Study of hyperpigmentation in human skin disorder using different electron microscopy techniques

There is a global trend of increase in the demand for three‐dimensional electron microscopy with high resolution. The ultrastructural change and related functional studies are necessary to investigate biological phenomena. In this study, currently available 3D reconstruction techniques of electron microscopes (serial block‐face scanning electron microscopy and focused ion beam—scanning electron microscopy) were used to investigate hyperpigmentary disorders in human skin. In the basal layer of the epidermis in the human skin, there are melanocytes that produce melanin and keratinocytes that act as a barrier against environmental damage. The 3D structure from serial images through scanning electron microscopy showed locations of melanosomes between melanocyte and keratinocyte in the hyperpigmentary disorder, in addition, the electron tomography showed pigment transfer through melanin instead melanosome. These results support the exocytosis‐endocytosis theory of pigment in human skin.     

X-ray microanalysis of cultured keratinocytes: methodological aspects and effects of the irritant sodium lauryl sulphate on elemental composition

Irritant substances have been shown to induce elemental changes in human and animal epidermal cells in situ . However, skin biopsies are a complicated experimental system and artefacts can be introduced by the anaesthesia necessary to take the biopsy. We therefore attempted to set up an experimental system for X-ray microanalysis (XRMA) consisting of cultured human keratinocytes. A number of methodological aspects were studied: different cell types, washing methods and different culture periods for the keratinocytes. It was also investigated whether the keratinocytes responded to exposure to sodium lauryl sulphate (SLS) with changes in their elemental composition. The concentrations of biologically important elements such as Na, Mg, P and K were different in HaCaT cells (a spontaneously immortalized non-tumorigenic cell line derived from adult human keratinocytes) compared to natural human epidermal keratinocytes. The washing procedure and time of culture influenced the intracellular elemental content, and rinsing with distilled water was preferred for further experiments. Changes in the elemental content in the HaCaT cells compatible with a pattern of cell injury followed by repair by cell proliferation were seen after treatment with 3.33 µ m and 33 µ m SLS. We conclude that XRMA is a useful tool for the study of functional changes in cultured keratinocytes, even though the preparation methods have to be strictly controlled. The method can conceivably be used for predicting effects of different chemicals on human skin.     

Real‐time in vivo confocal laser scanning microscopy of melanin‐containing cells: A promising diagnostic intervention

The use of noninvasive imaging techniques to evaluate different types of skin lesions is increasing popular. In vivo confocal laser scanning microscopy (CLSM) is a new method for high resolution non‐invasive imaging of intact skin in situ and in vivo. Although many studies have investigated melanin‐containing cells in lesions by in vivo CLSM, few studies have systematically characterized melanin‐containing cells based on their morphology, size, arrangement, density, borders, and brightness. In this study, the characteristics of melanin‐containing cells were further investigated by in vivo CLSM. A total of 130 lesions, including common nevi, giant congenital pigmented nevi, vitiligo, melasma, melanoma, and chronic eczema, were imaged by in vivo CLSM. This research helps dermatologists understand the characteristics of melanin‐containing cells and facilitate the clinical application of melanin‐containing cells in the investigation of dermatological disease. In summary, melanin‐containing cells include keratinocytes, melanocytes, macrophages, and melanocytic skin tumor cells. Our study presents the CLSM characteristics of melanin‐containing cells to potentially facilitate in vivo diagnosis based on shape, size, arrangement, density, borders, and brightness. Microsc. Res. Tech. 78:1121–1127, 2015. © 2015 Wiley Periodicals, Inc.     

Brief Note: Isolation,culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85± 4.6%), and a correspondingly low proportion K3 positive cells (15 ± 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean ± SE, n=10). Cultures reached full confluency after 17.3 ± 1.2 days when the medium was supplemented with human EGF, while 21.7 ± 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.     

Time‐dependent reduction of structural complexity of the buccal epithelial cell nuclei after treatment with silver nanoparticles

Recent studies have suggested that silver nanoparticles (AgNPs) may affect cell DNA structure in in vitro conditions. In this paper, we present the results indicating that AgNPs change nuclear complexity properties in isolated human epithelial buccal cells in a time‐dependent manner. Epithelial buccal cells were plated in special tissue culture chamber / slides and were kept at 37°C in an RPMI 1640 cell culture medium supplemented with L‐glutamine. The cells were treated with colloidal silver nanoparticles suspended in RPMI 1640 medium at the concentration 15 mg L^?1. Digital micrographs of the cell nuclei in a sample of 30 cells were created at five different time steps: before the treatment (controls), immediately after the treatment, as well as 15 , 30 and 60 min after the treatment with AgNPs. For each nuclear structure, values of fractal dimension, lacunarity, circularity, as well as parameters of grey level co‐occurrence matrix (GLCM) texture, were determined. The results indicate time‐dependent reduction of structural complexity in the cell nuclei after the contact with AgNPs. These findings further suggest that AgNPs, at concentrations present in today's over‐the‐counter drug products, might have significant effects on the cell genetic material.     

Platelet rich plasma–complexed hydrogel glue enhances skin wound healing in a diabetic rat model

Diabetic patients often exhibit delayed or incomplete progress in the healing of acute wounds, owing to poor blood perfusion. Platelet-rich plasma (PRP) has attracted much attention as a means to improve wound healing, because it contains high growth factor concentrations. However, the burst-like release of PRP growth factors results in a short half-life of these therapeutic proteins, thus greatly limiting the therapeutic effect. In this study, we prepared PRP from human umbilical cord blood and developed an in situ photocrosslinkable PRP hydrogel glue (HNPRP) by adding a photoresponsive hyaluronic acid (HA-NB) into PRP. The HNPRP hydrogel allowed for controlled release of platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β (TGF-β) for up to 28 days. In vitro cell culture showed that HNPRP promoted migration of fibroblasts and keratinocytes as well as PRP and did not reveal the advantages of HNPRP. However, in a diabetic rat skin wound model, HNPRP treatment promoted faster wound closure. Furthermore, the HNPRP group, compared with the control, PRP and hydrogel only groups, showed significantly greater re-epithelialization and numbers of both newly formed and mature blood vessels. The HNPRP group also displayed higher collagen formation than did the control group. In conclusion, HNPRP enhances angiogenesis and skin regeneration and consequently achieves faster wound healing, thus extending its potential for clinical applications to treat diabetic skin wounds.     

Melanization stimulating factor (MSF) and melanization inhibiting factor (MIF) in the integument of fish

The present studies were directed to demonstrate that adult fish skin contains putative factors that affect chromatophore and/or chromatoblast function. This hypothesis is based upon the possibility that hypo and/or hyperpigmented areas of the skin are so pigmented because of the localized expression of intrinsic factors that are either stimulatory or inhibitory to the differentiation of specific pigment cell types. In all the morphological and biochemical experiments carried out, we used culture media conditioned by dorsal (DCM) or ventral (VCM) skin from different species of fish. Both DCM and VCM were capable of stimulating differentiation of melanophores in neural crest explants. While the stimulation of melanization is an activity present in both dorsal and ventral skin, an inhibitory activity is also present in ventral skin at such a concentration that it overrides the stimulatory activity afforded by DCM. With biochemical assays, we demonstrated that the three important sequential enzymatic steps in melanogenesis are all stimulated by the conditioned media in a dose-dependent manner and this results in an increase in the amount of melanin present in cultured cells. The results of our investigations provide strong evidence that there are intrinsic pigment cell regulatory factors in the integument of fish, the inhibitory activity being stronger in the ventrum, and that those factors strongly influence, perhaps even determine, the pigment patterns of fish.     

Attachment and entry of Candida famata in monocytes and epithelial cells

Candida albicans is considered the main pathogenic yeast responsible for a multitude of infective disorders. However, other yeasts, such as Candida famata, are being recognized as potential emerging pathogens that cause several types of infections in humans and animals. Consequently, we have investigated the adhesion and internalization of Candida famata into monocytes and epithelial cells. The interaction of the yeast with the cells is very rapid and takes place during the first 15 min of injection. However, the affinity of the yeast for the cells varies, THP-1 (human monocytes) being the highest and followed in decreasing order by HeLa (human carcinoma), HaCaT, and Pam-212 (human and mouse keratinocytes, respectively). Heat inactivation or treatment with nystatin, significantly decreases yeast adhesion to cells. Immunofluorescence, as well as scanning and transmission electron microscopy, reveals that cell lines are able to internalize C. famata. At 48 h after infection, most of the yeasts located inside cells appear degraded, but some yeasts recovered from lysed cells, were still viable. Adhesion and internalization of C. famata into HeLa cells were found to be lower than those of C. albicans and C. glabrata, but higher than those of S. cerevisiae. In addition, infection with C. famata results in actin microfilaments rearrangement. This article presents novel data in the interaction of this pathogenic yeast with mammalian cells.     

Genotoxic effect of Physalis angulata L. (Solanaceae) extract on human lymphocytes treated in vitro

Physalis angulata L (Solanaceae) is a medicinal plant from North of Brazil, whose different extracts and infusions are commonly used in the popular medicine for the treatment of malaria, asthma, hepatitis, dermatitis and rheumatism. However, the genotoxic effects of P. angulata on human cells is not well known. The main purpose of the present study was to evaluate the in vitro genotoxic effects of aqueous extract of P. angulata using the comet assay and the micronucleus assay in human lymphocytes provided from 6 healthy donors. Treatments with P. angulata extracts were performed in vitro in order to access the extent of DNA damage. The comet assay has shown that treatments with P. angulata at 0.5, 1.0, 2.0, 3.0 and 6.0 microg/mL in culture medium were genotoxic. Lymphocytes treated with P. angulata at the concentrations of 3.0 and 6.0 microg/mL in culture medium showed a statistically significant increase in the frequency of micronucleus (p<0.05), however, the cytokinesis blocked proliferation index (CBPI) was not decreased after P. angulata treatment. In conclusion, the present work demonstrated the genotoxic effects of P. angulata extract on human lymphocytes in vitro.     

Single cell mechanics of keratinocyte cells

Keratinocytes represent the major cell type of the uppermost layer of human skin, the epidermis. Using AFM-based single cell compression, the ability of individual keratinocytes to resist external pressure and global rupturing forces is investigated and compared with various cell types. Keratinocytes are found to be 6–70 times stiffer than other cell types, such as white blood, breast epithelial, fibroblast, or neuronal cells, and in contrast to other cell types they retain high mechanic strength even after the cell’s death. The absence of membrane rupturing peaks in the force-deformation profiles of keratinocytes and their high stiffness during a second load cycle suggests that their unique mechanical resistance is dictated by the cytoskeleton. A simple analytical model enables the quantification of Young’s modulus of keratinocyte cytoskeleton, as high as 120–340 kPa. Selective disruption of the two major cytoskeletal networks, actin filaments and microtubules, does not significantly affect keratinocyte mechanics. F-actin is found to impact cell deformation under pressure. During keratinocyte compression, the plasma membrane stretches to form peripheral blebs. Instead of blebbing, cells with depolymerized F-actin respond to pressure by detaching the plasma membrane from the cytoskeleton underneath. On the other hand, the compression force of keratinocytes expressing a mutated keratin (cell line, KEB-7) is 1.6–2.2 times less than that for the control cell line that has normal keratin networks. Therefore, we infer that the keratin intermediate filament network is responsible for the extremely high keratinocyte stiffness and resilience. This could manifest into the rugged protective nature of the human epidermis.     

Skin regeneration stimulation: the role of PCL‐platelet gel nanofibrous scaffold

Skin is the largest organ of the human body. Thus far, tissue engineering of skin has developed rapidly and has used many types of growth factors and nanofibrous scaffolds. In this study, we differentiated neonate keratinocytes for epithelialization on the polycaprolactone‐Platelet gel (PCL‐PG) scaffold. Fabricated PCL nanofibers prepared by electrospinning technology and coated by platelet gel. Subsequently, the structure of the scaffold was evaluated by SEM, FTIR‐ATR, contact angle and tensile test assays. After seeding the neonate keratinocytes on neat PCL and PCL‐PG scaffolds, the epidermal maturation was tested by detecting cytokeratin 10 and loricrin determinants by immunocytochemistry; moreover, keratinocyte genes such as keratin 14, keratin 10, and Involucrin were investigated by real‐time PCR. The results of MTT assay indicated an increase in cell viability and cell proliferation of neonate keratinocytes on PCL‐PG nanofiber scaffolds compared with PCL. RT‐PCR and immunocytochemical analysis showed better cell differentiation on the PCL‐PG scaffolds than neat PCL. Furthermore, SEM microscopy images demonstrated that neo‐keratinocytes enhance adhesion and proliferation on PCL‐PG nanofiber scaffolds. We found that PG increases biocompatibility and wettability of scaffold, cell adhesion, and expression of keratinocyte markers. Overall, this procedure is recommended to be employed in skin tissue engineering and wounds healing.     

<i>In vitro</i> propagation of <i>Opuntia ellisiana</i> Griff. and acclimatization to field conditions

The genus Opuntia is a valuable forage resource in arid and semiarid lands during periods of drought and shortage of herbaceous plants. However, absolute minimum temperatures in the plains of Mendoza represent a limiting factor to cultivate several species.
Opuntia ellisiana is a cold hardy species, so the goals of this study were to massively propagate it using in vitro culture techniques, and then to acclimatize plantlets obtained to field conditions.
Different sterilization protocols were tested. Areoles were isolated in laminar airflow cabinet, and cultured on Murashige-Skoog medium, supplemented with sucrose and different BAP and IBA combinations. Explants were grown at 27±2ºC, under a 16-h photoperiod. The shoots produced were used in the rooting assay using different auxin combinations. In the most efficient growth treatment, plantlets reached 100% shooting after 35 days of culture, and a mean length of 10.2 mm after 49 days of culture. A 100% rooted plantlets was obtained on a medium containing 5 mg L^-1 IBA, after 12 days of culture. Acclimatization was achieved under greenhouse conditions, showing 100% plantlet survival.
This study suggests that O. ellisiana can be successfully micropropagated by areoles, and easily acclimatizated to field conditions.     

Freeze-substitution of plant tissues with a new medium containing dimethoxypropane*

Meristematic cells from root tips of Dahlia variabilis (L.) and highly vacuolated storage parenchyma cells of Helianthus tuberosus (L.) were fixed by the high-pressure freezing technique and freeze-substituted. A new water-free medium for freeze-substitution was developed to obtain better preservation of the ultrastructure of plant tissues. Adding dimethoxypropane (DMP) to the substitution medium gave two positive results: (1) water could be withdrawn from the samples without an exchange of the medium and without adding drying agents or working in a nitrogen atmosphere, and (2) the ultrastructural preservation of the substituted tissues was better than in a methanol medium and at least as good as in tissues fixed chemically at room temperature.     

A cryopreservation protocol for immature zygotic embryos of species of Ilex (Aquifoliaceae)

Tropical Ilex species have recalcitrant seeds. This work describes experiments demonstrating the feasibility of long-term conservation of Ilex brasiliensis, I. brevicuspis, I. dumosa, I. intergerrima, I. paraguariensis, I. pseudoboxus, I. taubertiana, and I. theezans through cryopreservation of zygotic rudimentary embryos at the heart developmental stage. The embryos were aseptically removed from the seeds and precultured (7 days) in the dark, at 27 +/- 2 degrees C on solidified (0.8% agar) 1/4MS medium, [consisting of quarter-strength salts and vitamins of Murashige and Skoog (1962) medium] with 3% sucrose and 0.1 mg/l Zeatin. The embryos were then encapsulated in 3% calcium alginate beads and pretreated at 24 h intervals in liquid medium supplemented with progressively increasing sucrose concentrations (0.5, 0.75 and 1 M). Beads were dehydrated for 5 h with silicagel to 25% water content (fresh weight basis) and then placed in sterile 5 ml cryovials. Then the beads were either plunged rapidly in liquid nitrogen were they were kept for 1 h (rapid cooling) or cooled at 1 degrees C min(-1) to -30 degrees C. Then the beads were immersed in liquid nitrogen for 1 h (slow cooling). The beads were rewarmed by immersion of the cryovials for 1 min in a water bath thermostated at 30 degrees C. Finally, beads were transferred onto culture medium (1/4MS, 3% sucrose, 0.1 mg/l zeatin, solidified with 0.8% agar) and incubated in a growth room at 27 +/- 2 degrees C under a 14 h light (116 micromol. m(-2) x s(-1))/ 10 h dark photoperiod. Maximum recovery percentages between 15 and 83% (depending on de the species and the treatment) were obtained with the cryopreserved embryos.     

Taxus globosa S. cell lines: Initiation,selection and characterization in terms of growth,and of baccatin III and paclitaxel production

Of the initial six cell lines originating from explants of Taxus globosa, or Mexican yew (stem internode, leaves and meristematic tissue), three were selected for their microbial and oxidation resistance, two from leaves and the other from stem internode. A study of their behavior, both in terms of cell growth, and of baccatin III and paclitaxel production, was developed in suspension cultures with an initially standardized biomass (fresh weight 0.23 g/L) using modified Gamborg’s B5 medium, and an elicitor (methyl jasmonate), on either the first or seventh day of culture, at several levels (0, 0.1, 1, 10, 100 μM). In most of the conditions used, the three cell lines showed growth associated baccatin III production. The cell line from stem internode was the highest producer of baccatin III using 1 μM elicitor, sampling at 10 days (p < 0.01, 6.45 mg/L). This same line also had the highest biomass production (6.85 g/L, p < 0.01) at 10 days of culture but at the higher elicitor concentration of 10 μM. All three cell lines did not produce paclitaxel under experimental conditions used.     

Organ culture of the bovine subcommissural organ: evidence for synthesis and release of the secretory material

The subcommissural organ (SCO) is a brain circumventricular organ formed by ependymal and hypendymal secretory cells. It secretes glycoproteins into the cerebrospinal fluid of the third ventricle where they condense into a thread-like structure known as Reissner's fiber (RF). The present study was designed to investigate whether or not the bovine SCO continues to synthesize and release glycoproteins after a long-term culture. Cultured explants of SCO survive for several months. The content of the secretory granules present in the cultured ependymocytes displayed immunoreactive and lectin-binding properties similar to those of the core glycosylated glycoproteins found in the bovine SCO. The explants actively incorporated (35)S-cysteine. In the cultured ependymocytes, the pattern of distribution of the radioactive label and that of the immunoreactive secretory material was similar, thus indicating that this material has been synthesized during culture. At the ultrastructural level, the cultured tissue exhibited a high degree of differentiation comparable to that of the bovine SCO in situ. A striking finding was the observation of similar results when cerebrospinal fluid was used as a culture medium. The addition of antibodies against RF-glycoproteins into the culture medium allowed visualization, by means of different immunocytochemistry protocols, deposits of extracellular immunoreactive secretory material on the free surface of the cultured ependymocytes, indicating that release of secretory glycoproteins into the culture medium does occur. Primary culture of dispersed SCO ependymocytes, obtained either from fresh or organ cultured bovine SCO, showed that these cells release RF-glycoproteins that aggregate in the vicinity of each cell. The present investigation has shown that: (1) two types of secretory ependymocytes become evident in the cultured SCO; (2) under culture conditions, the SCO cells increase their secretory activity; (3) explants of bovine SCO synthesize RF-glycoproteins and release them to the culture medium; (4) after release these proteins aggregate but do not form a RF; (5) a pulse of anti-RF antibodies into the culture medium blocks the secretion of RF-glycoproteins for several days.     

超低温弹簧蓄能密封圈动密封性能有限元分析与优化设计*

为进一步探究超低温工况下弹簧蓄能密封圈系统的动密封性能,建立弹簧蓄能密封圈系统的等效二维轴对称有限元模型;对密封圈轴向装配以及径向装配2种典型数值装配过程进行模拟、分析与比较,利用稳定装配后的构型计算在常温与超低温下密封圈在不同介质压力下的密封特性,并分析密封圈在不同工况下的动密封特性。基于数值模拟试验结果,对弹簧蓄能密封圈内唇表面结构、底座宽度以及弹簧刚度进行优化,并对3种优化策略进行评估验证。结果表明：在室温与超低温下,轴滑动的摩擦力都随介质压力的增大而增加;在超低温环境下夹套材料由于收缩而增大了对轴的压力,轴滑动时的摩擦力比常温下更大;随着介质压力增大,低温与室温下摩擦力差异减小。提出的3种优化方案都可在对密封性影响较小的前提下减小轴在工作过程中摩擦力,其中蓄能弹簧刚度的优化对于改善密封圈性能最为有效。     

Ionic liquid enables simple and rapid sample preparation of human culturing cells for scanning electron microscope analysis

Ionic liquid is a kind of salt that stays in a molten state even at room temperature. It does not vaporize at all in vacuum and facilitates electrical conductivity to the sample surfaces for observations with a scanning electron microscope (SEM). In this study, we used an ionic liquid in SEM for the first time to observe fixed human culture cells. The condition for the cell culture using wrapping sheets and SEM settings were varied to elucidate the optimized protocol. Compared to samples prepared by the conventional way, the ionic liquid‐treatment of samples gave SEM images of the cellular ultra structures in more detail, enabling observation of microvilli that made bridges between separated cells. In addition, the ionic liquid treatment is less time consuming as well as less laborious compared with the conventional way that includes dehydration, drying, and conductivity treatments. Totally, we concluded the ionic liquid is a useful reagent for SEM sample preparation. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Contact Mechanics and Friction on Dry and Wet Human Skin

The surface topography of the human wrist skin is studied using an optical method and the surface roughness power spectrum is obtained. The Persson contact mechanics theory is used to calculate the contact area for different magnifications, for both dry and wet condition of the skin. For dry skin, plastic yielding becomes important and will determine the area of contact observed at the highest magnification. The measured friction coefficient [M.J. Adams et al., Tribol Lett 26:239, 2007] on both dry and wet skin can be explained assuming that a frictional shear stress σ_f ≈ 15 MPa acts in the area of real contact during sliding. This frictional shear stress is typical for sliding on polymer surfaces, and for thin (nanometer) confined fluid films. The big increase in the friction, which has been observed for glass sliding on wet skin as the skin dries up, can be explained as resulting from the increase in the contact area arising from the attraction of capillary bridges. This effect is predicted to operate as long as the water layer is thinner than ～14 μm, which is in good agreement with the time period (of order 100 s) over which the enhanced friction is observed (it takes about 100 s for ～14 μm water to evaporate at 50% relative humidity and at room temperature). We calculate the dependency of the sliding friction coefficient on the sliding speed on lubricated surfaces (Stribeck curve). We show that sliding of a sphere and of a cylinder gives very similar results if the radius and load on the sphere and cylinder are appropriately related. When applied to skin the calculated Stribeck curve is in good agreement with experiment, except that the curve is shifted by one velocity-decade to higher velocities than observed experimentally. We explain this by the role of the skin and underlying tissues viscoelasticity on the contact mechanics.     

Comparison of the Friction Behavior of Occluded Human Skin and Synthetic Skin in Dry and Moist Conditions

The goal of this work was to assess the suitability of a commercial synthetic skin to simulate occluded human skin friction behavior in dry and moist skin conditions and under different applied surface pressures, with the view to using this material as a tribological test bed for health care and personal care devices that are in direct contact with the skin during use. A flat rotating ring friction measurement device, in which one part of the skin surface is continuously covered (i.e., occluded), was used to compare the friction behavior of human skin and the synthetic skin in controlled nominally dry and nominally moist skin conditions. Three loading levels were tested, simulating light, medium, and high skin pressures typical of many lifestyle- and personal health–related applications. The results showed that the friction behavior of the synthetic skin tested was notably different to that of human skin in vivo in terms of the effects of skin hydration, sliding time, and applied surface pressure. It is concluded that, for use as a tribological test bed, the tested synthetic skin model does not provide an acceptable alternative to in vivo tests using human skin.