Melanocyte Density Research Articles

Utilizing deep learning model for assessing melanocytic density in resection margins of lentigo maligna

BackgroundSurgical excision with clear histopathological margins is the preferred treatment to prevent progression of lentigo maligna (LM) to invasive melanoma. However, the assessment of resection margins on sun-damaged skin is challenging. We developed a deep learning model for detection of melanocytes in resection margins of LM.MethodsIn total, 353 whole slide images (WSIs) were included. 295 WSIs were used for training and 58 for validation and testing. The algorithm was trained with 3,973 manual pixel-wise annotations. The AI analyses were compared to those of three blinded dermatopathologists and two pathology residents, who performed their evaluations without AI and AI-assisted. Immunohistochemistry (SOX10) served as the reference standard. We used a dichotomized cutoff for low and high risk of recurrence (≤ 25 melanocytes in an area of 0.5 mm for low risk and > 25 for high risk).ResultsThe AI model achieved an area under the receiver operating characteristic curve (AUC) of 0.84 in discriminating margins with low and high recurrence risk. In comparison, the AUC for dermatopathologists ranged from 0.72 to 0.90 and for the residents in pathology, 0.68 to 0.80. Additionally, with aid of the AI model the performance of two pathologists significantly improved.ConclusionsThe deep learning showed notable accuracy in detecting resection margins of LM with a high versus low risk of recurrence. Furthermore, the use of AI improved the performance of 2/5 pathologists. This automated tool could aid pathologists in the assessment or pre-screening of LM margins.

Melanocyte Density in the Diagnosis of Melanoma In Situ in Sun-Damaged Skin.

Histologic differentiation between melanoma in situ in chronically sun-damaged skin (CSDS) [lentigo maligna (LM)] and CSDS without malignancy is difficult because signs of melanocyte activation and proliferation are found in both. A potentially reliable and quantifiable criterion is melanocyte density (MD). Here, we evaluated whether and to what extent MD allows the distinction between LM and CSDS, which is particularly relevant for the evaluation of borderline cases and surgical margins.Articles assessing MD in LM and/or CSDS were evaluated in a systematic review. The results were categorized and compared according to staining. Cutoff values were included whenever stated.Twenty articles matched the selection criteria. Six hundred forty-four samples of CSDS and 227 samples of LM were considered. In each individual study, mean MD scores were higher for LM than for CSDS. However, looking at the overall study situation, it becomes clear that the data are very heterogeneous and show overlaps. Therefore, no reliable orientation value can be derived. Only 1 article defined a cutoff value.The data of MD in LM in contrast to CSDS were sparse, and a defined cutoff value was only mentioned in 1 article for microphthalmia-associated transcription factor, which cannot yet be generalized. Especially regarding the importance for the definition of surgical resection margins, this unsatisfactory data set highlights the need for further studies. More precise diagnostic criteria could spare some patients extensive and possibly disfiguring surgery.

COMPARATIVE CHARACTERISTICS OF SKIN RECOVERY IN RATS AFTER ULTRAVIOLET IRRADIATION WITH AND WITHOUT ERBIUM LASER

Introduction. Ultraviolet radiation, which has a significant biological impact among the spectrum of sunlight, can cause skin aging and tumors, but in controlled use it also has therapeutic effects on the skin. Ultraviolet radiation with a wavelength of 311 nm is used for phototherapy of skin diseases. Melanin, contained in melanosomes (MS), is produced in melanocytes (MC) during melanogenesis. Phototherapy can cause clinical hyperpigmentation by provoking an increase in the number of MCs. Ablative lasers, such as Er:YAG, are used to manage hyperpigmentation, but at the same time have the risk of causing hyperpigmentation. The frequency of such side effects varies from 10 to 46%. The aim of the study was to determine changes in the number of MCs in the epidermis relative to the length of the basement membrane and the density of MSs in the epidermis in two groups of animals: after UV irradiation without and with an ablative laser. Materials and methods. The study began with an imitation of phototherapy of dermatoses with narrow-band UV radiation. The animals were divided into 2 experimental groups: free readaptation, using laser and a separate control group (intact rats). Skin samples were taken from 54 rats: 12 animals per checkpoint (6 for each group) on days 31, 45, 61, and 121, and 6 animals in the control group. The melanocytic and melanosomal components of the epidermis were evaluated, and comparisons were made between groups and with intact animals. Results. In the group of free readaptation on day 45, the density of MCs reached 0.78±0.16 cells per 100 mkm of basal membrane (units), which exceeded the normal value, but did not show statistical significance (p=0.37). On days 61 and 121, the density was 0.72±0.13 units and 0.70±0.17 units, respectively, which indicated a correlation with the elapsed time, r = -0.732, but at once was not significantly different from the control group. In the laser hypopigmentation group, the density of MCs on the basement membrane was 0.68±0.26, 0.70±0.26 and 0.69±0.23 units on days 45, 61 and 121, respectively, demonstrating stability. The correlation between MCs density and time after UV irradiation was not taken into account (r = ‑0.485). As for the analysis of MSs, in the control group, the area of MSs was 0.77±0.24% of the epidermis square. On day 31, the density of MSs reached a peak and amounted to 1.21±0.32%, statistically different from the control group (p=0.04). Subsequently, in the group of free readaptation, the area of MSs decreased to 1.03±0.16% on day 45, 0.91±0.13% on day 61, and 0.91±0.25% on day 121. An inverse correlation was observed between the density of MSs and the elapsed time, r = -0.735. Similar patterns were observed in the laser ablation group. On days 45, 61, and 121, the area of MSs was 1.07±0.28%, 0.92±0.22%, and 0.84±0.19%, respectively. The correlation between MSs density and time after UV irradiation was significant, r = -0.881. Comparison of the density of MSs in the epidermis between the experimental groups did not reveal statistical differences at any stage of the experiment. Conclusions. The described ultraviolet impact on the skin did not lead to a statistically significant increase in the number of MCs on the basal membrane (p=0.37), but significantly increased the area of MSs in the epidermis (p=0.04). Comparison of the density of MCs in the groups of laser hypopigmentation and free readaptation did not reveal statistically significant differences between each other and the control group. Laser ablation did not lead to either an increase or decrease in the average concentration of MSs in the epidermis compared to the free readaptation group.

Characterising the Effect of Wnt/β-Catenin Signalling on Melanocyte Development and Patterning: Insights from Zebrafish (Danio rerio).

Zebrafish (Danio rerio) is a well-established model organism for studying melanocyte biology due to its remarkable similarity to humans. The Wnt signalling pathway is a conserved signal transduction pathway that plays a crucial role in embryonic development and regulates many aspects of the melanocyte lineage. Our study was designed to investigate the effect of Wnt signalling activity on zebrafish melanocyte development and patterning. Stereo-microscopic examinations were used to screen for changes in melanocyte count, specific phenotypic differences, and distribution in zebrafish, while microscopic software tools were used to analyse the differences in pigment dispersion of melanocytes exposed to LiCl (Wnt enhancer) and W-C59 (Wnt inhibitor). Samples exposed to W-C59 showed low melanocyte densities and defects in melanocyte phenotype and patterning, whereas LiCl exposure demonstrated a stimulatory effect on most aspects of melanocyte development. Our study demonstrates the crucial role of Wnt signalling in melanocyte lineage and emphasises the importance of a balanced Wnt signalling level for proper melanocyte development and patterning.

Effect of Embryonic Alcohol Exposure on Craniofacial and Skin Melanocyte Development: Insights from Zebrafish (Danio rerio).

Alcohol is a common addictive substance and prenatal alcohol exposure could cause fetal alcohol spectrum disorder (FASD) and can lead to various birth defects. The small teleost zebrafish (Danio rerio) has been identified as a fine animal model in developmental biology and toxicological research. Zebrafish models are widely used to study the harmful effects of alcohol and limited studies are available on the craniofacial and skin malformations associated with FASD. The present study attempts to investigate the effect of alcohol on early zebrafish embryonic development. The effects of prenatal alcohol exposure on neural crest cell-derived organ formation, including pharyngeal dentition, palatal bones and skin melanocytes were analysed. Whole-mount cartilage and bone staining and imaging techniques were applied to determine the effects of alcohol on the above-mentioned structures. The tooth size and shape were affected by alcohol exposure, but the number of teeth in the pharyngeal dentition was not affected. Only first-generation teeth showed size differences. The alcohol-exposed ethmoid bone, which is homologous to the human hard palate, was smaller and less dense in cell arrangement compared with the control medial ethmoid bone. The skin pigmentation defects included reduced melanocyte density, melanin contraction, smaller melanocyte surface area and aberrations in melanosome dispersion, revealing that alcohol significantly influenced and downregulated each and every step of the melanocyte developmental process. This descriptive study summarises the effects of alcohol on the development of neural crest cell-derived structures and highlights the importance of zebrafish in studying the phenotypic characteristics of fetal alcohol spectrum disorder.

32329 Assessing the utility of Fontana-Masson and MART-1 stains in evaluating skin pigmentary changes induced by ultraviolet radiation and visible light

One of the observed effects of ultraviolet radiation (UVR) and visible light (VL) on melanocompetent skin is increased pigmentation, also known as skin darkening or tanning. Many methods are used to measure skin pigment, including the Fontana-Masson stain (FMS) and MART-1 immunostain, which histologically highlight melanin and melanocyte density, respectively. While these approaches may be adequate in assessing baseline skin pigment among individuals of different races and phototypes, detecting the skin darkening induced by UVR and VL within the same individual is a challenge, especially in darker skin phototypes. We conducted a literature search and analyzed 16 articles comparing skin pigmentation changes induced by VL and UVR to their corresponding FMS and MART-1 results. Notably, significant skin darkening could be detected by diffuse reflectance spectroscopy after irradiation in skin phototypes III-VI, but FMS and MART-1 were unable to significantly differentiate these changes and reliably enumerate them within the same studies. Outcome measurements of FMS and MART-1 may be affected by several factors across studies, such as varying spectrums of radiation, radiation doses, skin phototypes, and subjectivity of the rater. Our results indicate that objective instrumental assessments, such as spectroscopy, are likely superior to FMS and MART-1 for measuring skin pigmentary changes induced by UVR and VL. Further research is recommended to investigate the value in performing FMS and MART-1 stains in future studies evaluating skin tanning from UVR and VL.

Diagnosis and treatment of nail melanoma: a review of the clinicopathologic, dermoscopic, and genetic characteristics.

Nail melanoma (NM) is an important differential diagnosis in patients with longitudinal melanonychia. However, diagnosis is often challenging as it is difficult to differentiate from other pigmented nail disorders. The main challenge for diagnosis is obtaining adequate nail matrix biopsy specimens for histopathological assessment. Furthermore, the histopathological changes in the early stages of NM are subtle and contribute to a delay in diagnosis and care. Therefore, the integration of clinical and histopathological analyses is essential. Clinical and dermoscopic features, such as a broadened width of asymmetric bands in an irregular pattern, with multicolour pigmentation, periungual pigmentation, and continuous growth, are features that support the diagnosis of NM. The essential histological features that must be assessed are cellular morphology, architectural features, melanocyte density, and inflammatory changes. The reported mutations in NMs were BRAF (0-43%), NRAS (0-31%), KIT (0-50%), NF1 (0-50%), and GNAQ (0-25%). Surgery is the primary treatment for NM. The recommended treatment for insitu or minimally invasive NM is functional surgery, but cases with suspected bone invasion should be treated with amputation. Targeted therapy and immunotherapy are indicated for advanced stages of NM. This review summarizes the updated guidelines for the diagnosis and treatment of NM.

Morphological and transcriptional evaluation of multiple facial cutaneous hyperpigmented spots.

BackgroundMorphological characteristics of major facial hyperpigmented spots have been well documented. However, detailed alterations of respective transcriptional profile for each spot and in‐depth comparisons across multiple spot types have not been reported.ObjectivesTo comprehensively assess and compare multiple facial hyperpigmented spot types at the morphological and molecular levels by utilising transcriptional expression profiling with correlation to quantified histological features.MethodsMultiple types of facial spot biopsies were collected from Chinese women and compared to additional biopsies taken from adjacent healthy skin. The types of spots included Solar Lentigos with both elongated dermal‐epidermal junction (DEJ) (SL[E]) and flat DEJ (SL[F]), Seborrhoeic Keratosis (SK), Melasma, Freckles, Post‐inflammatory hyperpigmentation of resolving acne (PIH[A]) and other stimuli (PIH[O]). Combined histomorphometry, immunohistology, and transcriptome analysis for suprabasal‐epidermis, basal‐epidermis, and dermal compartments dissected by Laser Capture Microdissection (LCM) were conducted and compared across different spot types.ResultsEach spot type was confirmed to have the unique histological pathology already documented elsewhere. Most of the spot types except Melasma and PIH (A) revealed similar melanocyte density to adjacent skin. All spots exhibited increased melanin synthesis, melanosome transportation, as well as enhanced melanocyte dendricity, however, each spot revealed a distinct transcriptome regulation pattern in pigmentation pathways. Upregulation of pigmentation genes was also observed in the dermis of SL(F), SL(E), SK and PIH(O), associated with significant modulation of DEJ related genes in basal‐epidermis and/or dermal compartments, suggesting potential melanocyte infiltration into the dermis due to impaired DEJ quality. Beyond upregulated pigmentation, for most spots, gene expression in the suprabasal‐epidermis regulating keratinisation was significantly upregulated in conjunction with thickened stratum corneum. Furthermore, downregulation of tight junction related genes represented by claudin‐1 was observed in majority of spot types, suggesting compromised barrier function could be a similarity across spots. Additionally, Cyclin‐Dependent Kinase Inhibitor 2A (CDKN2A) was upregulated in all types of spots, indicating involvement of cell senescence as a common theme.ConclusionThis comprehensive and comparative study based on the histological and transcriptional analysis of three skin compartments provided unique insights into specific causations as well as differences and similarities across multiple hyperpigmented spot types.

Melanocytes as the source of the increased melanisation in pigmented epithelial tumours: a holistic approach.

We aimed to elucidate the causes of the increased melanisation in basal cell carcinoma (BCC) and seborrheic keratosis (SK), and the role of melanocytes in this process. This study was a retrospective-cohort study conducted in the pathology department of a university hospital between January 2019 and October 2020. Forty-nine SK and 30 pigmented BCC were included in our study. SRY-box transcription factor 10 (SOX10), CD68, and Masson-Fontana staining was used for analysis in all samples. A representative section of each specimen was photographed under ×400 magnification to facilitate the assessments of the morphology of the melanocytes and their following morphometric parameters: density, nuclear diameter, and distribution. The density of pigmented keratinocytes in the lesional epidermis was scored. The nuclear diameters of melanocytes located in the nonlesional epidermis, the density of the melanophages, and the presence or absence of ulceration and solar elastosis were also recorded. The morphometric findings confirmed a statistically significant increase in melanocyte density in the BCC group compared with that in the SK group (p < 0.001). Moreover, the nuclear minor diameters in the melanocytes of the BCC sections were significantly higher than those in the SK specimens (p < 0.001). The epidermal melanocytes were distributed diffusely in almost all BCC specimens (96.7%), whereas they were mainly limited to the basal layer in the majority of the SK sections (59.2%). The number of epidermal melanised keratinocytes with a score of 3 was significantly higher in the SK group (n = 31; 63.2%) than in the BCC group (n = 6; 20%) (p = 0.001), and they were the main cells representing the pigmented appearance of the tumours. No significant difference was found between both tumour groups in terms of their melanophage density scores (p = 0.206). This study is the first step towards an objective quantification of the melanocytes in pigmented epithelial tumours and may provide a morphological background for future studies on these skin lesions.

Analysis of interobserver reproducibility in grading dysplastic nevi: Results of the application of the 2018 World Health Organization grading criteria.

We aimed to determine whether the histopathological grading of dysplastic nevi is an objective endeavor, considering interobserver variability, according to 2018 World Health Organization (WHO) criteria. In total, 179 cases of dysplastic nevi, with high and moderate degree of atypia, diagnosed and graded according to the previous criteria were reviewed by three pathologists. Then, the observers graded the dysplastic nevi as low or high according to 2018 WHO criteria. Grading of dysplastic nevi was in complete agreement in 99 out of 179 cases across three observers with a fair level of overall interobserver agreement (multirater κfree : 0.40). The observers showed moderate to good agreement for most of the architectural features, except for criteria regarding focal continuous basal proliferation of melanocytes, density of non-nested junctional melanocytes, and presence of dyscohesive nests of intraepidermal melanocytes, whereas fair agreement was achieved for the cytological criteria. The 2018 WHO criteria for dysplastic nevus will ensure a common approach to the diagnosis and grading of dysplastic nevi. However, histopathological criteria, such as cytological features and focal continuous basal proliferation of melanocytes, should be improved so as to ensure a more accurate surgical approach and risk assessment.

Melanocytic hyperplasia associated with surgical scars in basal cell carcinoma re‐excision specimens: A single‐center retrospective study

Melanocytic hyperplasia (MH) overlying surgical scars has been reported but MH adjacent to the scar has not been previously addressed. We evaluated scar-associated MH in 489 re-excision specimens of basal cell carcinoma. Melanocytic density, confluence, atypia, suprabasal scatter, follicular extension, distance from the scar, correlation with clinicopathologic parameters, and PRAME expression were recorded. One hundred seventy-seven of 489 (36%) cases exhibited MH beyond the scar extending to 6.65 mm median distance, with a median density of 13 melanocytes/0.5 mm (6-24 mm), confluence in 74 (42%) cases, atypia in 14 (8%), suprabasal melanocytes in 25 (14%), and follicular extension in 21 (12%). Mean age of MH group was significantly lower than non-MH group (P< 0.0001). MH overlying the scar was significantly higher in specimens with elastosis than those without elastosis (P< 0.0001). PRAME expression was absent in 14 of 20 cases and present in rare melanocytes in 6 cases. Increased melanocytic density with confluence, mild atypia, suprabasal scatter, and superficial follicular extension may be observed in re-excision specimens adjacent to surgical scars in the absence of a melanocytic neoplasm. These observations are helpful for appropriate interpretation of melanoma re-excision specimens, to avoid potential pitfalls in diagnosis and unnecessary therapeutic measures.

Lentigo maligna: defining margins and predictors of recurrence utilizing clinical, dermoscopic, confocal microscopy and histopathology features.

Lentigo maligna (LM) is a subtype of melanoma insitu with poorly defined margins and a high recurrence rate. The biological behaviour of LM appears to differ widely between cases, from biologically indolent to biologically active variants, with some patients experiencing multiple recurrences. It is not known whether this is secondary to inadequate margins, field cancerization or the innate biology of the lesion itself. (a) Describe the margins of LM in detail by analysing LM in three zones, that is centre, edge and surround using reflectance confocal microscopy (RCM) and histopathology; (b) ascertain association of histological distance of LM and atypical melanocytic hyperplasia from the surgical margin with multi-recurrent (MR) disease and (c) identify features (clinical, dermoscopy, RCM and histopathology) associated with MR LM. (1) Descriptive observational study comparing the centre, edge and surround of LM on histopathology and RCM; (2) retrospective cohort study comparing parameters associated with MR and non-recurrent (NR) LM. 30 patients (median follow-up time 6.2years) were included. On histopathology, confluent or near confluent lentiginous proliferation, melanocyte density >15 per 0.5mm and adnexal spread were best for distinguishing surround from edge of LM. On RCM, predominant melanocytes, lentiginous proliferation and pleomorphism distinguished surround from centre/edge. MR patients had a median histological distance of LM from the surgical margin of 2mm (versus NR patients with an average distance of 4mm). MR patients had a greater proportion of more florid features, compared with NR on histopathology at both the centre and the edge but were similar in the surround. These data may help pathologists and confocalists better define margins of LM. More florid features in MR patients, despite a similar background of sun-damaged skin, suggest the innate biology of the lesion rather than the field of cancerization may explain MR LM.

Study of melanocyte density and epidermal thickness in vulvar lichen sclerosus lesions.

This study aimed to analyze changes in melanocyte density and epidermal thickness in vulvar lichen sclerosus (VLS). Vulvar skin tissues were collected from 15 VLS female patients in Beijing Hospital, classified into early (n=7) and late VLS (n=8) groups according to pathological manifestations. Melanocyte density and full epidermal and cell-layer (from the bottom of the stratum corneum to that of the basal layer) thickness were calculated using an image analysis software. The control group was normal vulvar skin tissues from 15 females after plastic surgery. The early VLS (0.170±0.071µm) and late VLS (0.110±0.035µm) groups had significantly lower densities of epidermal melanocytes than the control group (0.275±0.036) (F=36.426, P<0.001). The cell-layer thickness did not differ between the early VLS (154.603±121.984µm) and control (176.974±80.296µm) groups (P=0.899) but significantly decreased in the late VLS group (83.455±37.129µm) compared to the control group (P=0.003). Melanocyte density decreased in early and late VLS. The full epidermal and cell-layer thickness did not significantly change in early VLS, but the cell-layer thickness decreased in late VLS.

A Study of Normal Epidermal Melanocyte Distribution

Abstract Objective: To elucidate the epidermal melanocyte distribution according to sex, age, and body part with the goal of providing benchmark data for the diagnosis and therapeutic effect evaluation of pigmentary skin diseases. Methods: Epidermal melanocytes and keratinocytes were assessed using direct immunofluorescence staining, and the melanocyte density and epidermal thickness were calculated. The obtained data were statistically analyzed using SPSS Version 20.0 software. An independent-samples t test was used to compare the data between two groups, while data among three or more groups were compared by one-way analysis of variance. Data correlations were evaluated using Pearson correlation analysis. Results: Melanocytes were uniformly distributed among the keratinocytes in the basal layer, and the average ratio of melanocytes to keratinocytes was 1:7. Among them, the ratio in males was 1:6.5 and that in females was 1:7.4, with no significant difference (P = 0.127). The melanocyte density gradually declined as age increased; the ratio was 1:5.8 before 50 years of age without an obvious downtrend. The average melanocyte density was 1:7.9 within 51 to 65 years of age and 1:8.5 at &gt;65 years of age, and the difference was statistically significant (P &lt; 0.01). Obvious differences were found in the melanocyte density among different body parts; in descending order, these densities were as follows: face (1:4.0) &gt;neck (1:5.1) &gt;hip (1:5.7) &gt;upper limb (1:7.4) &gt;lower limb (1:8.3) &gt;lower back (1:9.2) &gt;thorax and abdomen (1:9.9). The melanocyte density was not related to the epidermal thickness. Conclusion: The melanocyte density showed a declining trend with age and significantly changed after 50 years of age. The melanocyte density was associated with body part; specifically, the density in the face, neck, and hip was higher than that in the limbs and torso. However, the melanocyte density was not associated with sex or epidermal thickness.

Quantitative Study of Human Scleral Melanocytes and Their Topographical Distribution

ABSTRACT Purpose While fibroblasts constitute the main cell component of the sclera, the purpose of the present study was to investigate the cell densities of melanocytes at different regions of the sclera, and to compare them with associated scleral fibroblast densities in human donor eye sections. Methods . Paraffin-embedded sections of sclera from 21 human eyes were stained with hematoxylin-eosin (H&E) and immunohistochemical staining (S-100/AEC). Scleral melanocyte and fibroblast numbers were counted in different regions of the sclera. The relationship between the melanocyte density and iris pigmentation was also analyzed. Results . Melanocytes were found in the posterior region of the sclera, especially around the vessels and nerves in emmissarial canals, whereas no or rare melanocytes were found in equatorial and anterior regions. In H&E sections, melanocyte densities in eyes with light-colored irides were significantly less than in eyes with medium or dark-colored irides (P < .05). In S-100-stained sections, more melanocytes could be detected than those in the H&E sections in light-colored eyes (P < .05), but not in medium or dark-colored eyes (P > .05). The numbers of scleral fibroblasts were relatively stable in different regions. In the posterior scleral region, the numbers of fibroblasts were slightly higher than the number of melanocytes, however, this differences were not statistically significant (P > .05). Conclusion . Notable numbers of melanocytes were present in the posterior sclera suggesting that these cells may play a role in ocular physiology and in the pathogenesis of various disorders of the sclera.

女阴硬化性苔藓皮损黑素细胞和表皮厚度分析

女阴硬化性苔藓皮损黑素细胞和表皮厚度分析

117 Light or Dark Pigmentation of Engineered Skin Substitutes Containing Melanocytes Protects Against UV-Induced DNA Damage in Vivo

Abstract Introduction Engineered skin substitutes (ESS) were developed to meet the need for prompt wound closure in patients with large full thickness burns. ESS containing autologous fibroblasts and keratinocytes were shown to provide stable wound closure in burn patients, but are limited by hypopigmentation. DNA damage caused by ultraviolet (UV) radiation is a known risk factor for development of skin cancer. In normal human skin, epidermal melanocytes provide pigmentation, helping to shield skin from UV-induced DNA damage. The current study investigated inclusion of human melanocytes (hM) and their role in the response of ESS to UV light in vivo. Methods Primary cells were isolated from skin of healthy de-identified human donors with IRB approval. Three groups of ESS were prepared with fibroblasts and keratinocytes, +/- hM, and were grafted orthotopically to immunodeficient mice: ESS without hM; ESS with light skin-derived (Caucasian) hM (ESS+hML); and ESS with dark skin-derived (African American) hM (ESS+hMD). After 8 weeks in vivo, grafts were irradiated with 135 mJ/cm2 UV, and mice were euthanized after 2 or 24 hours; non-UV treated mice served as controls. Pigmentation and erythema were measured with a Mexameter. Melanocytes and cyclobutane pyrimidine dimers (CPDs) were quantified by immunostaining with anti-TYRP1 and anti-CPD antibodies, respectively, followed by image analysis (Nikon Elements). Statistical analyses (SigmaPlot) utilized t-test or one-way ANOVA; P&amp;lt; 0.05 was considered significant. Results At 8 weeks post-grafting, mean hM density in ESS+hML and ESS+hMD was not significantly different from normal human skin samples. Pigmentation (in Mexameter units) before UV irradiation was significantly different among groups (ESS+hMD &amp;gt; ESS+hML &amp;gt; ESS no hM). UV irradiation did not increase erythema in any group, but resulted in significantly increased pigmentation in ESS+hML and ESS+hMD at 2 hours, but not 24 hours, post-UV. CPDs, the most prevalent form of UV-induced DNA damage, were significantly elevated 24 hours post-UV in ESS without hM. DNA damage was significantly lower 24 hours post-UV in ESS+hML and ESS+hMD compared with ESS without hM. No differences in DNA damage were observed between ESS+hML and ESS+hMD. Conclusions Pigmentation of ESS+hML and ESS+hMD in vivo varied according to the skin phototype of the hM donor, with no difference in melanocyte density, which was similar to normal human skin. Inclusion of either light or dark hM decreased UV-induced DNA damage, suggesting that hM in ESS play a photoprotective role, as in normal human skin. Applicability of Research to Practice Protection against UV-induced DNA damage may reduce the risk of skin cancer in patients grafted with ESS containing melanocytes.

Light or Dark Pigmentation of Engineered Skin Substitutes Containing Melanocytes Protects Against Ultraviolet Light-Induced DNA Damage In Vivo.

Engineered skin substitutes (ESS) containing autologous fibroblasts and keratinocytes provide stable wound closure in patients with large, full-thickness burns, but are limited by hypopigmentation due to absence of added melanocytes. DNA damage caused by ultraviolet radiation (UV) increases risk for skin cancer development. In human skin, melanocytes provide pigmentation that protects skin from UV-induced DNA damage. This study investigated whether inclusion of human melanocytes (hM) affects the response of ESS to UV in vivo. Specifically, pigmentation and formation of cyclobutane pyrimidine dimers (CPDs), the most prevalent UV-induced DNA photoproduct, were analyzed. Three groups of ESS were prepared with fibroblasts and keratinocytes, ± melanocytes, and grafted orthotopically to immunodeficient mice: ESS without melanocytes (ESS-hM), ESS with light skin-derived (Caucasian) melanocytes (ESS+hM-L), and ESS with dark skin-derived (African-American) melanocytes (ESS+hM-D). Pigmentation of ESS+hM-L and ESS+hM-D increased significantly after grafting; pigmentation levels were significantly different among groups. Mean melanocyte densities in ESS+hM-L and ESS+hM-D were similar to each other and to densities in normal human skin. After 8 weeks in vivo, grafts were irradiated with 135 mJ/cm2 UV; non-UV-treated mice served as controls. UV modestly increased pigmentation in the ESS+hM groups. UV significantly increased CPD levels in ESS-hM, and levels in ESS-hM were significantly greater than in ESS+hM-L or ESS+hM-D. The results demonstrate that light or dark melanocytes in ESS decreased UV-induced DNA damage. Therefore, melanocytes in ESS play a photoprotective role. Protection against UV-induced DNA damage is expected to reduce skin cancer risk in patients grafted with ESS containing autologous melanocytes.

Efficacy of liquid nitrogen and electrocautery assisted gingival depigmentation in term of patient's perception, histological wound healing - A randomized triple blind clinical trial.

Background:Hyper-melanin pigmentation of the gingiva (GMP) is one of the imperative contributory factors for smile-sensitive individuals. Numerous gingival depigmentation (GD) procedures have been attempted in the literature to evaluate the clinical outcome mostly. Hence, a randomized clinical-histopathological triple-blinded trial was planned to evaluate the pain experienced by the patient, gingival wound healing, and density of melanocytes following liquid nitrogen-assisted GD (LNAGD) and electrocautery-assisted GD (ECAGD) procedures.Materials and Methods:Thirty-two arches with bilateral physiologic labial/buccal GMP extending from distal aspect tooth #14–24 and #34–44 in 16 healthy individuals were selected and were equally treated with LNAGD and ECAGD techniques. Dummett oral pigmentation index and Hedin melanin index were evaluated at baseline and 3 months' postoperatively (PO). The visual analog scale was utilized for the intensity of pain assessment at baseline (immediately after treatment) and 1st day and 7th day PO. Histological wound healing and density of melanocytes were evaluated using Gal et al.'s wound-healing assessment index and Patsakas et al.'s criterion, at baseline (0), 8, 24, 72, and 96 h; 1st, 2nd, 3rd, and 4th week; and 3 months and at 0 and 3 months' PO, respectively. Statistical analysis was done using one-way ANOVA, post hoc Tukey, unpaired, and paired “t” test.Results:Both groups showed a statistically significant influence on the parameters evaluated.Conclusion:The LNAGD had a substantial superior result in terms of early wound healing, reduction in density of melanocytes, reduction in pain experienced by the patient, with reduction and delay in the recurrence of GMP.

Melanocytic aggregates with unique morphology associated with regression of basal cell carcinoma.

Spontaneous regression of basal cell carcinomas (BCC) is a well-documented phenomenon. In practice, we have observed melanocytic aggregates associated with BCC at various stages of regression showing unique morphologic features. Fourteen cases featuring melanocytic aggregates were retrospectively identified through a pathology database search. Clinical and histopathologic features were systematically evaluated, and additional immunohistochemical studies were performed. Melanocyte density within tumor nodules was compared to a group of control BCCs. All cases showed BCC at various stages of regression with associated melanocytic aggregates, as highlighted by Melan-A and SOX10 immunostains. Three of 14 cases (21.4%) had only dermal melanocytic nests, while 11 (78.6%) had both junctional and dermal nests. The melanocytic aggregates all had similar asymmetrical architecture and lacked maturation. The melanocytes were small, uniform, bland, and had minimal cytoplasm. Their nuclei were overlapping and hyperchromatic, and had inconspicuous nucleoli. None of the melanocytic aggregates stained for BRAFV600E by immunohistochemistry. No patient developed a recurrent or metastatic melanocytic lesion (median follow-up 42 months). Melanocyte density was higher in the case series than in the control BCCs (P = 0.0008). We described the unique morphology of melanocytic aggregates associated with BCC regression.