A three‐dimensional skin equivalent reflecting some aspects of in vivo aged skin

Human skin undergoes morphological, biochemical and functional modifications during the ageing process. This study was designed to produce a 3‐dimensional (3D) skin equivalent in vitro reflecting some aspects of in vivo aged skin. Reconstructed skin was generated by co‐culturing skin fibroblasts and keratinocytes on a collagen–glycosaminoglycan–chitosan scaffold, and ageing was induced by the exposition of fibroblasts to Mitomycin‐C (MMC). Recently published data showed that MMC treatment resulted in a drug‐induced accelerated senescence (DIAS) in human dermal fibroblast cultures. Next to established ageing markers, histological changes were analysed in comparison with in vivo aged skin. In aged epidermis, the filaggrin expression is reduced in vivo and in vitro. Furthermore, in dermal tissue, the amount of elastin and collagen is lowered in aged skin in vivo as well as after the treatment of 3D skin equivalents with MMC in vitro. Our results show histological signs and some aspects of ageing in a 3D skin equivalent in vitro, which mimics aged skin in vivo.     

In vivo skin measurements with a novel probe head for simultaneous skin impedance and near‐infrared spectroscopy

Background/purpose: Near‐infrared (NIR) spectroscopy and skin impedance (IMP) measurements are useful techniques for objective diagnostics of various skin diseases. Here, we present a combined probe head for simultaneous, time‐saving NIR spectroscopy and skin impedance measurements. The probe also ensures that both measurements are performed under equal conditions and at the same skin location. Methods: Finite element method simulations were performed for evaluation of the impedance. In vivo skin measurements were performed and combined NIR and impedance spectra were analysed by means of multivariate methods with respect to body location, age and gender. The classification rate was determined by a planar discriminant analysis. Reproducibility was investigated by calculation of scatter values and statistical significance between overlapping groups was assessed by the calculation of intra‐model distances, q. Results: The novel probe yielded rapid reproducible results and was easy to manage. Significant differences between skin locations and to a lesser extent age groups and gender were demonstrated. Conclusion: With the novel probe, statistically significant differences between overlapping classes in score plots can be confirmed by calculating intra‐model distances. The influence of molecular differences in the skin at different body locations is larger than the influence of gender or age and therefore relevant reference measurements are discussed.     

Morphological skin ageing criteria by multiphoton laser scanning tomography: non-invasive in vivo scoring of the dermal fibre network

Background: Morphological changes in the dermal collagen and elastin fibre network are characteristic for skin ageing and for pathological skin conditions of the dermis. Objectives: To characterize pathological and physiological conditions by multiphoton laser scanning tomography (MLT) in vivo, it is necessary to investigate and identify morphological alterations related to ageing. Methods: In vivo MLT was used to image two‐photon excited autofluorescence (AF) and second harmonics generation (SHG) in human dermis of 18 volunteers of different ages. Criteria for the evaluation of age‐dependent morphological changes in MLT images were fibre tension and morphology, network pattern, clot formation and image homogeneity. These criteria were weighted and a score was calculated. Results: The resulting MLT‐based Dermis Morphology Score is correlated with age (R² = ?0.90) and with the previously published SHG to AF Ageing Index of Dermis (R² = 0.66). The two groups of young (age 21–38) and old (age 66–84) volunteers showed a significant difference in MLT score values (P < 0.001). Conclusions: We could demonstrate an in vivo relationship between morphological characteristics of human dermis assessed by MLT and age. The present score allows the semi‐quantitative evaluation of specific morphological changes of the dermal fibre network in ageing skin by in vivo AF and SHG imaging. This method will be useful for diagnostics of pathological conditions and their differentiation from ageing effects.     

Efficacy of anti‐wrinkle products in skin surface appearance: a comparative study using non‐invasive methods

Background/purpose: Age has a huge influence on skin roughness; with increasing age, the number of collagen and elastine fibers is reduced and elasticity decreases significantly. Pharmaceutical and cosmetics, environmental factors and lifestyle have an important effect on skin. In this study, the efficacy of 12 commercial anti‐wrinkle products was evaluated using a direct non‐invasive method to measure the skin surface morphology. Four clinical parameters surface evaluation of the living skin (SELS) (Ser, Sesc, Sesm, and Sew) were evaluate using Visioscan^® VC98. Methods: Two hundred and forty‐eight healthy female volunteers, aged between 30 and 70 years, were chosen for this study. The duration of treatment was 28 days. Skin microrelief parameters were evaluated using the Visioscan^® VC98 – SELS 2000 from Courage+Khazaka. Measurements were made in the crow's feet area and the differences were evaluated for statistical significance. Results: Significant differences were found for some of the SELS parameters. According to the results obtained for SELS Sew, products that showed to be more effective against aging were V, M, N, T, P, R and L. We think this methodology may be considered very useful for the direct study of the skin surface and may be suitable as a routine method in wrinkle evaluation.     

The perilesional skin in vitiligo: a colorimetric in vivo study of 25 patients

Background: The aim of this work was to study in vivo the perilesional skin in vitiligo with a colorimetric method. Methods: Twenty‐five patients affected by vitiligo were included. For each patient, three different areas were considered: the lesional, the perilesional and the normal skin as far as 5 cm from the nearest vitiligo spot. Skin pigmentation measurements were performed with a chromameter. Results: The results showed that luminance L^* decreased significantly in relation to increasing distance from the vitiligo spot. As expected, L^* in the vitiligo spot was significantly higher than in the perilesional (P<0.0001) and normal skin (P<0.0001). There was a small difference in L^* between normal skin as far as 5 cm from the nearest vitiligo spot and perilesional skin. In contrast, the pigmentation index (b^*) gradually increased from lesional to perilesional to normal skin. Furthermore, the comparison of the b^* value between the normal skin as far as 5 cm from the nearest vitiligo spot was higher than perilesional skin and it was statistically significant (P<0.0001). Conclusion: Our results in vivo underline that the perilesional skin near the vitiligo spot is lighter than normal skin as far as 5 cm from the vitiligo spot.     

Two‐photon microscopy of dermal innervation in a human re‐innervated model of skin

When skin is injured, innervation can be severely disrupted. The subsequent re‐innervation processes are poorly understood notably because of the inability to image the full meandering course of nerves with their ramifications and endings from histological slices. In this letter, we report on two‐photon excitation fluorescence (TPEF) microscopy of entire human skin explants re‐innervated by rodent sensory neurons labelled with the styryl dye FM1‐43. TPEF imaging of nerve fibres to a depth up to roughly 300 μm within the dermis was demonstrated, allowing three‐dimensional reconstruction of the neural tree structure. Endogenous second‐harmonic imaging of type I fibrillar collagen was performed in parallel to TPEF imaging using the same nonlinear microscope, revealing the path of the nerves through the dermis.     

Long non‐coding RNA TSIX is upregulated in scleroderma dermal fibroblasts and controls collagen mRNA stabilization

Long non‐coding RNAs (lncRNAs) are thought to have various functions other than RNA silencing. We tried to evaluate the expression of lncRNAs in patients with systemic sclerosis (SSc) and determined whether lncRNAs controls collagen expression in dermal fibroblasts. lncRNA expression was determined by real‐time PCR and in situ hybridization. Protein and mRNA levels of collagen were analysed using immunoblotting and real‐time PCR. We found TSIX, one of the lncRNAs, was overexpressed in SSc dermal fibroblasts both in vivo and in vitro, which was inhibited by the transfection of transforming growth factor (TGF)‐β1 siRNA. TSIX siRNA reduced the mRNA expression of type I collagen in normal and SSc dermal fibroblasts, but not the levels of major disease‐related cytokines. In addition, TSIX siRNA significantly reduced type I collagen mRNA stability, but not protein half‐lives. Furthermore, we first investigated serum lncRNA levels in patients with SSc, and serum TSIX levels were significantly increased in SSc patients. TSIX is a new regulator of collagen expression which stabilizes the collagen mRNA. The upregulation of TSIX seen in SSc fibroblasts may result from activated endogenous TGF‐β signalling and may play a role in the constitutive upregulation of collagen in these cells. Further studies on the regulatory mechanism of tissue fibrosis by lncRNAs in SSc skin lead to a better understanding of the pathogenesis, new diagnostic methods by their serum levels and new therapeutic approaches using siRNAs.     

Objective and non‐invasive evaluation of photorejuvenation effect with intense pulsed light treatment in Asian skin

Background Intense pulsed light (IPL) has been widely used for photorejuvenation. Although previous literature has shown clinical effectiveness of IPL treatments on cutaneous photoaging, the associated changes in the biophysical properties of the skin following IPL treatments have not been fully elucidated. Objective The aim of this study was to evaluate changes in skin biophysical properties in patients with photoaging after IPL treatments, using non‐invasive, objective skin measuring devices. Patients and methods A total of 26 Korean women with facial dyschromias underwent three sessions of IPL treatment at 4‐week intervals. Outcome assessments included standardized photography, global evaluation by blinded investigators, patients’ self‐assessment and objective measurements of colour (Mexameter MX18, Chromatometer), elasticity (Cutometer), roughness (Visiometer), sebum (Sebumeter) and skin hydration (Corneometer). Results Intense pulsed light treatments produced a 15% decrease in the size of representative pigmented lesions (P < 0.05). Patients’ self‐assessment revealed that 84% and 58% of subjects considered their pigmented lesions and wrinkles were improved respectively. Objective colorimetric measurement demonstrated significant improvements following IPL treatments that were most remarkable after one session of IPL. Moreover, skin elasticity showed significant improvements at the end of the study. Skin wrinkles as measured using Visiometer showed a mild improvement without statistical significance. Sebum secretion and water content of skin remained unchanged. Conclusions Intense pulsed light provided significant improvement in the appearance of facial pigmented lesions in Korean patients. These effects appeared to be more remarkable in improving pigmentation, skin tone and elasticity.     

Retinoids suppress cysteine-rich protein 61 (CCN1), a negative regulator of collagen homeostasis, in skin equivalent cultures and aged human skin in vivo

Alterations in connective tissue collagen are prominent features of both chronologically aged and photoaged (ageing because of sun exposure) human skin. These age-related abnormalities are mediated in part by cysteine-rich protein 61 (CCN1). CCN1 is elevated in the dermis of both chronologically aged and photoaged human skin in vivo and promotes aberrant collagen homeostasis by down-regulating type I collagen, the major structural protein in skin, and promoting collagen degradation. Vitamin A and its metabolites have been shown to improve chronologically aged and photoaged skin by promoting deposition of new collagen and preventing its degradation. Here, we investigated regulation of CCN1 expression by retinoids in skin equivalent cultures and chronologically aged and photoaged human skin in vivo. In skin equivalent cultures, all-trans retinoic acid (RA), the major bioactive form of vitamin A in skin, significantly increased type I procollagen and reduced collagenase (matrix metalloproteinases-1, MMP-1). Addition of recombinant human CCN1 to skin equivalent cultures significantly reduced type I procollagen and increased MMP-1. Importantly, RA significantly reduced CCN1 expression in skin equivalent cultures. Topical treatment with retinol (vitamin A, 0.4%) for 7days significantly reduced CCN1 mRNA and protein expression in both chronologically aged (80+years) and photoaged human skin in vivo, compared to vehicle-treated skin. These data indicate that the mechanism by which retinoids improve aged skin, through increased collagen production, involves down-regulation of CCN1.     

Expression of extracellular matrix proteins in reticular variant of mid‐dermal elastolysis

Background Mid‐dermal elastolysis (MDE) is a rare disorder of the elastic tissue that is characterized histopathologically by selective loss of elastic fibres in the mid dermis. Objective We aimed to investigate the protein and mRNA expression of extracellular matrix‐related proteins and growth factors in the skin (lesional and non‐lesional) of a female patient with the reticular variant of MDE. Methods Real‐time RT‐PCR and immunohistochemistry was performed for matrix metalloproteinase‐1 (MMP‐1), tissue inhibitor of metalloproteinase‐1, decorin, biglycan, versican, fibronectin, elastin, extracellular matrix protein 1, cathepsin G, transforming growth factor ß1 and connective tissue growth factor. Results Although protein expression of decorin, biglycan and versican was reduced in the mid dermis of lesional skin, mRNA expression did not differ between lesional and non‐lesional skin. As expected, elastin expression was significantly diminished in the mid dermis of lesional skin, whereas mRNA expression levels of elastin were equal to non‐lesional skin. Immunoreactivity of MMP‐1 was increased in lesional upper and mid dermis. Accordingly, MMP‐1 mRNA was also significantly higher expressed in MDE when compared with non‐lesional skin. Conclusions The results of this study confirm data of the previous investigations indicating that increased MMP‐1 activity followed by elastin degradation seems to constitute the pathogenetic background of MDE.     

Evaluation of photoaging in facial skin by multiphoton laser scanning microscopy

Background/purpose: It has been reported that autofluorescence (AF) and second harmonic generation (SHG) generated in the upper dermis are related with skin photoaging. In this study, we assessed the photoaging of facial skin exposed to daily sunlight using in vivo multiphoton laser microscopy to measure AF and SHG. Methods: The intensities of AF and SHG in the upper dermis of cheek skin of 56 healthy volunteers aged 20–69 years were measured using a commercially available multiphoton laser microscope (DermaInspect^®). Correlations between the photo‐signals and volunteer age were calculated. Results: The intensity of SHG and the SHG‐to‐AF aging index of dermis (SAAID) correlated significantly with age (r=−0.48, −0.67, respectively). Conclusion: Our results suggest that SHG and the SAAID index are useful indicators of facial skin aging in vivo.     

Follow‐up of actinic keratoses after shave biopsy by in‐vivo reflectance confocal microscopy – a pilot study

Background Monitoring of treatment efficacy after shave biopsy of actinic keratoses (AK) is often difficult, as clinical and dermoscopic features may not be reliable. Objectives  We investigated the applicability of in‐vivo reflectance confocal microscopy (RCM) for the follow‐up of AK after shave biopsy. Methods A total of 10 lesions were investigated by RCM before shave biopsy, after 3 and 12 months by two observers in agreement blinded to location, patients and time interval. Results At baseline all lesions showed typical clinical, dermoscopic and RCM criteria of AK. Three months after shave biopsy, all lesions presented clinically as normal skin (NS), but two lesions showed features suspicious for AK by RCM. After 12 months, one lesion of these two lesions changed into NS in RCM, whereas the other lesion progressed into clinical visible AK. At baseline, the two observers diagnosed 10 of 10 lesions correctly in RCM, after 3 months eight of 10 lesions and after 12 months all lesions were diagnosed correctly. Conclusions Our results suggest that RCM might be a useful tool in the follow‐up of AK after shave biopsy and might be used in inconclusive clinical and dermoscopic presentations of lesions after surgery or other treatment modalities.