PDGF-A and sonic hedgehog in development of mesenchymal components of the hair follicle.

Skin appendages, such as hair, develop as a result of complex reciprocal signaling between epithelial and mesenchymal cells. These interactions are not well understood at the molecular level. Platelet-derived growth factor-A (PDGF-A) is expressed in the developing epidermis and hair follicle epithelium, and its receptor PDGF-Ralpha is expressed in associated mesenchymal structures. Here we have characterized the skin and hair phenotypes of mice carrying a null mutation in the PDGF-A gene. Postnatal PDGF-A-/- mice developed thinner dermis, misshapen hair follicles, smaller dermal papillae, abnormal dermal sheaths and thinner hair, compared with wild-type siblings. BrdU labeling showed reduced cell proliferation in the dermis and in the dermal sheaths of PDGF-A-/- skin. PDGF-A-/- skin transplantation to nude mice led to abnormal hair formation, reproducing some of the features of the skin phenotype of PDGF-A-/- mice. Taken together, expression patterns and mutant phenotypes suggest that epidermal PDGF-A has a role in stimulating the proliferation of dermal mesenchymal cells that may contribute to the formation of dermal papillae, mesenchymal sheaths and dermal fibroblasts. Finally, we show that sonic hedgehog (shh)-/- mouse embryos have disrupted formation of dermal papillae. Such embryos fail to form pre-papilla aggregates of postmitotic PDGF-Ralpha-positive cells, suggesting that shh has a critical role in the assembly of the dermal papilla.     

Quantitative and qualitative effects of chemical peeling on photo-aged skin: an experimental study

Chemical peel reverses the visible stigmata of photo aging in human skin. The qualitative and, in particular, the quantitative changes in the dermis that effect this transformation are unclear. This study used a recognized photo-aged animal model, the Skh:HR-1 hairless mouse, to quantify and qualify the changes that occurred in collagen and glycosaminoglycan content after chemical peel. One hundred Skh:HR-1 hairless mice were photo-aged by use of chronic ultraviolet B irradiation for 14 weeks. After irradiation the animals were randomly distributed into five groups of 20 mice each: group 1, control; group 2, 50% glycolic acid peel; group 3, 30% trichloroacetic acid peel; group 4, 50% trichloroacetic acid peel; group 5, phenol peel (Baker-Gordon formula). The respective peeling agent was applied to the dorsal skin of each animal while it was fully anesthetized. Punch biopsies were taken at several times after peel for histological and biochemical analysis. Glycosaminoglycan content was assessed at 14, 28, and 60 days using a colorimetric assay. Collagen content per unit volume increased initially 3 days after the procedure in all chemical peel groups, declining on day 7, and peaking again on day 28. Significant elevations (p < 0.04) were seen in the 30% trichloroacetic acid, 50% trichloroacetic acid, and phenol peels on days 3 and 28 in comparison with controls. This increase in collagen content was not maintained and returned to control values by 60 days. Glycosaminoglycan content per unit volume was elevated initially after peel with significant elevation (p < 0.02) in the 50% trichloroacetic acid and phenol groups on days 14 and 28. This increase in glycosaminoglycan content was not maintained beyond 28 days and declined to control values by day 60 in all groups. Histological examination demonstrated an increase in dermal thickness in the 50% trichloroacetic acid and phenol groups in comparison with controls by day 60. Under polarized light all chemical peel groups at day 60 demonstrated a reorganization of collagen in the reticular and papillary dermis. The elastotic masses that are pathognomonic of photo aging were present in the control group but were absent in the peel groups and demonstrated a reorganization of the elastic fibers in the dermis. This effect was deeper in the dermis in the deeper peel groups (50% trichloroacetic acid and phenol peel). The beneficial effects of chemical peel were due to a combination of two findings; a reorganization in dermal structural elements and an increase in dermal volume. These effects were more pronounced in the deeper peel groups.     

A selective histochemical method for the quantitative estimation of elastic fibers by computerized morphometric analysis. Effect of colchicin treatment

A morphometric technique is reported that uses a new selective staining of the elastic system fibers in skin biopsy specimens to facilitate the quantitative evaluation of the volume fraction occupied by these elastic fibers in the tissue. The study of elastic fibers in the dermis of 30 patients, before and after six months of treatment with Colchicin, was carried out with a Quantimet 720 system. Preelastic (oxytalan and elaunin) fibers and mature elastic fibers were quantitated separately. Compared to the average volume fraction (surface occupied by the elastic fibers) before treatment with Colchicin (1.449 +/- 0.64%), the mean values after treatment were significantly increased (2.076 +/- 0.61%). The same results were found for the preelastic fibers: 0.807 +/- 0.51% before treatment and 1.025 +/- 0.54% after treatment. These results demonstrate the advantages of our monochromatic staining method for automatic quantitation of elastic fibers as well as the possibilities of the quantitative study of the elastic fibers in human dermis. This methodology should be applicable to other inherited or acquired diseases affecting skin elastic fibers as well as to other tissues containing elastic fibers.     

Comparison of Long‐Term Survival of Pigmented Epidermal Reconstructs Cultured In Vitro vs. Xenografted on Nude Mice

Epidermal reconstructs incorporating pigment cells have been used in vitro over the last decade to study the physiology of the epidermal melanin unit. However, the major limitation of this technology is the duration of the assays, which need to be completed within 2–3 weeks to obviate the problem of epidermal senescence and excessive terminal differentiation. This becomes a major problem for studying long‐term biological phenomena in photoprotection and epidermal skin cancers. We report here a simplified surgical technique in immunotolerant mice allowing long‐term studies. The creation of a vascularized mouse skin flap is the key point of the surgical procedure. Long‐term pigmentation of the xenografts seemed macroscopically successful, but surprisingly microscopy at 11 and 16 weeks postgrafting showed mostly dermal pigment aggregates and rare Melan‐A positive dermal and epidermal pigment cells. In the same reconstructs maintained in vitro, dermal pigment and dermal pigment cells were never noted. It could be speculated that in our model, the colonization of the xenografted dead human dermis by murine cells influences melanocyte survival.     

The exclusion of human serum albumin by human dermal collagenous fibres and within human dermis.

Preparations of dermal collagenous fibres and slices of human dermis have been equilibrated with 125I-labelled monomeric human serum albumin. The space inaccessible to the albumin in the fibres and in the dermis was determined by subtraction of the accessible space, calculated from the radioactivity of the specimen, from its total fluid. For a fibre preparation examined in detail, the fluid exclusion was independent of the concentration of either albumin or collagen. Binding of albumin to the fibres was not demonstrable. Three fibre preparations excluded albumin from 3.75 +/- 0.96, 3.55 +/- 0.67, and 2.05 +/- 0.39 g of fluid/g of collagen (+/-S.D.). Slices from three specimens of dermis excluded albumin from 1.45 +/- 0.08 g of fluid/g of insoluble solids or 1.57 +/- 0.11 g of fluid/g of collagen (+/-S.D.). Thus the exclusion of albumin by dermis was much less than expected from its content of collagenous fibres. On the basis of these data and the published composition of dermis, the concentration of albumin in the accessible interstitial space was estimated to be close to that in the plasma.     

Pharmacotoxicological applications of an equivalent dermis: three measurements of cytotoxicity

The legal procedure for evaluating the toxicity of cosmetic, household, chemical and pharmaceutical products is still the irritancy Draize test on rabbits. Various irritation tests are currently being developed as alternatives toin vivo animal testing. Ourin vitro model system is composed of 24 equivalent dermis (ED) comprising a chitosan-cross-linked collagen-glycosaminoglycan matrix populated by foreskin fibroblasts. In evaluating this system for irritancy testing, three different measures of toxicity were used: MTT (dimethylthiazol diphenyltetrazolium bromide) reduction, and lactate dehydrogenase and interleukin-6 release. The experiments described herein represent a preliminary evaluation to determine the usefulness and predictive value of our 24 ED kit as an alternative method for the prediction of human dermal reaction, versus three chemical products: cadmium chloride, lauryl sulfate, and benzalkonium chloride. Preliminary results suggest that the ED may be a usefulin vitro model for the prediction of cutaneous and ocular toxicity and allow the development of a 24-skin-equivalent kit realized by seeding human normal keratinocytes onto the equivalent dermis.Abbreviations ED equivalent dermis - ECM extracellular matrix - FCM fibroblast culture medium - LDH lactate dehydrogenase - IL-6 interleukin-6 - MTT dimethylthiazol diphenyltetrazolium bromide     

Hair follicle destruction and regeneration in guinea pig skin after cutaneous freeze injury

We have investigated the histological changes in hair follicles in guinea pig skin after standardized moderate and severe cryosurgery injuries. Hair follicles were permanently destroyed by cryosurgery, but more than one mechanism may be operative during follicle destruction and shedding. The mechanism depends upon the severity of the freeze. After a light freeze injury, the changes are predominantly within the hair follicle. The hair is shed at the surface and there is selective autolysis of follicular cells, but dermal connective tissue is preserved and there is little surrounding damage. However, after a severe cryoinjury as used in "tumor doses," there is destruction of dermal connective tissue and dermal scarring. The necrotic dermis is shed, taking with it the dead follicles and morphologically normal elastic tissue.     

A tissue-engineered human dermal construct utilizing fibroblasts and transforming growth factor β1 to promote elastogenesis

Numerous studies have shown that extracellular matrix (ECM)-based scaffolds are suitable for dermal constructs for the differentiation of various cell types in vitro and for constructive tissue remodeling after implantation in vivo. However, a shortcoming of these ECM materials is its limited elastogenesis. Elastic fibers constitute an essential component of mammalian connective tissue and the presence of elastic fibers is crucial for the proper function of the cardiovascular, pulmonary, and intestinal systems. Since it is still largely unknown how cells coordinate the molecular events of elastic-fiber assembly, understanding the ability to regenerate elastic fibers in tissues remains a significant challenge. For this reason, human neonatal dermal fibroblasts (HDFneo) were analyzed for their potential to serve as a cell culture model for elastic fiber assembly. Using optical technologies such as multiphoton laser-scanning microscopy (MPSLM) we demonstrate that HDFneo stimulated with transforming growth factor β1 (TGF-β1) are able to produce a distinct and complex elastic fiber system in vitro. As shown by the desmosine and isodesmosine content, crosslinked elastic fibers were formed within the 3D ECM-based scaffold. This tissue-engineered dermal construct may prove to be an effective template for the development of medicinal approaches in regenerative soft skin tissue reconstruction through TGF-β1 induction.