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.     

The lamellar peel: A sequential nonablative and ablative laser treatment for facial skin rejuvenation

Objectives. Ablative and nonablative laser treatments have established themselves independently in the rejuvenation of aging skin. This study was designed to determine the effects of sequential nonablative and ablative laser treatments on facial skin.

Materials and methods. Twelve patients, ages 32–56 years, with skin phototypes I‐IV, received three treatments spaced six weeks apart. Each treatment consisted of one pass with 1319‐nm Nd:YAG laser at 16 J/cm², 50 ms pulse duration. This was immediately followed by one pass of 2940‐nm Er:YAG laser at 5 J/cm². Photographs and biopsies were taken prior to the first treatment and following the third treatment.

Results. Clinical improvements in facial skin tone and texture, acne scarring and dyschromia were noted in all patients. Histologic changes included a more compact epidermal granular layer and lamellar collagen formation with decreased solar elastosis in the dermis. Compared to controls, treated skin had a thicker, more homogeneous papillary dermis.

Conclusion. The sequential use of nonablative and superficial ablative laser treatments clinically and histologically improved photodamaged and chronologically aged skin. This occurred with minimal recovery and little morbidity, demonstrating this laser application to be a safe and effective method for facial rejuvenation.     

Superficial erbium:YAG laser resurfacing of photodamaged skin

Background: Light chemical peels and microdermabrasion have enjoyed recent popularity for the treatment of mild photoaging. However, clinical improvement from these modalities is often minimal from both a patient's and physician's perspective. Erbium:YAG lasers have been effective in treating mild to moderate photoaging, but the need for either regional or general anesthesia, as well as the significant post‐treatment recovery period has limited its use.

Objective: We sought to utilize a very low fluence approach to erbium:YAG laser resurfacing, with topical anesthesia, to ascertain its efficacy in treating mild to moderate photoaging.

Methods: A total of 250 subjects aged 28–80 years with skin types 1–4 and mild to moderate facial rhytids were treated with topical anesthesia and subsequently one pass of a 2940?nm erbium:YAG laser, using between 5 and 17.5?J/cm². In addition, 58 of the treated facial subjects underwent neck resurfacing with fluences between 5 and 15?J/cm² and eight treated facial subjects underwent upper chest resurfacing at fluences of 5–7?J/cm². A single treatment was received by 246 subjects; four subjects were treated a second time after a 1‐month interval.

Results: Most subjects completely re‐epithelialized by 3–4 days; healing time was depth dependent. Most subjects were able to start skin care regimens within 1–2 weeks after the procedure. Results were judged to be excellent in individuals with thin skin and good in subjects with thicker skin.

Conclusions: One pass of low fluence erbium:YAG resurfacing, under topical anesthesia, was effective for the treatment of mild to moderate photoaging.     

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.

     

In vivo measurement of the human epidermal thickness in different localizations by multiphoton laser tomography

Background: The in vivo measurement of epidermal thickness is still challenging. While ultrasound, optical coherence tomography and confocal laser microscopy are used with moderate success, this issue has not been addressed by multiphoton laser tomography. Objectives: In the present study, an in vivo measurement of four different morphometric epidermal parameters is performed. Methods: Thirty healthy volunteers aged 21–82 years were included in the study after informed consent and approval of the local ethics committee. At the dorsal forearm and the dorsum of the hand, the thicknesses of the total epidermis, viable epidermis and stratum corneum and the depth of the papillary dermis were calculated from depth‐resolved intensity curves after correlation with multiphoton images. Results: We have shown consistently that in all age groups, the four morphometric parameters are significantly higher at the hand compared with the forearm, while there were no differences between age groups. This is consistent with most previous findings. Conclusion: The method presented here provides a novel in vivo investigation tool for the measurement of epidermal morphometric parameters that may be useful for the observation of epidermal changes over time in skin disorders, therapy side effects or in cosmetic science.     

Folic acid and creatine improve the firmness of human skin in vivo

Background The decrease in firmness is a hallmark of skin aging. Accelerated by chronic sun exposure, fundamental changes occur within the dermal extracellular matrix over the years, mainly impairing the collagenous network. Aims Based on the qualitative and quantitative assessment of skin firmness, in vitro and in vivo studies were carried out to elucidate the effects of topical folic acid and creatine to counteract this age‐dependent reduction in the amount of collagen. Patients/Methods Topical application of a commercially available formulation containing folic acid and creatine was performed to study effects on skin firmness in vivo using cutometric analysis. Imaging and quantification of collagen density were carried out using multiphoton laser scanning microscopy (MPLSM). To investigate the effects of these compounds on collagen gene expression, procollagen synthesis, and collagen fibril organization, complementary in vitro studies on cultured fibroblast‐populated collagen gels were carried out. Results The underlying structural changes in the collagen network of young and aged sun‐exposed facial skin in vivo were visualized by MPLSM. Topical application of a folic acid‐ and creatine‐containing formulation significantly improved firmness of mature skin in vivo. Treatment of fibroblast‐populated dermal equivalents with folic acid and creatine increased collagen gene expression and procollagen levels and improved collagen fiber density, suggesting that the in vivo effects are based on the overall improvement of the collagen metabolism. Conclusions Employing MPLSM, dermal changes occurring in photo‐aged human skin were visualized in an unprecedented manner and correlated to a loss of firmness. Treatment of aged skin with a topical formulation containing folic acid and creatine counteracted this age‐dependent decline by exerting sustained effects on collagen metabolism. Our results support previous findings on the efficacy of these actives.     

Cutaneous photoaging treated with a combined 595/1064 nm laser

Background and objectives: The pulsed dye laser (PDL) has been used for a variety of vascular changes associated with photoaging. The Nd:YAG laser has been shown to be effective in the treatment of deeper facial vessels, as well as to stimulate new dermal collagen deposition. This study was undertaken to evaluate the safety and efficacy of sequential dual‐wavelength PDL and Nd:YAG laser treatment of photoaged facial skin. Methods: Fifteen individuals, between the ages of 38 and 66 years old, with various stigmata of facial photoaging, were entered into the study. Five sequential combined PDL/Nd:YAG (Cynergy, Cynosure Inc., Westford, MA, USA) treatments were performed using a 10‐mm handpiece at monthly intervals. PDL parameters included a 10‐ms pulse duration and a fluence set at 1?J/cm² below the purpura threshold. Nd:YAG parameters were set at a 50‐ms pulse duration with fluences varying between 35 and 50?J/cm², depending on patient comfort. Improvement was determined by evaluation of photographs taken before the first treatment and at 1 and 3 months following the last treatment, as well as the individual's self‐assessment. Results: Individuals tolerated treatments well with no serious, long‐term, or permanent adverse effects. Improvement was most pronounced in telangiectasias and diffuse erythema, followed by epidermal dyspigmentation and lentigines. Some individuals were also noted to have improved smoothness, radiance, or pore size. The improvement was generally maintained at the 3‐month follow‐up. In addition, the average self‐reported improvement was 1.7 (on a 0–4 scale) at 1 month and 2.4 at 3 months following the last treatment. Conclusion: A novel combined sequential PDL/Nd:YAG laser can be used to treat a variety of cutaneous changes associated with photoaging. Further studies may determine the relative impact of each laser and whether the total effect is simply additive or synergistic.     

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.     

Melanin and facial skin fluorescence as markers of yellowish discoloration with aging

Background: Although one clinical sign of aging and/or photoaging is a yellowish discoloration of the facial skin, little is known about the cause of this change. In addition to the increase in the epidermal melanin content, it has been suggested that advanced glycation end products (AGEs), which are known to accumulate in photoaged skin, may affect this discoloration. Aim: The objective of this pilot study was to non‐invasively investigate the roles of melanin and AGEs in this yellowish discoloration of the facial skin. Methods: We examined the spectral reflectance at the cheek in 40 healthy Japanese women of various ages (mean age, 38.1 years) using a reflectance spectrophotometer and a spectrofluorimeter. The degree of yellowish tint was evaluated in terms of b^*. The amount of melanin in the skin was evaluated by calculating the melanin index (MI) A₆₄₀–A₆₇₀ [A_λ: log₁₀ (1/reflectance) at a wavelength of λ]. The amount of AGEs was roughly evaluated using the AGEs index, which is thought to linearly correlate with the amount of intrinsic fluorescence markers irrespective of the concentration of melanin and is defined as follows: AGEs index=I₅/SQR (I₁×I₂). In this equation, the intensities of reflectance are I₁ at an excitation wavelength of 335 nm, I₂ at an emission wavelength of 390 nm and I₅ at 390 nm under an excitation wavelength of 335 nm. Results: Both b^* and the AGEs index were significantly correlated with subject age (r=0.34, P<0.05 and r=0.68, P<0.0001, respectively). Significant correlations were also observed between MI and b^* (r=0.63, P<0.0001) and between the AGEs index and b^* (r=0.53, P<0.0005). However, no significant correlations were seen between MI and the AGEs index. Conclusion: The AGEs index does not appear to be influenced by the amount of melanin and may be utilized as an indicator of the amount of AGEs in the skin. AGEs are likely to play a role in the yellowish discoloration of skin with aging.     

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.     

Multiphoton laser scanning microscopy of localized scleroderma

Background/purpose: A real-time, non-invasive method will confer a benefit for the diagnosis and treatment of localized scleroderma (LS) in the clinic. The aim of this work was to demonstrate the potential of multiphoton laser scanning microscopy (MPLSM) for diagnosing LS and monitoring the treatment response in vivo .
Methods: Three sclerodermatous skin specimens and two normal skin specimens were investigated using MPLSM based on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG). MPLSM consists of a femtosecond Ti:sapphire laser and a scanning inverted microscope. Several parameters such as the epidermal thickness, the orientation ratio index of collagen bundles (ORICB), the spacing of collagen fibrin as well as the SHG to TPEF index of the dermis (STID) were developed to quantitatively discriminate the sclerodermatous skin from the normal skin.
Results: The morphological differences were visualized obviously in the TPEF/SHG images of human skin (normal and sclerodermatous). The values of the developed parameters in normal skin were significantly different from that in sclerodermatous skin ( P <0.05).
Conclusion: MPLSM could discriminate the sclerodermatous skin from the normal skin. With the advent of the clinical portability of typical MPLSM, this technique has great potential for application in the in vivo diagnosis of LS as well as for monitoring the treatment response.     

Visualizing radiofrequency-skin interaction using multiphoton microscopy in vivo

Background

Redundant skin laxity is a major feature of aging. Recently, radiofrequency has been introduced for nonablative tissue tightening by volumetric heating of the deep dermis. Despite the wide range of application based on this therapy, the effect of this technique on tissue and the subsequent tissue remodeling have not been investigated in detail.

Objective

Our objective is to evaluate the potential of non-linear optics, including multiphoton autofluorescence and second harmonic generation (SHG) microscopy, as a non-invasive imaging modality for the real-time study of radiofrequency-tissue interaction.

Methods

Electro-optical synergy device (ELOS) was used as the radiofrequency source in this study. The back skin of nude mouse was irradiated with radiofrequency at different passes. We evaluated the effect on skin immediately and 1 month after treatment with multiphoton microscopy.

Results

Corresponding histology was performed for comparison. We found that SHG is negatively correlated to radiofrequency passes, which means that collagen structural disruption happens immediately after thermal damage. After 1 month of collagen remodeling, SHG signals increased above baseline, indicating that collagen regeneration has occurred. Our findings may explain mechanism of nonablative skin tightening and were supported by histological examinations.

Conclusions

Our work showed that monitoring the dermal heating status of RF and following up the detailed process of tissue reaction can be imaged and quantified with multiphoton microscopy non-invasively in vivo.     

A randomized,single-blind,study evaluating a 755-nm picosecond pulsed Alexandrite laser vs. a non-ablative 1927-nm fractionated thulium laser for the treatment of facial photopigmentation and aging

Background: Laser toning is one of the most popular strategies to treat facial photopigmentation and aging. Several laser modalities, including fractional non-ablative, Q-switched (QS) lasers and new generation picosecond lasers have been used for this indication. However, there is paucity of head to head comparisons of older generation of lasers with new ones.

Objective: To compare a 755 nm picosecond pulsed alexandrite laser with a non-ablative 1927 nm fractionated thulium laser for the treatment of facial photopigmentation and aging through a randomized, single-blind study.

Materials and methods: 20 subjects (skin types I–IV) were randomized to receive either four 755-nm picosecond alexandrite laser treatments, spaced 3 weeks apart, or two dual wavelength thulium fiber fractionated 1550/1927 nm laser treatments, spaced 6 weeks apart. Follow-up assessment visits occurred 4 and 12 weeks after the last study treatment.

Results: At the 4- and 12-week follow-up, both groups showed significant improvement of photoaging, pigmentation, skin quality according to the investigator and subjects assessments. When comparing the two groups, subjects in 755 nm group had statistically significant greater improvement in investigator assessments of photoaging/skin quality and subject satisfaction than those in the 1927 nm group.

Conclusion: Both the non-ablative 1927 and 755 nm picosecond laser can improve facial photopigmentation, but the latter can yield superior results with less pain and side effects according to patient and investigator assessments.     

Relevance of the directionality of skin elasticity to aging and sagging of the face

Background: Forces acting in facial skin have been suggested to show directionality. Non‐invasive methods of measuring this directionality may thus provide information related to aging processes. The Reviscometer^® RVM600 device is capable of measuring directionality of forces on the skin. This device has not been used previously in a published study to evaluate changes in directionality of forces on facial skin with aging. Aim: The first objective of this pilot study was to investigate relationships between mechanical directionality using the Reviscometer^® RVM600, the Cutometer^® MPA580, and aging of the facial skin in a supine position. In addition, the study investigated relationships between mechanical directionality and ‘skin sagging,’ which may be caused by gravity. To validate this as a new measurement of mechanical directionality, we also performed double‐blinded trials on two groups of subjects, with one group using a product containing an anti‐aging substance and the other group using a placebo product without an anti‐aging substance. Methods: We examined 91 healthy Japanese women with a mean age of 48.5 years (range, 20–79 years) at the three sites on the face using the Reviscometer^® RVM600 and the Cutometer^® MPA580, and evaluation was performed for skin sagging in September and November 2008, and January 2009. The Reviscometer^® RVM600 was used to measure resonance‐running time (RRT) every 10° from 0° to 350°. Evaluation of skin sagging was undertaken by making marks on the face and using face photographs taken in both sitting and supine positions to calculate the sagging index. Usage testing was conducted on 38 healthy Japanese women in a double‐blinded study with one group, using a preparation containing Yomogi AGEs Clearing (YAC) extract and another group using the same preparation without the YAC extract from October 2008 to April 2009. Mean age of these subjects was 44.0 years (range, 30–60 years). Measurements were taken at the three sites on the face using the Reviscometer^® RVM600 and the Cutometer^® MPA580 and sagging index. Results: A significant correlation was identified between RRT parameters and subject age at all three measurement sites. Significant correlations between sagging index and RRT values were found for 110–170° and 290–350° only at the center of the cheek. Significant differences in RRT values were noted for 110–150° and 300–350° at this site between subjects with and without the use of YAC extract. A similar trend was found in sagging index for this site alone between subjects with and without YAC extract. Conclusion: The use of non‐invasive procedures to measure skin mechanical parameters on the face in all directions may evaluate aging and effective preventive and restorative support.     

Gadolinium toxicity: epidermis thickness measurement by magnetic resonance imaging at 500 MHz

Purpose: Regional epidermal thickening and hair follicle width measurement by delayed gadolinium contrast magnetic resonance imaging (MRI) may assess the contrast agent gadolinium toxicity on mice skin. Materials and methods: Delayed contrast in vivo MRI was performed in mice. Six mice skin samples were removed and exposed to a gadolinium contrast agent at different times after 2, 4, 6 and 8 h. The relaxation constants of each skin structure were measured. The thickness of the epidermis and hair follicle on follow‐up ex vivo delayed‐contrast MRI served as an index of gadolinium toxicity on the skin. Results: In vivo MRI by fast low‐angle shot imaging technique showed distinct skin layers. High‐resolution gradient echo T1‐weighted and multislice multiecho proton density‐weighted MRI intensities in the epidermis and hair follicle showed a positive correlation with delayed gadolinium‐enhanced MRI hyperintensities (Pearson's correlation coefficient r²=0.81, P<0.0001) in the excised mice skin tissues. Delayed contrast‐enhanced mice skin MRI after 2–4 h showed epidermis swelling and hair follicle regions with a size measurement accuracy of 65%, a sensitivity of 95%, a specificity of 25%, a positive predictive value of 65% and a negative predictive value of 65%. Areas under the receiver operating characteristic curves by MRI were 0.92–0.94 for hair and epidermis as good discriminators. MRI visualized distinct relaxation constants of the epidermis, sebaceous gland, skin papillary and reticular dermis layers and hair follicle. Conclusion: Gadolinium contrast‐enhanced MRI may visualize the thickening of the epidermis wall and hair follicle as an index of viable mice skin. Gadolinium enhanced the MRI visibility of skin structures. Gadolinium treatment showed skin toxicity as epidermis thickening the first time due to the undesirable use of high concentrations of gadolinium in microimaging.     

High-fluence fractional treatment of photodamaged facial skin using a 2940 nm erbium:yttrium-aluminum-garnet laser

Abstract

Introduction: Fractional resurfacing with an Erbium:yttrium-aluminum-garnet (Er:YAG) 2940 nm laser is an increasingly popular option for the treatment of the signs of facial photoaging, which include wrinkles as well as pigmentation issues and unwanted textural changes. Fractional treatment has produced favorable clinical responses, but with less complications and shorter recovery times than traditional laser resurfacing. This study was conducted to evaluate a fractionated Er:YAG treatment regimen of 1–2 higher fluence sessions with a multiple-pass technique. Materials and methods: Eight subjects with moderate to severely photodamaged facial skin received one to two full-face laser treatments. Multiple-pass (MP) treatment results were evaluated in terms of procedure time, discomfort, social downtime and effectiveness. A photographic evaluation, subject improvement assessments and a subject satisfaction assessment were performed. Results: An investigator's photographic review showed a 26–75% improvement in the signs of overall photoaging. Subjects treated with the MP technique exhibited a relatively short 3–4 day downtime and ratings of mostly moderate discomfort with the use of topical anesthetic cream only. Subjects treated with higher fluences demonstrated the highest average improvement in specific features of photoaging. Two laser treatments resulted in substantially higher improvement scores than those received just one laser treatment.     

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.     

In vivo fluorescence of human skin. A potential marker of photoaging

Fluorescence is a feature of elastin and collagen, both major compounds of human dermis that are altered by age and photoexposure. We studied the intrinsic fluorescence of skin in vivo in 28 human volunteers to determine whether photoaging and chronologic aging of the skin could be evaluated by this noninvasive technique. We demonstrate that the excitation of skin autofluorescence by laser ultraviolet radiation yields characteristic tissue fluorescence spectra that are unrelated to age, pigmentation, or skin thickness. The differences in skin autofluorescence appear to be related to photoexposure. Thus, laser-induced fluorimetry, a noninvasive technique, may be adaptable as a marker of photoaging.     

Laser skin resurfacing with a novel portable erbium:YAG laser

Background and objective. The erbium:YAG laser is a popular modality for laser skin resurfacing (LSR). This study was performed to evaluate the safety and efficacy of a new portable Er:YAG laser in the treatment of photo‐damaged skin.

Methods. Nine patients with skin types I–III were treated for rhytides, large pores, pigmented lesions, lentigines and photo‐damage. Small facial areas such as the periorbital area, nose, cheeks, and upper lip were treated with one to six passes at 5–6 J/cm² with a new portable Er:YAG laser. Topical and local anesthesia was used.

Results. All treated areas showed improvement and, depending upon the number of passes, re‐epithelialization was complete within 2–7 days. The intense erythema resolved within 7 days and there was blending of treated and untreated areas within 2 weeks.

Conclusion. The technique of applying a tailored number of 5–6 J/cm², 300 µs pulses of a new portable Er:YAG laser to small areas appears to be safe and effective. There was minimal discomfort and a high level of patient satisfaction after a relatively short recovery time.