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.     

Texture analysis of second‐harmonic‐generation images for quantitative analysis of reticular dermal collagen fibre in vivo in human facial cheek skin

Second‐harmonic‐generation (SHG) microscopy is a powerful tool for in vivo visualisation of collagen fibres in human skin because of its specific collagen selectivity without the need for staining, non‐invasiveness and high‐resolution three‐dimensional imaging. Although texture analysis of SHG images is a promising method for the quantitative analysis of well‐orientated collagen fibre structure in the tendon and cornea, there are few attempts to assess cutaneous ageing. In this study, we applied two texture analysis techniques, namely autocorrelation (2D‐AC) analysis and two‐dimensional Fourier transform (2D‐FT), to evaluate the age‐dependent changes in reticular dermal collagen fibres in in vivo human cheek skin. Age‐dependent changes in the reticular dermal collagen fibres of female subjects in their 20s, 40s and 60s clearly appeared in these texture analyses. Furthermore, the parameter from 2D‐AC analysis showed a significantly higher correlation with skin elasticity measured by a Cutometer^®. These results clearly indicate that 2D‐AC analysis of SHG images is highly promising for the quantitative evaluation of age‐dependent change in facial collagen fibres as well as skin elasticity. An appropriate texture analysis will help to provide quantitative insight into collagen fibre structure and will be useful for the diagnosis of pathological conditions as well as cutaneous ageing in skin.     

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 measurement of the water content in the dermis by confocal Raman spectroscopy

Background/purpose: Dermal water plays an important role in the physical properties of the skin. Recently, researchers have attempted to directly measure the dermal water content in vivo using magnetic resonance imaging, near infrared spectroscopy, and Raman spectroscopy. However, these methods have limitations. Although confocal Raman spectroscopy has been developed to measure the water content in the skin, no reports have suggested that this instrument can measure the dermal water content. This report describes a method for measuring the dermal water content in vivo using confocal Raman spectroscopy. Methods: We used a confocal Raman spectrometer and adjusted the laser exposure time and depth increments according to the skin depth. Age‐related changes in the dermal water content of the forearm were examined in 30 young and 30 elderly male subjects. Diurnal changes in the dermal water content of the forearm were examined in 12 elderly male subjects. Results: Adjusting the exposure time and depth increment dramatically improved the signal‐to‐noise ratios of the Raman spectra. Elderly dermis had significantly higher water content than young dermis. Moreover, the dermal water content displayed a diurnal change. Conclusion: This study demonstrates that the dermal water content can be measured in vivo using confocal Raman spectroscopy.     

Keratinocytes express fibrillin and assemble microfibrils: implications for dermal matrix organization

Fibrillin–containing microfibrils are key architectural structures of the upper dermis and integral components of the dermal elastic fibre network. Microfibril bundles intercalate into the dermal—epithelial junction and provide an elastic connection between the dermal elastic fibre network and the epidermis. Immunohistochemical studies have suggested that they are laid down both at the dermal—epithelial junction and in the deep dermis. While dermal fibroblasts are responsible for deposition of the elastin and microfibrillar components that comprise the elastic fibres of the deep dermis, the cellular origin of the microfibril bundles that extrude from the dermal—epithelial junction is not well defined. We have used fresh tissues, freshlyisolated epidermis and primary human and porcine keratinocyte cultures to investigate the possibility that keratinocytes are responsible for deposition of these microfibrils. We have shown that keratinocytes in vivo and in vitro synthesize both fibrillin-1 and fibrillin-2, and assemble beaded microfibrils concurrently with expression of basement membrane collagen. These observations suggest that keratinocytes co-ordinate the secretion, deposition and assembly of these distinct structural elements of the dermal matrix, and have important implications for skin remodelling.     

Evaluation of unique elastic aggregates (elastic globes) in normal facial skin by multiphoton laser scanning tomography

Background

There is no reliable marker to estimate the degree of skin aging in vivo. It now has become possible to quantitatively determine the dermal characteristics of the extracellular matrix (ECM) in vivo using multiphoton laser tomography (MLT).

Methods

Fifty-seven healthy Japanese female volunteers, aged from 20 to 60 years old, were examined using multiphoton depth-resolved measurements of autofluorescence (AF) and second harmonic generation (SHG) at three sites on their right cheek. Paraffin-embedded skin specimens obtained from the faces of 12 normal individuals aged 38-68 years old were stained with Elastica van Gieson (EVG).

Results

We found unique elastic aggregates at a 20µm depth from the dermo-epidermal junction (DEJ) in vivo which increased in size with aging of subjects from 20 to 60 years old. SHG fibers seemed to surround those elastic aggregates. Histological examination of specimens from normal individuals stained with EVG confirmed the occurrence of elastic aggregates with varied sizes just beneath the epidermis or hair follicles.

Conclusions

The elastic aggregates are morphologically similar to previously described ‘elastic globes’ and can serve as a marker of the early stage of photoaging. MLT will contribute to determine age-related dermal changes using a non-invasive technique.

     

Subpopulations of dermal skin fibroblasts secrete distinct extracellular matrix: implications for using skin substitutes in the clinic

Chronic wounds affect 1–2% of the world's population at any given time. These can be as a result of burns, or ulceration, and are essentially wounds which do not close. To facilitate closure, there are a number of biological products available which can be used as temporary skin replacements, or to promote tissue repair. These products usually replicate the two main layers found in human skin: the epidermis and dermis. Within the skin dermis the most abundant cell type are fibroblasts, whose primary role is to secrete extracellular matrix and support growth of cells in the adjacent epidermal layer. As fibroblasts within the skin are highly varied, the extracellular matrix in distinct locations of the dermis is also different; however skin substitutes do not usually reflect this diversity. In this study, from the UK, the researchers isolated three fibroblast sub‐types from human scalp skin dermis, and set about to characterise the extracellular matrix which the different sub‐types of fibroblasts synthesised in culture (i.e. developed in the lab, rather than on living skin). They found that the different fibroblast sub‐types produced extracellular matrix in culture reflective of the extracellular matrix found in distinct dermal locations in vivo (in living skin). They also found that certain fibroblast sub‐types were more proficient at supporting adjacent epithelial cells than others, which reflected the sub‐anatomical location from which the fibroblast sub‐types were originally isolated. The authors concluded that inspiration should be taken from the extracellular matrix which fibroblasts secrete to improve the design of biomimetic skin substitutes with improved therapeutic potential for skin tissue engineering.     

Dermal penetration of creatine from a face-care formulation containing creatine, guarana and glycerol is linked to effective antiwrinkle and antisagging efficacy in male subjects

Background The dermal extracellular matrix provides stability and structure to the skin. With increasing age, however, its major component collagen is subject to degeneration, resulting in a gradual decline in skin elasticity and progression of wrinkle formation. Previous studies suggest that the reduction in cellular energy contributes to the diminished synthesis of cutaneous collagen during aging. Aims To investigate the potential of topically applied creatine to improve the clinical signs of skin aging by stimulating dermal collagen synthesis in vitro and in vivo. Patients/Methods Penetration experiments were performed with a pig skin ex vivo model. Effects of creatine on dermal collagen gene expression and procollagen synthesis were studied in vitro using cultured fibroblast‐populated collagen gels. In a single‐center, controlled study, 43 male Caucasians applied a face‐care formulation containing creatine, guarana extract, and glycerol to determine its influence on facial topometric features. Results Cultured human dermal fibroblasts supplemented with creatine displayed a stimulation of collagen synthesis relative to untreated control cells both on the gene expression and at the protein level. In skin penetration experiments, topically applied creatine rapidly reached the dermis. In addition, topical in vivo application of a creatine‐containing formulation for 6 weeks significantly reduced the sagging cheek intensity in the jowl area as compared to baseline. This result was confirmed by clinical live scoring, which also demonstrated a significant reduction in crow’s feet wrinkles and wrinkles under the eyes. Conclusions In summary, creatine represents a beneficial active ingredient for topical use in the prevention and treatment of human skin aging.     

An investigation of striae distensae using reflectance confocal microscopy

Background: Striae distensae, otherwise known as stretch marks, are white or red scar‐like streaks on the skin. Although they are not associated with adverse health outcomes, striae are associated with significant cosmetic morbidity. While they have been well characterised histopathologically, a non‐invasive method of microscopic lesion assessment of striae would be welcome. Methods: To gain insight into the small‐scale morphological features associated with striae we undertook an in vivo investigation of nine patients with striae alba and one with striae rubra utilising reflectance confocal microscopy (RCM). Results: Here we demonstrate that features known to be present using light microscopy, such as parallel collagen bundles in the dermis, and some features that are not well recognised by light microscopy, including distortion of dermal papillae, are demonstrable using RCM. Conclusions: Characterising the features of early and established striae distensae with confocal microscopy is an important foundation for future work. The potential ability to reliably identify the earliest pathological changes in skin in early lesions or before clinically manifest striae develop – a task facilitated by our findings – will increase the understanding of their pathogenesis and will have significant practical utility in monitoring the impact of future preventative interventions.     

Thrombomodulin expression on dermal cells in normal and psoriatic skin

Abstract The various subsets of dermal cells with a dendritic appearance can be identified by phenotypic differences in cell markers. We report on the morphology and tissue distribution of dermal cells detected with a monoclonal antibody against thrombomodulin in histological sections of normal arm and scalp skin and psoriatic skin. Double staining with antibodies to factor XIIIa, CD34 and CD68 was also employed in scalp biopsies to elucidate the relationship between thrombomodulin⁺ dermal cells and dermal dendrocytes and macrophages described by others. Thrombomodulin⁺ dermal cells in normal arm skin had little cytoplasm with fine branched dendrites and tended to be localized just beneath the epidermis. In scalp skin these cells had longer, more numerous dendrites and were distributed in the papillae and perivascular adventitial dermis primarily in the upper and central reticular dermis. In psoriatic skin, thrombomodulin⁺ dermal cells had an increased cytoplasmic volume with stout, less branched dendrites and appeared in the papillae and among inflammatory cells. Dermal cells detectable by thrombomodulin expression were factor XIIIa^–, CD34^– and CD68^–, and seemed to represent a distinct subset of dermal cells which may function in tissue repair. However, thrombomodulin⁺ dermal cells and factor XIIIa⁺ dendrocytes were frequently seen close together and could act cooperatively to regulate extravascular thrombin homeostasis in both normal and pathological dermal environments. Received: 26 May 1997     

Papillary fibroblasts differentiate into reticular fibroblasts after prolonged in vitro culture

The dermis can be divided into two morphologically different layers: the papillary and reticular dermis. Fibroblasts isolated from these layers behave differently when cultured in vitro. During skin ageing, the papillary dermis decreases in volume. Based on the functional differences in vitro, it is hypothesized that the loss of papillary fibroblasts contributes to skin ageing. In this study, we aimed to mimic certain aspects of skin ageing by using high‐passage cultures of reticular and papillary fibroblasts and investigated the effect of these cells on skin morphogenesis in reconstructed human skin equivalents. Skin equivalents generated with reticular fibroblasts showed a reduced terminal differentiation and fewer proliferating basal keratinocytes. Aged in vitro papillary fibroblasts had increased expression of biomarkers specific to reticular fibroblasts. The phenotype and morphology of skin equivalents generated with high‐passage papillary fibroblasts resembled that of reticular fibroblasts. This demonstrates that papillary fibroblasts can differentiate into reticular fibroblasts in vitro. Therefore, we hypothesize that papillary fibroblasts represent an undifferentiated phenotype, while reticular fibroblasts represent a more differentiated population. The differentiation process could be a new target for anti‐skin‐ageing strategies.     

Changes of human skin in subepidermal wound healing process

Background/purpose: The wound healing process involves unexplained mechanisms. An aberration in this process is known to cause dermal disorders such as keloid or hypertrophic scars, but the mechanism by which these scars are formed remains to be elucidated. Here we examined the usefulness of a non‐invasive optical imaging device to clarify mechanisms of wound healing and of scar formation. Methods: An 8 mm experimental wound was made in the forearms of six subjects by a suction blister method. To observe chronological changes associated with wound healing, horizontal cross‐sectional images were non‐invasively obtained of the wounded area from the skin surface down to 129 μm below at 21.5 μm intervals using in vivo laser confocal scanning microscopy (LCSM). Results: The wounds were covered with a new epidermis by week 2, at which time the dermal papilla count decreased while the thickness from the skin surface to the apex of the dermal papilla increased. The count and the thickness returned to the initial levels when the wound was healed. In two out of six subjects, fibrous tissues were observed in the upper dermis, whereas in one other subject, melanocyte‐like dendritic cells were observed in the epidermis–dermis border in later phases of wound healing. Conclusion: This non‐invasive method using in vivo LCSM revealed chronological changes in the dermis and epidermis during wound healing. In addition, although a scar was not formed in any of study subjects, this microscopy revealed aspects similar to the fibrous tissue overgrowth or to melanocyte migration, both of which may relate to wound healing. These results indicate the usefulness of this non‐invasive method in studies of wound healing and of scar formation.     

Non‐invasive imaging of mid‐dermal elastolysis

Mid‐dermal elastolysis (MDE) is a rare disease that is characterized histopathologically by selective loss of elastic tissue in the mid dermis. We aimed to assess MDE using noninvasive skin imaging techniques in vivo. We examined both the lesional and adjacent healthy skin of a woman with the reticular variant of MDE, using confocal scanning laser microscopy, optical coherence tomography (OCT) and high‐frequency ultrasound (HFUS). The median diameter of blood vessels at the top of dermal papillae was significantly increased in erythematous lesional skin compared with healthy skin. The mid‐dermal signal intensity detected by OCT was higher in healthy skin than in lesional skin. With HFUS, mid‐dermal density values were significantly higher in healthy skin than in lesional skin. Our preliminary findings indicate that noninvasive skin imaging methods such as OCT and HFUS might be suitable techniques for the evaluation and monitoring of elastolytic skin disorders such as MDE.     

Molecular imaging reveals time course of matrix metalloproteinase activity in acute cutaneous vasculitis in vivo

Matrix metalloproteinases (MMPs) play a critical role in various pathological conditions including cutaneous inflammation. Thus far, serial assessment of MMP activity in ongoing inflammation is hampered due to technical limitations. Here, we present an innovative method for longitudinal detection of MMP activity by in vivo imaging. First, we analysed skin sections from patients suffering from leucocytoclastic vasculitis (LcV) and detected a significant MMP signal via immunofluorescence staining. Then, we mimicked LcV in mice in a well‐studied model of immune complex‐mediated vasculitis (ICV). This acute inflammatory process was serially visualized in vivo using the fluorescence‐labelled MMP tracer Cy5.5‐AF443. The deposition of fluorescence‐labelled immune complexes and MMP tracer distribution was visualized repeatedly and non‐invasively by fluorescence reflectance imaging. In correlation with the presence of MMP‐2 and MMP‐9 in immunofluorescence stainings, Cy5.5‐AF443 accumulated in ICV spots in the skin of C57BL/6 mice. This tracer accumulation could also be observed in mice equipped with a dorsal skinfold chamber, where microscopic observations revealed an increased recruitment of fluorescence‐labelled leucocytes during ICV. The specificity of the MMP tracer was supported by (i) analysis of mice deficient in functional β₂‐integrins (CD18^?/?) and (ii) subsequent MMP immunofluorescence staining. These findings let us conclude that MMP accumulation in the acute phase of ICV depends on β₂‐mediated leucocyte recruitment. In summary, we show that MMPs are involved in ICV as determined by Cy5.5‐AF443, a new optical marker to longitudinally and non‐invasively follow MMP activity in acute skin inflammation in vivo.     

Heat shocks enhance procollagen type I and III expression in fibroblasts in ex vivo human skin

Background: The well‐known characteristics of aging skin are the development of fine lines and wrinkles, but changes in skin tone, skin texture, thickness and moisture content are also aspects of aging. Rejuvenation of the skin aims at reversing the signs of aging and can be established in the epidermis as well as in the dermis. Aged dermis, in fact, has a degenerated collagen matrix. To regenerate this matrix, fibroblasts need to be stimulated into synthesizing new collagen. Aims: In this study, the effects of heat shocks of different temperatures on human dermal fibroblasts in ex vivo skin on the expression of procollagen 1, procollagen 3, heat shock protein (hsp)27, hsp47, and hsp70 are investigated. Materials and methods: The heat shocks were applied on ex vivo skin samples by immersing the samples in heated phosphate‐buffered saline of 45 °C or 60 °C. Metabolic activity was measured and at similar time points propidium–iodide–calceine staining was performed to establish cell viability. Quantitative polymerase chain reaction (qPCR) was performed after the heat shock to determine gene expression levels relative to the reference temperature. Furthermore, PicroSirius Red and hematoxylin stainings were performed to visualize the collagen network and the cells. Results: The skin samples were shown to be viable and metabolically active. Histology indicated that the heat shocks did not influence the structure of the collagen network or cell appearance. qPCR results showed that in contrast to the 45 °C heat shock the 60 °C heat shock resulted in significant upregulations of procollagen type I and III, hsp70 and hsp47. Conclusion: A 60 °C, heat shock stimulates the human dermal fibroblasts in ex vivo skin to upregulate their procollagen type I and type III expression.     

Recellularizing of human acellular dermal matrices imaged by high‐definition optical coherence tomography

High‐definition optical coherence tomography (HD‐OCT) permits real‐time 3D imaging of the impact of selected agents on human skin allografts. The real‐time 3D HD‐OCT assessment of (i) the impact on morphological and cellular characteristics of the processing of human acellular dermal matrices (HADMs) and (ii) repopulation of HADMs in vitro by human fibroblasts and remodelling of the extracellular matrix by these cells. Four different skin decellularization methods, Dispase II/Triton X‐100, Dispase II/SDS (sodium dodecyl sulphate), NaCl/Triton X‐100 and NaCl/SDS, were analysed by HD‐OCT. HD‐OCT features of epidermal removal, dermo‐epidermal junction (DEJ) integrity, cellularity and dermal architecture were correlated with reflectance confocal microscopy (RCM), histopathology and immunohistochemistry. Human adult dermal fibroblasts were in vitro seeded on the NaCl/Triton X‐100 processed HADMs, cultured up to 19 days and evaluated by HD‐OCT in comparison with MTT proliferation test and histology. Epidermis was effectively removed by all treatments. DEJ was best preserved after NaCl/Triton X‐100 treatment. Dispase II/SDS treatment seemed to remove all cellular debris in comparison with NaCl/Triton X‐100 but disturbed the DEJ severely. The dermal micro‐architectural structure and vascular spaces of (sub)papillary dermis were best preserved with the NaCl/Triton X‐100. The impact on the 3D structure and vascular holes was detrimental with Dispase II/SDS. Elastic fibre fragmentation was only observed after Dispase II incubation. HD‐OCT showed that NaCl/Triton X‐100 processed matrices permitted in vitro repopulation by human dermal fibroblasts (confirmed by MTT test and histology) and underwent remodelling upon increasing incubation time. Care must be taken in choosing the appropriate processing steps to maintain selected properties of the extracellular matrix in HADMs. Processing HADMs with NaCl/Triton X‐100 permits in vitro the proliferation and remodelling activity of human dermal fibroblasts. HD‐OCT provides unique real‐time and non‐invasive 3D imaging of tissue‐engineered skin constructs and complementary morphological and cytological information.     

Clinical study on the effects of a cosmetic product on dermal extracellular matrix components using a high‐resolution multiphoton tomograph

Background/purpose: The aim of this study was to demonstrate the effects of selected plant extracts in a cosmetic cream on the dermal network components after a 3‐month treatment using an in vivo multiphoton tomographic device. Methods: Twenty‐four Caucasian women aged between 45 and 65 applied randomly a cosmetic emulsion B containing active ingredients (soy and jasmine) twice a day on one arm and its vehicle A (without active ingredients) on the other arm during 3 months. Measurements were performed on the internal side of the forearm before starting the treatment (T0), after 4 week (T4) and 12 weeks (T12) treatment. Measurements consisted in a multi‐layers acquisitions using a multiphoton tomograph with subcellular resolution. Optical sections (about 6 μm thick) were recorded from 0 to about 200 μm using two different wavelengths: 760 and 820 nm. To compare the series of images and obtain an objective quantification of the signal of second harmonic generation (SHG) and autofluorescence, the method used consisted in taking the integrated brightness of an image (same rectangular area for all images) as a measure of the signal. Following this step a ratio between brightness of images from the area treated with cream A or B and brightness of untreated area was calculated and used as an assessment of treatment efficacy. The parameter used for statistical analysis (variance analysis) is the difference before and after 12 weeks of treatment by either cream A or B of the signal ratios calculated in the upper dermis (118–130 μm) and those from a deeper region of the upper dermis (165–178 μm). Results: Signals (autofluorescence+SHG) of extracellular matrix do not change significantly with time (weeks 0, 4 and 12) when cream A (vehicle with no active ingredient) is applied. Treatment with cream B results in an enhancement in the signal level of extracellular matrix at week 12. The comparison of signals, in both areas (118–130 μm and 160–178 μm), show an higher increase in the deeper region than in the more superficial one for product B while we do not notice any change with product A. Conclusion: The multiphoton tomograph provided excellent high‐resolution images, which describe clearly the different skin layers, single cells and extracellular matrix components in all the 24 volunteers. Statistic analyses reveal a real effect for product B with selected plant extracts, known to increase collagen synthesis. Changes observed are characteristics of modifications in dermal collagen and elastin content. To our knowledge, it is the first time that it was possible to demonstrate in vivo the effect of a cosmetic product on the superficial dermal layer, in a non‐invasive and non‐destructive process, i.e. without cutting the skin.     

Factors influencing skin ageing in a Mediterranean population from Turkey

Background. Skin ageing is a continuous process, with intrinsic factors determining which extrinsic factors (chronic sun exposure and other environmental factors, particularly smoking) have the greatest effect. Aim. To investigate the effects of lifestyle and environmental factors on skin ageing in a Mediterranean population from Ankara, Turkey. Methods. In total, 574 (337 women, 237 men; age range 18–89 years) were enrolled into the study. Data were collected on age, gender, weight, height, body mass index (BMI), skin phototype, smoking status, consumption of alcohol (> 3 units/week) and coffee (> 1 cup/day), sun exposure, use of sunscreen and sunglasses, and involvement in sports and physical activities. The Daniell skin‐wrinkling grading system was used as a marker of skin ageing. Results. We found that male gender, chronic sun exposure and number of pack‐years of cigarette smoking significantly contributed to the formation of facial wrinkles. There was a negative correlation between facial wrinkling and the use of sunscreen and sunglasses and facial wrinkling (P < 0.001 for both). We did not find any significant association between wrinkling score and alcohol consumption, coffee consumption, sports participation or d skin phototype. Moreover, wrinkling score was significantly higher in patients with a BMI < 25 kg/m² than in patients with a BMI > 25 kg/m² (P < 0.018). Multiple logistic regression analysis was conducted after adjusting for age, gender, smoking status, alcohol consumption, skin phototype, sun exposure, and use of sunglasses and topical sun protection. We found that gender and age were significantly associated with skin ageing (P < 0.014 and < 0.001, respectively). Conclusion. In this study, older age, male gender, low BMI, smoking and chronic sun exposure had a negative influence on skin ageing in a Turkish population.     

Interdigitation index – a parameter for differentiating between young and older skin specimens

Background/aims: Quantitative morphometry developed rapidly during the last decade due to advances is computers and software. We wish to establish a simple baseline for the morphometric differences due to intrinsic ageing between young and old cohorts: the interdigitation index. It is an expression of the shape of the border between the epidermis and dermis. Methods: We used volar forearm biopsies of women, since the volar forearm is usually not photodamaged. The biopsies were fixed in buffered formalin, embedded in paraffin and sectioned. Separate sections were stained by hematoxylin‐eosin, by orcein and by dimethyl‐methylene blue. We had seven female volunteers in each group; the young cohort had a mean age of 26.6 years, the older cohort 50.9 years. We chose a cohort that was just about postmenopausal, since in the future we wish to evaluate the effect of externally‐applied agents on postmenopausal female skin and the earlier it is applied the better its chance of being effective. Results: We found no difference between the young and older cohort with regard to epidermal thickness. We found a decrease of glycosaminoglycen (GAG) as measured by dimethyl‐methylene blue staining. The results of the elastic staining by orcein, although in line with the reports in the literature, are not useful for evaluating the intrinsic ageing process, at least not by the simple percentage of area stained procedure. We introduced a new parameter: the interdigitation index. It is a simple measurement of the interdigitation in the epidermal‐dermal junction, known to be diminished by age. This index was diminished by about 20% between the young and older cohort. Conclusions: Quantitative morphometry using simple epidermal and dermal measurements on biopsies of the volar forearm of women is suitable for following intrinsic ageing of the skin and offers a simple objective method for following the ageing process of the skin.     

Decellularized dermis in combination with cultivated keratinocytes in a short- and long-term animal experimental investigation

Decellularized human dermis as a potentially ideal scaffold for dermal substitution in severe burns was examined in a two‐staged animal experiment. In an initial step, an in vitro generated composite graft consisting of human keratinocytes and decellularized dermis (AlloDerm®) was transplanted onto nude mice in a short‐term trial (n = 20, 14 days). Subsequently, a combined one‐step grafting of full thickness wounds with both decellularized dermis (in part preincubated with fibroblasts) and cultivated autologous keratinocytes as a cell suspension in fibrin glue was done in a long‐term porcine animal model (n = 10, 6 months). In both series, macroscopic wound healing was evaluated by planimetry. Histological investigations included morphological as well as immunohistochemical parameters. The short‐term study showed both successful integration of the composite grafts and reduction of wound contraction compared with the control group (epithelial grafts). The long‐term porcine study displayed reduced myofibroblast formation and contraction in the wounds that had been treated with fibroblast‐preincubated dermis. After 4 weeks, a decline of the structural integrity of the dermal matrix could be noticed. The utility of decellularized dermis as template for both dermal reconstitution and keratinocyte delivery vehicle was shown. The closure of full thickness wounds by a single‐step combination of an autologous keratinocyte fibrin sealant suspension and acellular dermis in a pig animal model could be shown. Incorporation of fibroblasts led to reduced wound contraction but could not prevent the loss of dermal integrity. The engineered ‘skin’ remained viable and stable over a period of 6 months.