Photobiomodulation of human dermal fibroblasts in vitro: decisive role of cell culture conditions and treatment protocols on experimental outcome

YesPhotobiomodulation-based (LLLT) therapies show tantalizing promise for treatment of skin diseases. Confidence in this approach is blighted however by lamentable inconsistency in published experimental designs, and so complicates interpretation. Here we interrogate the appropriateness of a range of previously-reported treatment parameters, including light wavelength, irradiance and radiant exposure, as well as cell culture conditions (e.g., serum concentration, cell confluency, medium refreshment, direct/indirect treatment, oxygen concentration, etc.), in primary cultures of normal human dermal fibroblasts exposed to visible and near infra-red (NIR) light. Apart from irradiance, all study parameters impacted significantly on fibroblast metabolic activity. Moreover, when cells were grown at atmospheric O2 levels (i.e. 20%) short wavelength light inhibited cell metabolism, while negligible effects were seen with long visible and NIR wavelength. By contrast, NIR stimulated cells when exposed to dermal tissue oxygen levels (approx. 2%). The impact of culture conditions was further seen when inhibitory effects of short wavelength light were reduced with increasing serum concentration and cell confluency. We conclude that a significant source of problematic interpretations in photobiomodulation reports derives from poor optimization of study design. Further development of this field using in vitro/ex vivo models should embrace significant standardization of study design, ideally within a design-of-experiment setting

Does blue light restore human epidermal barrier function via activation of Opsin during cutaneous wound healing?

YesBackground and Objective Visible light has beneficial effects on cutaneous wound healing, but the role of potential photoreceptors in human skin is unknown. In addition, inconsistency in the parameters of blue and red light‐based therapies for skin conditions makes interpretation difficult. Red light can activate cytochrome c oxidase and has been proposed as a wound healing therapy. UV‐blue light can activate Opsin 1‐SW, Opsin 2, Opsin 3, Opsin 4, and Opsin 5 receptors, triggering biological responses, but their role in human skin physiology is unclear. Materials and Methods Localization of Opsins was analyzed in situ in human skin derived from face and abdomen by immunohistochemistry. An ex vivo human skin wound healing model was established and expression of Opsins confirmed by immunohistochemistry. The rate of wound closure was quantitated after irradiation with blue and red light and mRNA was extracted from the regenerating epithelial tongue by laser micro‐dissection to detect changes in Opsin 3 (OPN3) expression. Retention of the expression of Opsins in primary cultures of human epidermal keratinocytes and dermal fibroblasts was confirmed by qRT‐PCR and immunocytochemistry. Modulation of metabolic activity by visible light was studied. Furthermore, migration in a scratch‐wound assay, DNA synthesis and differentiation of epidermal keratinocytes was established following irradiation with blue light. A role for OPN3 in keratinocytes was investigated by gene silencing. Results Opsin receptors (OPN1‐SW, 3 and 5) were similarly localized in the epidermis of human facial and abdominal skin in situ. Corresponding expression was confirmed in the regenerating epithelial tongue of ex vivo wounds after 2 days in culture, and irradiation with blue light stimulated wound closure, with a corresponding increase in OPN3 expression. Expression of Opsins was retained in primary cultures of epidermal keratinocytes and dermal fibroblasts. Both blue and red light stimulated the metabolic activity of cultured keratinocytes. Low levels of blue light reduced DNA synthesis and stimulated differentiation of keratinocytes. While low levels of blue light did not alter keratinocyte migration in a scratch wound assay, higher levels inhibited migration. Gene silencing of OPN3 in keratinocytes was effective (87% reduction). The rate of DNA synthesis in OPN3 knockdown keratinocytes did not change following irradiation with blue light, however, the level of differentiation was decreased. Conclusions Opsins are expressed in the epidermis and dermis of human skin and in the newly regenerating epidermis following wounding. An increase in OPN3 expression in the epithelial tongue may be a potential mechanism for the stimulation of wound closure by blue light. Since keratinocytes and fibroblasts retain their expression of Opsins in culture, they provide a good model to investigate the mechanism of blue light in wound healing responses. Knockdown of OPN3 led to a reduction in early differentiation of keratinocytes following irradiation with blue light, suggesting OPN3 is required for restoration of the barrier function. Understanding the function and relationship of different photoreceptors and their response to specific light parameters will lead to the development of reliable light‐based therapies for cutaneous wound healing.European Commission 7th Framework Programme for Research and Technical Development - Marie Curie Innovative Training Networks (ITN), Grant agreement no.: 60788

Development of a Novel Approach to Studying Corneodesmosomes and Stratum Corneum Adhesion: Extending Knowledge on the Pathophysiology of Sensitive Skin

BACKGROUND/AIMS: Aberrant skin barrier and intercorneocyte adhesion are potential contributors to the pathomechanism of sensitive skin (SS). Here we aimed to develop a novel and easy-to-apply method to analyze corneodesmosomes and to interrogate potential differences between corneocytes of subjects with SS and non-SS (NSS). METHODS: Corneocytes of the volar forearm and upper outer quadrant of the left buttock of SS (n = 10) and NSS (n = 8) subjects were extracted as a function of depth using adhesive tape and stained with anti-desmoglein 1 (DSG1) antibody. The total area of corneocytes and the number and average size of cells per tape was estimated using image processing. RESULTS: The total area of extracted corneocytes and the quantity of DSG1 decreased with depth. The level of decrease, total area of corneocytes, and average area of individual cells differed between anatomical locations. In SS, a larger total area of extracted corneocytes and a larger average cell size per tape was found at all inspected depths. CONCLUSION: The developed novel and easy-to-apply approach allows investigation of corneodesmosome components. We confirm a role of altered corneocytes in the pathomechanism of SS. The disclosed protocol can further be optimized in studies of skin conditions with strongly affected corneodesmosomes

Sensitive skin and the influence of female hormone fluctuations: results from a cross-sectional digital survey in the Dutch population

Sensitive skin is a widespread condition, which is most frequently reported by women. Changing hormone levels during the menstrual cycle and menopause have been suggested among the stimuli triggering sensitive skin. To investigate the perceived influence of fluctuating hormone levels on self-assessed sensitive skin, including symptoms and stimuli linked to skin sensitivity, as well as potential changes in facial and body skin and sensitive body parts, depending on hormonal status. A digital questionnaire was distributed to a population of women aged 20-65 years old. A total of 278 women were included in the analysis. About 42+ACU- premenopausal women declared a perception of (increased) skin sensitivity just before and during the menstrual cycle, while this was reported by almost 32+ACU- of peri- and postmenopausal women following the menopause. The majority of reported symptoms included the presence of bumps/pimples, dryness, itching, and redness, and the majority of reported stimuli were shaving, weather, toiletries, and emotions. No differences emerged regarding characteristics of facial and body skin across different hormonal status. Significant differences in sensitivity of body parts emerged for the face and feet, reported by a larger percentage of premenopausal women and peri- and postmenopausal women, respectively. The prevalence of the perceived effects of fluctuating hormone levels on self-assessed sensitive skin in women is high. These effects should be taken into consideration in skin testing and dermatological practice, and support the need for selecting personal care routine or treatment during the menstrual cycle and menopause

Risk factors associated with sensitive skin and potential role of lifestyle habits: a cross-sectional study

Item does not contain fulltextSensitive skin (SS) is a widespread condition, but still not completely understood. To identify risk factors that increase the likelihood of SS, 258 women aged between 20 and 65 years old and resident in the Netherlands were surveyed by questionnaire, which included questions on sociodemographic characteristics (age group, Fitzpatrick skin type, hormonal status), health state (atopic predisposition, skin diseases) and lifestyle habits (history of smoking and of sun exposure, frequency of physical exercise). Analysis of the responses confirmed that atopic predisposition, presence of skin diseases and Fitzpatrick skin types I and II are risk factors significantly associated with SS. In addition, as current or past smoking and a history of low sun exposure showed a trend to increase the likelihood of reporting SS, we suggest that the potential role of lifestyle factors in the onset or exacerbation of SS should be investigated further

Effects of red light on inflammation and skin barrier recovery following acute perturbation. Pilot study results in healthy human subjects

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A model for perception-based identification of sensitive skin

Contains fulltext : 169643.pdf (publisher's version ) (Closed access)BACKGROUND: With high prevalence of sensitive skin (SS), lack of strong evidence on pathomechanisms, consensus on associated symptoms, proof of existence of 'general' SS and tools to recruit subjects, this topic attracts increasing attention of research. OBJECTIVE: To create a model for selecting subjects in studies on SS by identifying a complete set of self-reported SS characteristics and factors discriminatively describing it. METHODS: A survey (n +AD0- 3058) was conducted, comprising questions regarding socio-demographics, atopy, skin characteristics, personal care, degree of self-assessed SS and subjective and objective reactions to endogenous and exogenous factors. Exploratory factor analysis on 481 questionnaires was performed to identify underlying dimensions and multivariate logistic regression to find contributing variables to the likelihood of reporting SS. RESULTS: The prevalence of SS was found to be 41+ACU-, and 56+ACU- of SS subjects reports a concomitant atopic condition. The most discriminative were the eliciting factors toiletries and emotions, and not specific skin symptoms in general. CONCLUSION: Triggers of different origins seem to elicit SS, it is not defined by concomitant skin diseases only, suggesting existence of 'general' SS. A multifactorial questionnaire could be a better diagnostic than a one-dimensional provocative test

Effects of blue light on inflammation and skin barrier recovery following acute perturbation. Pilot study results in healthy human subjects

Contains fulltext : 192145.pdf (publisher's version ) (Closed access

Clinical, biophysical and immunohistochemical analysis of skin reactions to acute skin barrier disruption - a comparative trial between participants with sensitive skin and those with nonsensitive skin

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Responses to sodium dodecyl sulphate as an in vivo human model to study the pathomechanisms underlying sensitive skin

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