Tattoo Pigments Are Localized Intracellularly in the Epidermis and Dermis of Fresh and Old Tattoos: In vivo Study Using Two-Photon Excited Fluorescence Lifetime Imaging.

BACKGROUND: The knowledge about the location and kinetics of tattoo pigments in human skin after application and during the recovery is restricted due to the limitation of in vivo methods for visualizing pigments. Here, the localization and distribution of tattoo ink pigments in freshly and old tattooed human skin during the regeneration of the epidermis and dermis were investigated in vivo. METHODS: Two-photon excited fluorescence lifetime imaging (TPE-FLIM) was used to identify tattoo ink pigments in human skin in vivo down to the reticular dermis. One subject with a freshly applied tattoo and 10 subjects with tattoos applied over 3 years ago were investigated in the epidermal and dermal layers in vivo. One histological slide of tattooed skin was used to localize skin-resident tattoo pigment using light microscopy. RESULTS: The carbon black particles deposited around the incision have still been visible 84 days after tattoo application, showing delayed recovery of the epidermis. The TPE-FLIM parameters of carbon black tattoo ink pigments were found to be different to all skin components except for melanin. Distinction from melanin in the skin was based on higher fluorescence intensity and agglomerate size. Using TPE-FLIM in vivo tattoo pigment was found in 75% of tattoos applied up to 9 years ago in the epidermis within keratinocytes, dendritic cells, and basal cells and in the dermis within the macrophages, mast cells, and fibroblasts. Loading of highly fluorescent carbon black particles enables in vivo imaging of dendritic cells in the epidermis and fibroblasts in the dermis, which cannot be visualized in native conditions. The collagen I structures showed a higher directionality similar to scar tissue resulting in a greater firmness and decreased elasticity of the tattooed skin. CONCLUSIONS: Here, we show the kinetics and location of carbon black tattoo ink pigment immediately after application for the first time in vivo in human skin. Carbon black particles are located exclusively intracellularly in the skin of fresh and old tattoos. They are found within macrophages, mast cells, and fibroblasts in the dermis and within keratinocytes, dendritic cells, and basal cells in the continuously renewed epidermis even in 9-year-old tattoos in skin showing no inflammation.

Subcutaneous fat infiltration into the dermal layer induces wrinkle formation.

BACKGROUND: Wrinkles appear with aging, producing an aged impression, but the mechanism of wrinkle formation has not yet been fully elucidated. We recently reported that subcutaneous fat infiltrates into the dermal layer with aging and impairs skin elasticity, but the contribution of this process to wrinkle formation is still unclear. PURPOSE: We aimed to clarify the contribution of dermal fat infiltration to wrinkle formation by analyzing the relationship between them in the forehead of female volunteers. METHODS: We measured the severity of fat infiltration in the forehead of 29 middle-aged female volunteers by means of ultrasonography. Fixed wrinkles present when the eyes were closed and wrinkles transiently formed when the eyes were open were evaluated using a photograph-based 6-grade evaluation system for each type of wrinkle. RESULTS: Fat infiltration at the forehead area was observed similarly to that in the cheek area as we reported previously. We found that opening the eyes induced the formation of stable transient wrinkles, the grade of which was significantly related to fat infiltration severity. Furthermore, fat infiltration was also significantly related to the severity of fixed wrinkles. Moreover, the severity of transient wrinkles was significantly related to that of fixed wrinkles. CONCLUSIONS: Our results suggest that fat infiltration into the dermal layer enhances transient wrinkle formation during facial expression by impairing the ability of the skin to resist deformation, thereby promoting fixed wrinkle formation. Therefore, fat infiltration is a critical cause of wrinkle formation.

Application of Cellular Resolution Full-Field Optical Coherence Tomography in vivo for the Diagnosis of Skin Tumours and Inflammatory Skin Diseases: A Pilot Study.

BACKGROUND: Optical coherence tomography (OCT) has been shown to provide non-invasive diagnosis of common skin neoplasms, especially basal cell carcinoma. OCT produces a cross-sectional view of the tissue, similar to a traditionally sectioned histopathological view, but the resolution of conventional OCT is low and thus limits clinical application. OBJECTIVES: This study aimed to investigate the application ability of a full-field (FF)OCT system which was newly developed to scan the skin at the cellular level. METHODS: Patients with skin tumours or inflammatory lesions warranting biopsy were consecutively enrolled. All lesions underwent clinical, dermoscopic, and OCT assessment, followed by routine biopsy. The adjacent normal skin was scanned for comparison. OCT images were interpreted (blinded to the biopsy results) and then compared with the histopathological diagnosis. RESULTS: A total of 111 patients with 115 lesions completed the protocol, including 80 skin tumours, 28 inflammatory diseases, and 7 other diseases. Of the OCT images, 43.5% were of good quality and show expected features. Identifiable features of actinic keratosis, Bowen's disease, basal cell carcinoma, extramammary Paget's disease, seborrheic keratosis, large cell acanthoma, bullous pemphigoid, interface dermatitis, lichenoid tissue reaction, and psoriasis were demonstrated. Lesions are located deeply, and so some features were out of the field of view, accounting for 40.0% (46/115). CONCLUSIONS: This study expanded the ability of FFOCT for the clinical diagnosis of various skin conditions. This new optical technique can clearly visualise skin lesions located in the epidermis and upper dermis. It provided an effective way to perform digital skin biopsy in superficial skin diseases.

Infiltration of subcutaneous adipose layer into the dermal layer with aging.

BACKGROUND: The elasticity of the dermal layer decreases with aging, leading to ulcer formation and wrinkling, but the mechanism of this change is not fully understood, because it is difficult to access the complex three-dimensional (3D) internal structure of the dermis. OBJECTIVE: To clarify age-dependent changes in the overall 3D structure of the dermal layer by means of 3D analysis technology. METHODS: We observed sun-protected human skin by means of X-ray micro CT, identified the layers of the skin, and reconstructed the 3D structure on computer. Age-dependent structural changes of the dermal layer were evaluated by statistical comparison of young and aged skin. RESULTS: Histological observations suggested the presence of two types of ectopic fat deposits, namely infiltrated subcutaneous fat and isolated fat, in the lower region of the reticular dermal layer in aged skin. To elucidate their nature, we observed skin specimens by X-ray microCT. The epidermis, dermal layer, and subcutaneous adipose layer were well differentiated on CT images, and 3D skin was digitally reconstructed on computer. This method clearly showed that the isolated fat observed histologically was in fact connected to the subcutaneous fat, namely all ectopic fat is connected to the subcutaneous adipose layer. Statistical analysis showed that the severity of fat infiltration into dermal layer is significantly increased in aged skin compared with young skin. CONCLUSION: Our findings indicate that subcutaneous fat infiltrates into the dermal layer of aged skin. Our 3D analysis approach is advantageous to understand changes of complex internal skin structures with aging.

Improving dermal level images from reflectance confocal microscopy using wavelet-based transformations and adaptive histogram equalization.

OBJECTIVES: Reflectance confocal microscopy (RCM) generates scalar image data from serial depths in the skin, allowing in vivo examination of cellular features. The maximum imaging depth of RCM is approximately 250 µm, to the papillary dermis, or upper reticular dermis. Frequently, important diagnostic features are present in the dermis, hence improved visualization of deeper levels is advantageous. METHODS: Low contrast and noise in dermal images were improved by employing a combination of wavelet-based transformations and contrast-limited adaptive histogram equalization. RESULTS: Preserved details, noise reduction, increased contrast, and feature enhancement were observed in the resulting processed images. CONCLUSIONS: Complex and combined wavelet-based enhancement approaches for dermal level images yielded reconstructions of higher quality than less sophisticated histogram-based strategies. Image optimization may improve the diagnostic accuracy of RCM, especially for entities with dermal findings.

Seeing the unseen with superb microvascular imaging: Ultrasound depiction of normal dermis vessels.

PURPOSE: Current color- and power-Doppler techniques cannot demonstrate vascularization of the dermis. Aim of this prospective study was to investigate whether the new superb vascular imaging (SMI) technique improves the ultrasound (US) depiction of dermis vessels in healthy volunteers. SMI was compared side-by-side to conventional power-Doppler (PD) imaging. METHODS: Thirty adult subjects (18 men and 12 women, mean age 45 years old) were evaluated with US at level of five body areas: forehead, forearm, palm, buttock, and thigh. The vascular index (VI) was employed to objectively quantify the difference between SMI and PD imaging in terms of dermis flow amount. RESULTS: Forehead VI was higher for SMI than for PD in 93% of cases, forearm VI was higher for SMI than for PD in 97% of cases, palm VI was higher for SMI than for PD in 87% of cases, buttock VI was higher for SMI than for PD in 100% of cases, thigh VI was higher for SMI than for PD in 100% of cases. SMI-detected vascular signals in 100% of the body areas. PD failed to show any flow signals from the forehead in 23% of cases, forearm in 37% of cases, palm in 33% of cases, buttock in 47% of cases, and thigh in 50% of cases. CONCLUSION: SMI can demonstrate normal dermis vascularization whereas conventional PD cannot. SMI is a sensitive and promising technique in the study of dermis abnormalities, particularly when quantifying the disease activity is important.

Examination of age-related changes of viscoelasticity in the dermis and subcutaneous fat layer using ultrasound elastography.

BACKGROUND: Using ultrasound elastography, the present study aimed to measure the viscoelasticity in each skin layer and to determine the relationship between the measured value, age, and body mass index (BMI). MATERIALS AND METHODS: The present study included 77 Japanese women. We calculated the BMI and measured the facial cheek via elastography. With the use of the elastographic image, the dermis was divided into two layers, and the subcutaneous fat layer was divided into five equal sections according to the depth, ultimately obtaining seven layers. Furthermore, the thickness and viscoelasticity of each divided layer were measured. RESULTS: The analysis of echo images revealed that the thickness of the upper dermis layer decreased with age, whereas that of the subcutaneous fat layer tended to increase with age and BMI. As measured by elastography, the viscoelasticity of both the lower dermis and the upper subcutaneous fat layer decreased with age. As the BMI increased, the viscoelasticity of the lower subcutaneous layer also increased, but that of the upper subcutaneous layer decreased. CONCLUSIONS: The present study revealed the relationship between aging and viscoelasticity in the lower dermis and the relationship between aging, BMI, and viscoelasticity in the upper subcutaneous fat layer.

Intravital imaging of cutaneous immune responses.

Various immune cells are present in the skin and modulate the cutaneous immune response. In order to capture such dynamic phenomena, intravital imaging is an important technique and there is a possibility to provide substantial information that is not available using conventional histological analysis. Multiphoton microscope enable direct, three-dimensional, minimally invasive imaging of biological samples with high spatiotemporal resolution, and now become the main method for intravital imaging studies. Here, we will introduce the latest knowledge obtained by intravital imaging of the skin.

Intravital imaging of skin infections.

Intravital imaging of cutaneous immune responses has revealed intricate links between the skin's structural properties, the immune cells that reside therein, and the carefully orchestrated migratory dynamics that enable rapid sensing and subsequent elimination of skin pathogens. In particular, the development of 2-photon intravital microscopy (2P-IVM), which enables the excitation of fluorescent molecules within deep tissue with minimal light scattering and tissue damage, has proven an invaluable tool in the characterization of different cell subset's roles in skin infection. The ability to visualize cells, tissue structures, pathogens and track migratory dynamics at designated times following infection, or during inflammatory responses has been crucial in defining how immune responses in the skin are coordinated, either locally or in concert with circulating immune cells. Skin pathogens affect millions of people worldwide, and skin infections leading to cutaneous pathology have a considerable impact on the quality of life and longevity of people affected. In contrast, pathogens that infect the skin to later cause systemic illness, such as malaria parasites and a variety of arthropod-borne viruses, or infection in distant anatomical sites are a significant cause of morbidity and mortality worldwide. Here, we review recent advances and seminal studies that employed intravital imaging to characterize key immune response mechanisms in the context of viral, bacterial and parasitic skin infections, and provide insights on skin pathogens of global significance that would benefit from such investigative approaches.

Comparison of line-field confocal optical coherence tomography images with histological sections: Validation of a new method for in vivo and non-invasive quantification of superficial dermis thickness.

BACKGROUND: Line-field confocal optical coherence tomography (LC-OCT) is an imaging technique providing "optical biopsies" of the skin in real time and non-invasively. At a center optical wavelength of 1.3 µm, this innovative technology can be applied to dermo-cosmetic product development due to both high image resolution (~2 µm) and sufficient penetration (~0.5 mm). Nevertheless, the precise dermal area analyzed with LC-OCT has never been identified. In this study, the objective was to compare LC-OCT images with histological sections of the same area, in order to validate a new method for in vivo and non-invasive quantification of superficial dermis thickness. Once validated, this standardized and quantitative method was used to assess age-related changes of the superficial dermis. MATERIALS AND METHODS: Ex vivo LC-OCT acquisitions and hematoxylin-eosin-safran staining were performed on a panel of four healthy Caucasian female volunteers. In vivo LC-OCT study of skin aging was performed on a panel of 37 healthy Caucasian female divided into five different age-groups. RESULTS: Comparison with histological sections revealed that LC-OCT images allow the visualization and the quantification of the superficial portion of papillary dermis. Applied to different age-group of volunteers, LC-OCT images show a constant decrease in this superficial dermis thickness with age. CONCLUSIONS: In conclusion, we have introduced LC-OCT as a novel technique for in vivo and non-invasive evaluation of superficial dermis thickness. This approach could be used in the future to demonstrate visually and quantitatively the capacity of a dermo-cosmetic active ingredient to renormalize the structural properties of the dermis.

CT and MR imaging findings of solitary nevus lipomatosus cutaneous superficialis: radiological-pathological correlation.

OBJECTIVE: This study assessed the CT and MRI findings of solitary nevus lipomatosus cutaneous superficialis (NLCS). MATERIALS AND METHODS: Eleven patients with histopathologically and clinically confirmed solitary NLCS who underwent CT and/or MRI were enrolled. Radiological and histopathological findings of elevated lesions located above the level of the surrounding normal skin surface and coexisting subcutaneous lipoma-like lesions were assessed retrospectively. RESULTS: Elevated skin lesions were observed in all 11 patients; these lesions were pedunculated in 4 patients (36%) and broad-based in 7 (64%). The CT attenuation of elevated lesions was fat attenuation in 2 out of 7 patients (29%), slightly increased fat attenuation in 4 out of 7 (57%), and combined fat and soft-tissue attenuation in 1 out of 7 (14%). The MR signal intensity of elevated lesions on T1-weighted images was fat signal intensity in 2 out of 6 patients (33%), slightly decreased fat signal intensity in 3 out of 6 (50%), and combined fat signal intensity and hypointensity in 1 out of 6 (17%). Subcutaneous lipoma-like lesions with fat attenuation and/or fat signal intensity were observed in 6 out of 11 patients (55%). Histopathologically, various amounts of fatty tissue and collagenous fiber were observed within the elevated lesions in all 11 patients. CONCLUSION: The CT and MRI features of solitary NLCS were the broad-based or pedunculated elevated lesions, including fatty components. Additionally, subcutaneous lipoma-like lesions were frequently observed.

Reflectance confocal microscopy as a new diagnostic tool in transformed mycosis fungoides.

Patients with mycosis fungoides typically experience an indolent disease. In some cases, the disease undergoes a process of large cell transformation which often heralds a more aggressive course with shortened overall survival. In order to rule out large cell transformation, biopsy specimens are often collected from patients with established disease who develop new papules, plaques or tumours. In some cases, multiple biopsies are needed and scar, infection and sampling error can occur. Our aim was to evaluate lesions suggestive of large cell transformation using in vivo reflectance confocal microscopy and to correlate confocal features with histopathologic findings in three patients with biopsy-proven mycosis fungoides who developed new lesions during follow-up. A total of six lesions, two lesions per patient, were examined. Reflectance confocal microscopy demonstrated large bright roundish pleomorphic cells in the epidermis, dermoepidermal junction, dermis and hair follicle in 5 of 6 lesions. The same 5 lesions were confirmed as large cell transformation by histopathology. Dermoepidermal junction obscuration, Pautrier microabscesses, epidermal disarray, spongiosis and dendritic cells were also detected by reflectance confocal microscopy and correlated to histopathology. In conclusion, reflectance confocal microscopy is useful in identifying large cell transformation within mycosis fungoides lesions. Reflectance confocal microscopy can therefore be of value in targeting the biopsy site, thereby reducing the chance of a false-negative histopathological finding.

"Virtual Biopsies" of Normal Skin and Thermal and Chemical Burn Wounds.

The search for noninvasive methods to image and measure the mechanical properties of skin has been a frequent subject of research for many years. Although suction testing, elastography, and other testing can be noninvasive, these tests fail to yield comparable results to destructive tests such as uniaxial tensile testing. Accordingly, researchers have developed a technique to combine optical coherence tomography with vibrational analysis (vibrational optical coherence tomography) to image and analyze the biomechanical properties of tissues noninvasively and nondestructively. The result of this analysis is a "virtual biopsy" of skin, along with a physical analysis of the major components of the epidermis and dermis.In this study, the authors compare virtual biopsies of thermal and chemical burns to that of normal skin. They conclude that the enhanced optical coherence tomography images and measurements of the resonant frequency after thermal or chemical burns exhibit large differences when compared with the morphology and moduli of normal skin. Using vibrational optical coherence tomography, it is possible to follow changes in the morphology and physical properties of the epidermis and dermis associated with skin diseases and therapeutic treatments in situ.

Objective assessment of dermal fibrosis in cutaneous scarring, using optical coherence tomography, high-frequency ultrasound and immunohistomorphometry of human skin.

BACKGROUND: Noninvasive quantitative assessment of dermal fibrosis remains a challenge. Optical coherence tomography (OCT) and high-frequency ultrasound (HFUS) can accurately measure structural and physiological changes in skin. OBJECTIVES: To perform quantitative analysis of cutaneous fibrosis. METHODS: Sixty-two healthy volunteers underwent multiple sequential skin biopsies (day 0 and 1-8 weekly thereafter), with OCT and HFUS measurements at each time point supported with immunohistomorphometry analysis. RESULTS: HFUS and OCT provided quantitative measurements of skin thickness, which increased from uninjured skin (1·18 and 1·2 mm, respectively) to week 1 (1·28 mm, P = 0·01; 1·27 mm, P = 0·02), and compared favourably with haematoxylin and eosin. Spearman correlation showed good agreement between techniques (P < 0·001). HFUS intensity corresponded to dermal density, with reduction from uninjured skin (42%) to week 8 (29%) (P = 0·02). The OCT attenuation coefficient linked with collagen density and was reduced at week 8 (1·43 mm, P < 0·001). Herovici analysis showed that mature collagen levels were highest in uninjured skin (72%) compared with week 8 (42%, P = 0·04). Fibronectin was greatest at week 4 (0·72 AU) and reduced at week 8 (0·56 AU); and α-smooth muscle actin increased from uninjured skin (11·5%) to week 8 (67%, P = 0·003). CONCLUSIONS: Time-matched comparison images between haematoxylin and eosin, OCT and HFUS demonstrated that epidermal and dermal structures were better distinguished by OCT. HFUS enabled deeper visualization of the dermis including the subcutaneous tissue. Choice of device was dependent on the depth of scar type, parameters to be measured and morphological detail required in order to provide better objective quantitative indices of the quality and extent of dermal fibrosis.

Assessment of the skin tissue structure using an ultrasonic diagnostic imaging device in patients undergoing open surgery.

BACKGROUND: Perioperative skin injury is a major issue; therefore, several preventative measures have been developed. However, no previous studies have visualized the effects of stromal edema caused by surgical invasion of skin tissue, and therefore, the details remain unknown. We used an ultrasonic diagnostic imaging device to clarify changes in the skin tissue structure of patients after open surgery. MATERIALS AND METHODS: Twenty subjects who underwent open hepatectomy were enrolled. We selected the lateral abdomen, upper arms, and lower legs as ultrasonic imaging measurement sites. We performed measurements on the day before surgery and on postoperative days 1, 3, and 5. We calculated the epidermal/dermal tissue thickness, subcutaneous tissue thickness, and skin tissue thickness. We performed a one-way analysis of variance with repeated measurements for each of the postoperatively measured values on the basis of the preoperative values. Significantly different variables were subjected to the Bonferroni method. We evaluated ultrasonic imaging findings and skin injury. RESULTS: Epidermal/dermal tissue thickness at all measurement sites exhibited sustained thickening on postoperative day 5 compared to that preoperatively. The lateral abdomen exhibited thickening of the subcutaneous tissue and skin tissue on postoperative day 1. In addition, increased echogenicity, increased opacity of the layer structure, and a cobblestone appearance occurred during the postoperative course. Postoperatively, 80% of subjects exhibited skin injury. CONCLUSION: We evaluated the effects of surgical invasion on skin tissue over time. Continual observation and protective skincare are necessary near the surgical wound, where significant invasiveness occurs. Prevention of skin injury due to skin tissue thickening requires further study.

Comparative "virtual biopsies" of normal skin and skin lesions using vibrational optical coherence tomography.

BACKGROUND: Increased tissue stiffness (also termed modulus) has been shown to be a characteristic of potential tumor metastasis. Measured values of the stiffness of tumors and cancer cells are reported in the literature to increase compared to neighboring normal tissues. Yet the relationship between the mechanical properties of cells and the extracellular matrix has yet to be correlated with the histopathology of cancerous lesions. MATERIALS AND METHODS: We have developed a technique to do virtual biopsies of skin lesions by combining images made using optical coherence tomography with stiffness measurements made simultaneously using vibrational analysis. The technique is termed vibrational optical coherence tomography (VOCT). RESULTS: In this paper, we report that precancerous and cancerous lesions are characterized by changes in both the morphology and stiffness of the cellular components of the skin. The ratio of the peak heights that correspond to the epidermal (40-60Hz) and dermal (140-160 Hz) resonant frequencies appear to be different for benign and cancerous or precancerous lesions compared with normal skin and scar. CONCLUSIONS: Cell-to-cell and epidermal-to-dermal interactions may be very important in evaluating the potential of skin lesions to become malignant. These interactions can be evaluated using VOCT, a new technique for performing "virtual biopsies" of skin lesions.

Characterization of Laser-Resistant Port Wine Stain Blood Vessels Using In Vivo Reflectance Confocal Microscopy.

BACKGROUND AND OBJECTIVES: Port wine stain (PWS) is a congenital vascular malformation of the human skin. Laser is the treatment of choice for PWS. Laser-resistant PWS is one crucial factor accounting for inadequate treatment outcome, which needs to be fully characterized. This study aims to quantitatively characterize the morphology of laser-resistant PWS blood vessels in the upper papillary dermis using in vivo reflectance confocal microscopy (RCM). STUDY DESIGN/MATERIALS AND METHODS: A total of 42 PWS subjects receiving laser treatment from August 2016 through July 2018 were enrolled into this study. Thirty-three subjects had facial PWS; nine had extremity PWS. All subject's PWS received multiplex 585/1,064 nm laser treatment. RCM images were taken before and after treatment. The density, diameter, blood flow, and depth of PWS blood vessels were analyzed. RESULTS: We found 44.4% PWS on the extremities (four out of nine subjects) were laser-resistant, which was significantly higher (P < 0.001) when compared with those PWS on the face (15.2%, 5 out of 33 subjects). The laser-resistant facial PWS blood vessels had significantly higher blood flow (1.35 ± 0.26 U vs. 0.89 ± 0.22 U, P < 0.001), larger blood vessel diameters (109.60 ± 18.24 µm vs. 84.36 ± 24.04 µm, P = 0.033) and were located deeper in the skin (106.01 ± 13.87 µm vs. 87.82 ± 12.57 µm, P < 0.001) in the skin when compared with laser-responsive PWS on the face. The average PWS blood vessel density (17.01 ± 4.63/mm2 vs. 16.61 ± 4.44/mm2 , P = 0.857) was not correlated to the laser resistance. CONCLUSIONS: Laser-resistant PWS blood vessels had significantly higher blood flow, larger diameters, and were located deeper in the skin. RCM can be a valuable tool for a prognostic evaluation on laser-resistant lesions before treatment, thereby providing guidance for tailored laser treatment protocols, which may improve the therapeutic outcome. The limitations for this study include relative small sample size and acquisitions of different blood vessels before and after 2 months of treatment. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Correlation between circulating fibrocytes and dermal thickness in limited cutaneous systemic sclerosis patients: a pilot study.

The objective is to detect any possible correlation between the modified Rodnan skin score (mRSS) and dermal thickness (DT) measured by skin high-frequency ultrasound (US) and the percentage of circulating fibrocytes in patients with limited cutaneous systemic sclerosis (lcSSc). Eight lcSSc patients and five healthy subjects (control group, CNT) were enrolled. The skin involvement was evaluated by mRSS and US (18 and 22 MHz probes) in all 13 subjects in the 17 standard skin areas evaluated by mRss. Circulating fibrocytes were isolated from the peripheral blood mononuclear cells (PBMCs) of all lcSSc patients and the CNT group to analyze their percentage at baseline time (T0) when the experiments started with PBMCs' isolation and collection and after 8 days of culture (T8). Non-parametric tests were used for the statistical analysis. A positive correlation between the percentage of circulating fibrocytes at T0, mRSS (p = 0.04 r = 0.96), and DT-US, evaluated by the 22 MHz and the 18 MHz probes (p = 0.03, r = 0.66 and p = 0.05, r = 0.52, respectively), was observed in lcSSc patients. Conversely, at T8, there was no correlation (p > 0.05) between these parameters in lcSSc group. In the CNT group, no correlations between mRSS or DT-US and the percentage of circulating fibrocytes were observed both at T0 and T8. The study shows the presence of a significant relationship between the percentage of circulating fibrocytes and DT, as evidenced by both mRSS and US, in limited cutaneus SSc. This observation may well suggest the reasonable hypothesis of a crucial contribution of circulating fibrocytes to skin fibrosis progression, which might be considered as further biomarkers.

Clinical Evidence of Dermal and Epidermal Restructuring from a Biologically Active Growth Factor Serum for Skin Rejuvenation

Background: Topical skin care products use various technologies to promote skin repair. Growth factors of human, animal, and plant-derived origins have clinically demonstrated the ability to repair skin by promoting collagen, elastin, and glycosaminoglycan (GAG) production to reconstruct and reinforce skin's extracellular matrix (ECM). Human skin cells respond to instructions from highly specialized proteins or hormones referred to as growth factors. These growth factors initiate cellular communication that instigates cellular replication, production, or proliferation. The production of elastin and collagen dermal connective fibers slows, and, with age, the regenerative rates of GAGs become delayed. These biological issues can be exacerbated by extrinsic factors such as sun exposure, pollutants, and various other factors. Growth factor-based products have become important topical treatment modalities for addressing signs of skin aging such as fine lines, deep wrinkles, dryness, laxity, and textural irregularities. Objective: The aim of a 12-week clinical trial of a growth factor composition was to assess its effectiveness at restoring skin health through dermal and epidermal restructuring of aged skin. Results: Data from expert grading, and from corneometer and cutometer evaluations, as well as 2D and 3D image analysis, reflected significant improvements in facial skin appearance, firmness, elasticity, and hydration. Elements that improved most dramatically in investigators' assessments included radiance, firmness, tactile elasticity, textural smoothness, overall appearance, and crow's feet. Ultrasound imaging showed continual increases in dermal and epidermal restructuring throughout the study duration. Subject assessments reflected positive product tolerability and positive perception across a broad range of efficacy attributes through 12 weeks of usage. Conclusion: The results verified the ability of a multi-modal plant and enzymatically derived growth factor-based product to achieve skin rejuvenation improvements by stimulating dermal ECM and fibrous tissue regeneration to reduce fine lines and coarse wrinkles, and improve skin firmness and elasticity, while restoring skin to a properly hydrated state. J Drugs Dermatol. 2019;18(3):290-295.

Atomic force microscopy for biomechanical and structural analysis of human dermis: A complementary tool for medical diagnosis and therapy monitoring.

Skin mechanical properties are usually measured considering the entire skin thickness and very little is known about the mechanical behaviour of individual skin layers. We propose atomic force microscopy (AFM) as a tool to quantify nanoscale changes in the biomechanical properties and ultrastructure of human papillary dermis exposed to different mechanical and physical stimuli. Samples from 3 human skin biopsies were studied: one stretched by obesity, one subjected to a high level of sun exposure and normal skin as control. Slices of the papillary dermis layer were harvested at controlled depths from each skin biopsy and 25 µm2 areas of each slice were imaged and D-periodicity of collagen fibres measured by AFM, together with their stiffness. Standard histological analysis was also carried out to correlate biochemical properties and their distribution with stiffness and topography. We obtained similar stiffness values between the sample affected by obesity and the control sample at any depth level into the dermis, while the sun-exposed sample presented a significantly lower stiffness. Additionally, all samples presented an increase in the stiffness at higher depths into the papillary dermis layer. Collagen fibres close to the epidermis of sample affected either by obesity and sun exposure-the former even more than the latter-are thicker and present a larger D-period than those in the control sample. Our results open the possibility to use structural and mechanical analysis based on AFM as a complementary tool for medical diagnosis and therapy monitoring.