Morphological pattern of the dermal denticles of the Southern sawtail catshark Galeus mincaronei Soto, 2001.

Dermal denticles cover the skin of sharks for protection and may have additional functions, depending on the environment. The aim of the present study was to analyze the morphological pattern of dermal denticles across the body of the poorly known deep-water catshark Galeus mincaronei, which is endemic to Southern Brazil. Additionally, we aimed to identify differences between sexes and stages of sexual maturity. For this purpose, we analyzed patches of eight skin samples from body regions, that is, the head, body, and tail. Dermal denticles were imaged using scanning electron microscopy. Measurements of crown morphological variations (length and width) and density of dermal denticles were performed. Nine characters of dermal denticle morphology were selected to describe a morphological pattern using principal component analysis (PCA). The first PCA axis explained 45.2% of the variation, the second explained 33%, and the third explained 14.3%, together explaining 92.5% of the total variation in denticle morphology. We identified three groups of dermal denticle crown morphologies and their possible functions. The first was composed of nostril and snout denticles, which might suggest abrasion strength functions; the second was composed of most body denticles, such as those in the cranium, on the left side of the body, and on the pectoral, dorsal, and caudal fins, which might be related to generalized functions; and the third was composed of only caudal crest denticles, which might indicate defense functions with hydrodynamic properties in G. mincaronei. Likewise, our results reveal variations in dermal denticle morphology and arrangement along the three maturity stages considered in both sexes. Similarly, sexual dimorphism in the crown width and morphology of dermal denticles was observed in developing and mature individuals. Our results are the first to reveal the morphological variation of dermal denticles in relation to function, sex, and ontogeny of individual G. mincaronei.

A mechanistic view on the aging human skin through ex vivo layer-by-layer analysis of mechanics and microstructure of facial and mammary dermis.

Age-related changes in skin mechanics have a major impact on the aesthetic perception of skin. The link between skin microstructure and mechanics is crucial for therapeutic and cosmetic applications as it bridges the micro- and the macro-scale. While our perception is governed by visual and tactile changes at the macroscopic scale, it is the microscopic scale (molecular assemblies, cells) that is targeted by topical treatments including active compounds and energies. We report here a large dataset on freshly excised human skin, and in particular facial skin highly relevant for cosmetics and aesthetic procedures. Detailed layer-by-layer mechanical analysis revealed significant age-dependent decrease in stiffness and elastic recoil of full-thickness skin from two different anatomical areas. In mammary skin, we found that the onset of mechanical degradation was earlier in the superficial papillary layer than in the deeper, reticular dermis. These mechanical data are linked with microstructural alterations observed in the collagen and elastic networks using staining and advanced imaging approaches. Our data suggest that with ageing, the earliest microstructural and mechanical changes occur in the top-most layers of dermis/skin and then propagate deeper, providing an opportunity for preventive topical treatments acting at the level of papillary dermis.

Genetic and correlative light and electron microscopy evidence for the unique differentiation pathway of erythrophores in brown trout skin.

Based on their cell ultrastructure, two types of erythrophores in the spotted skin regions of brown trout (Salmo trutta) were previously described. To test the hypothesis regarding the origin of a new cell type following genome duplication, we analysed the gene and paralogue gene expression patterns of erythrophores in brown trout skin. In addition, the ultrastructure of both erythrophore types was precisely examined using transmission electron microscopy (TEM) and correlative light microscopy and electron microscopy (CLEM). Ultrastructural differences between the sizes of erythrophore inclusions were confirmed; however, the overlapping inclusion sizes blur the distinction between erythrophore types, which we have instead defined as cell subtypes. Nevertheless, the red spots of brown trout skin with subtype 2 erythrophores, exhibited unique gene expression patterns. Many of the upregulated genes are involved in melanogenesis or xanthophore differentiation. In addition, sox10, related to progenitor cells, was also upregulated in the red spots. The expressions of paralogues derived from two genome duplication events were also analysed. Multiple paralogues were overexpressed in the red spots compared with other skin regions, suggesting that the duplicated gene copies adopted new functions and contributed to the origin of a new cell subtype that is characteristic for red spot. Possible mechanisms regarding erythrophore origin are proposed and discussed. To the best of our knowledge, this is the first study to evaluate pigment cell types in the black and red spots of brown trout skin using the advanced CLEM approach together with gene expression profiling.

Evaluation of Device-Based Cutaneous Channels Using Optical Coherence Tomography: Impact for Topical Drug Delivery.

BACKGROUND: Topical medications play a large role in the management of cutaneous diseases, but penetration is limited. Device-assisted drug delivery using mechanical destruction, lasers, and other energy-based modalities can increase penetration and absorption through creation of transcutaneous channels. OBJECTIVE: To examine real-time, in vivo cutaneous changes in response to various devices used to improve topical drug delivery through optical coherence tomography (OCT) imaging. METHODS AND MATERIALS: Treatment was performed with 8 medical devices, including mechanical destruction, lasers, and other energy-based modalities. Optical coherence tomography was used for real-time, noninvasive, in vivo imaging. RESULTS: Using OCT, microneedling and radiofrequency microneedling demonstrated no cutaneous channels. Both low-energy, low-density, fractional nonablative lasers produced transient channels, which closed within hours. The fractional nonablative 1,927-nm thulium fiber and 1,550-nm erbium fiber lasers created channels with epidermal debris within, which were still closing at 24 hours. The fractional thermomechanical ablative device and the fractional ablative CO2 laser produced channels that were still open at 24 hours. CO2 laser channels had thick rims of coagulated tissue and remained open for longer. CONCLUSION: Demonstrable differences among the devices were seen, and only some can produce observable channels, the characteristics of which vary with each technology.

Long-Term Evaluation of Poly(lactic acid) (PLA) Implants in a Horse: An Experimental Pilot Study.

In horses, there is an increasing interest in developing long-lasting drug formulations, with biopolymers as viable carrier alternatives in addition to their use as scaffolds, suture threads, screws, pins, and plates for orthopedic surgeries. This communication focuses on the prolonged biocompatibility and biodegradation of PLA, prepared by hot pressing at 180 °C. Six samples were implanted subcutaneously on the lateral surface of the neck of one horse. The polymers remained implanted for 24 to 57 weeks. Physical examination, plasma fibrinogen, and the mechanical nociceptive threshold (MNT) were performed. After 24, 28, 34, 38, and 57 weeks, the materials were removed for histochemical analysis using hematoxylin-eosin and scanning electron microscopy (SEM). There were no essential clinical changes. MNT decreased after the implantation procedure, returning to normal after 48 h. A foreign body response was observed by histopathologic evaluation up to 38 weeks. At 57 weeks, no polymer or fibrotic capsules were identified. SEM showed surface roughness suggesting a biodegradation process, with an increase in the median pore diameter. As in the histopathological evaluation, it was not possible to detect the polymer 57 weeks after implantation. PLA showed biocompatible degradation and these findings may contribute to future research in the biomedical area.

Scleroderma-like Impairment in the Network of Telocytes/CD34+ Stromal Cells in the Experimental Mouse Model of Bleomycin-Induced Dermal Fibrosis.

Considerable evidence accumulated over the past decade supports that telocytes (TCs)/CD34+ stromal cells represent an exclusive type of interstitial cells identifiable by transmission electron microscopy (TEM) or immunohistochemistry in various organs of the human body, including the skin. By means of their characteristic cellular extensions (telopodes), dermal TCs are arranged in networks intermingled with a multitude of neighboring cells and, hence, they are thought to contribute to skin homeostasis through both intercellular contacts and releasing extracellular vesicles. In this context, fibrotic skin lesions from patients with systemic sclerosis (SSc, scleroderma) appear to be characterized by a disruption of the dermal network of TCs, which has been ascribed to either cell degenerative processes or possible transformation into profibrotic myofibroblasts. In the present study, we utilized the well-established mouse model of bleomycin-induced scleroderma to gain further insights into the TC alterations found in cutaneous fibrosis. CD34 immunofluorescence revealed a severe impairment in the dermal network of TCs/CD34+ stromal cells in bleomycin-treated mice. CD31/CD34 double immunofluorescence confirmed that CD31-/CD34+ TC counts were greatly reduced in the skin of bleomycin-treated mice compared with control mice. Ultrastructural signs of TC injury were detected in the skin of bleomycin-treated mice by TEM. The analyses of skin samples from mice treated with bleomycin for different times by either TEM or double immunostaining and immunoblotting for the CD34/α-SMA antigens collectively suggested that, although a few TCs may transition to α-SMA+ myofibroblasts in the early disease stage, most of these cells rather undergo degeneration, and then are lost. Taken together, our data demonstrate that TC changes in the skin of bleomycin-treated mice mimic very closely those observed in human SSc skin, which makes this experimental model a suitable tool to (i) unravel the pathological mechanisms underlying TC damage and (ii) clarify the possible contribution of the TC loss to the development/progression of dermal fibrosis. In perspective, these findings may have important implications in the field of skin regenerative medicine.

Controlled Transdermal Iontophoresis of Insulin from Water-Soluble Polypyrrole Nanoparticles: An In Vitro Study.

The iontophoresis delivery of insulin (INS) remains a serious challenge due to the low permeability of the drug through the skin. This work aims to investigate the potential of water-soluble polypyrrole nanoparticles (WS-PPyNPs) as a drug donor matrix for controlled transdermal iontophoresis of INS. WS-PPyNPs have been prepared via a simple chemical polymerization in the presence of sodium dodecyl sulfate (SDS) as both dopant and the stabilizing agent. The synthesis of the soluble polymer was characterized using field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS), fluorescence spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. The loading mechanism of INS onto the WS-PPyNPs is based on the fact that the drug molecules can be replaced with doped dodecyl sulfate. A two-compartment Franz-type diffusion cell was employed to study the effect of current density, formulation pH, INS concentration, and sodium chloride concentration on anodal iontophoresis (AIP) and cathodal iontophoresis (CIP) of INS across the rat skin. Both AIP and CIP delivery of INS using WS-PPyNPs were significantly increased compared to passive delivery. Furthermore, while the AIP experiment (60 min at 0.13 mA cm-2) show low cumulative drug permeation for INS (about 20.48 µg cm-2); the CIP stimulation exhibited a cumulative drug permeation of 68.29 µg cm-2. This improvement is due to the separation of positively charged WS-PPyNPs and negatively charged INS that has occurred in the presence of cathodal stimulation. The obtained results confirm the potential applicability of WS-PPyNPs as an effective approach in the development of controlled transdermal iontophoresis of INS.

Induction of Apoptotic Temperature in Photothermal Therapy under Various Heating Conditions in Multi-Layered Skin Structure.

Recently, photothermal therapy has attracted attention as an alternative treatment to conventional surgical techniques because it does not lead to bleeding and patients quickly recover after treatment compared to incisional surgery. Photothermal therapy induces tumor cell death through an increase in the temperature using the photothermal effect, which converts light energy into thermal energy. This study was conducted to perform numerical analysis based on heat transfer to induce apoptosis of tumor tissue under various heating conditions in photothermal therapy. The Monte Carlo method was applied to evaluate a multi-layered skin structure containing squamous cell carcinoma. Tissue-equivalent phantom experiments verified the numerical model. Based on the effective apoptosis retention ratio, the numerical analysis results showed the quantitative correlation for the laser intensity, volume fraction of gold nanorods injected into the tumor, and cooling time. This study reveals optimal conditions for maximizing apoptosis within tumor tissue while minimizing thermal damage to surrounding tissues under various heating conditions. This approach may be useful as a standard treatment when performing photothermal therapy.

Spectroscopic and microscopic comparisons of cell topology and chemistry analysis of mouse embryonic stem cell, somatic cell and cancer cell.

While embryonic stem cells and cancer cells are known to have many similarities in signalling pathways, healthy somatic cells are known to be different in many ways. Characterization of embryonic stem cell is crucial for cancer development and cancer recurrence due to the shared signalling pathways and life course with cancer initiator and cancer stem cells. Since embryonic stem cells are the sources of the somatic and cancer cells, it is necessary to reveal the relevance between them. The past decade has seen the importance of interdisciplinary studies and it is obvious that the reflection of the physical/chemical phenomena occurring on the cell biology has attracted much more attention. For this reason, the aim of this study is to elementally and topologically characterize the mouse embryonic stem cells, mouse lung squamous cancer cells, and mouse skin fibroblast cells by using Atomic Force Microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM) supported with Electron Dispersive Spectroscopy (EDS) techniques in a complementary way. Our AFM findings revealed that roughness data of the mouse embryonic stem cells and cancer cells were similar and somatic cells were found to be statistically different from these two cell types. However, based on both XPS and SEM-EDS results, surface elemental ratios vary in mouse embryonic stem cells, cancer cells and somatic cells. Our results showed that these complementary spectroscopic and microscopic techniques used in this work are very effective in cancer and stem cell characterization and have the potential to gather more detailed information on relevant biological samples.

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.

Prediction of skin disease using a new cytological taxonomy based on cytology and pathology with deep residual learning method.

With the development of artificial intelligence, technique improvement of the classification of skin disease is addressed. However, few study concerned on the current classification system of International Classification of Diseases, Tenth Revision (ICD)-10 on Diseases of the skin and subcutaneous tissue, which is now globally used for classification of skin disease. This study was aimed to develop a new taxonomy of skin disease based on cytology and pathology, and test its predictive effect on skin disease compared to ICD-10. A new taxonomy (Taxonomy 2) containing 6 levels (Project 2-4) was developed based on skin cytology and pathology, and represents individual diseases arranged in a tree structure with three root nodes representing: (1) Keratinogenic diseases, (2) Melanogenic diseases, and (3) Diseases related to non-keratinocytes and non-melanocytes. The predictive effects of the new taxonomy including accuracy, precision, recall, F1, and Kappa were compared with those of ICD-10 on Diseases of the skin and subcutaneous tissue (Taxonomy 1, Project 1) by Deep Residual Learning method. For each project, 2/3 of the images were included as training group, and the rest 1/3 of the images acted as test group according to the category (class) as the stratification variable. Both train and test groups in the Projects (2 and 3) from Taxonomy 2 had higher F1 and Kappa scores without statistical significance on the prediction of skin disease than the corresponding groups in the Project 1 from Taxonomy 1, however both train and test groups in Project 4 had a statistically significantly higher F1-score than the corresponding groups in Project 1 (P = 0.025 and 0.005, respectively). The results showed that the new taxonomy developed based on cytology and pathology has an overall better performance on predictive effect of skin disease than the ICD-10 on Diseases of the skin and subcutaneous tissue. The level 5 (Project 4) of Taxonomy 2 is better on extension to unknown data of diagnosis system assisted by AI compared to current used classification system from ICD-10, and may have the potential application value in clinic of dermatology.

Nile tilapia skin (Oreochromis niloticus) for burn treatment: ultrastructural analysis and quantitative assessment of collagen.

Nile tilapia (Oreochromis niloticus) skin is a well-known biomaterial used as an occlusive dressing for burn treatment. It is also an inexpensive and important source of collagen. This study aims to describe the ultrastructural aspects of Nile tilapia skin, assess its collagen amount and organization, and compare quantitative methods of histochemical and immunohistochemical analysis (in all sterilization steps for use in burn dressings). One sample (0.5 × 0.5 cm) of ten different fish skins was divided in four groups: in natura skin (IN), chemical sterilization (CH), additional irradiation (30 kGy) (IR), and skins used in burn treatment (BT) to compare histochemical and immunohistochemical findings of collagen amount and describe ultrastructural aspects through scanning electron microscopy. The amount of type I collagen decreased during sterilization and clinical use owing to gradual reduction of immunostaining (anti-collagen-I) and decreasing fiber thickness of the collagen, when compared to type III (Picrosirius-red-polarized light). The collagen fibers were rearranged at each sterilization step, with a low collagen percentage and large structural disorganization in BT. The amount of type-I collagen was further reduced after BT (p < 0.05). Both the methods did not exhibit a quantified value difference (p = 0.247), and a positive correlation (r = 0.927; 95 % CI = 0.720-0.983) was observed between them, with concordance for collagen quantification in similar samples, presenting a low systematic error rate (Dalberg coefficient: 6.70). A significant amount of type-I collagen is still observed despite sterilization, although clinical application further reduces type I collagen. Its quantification can be performed both by immunohistochemistry and/or Picrosirius Red reliably.

Skin Ultrastructure and the Changes of HIF-2α, H-FABP Expression in the Myocardium of Electric Shock Death Rats.

ABSTRACT: Objective To observe the skin ultrastructure change of electric shock death rats and to test the expression changes of hypoxia-inducible factor-2α （HIF-2α） and heart type-fatty acid-binding protein （H-FABP） of myocardial cells, in order to provide basis for forensic identification of electric shock death. Methods The electric shock model of rats was established. The 72 rats were randomly divided into control group, electric shock death group and postmortem electric shock group. Each group was divided into three subgroups, immediate （0 min）, 30 min and 60 min after death. The skin changes of rats were observed by HE staining, the changes of skin ultrastructure were observed by scanning electron microscopy, and the expression of HIF-2α and H-FABP in rats myocardium was tested by immunohistochemical staining. Results The skin in the electric shock death group and postmortem electric shock group had no significant difference through the naked eye or by HE staining. Under the scanning electron microscope, a large number of cellular debris, cells with unclear boundaries, withered cracks, circular or elliptical holes scattered on the cell surface and irregular edges were observed. A large number of spherical foreign body particles were observed. Compared with the control group, the expression of HIF-2α in all electric shock death subgroups increased, reaching the peak immediately after death. In the postmortem electric shock group, HIF-2α expression only increased immediately after death, but was lower than that of electric shock death group （P<0.05）. Compared with the control group, the expression of H-FABP in all subgroups of electric shock death group and postmortem electric shock group significantly decreased. The expression of H-FABP in all subgroups of electric shock death group was lower than that of the postmortem electric shock group （P<0.05）. Conclusion Electric shock can increase HIF-2α expression and decrease H-FABP expression in the myocardium, which may be of forensic significance for the determination of electric shock death and identification of antemortem and postmortem electric shock.

Skin biological responses to urban pollution in an ex vivo model.

The skin epidermis is continuously exposed to external aggressions, including environmental pollution. The cosmetic industry must be able to offer dedicated products to fight the effects of pollutants on the skin. We set up an experimental model that exposed skin explants maintained in culture to a pollutant mixture. This mixture P representing urban pollution was designed on the basis of the French organization 'Air Parif' database. A chamber, called Pollubox®, was built to allow a controlled nebulization of P on the cultured human skin explants. We investigated ultrastructural morphology by transmission electron microscopy of high pressure frozen skin explants. A global transcriptomic analysis indicated that the pollutant mixture was able to induce relevant xenobiotic and antioxidant responses. Modulated detoxifying genes were further investigated by laser micro-dissection coupled to qPCR, and immunochemistry. Both approaches showed that P exposure correlated with overexpression of detoxifying genes and provoked skin physiological alterations down to the stratum basale. The model developed herein might be an efficient tool to study the effects of pollutants on skin as well as a powerful testing method to evaluate the efficacy of cosmetic products against pollution.

Line-field confocal optical coherence tomography of xanthogranuloma: Correlation with vertical and horizontal histopathology.

Line-field confocal optical coherence tomography (LC-OCT) is a new noninvasive technique for a real-time, vertical, and horizontal imaging of the skin at cellular resolution. A 47-year-old female presented with a 6-month history of an asymptomatic yellowish papule. LC-OCT evaluation was able to show the diagnostic microscopic features of xanthogranuloma and showed an excellent correlation with vertical and horizontal histopathological sections by revealing enlarged dermal papillae containing multiple, bright roundish giant cells, corresponding to foamy histiocytes, and giant cells characterized by a dark center surrounded by a highly hyper-refractile peripheral ring, corresponding to Touton cells. LC-OCT may represent a valid, noninvasive alternative to histopathological examination in clinically atypical cases of xanthogranuloma.

Observation of a tight junction structure generated in LbL-3D skin reconstructed by layer-by-layer cell coating technique.

Tissue-engineered skin equivalents are reconstructed the functions of human skin and can be used as an alternative to animal experiments in basic study or as cultured skin for regenerative medicine. Recent studies confirmed that epidermal tight junctions (TJs), which are complex intercellular junctions formed in the stratum granulosum of human skin, play an important part in the formation of the skin barrier function. In well-formed reconstructed human skin models, there are several reports on the expression of TJ proteins and their localization in epidermal layer, however, the morphological features of TJ, showing tight junctional contacts and the process of TJ formation have yet to be investigated. In this study, we systematically examined and identified TJ-related proteins and TJ structure in three-dimensional (3D) human skin equivalents reconstructed by layer-by-layer (LbL) cell coating technique (LbL-3D Skin). We demonstrate localization of TJ-related proteins and time course of formation of TJ structure with typical junctional morphology in LbL-3D Skin. These data provide evidence that the LbL-3D Skin is an in vitro model with structure and function extremely similar to living skin.

A method to analyze the influence of mechanical strain on dermal collagen morphologies.

Collagen fibers and their orientation play a major role in the mechanical behavior of soft biological tissue such as skin. Here, we present a proof-of-principle study correlating mechanical properties with collagen fiber network morphologies. A dedicated multiphoton stretching device allows for mechanical deformations in combination with a simultaneous analysis of its collagen fiber network by second harmonic generation imaging (SHG). The recently introduced Fiber Image Network Evaluation (FINE) algorithm is used to obtain detailed information about the morphology with regard to fiber families in collagen network images. To demonstrate the potential of our method, we investigate an isotropic and an anisotropic ex-vivo dorsal pig skin sample under quasi-static cyclic stretching and relaxation sequences. Families of collagen fibers are found to form a partially aligned collagen network under strain. We find that the relative force uptake is accomplished in two steps. Firstly, fibers align within their fiber families and, secondly, fiber families orient in the direction of force. The maximum alignment of the collagen fiber network is found to be determined by the largest strain. Isotropic and anisotropic samples reveal a different micro structural behavior under repeated deformation leading to a similar force uptake after two stretching cycles. Our method correlates mechanical properties with morphologies in collagen fiber networks.

Embryonic cuticle from artemia cyst shell displays amyloid-like characteristics and nontoxicity after oral consumption.

Artemia cysts are the essential food product for industrial larviculture of fishes. The cyst shell protects the artemia embryo from mechanical damage, ultraviolet light, excessive water loss, thermal variation and anoxia condition. However, the underlying mechanism of such environmental protection is largely unclear. The embryonic cuticle of cyst shell mainly constitutes chitin and proteins. Absence of cyst shell proteins compromises embryo survival. In literature, there are few examples of functional amyloids where proteins adapt amyloid-like structures and act as protective covering. We hypothesized that the proteins from the embryonic cuticle of artemia cyst shell may have amyloid-like properties. Using FTIR and CD analysis, we found that proteins in embryonic cuticle have high ß-sheet secondary structures. Embryonic cuticles displayed high Congo red binding affinity and stained samples showed apple-green birefringence under polarized light, confirming the presence of amyloid-like structures. Amyloid structures have a tendency to propagate and cause amyloidosis. However, feeding of amyloid rich embryonic cuticles to zebrafish did not show any signs of discomfort or morbidity and amyloid deposition. Taken together, the study reveals that amyloid-like structures are present in embryonic cuticle of artemia cyst and their consumption does not induce amyloidosis in zebrafish.