Contribution of Palmitic Acid to Epidermal Morphogenesis and Lipid Barrier Formation in Human Skin Equivalents

The outermost barrier layer of the skin is the stratum corneum (SC), which consists of corneocytes embedded in a lipid matrix. Biosynthesis of barrier lipids occurs de novo in the epidermis or is performed with externally derived lipids. Hence, in vitro developed human skin equivalents (HSEs) are developed with culture medium that is supplemented with free fatty acids (FFAs). Nevertheless, the lipid barrier formation in HSEs remains altered compared to native human skin (NHS). The aim of this study is to decipher the role of medium supplemented saturated FFA palmitic acid (PA) on morphogenesis and lipid barrier formation in HSEs. Therefore, HSEs were developed with 100% (25 μM), 10%, or 1% PA. In HSEs supplemented with reduced PA level, the early differentiation was delayed and epidermal activation was increased. Nevertheless, a similar SC lipid composition in all HSEs was detected. Additionally, the lipid organization was comparable for lamellar and lateral organization, irrespective of PA concentration. As compared to NHS, the level of monounsaturated lipids was increased and the FFA to ceramide ratio was drastically reduced in HSEs. This study describes the crucial role of PA in epidermal morphogenesis and elucidates the role of PA in lipid barrier formation of HSEs.Drug Delivery Technolog

Human skin equivalents cultured under hypoxia display enhanced epidermal morphogenesis and lipid barrier formation

Human skin equivalents (HSEs) are three-dimensional cell models mimicking characteristics of native human skin (NHS) in many aspects. However, a limitation of HSEs is the altered in vitro morphogenesis and barrier formation. Differences between in vitro and in vivo skin could have been induced by suboptimal cell culture conditions, of which the level of oxygen in vitro (20%) is much higher than in vivo (0.5-8%). Our aim is to study how external oxygen levels affect epidermal morphogenesis and barrier formation in HSEs. In the present study, fibroblast and keratinocyte monocultures, and HSEs were generated under 20% (normoxia) and 3% (hypoxia) oxygen level. In all cultures under hypoxia, expression of hypoxia-inducible factor target genes was increased. Characterization of HSEs generated under hypoxia using immunohistochemical analyses of morphogenesis biomarkers revealed a reduction in epidermal thickness, reduced proliferation, similar early differentiation, and an attenuated terminal differentiation program compared to normoxia, better mimicking NHS. The stratum corneum ceramide composition was studied with liquid chromatography coupled to mass spectrometry. Under hypoxia, HSEs exhibited a ceramide composition that more closely resembles that of NHS. Consequently, the lipid organization was improved. In conclusion, epidermal morphogenesis and barrier formation in HSEs reconstructed under hypoxia better mimics that of NHS.Drug Delivery Technolog

Excess Quantum Noise due to Nonorthogonal Polarization Modes

We show that the quantum-limited linewidth of a laser can be enhanced when the polarization eigenmodes of the laser resonator are nonorthogonal. For the theoretical description of this phenomenon we introduce a simple coupled two-mode model. Experimentally, we observed an enhancement of the quantum noise by a factor of 60 in a He-Xe gas laser

A multidirectional approach to optimize morphogenesis and barrier characteristics of human skin equivalents

Cell-based in vitro developed human skin equivalents facilitate screenings of compounds for therapeutic potential or toxicity and enable scientific research expanding knowledge on skin physiology and pathophysiology. Human skin equivalents resemble key features of native human skin, including the dermal and epidermal architecture. However, a limitation of human skin equivalents is the altered lipid barrier formation, which leads to a decreased barrier functionality. This could be induced by suboptimal cell culture conditions or the different cell microenvironment. The primary aim of this dissertational research was to enhance the morphogenesis and barrier formation of human skin equivalents to better mimic that of native human skin. The results indicate that modification of the dermal extracellular matrix by the biopolymer chitosan enhanced epidermal morphogenesis and barrier formation. Furthermore, by better resembling native skin conditions in vitro, primarily through a reduction in oxygen level, the epidermal morphogenesis and lipid barrier formation was improved. Finally, using a combinatory approach of optimized cell culture conditions and enhanced cell culture medium, the epidermal morphogenesis and barrier formation of human skin equivalents resembled that of native human skin more closely.TTW Stichting ProefdiervrijDrug Delivery Technolog

An ex vivo human skin model for studying skin barrier repair

In the studies described in this study, we introduce a novel ex vivo human skin barrier repair model. To develop this, we removed the upper layer of the skin, the stratum corneum (SC) by a reproducible cyanoacrylate stripping technique. After stripping the explants, they were cultured in vitro to allow the regeneration of the SC. We selected two culture temperatures 32 °C and 37 °C and a period of either 4 or 8 days. After 8 days of culture, the explant generated SC at a similar thickness compared to native human SC. At 37 °C, the early and late epidermal differentiation programmes were executed comparably to native human skin with the exception of the barrier protein involucrin. At 32 °C, early differentiation was delayed, but the terminal differentiation proteins were expressed as in stripped explants cultured at 37 °C. Regarding the barrier properties, the SC lateral lipid organization was mainly hexagonal in the regenerated SC, whereas the lipids in native human SC adopt a more dense orthorhombic organization. In addition, the ceramide levels were higher in the cultured explants at 32 °C and 37 °C than in native human SC. In conclusion, we selected the stripped ex vivo skin model cultured at 37 °C as a candidate model to study skin barrier repair because epidermal and SC characteristics mimic more closely the native human skin than the ex vivo skin model cultured at 32 °C. Potentially, this model can be used for testing formulations for skin barrier repair.Drug Delivery Technolog

Multitargeted approach for the optimization of morphogenesis and barrier formation in human skin equivalents

In vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to mimic native human skin in many fundamental aspects. However, the epidermal lipid barrier formation, which is essential for the functionality of the skin barrier, remains compromised. Recently, HSEs with an improved lipid barrier formation were generated by (i) incorporating chitosan in the dermal collagen matrix, (ii) reducing the external oxygen level to 3%, and (iii) inhibiting the liver X receptor (LXR). In this study, we aimed to determine the synergic effects in full-thickness models (FTMs) with combinations of these factors as single-, double-, and triple-targeted optimization approaches. The collagen-chitosan FTM supplemented with the LXR inhibitor showed improved epidermal morphogenesis, an enhanced lipid composition, and a better lipid organization. Importantly, barrier functionality was improved in the corresponding approach. In conclusion, our leading optimization approach substantially improved the epidermal morphogenesis, barrier formation, and functionality in the FTM, which therefore better resembled native human skin.Drug Delivery Technolog

Shedding light on the effects of 1,25-dihydroxyvitamin D3 on epidermal lipid barrier formation in three-dimensional human skin equivalents

Human skin equivalents (HSEs) are three dimensional models resembling native human skin (NHS) in manyaspects. Despite the manifold similarities to NHS, a restriction in its applications is the altered in vitro lipidbarrier formation, which compromises the barrier functionality. This could be induced by suboptimal cell culturingconditions, which amongst others is the diminished activation of the vitamin D receptor (VDR) signallingpathway. The active metabolite of this signalling pathway is 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). An interactingrole in the formation of the skin barrier has been ascribed to this pathway, although it remains unresolvedto which extent this pathway contributes to the (mal-)formation of the epidermal barrier in HSEs. Ouraim is to study whether cell culture medium enriched with 1,25(OH)2D3 affects epidermal morphogenesis andlipid barrier formation in HSEs. Addition of 20 nM 1,25(OH)2D3 resulted in activation of the VDR signallingpathway by inducing transcription of VDR target genes (CYP24A and LL37) in keratinocyte monocultures and inHSEs. Characterization of HSEs supplemented with 1,25(OH)2D3 using immunohistochemical analyses revealeda high similarity in epidermal morphogenesis and in expression of lipid processing enzymes. The barrier formationwas assessed using state-of-the art techniques analysing lipid composition and organization. Addition of1,25(OH)2D3 did not alter the composition of ceramides. Additionally, the lateral and lamellar organization ofthe lipids was similar, irrespective of supplementation. In conclusion, epidermal morphogenesis and barrierformation in HSEs generated in presence or absence of 1,25(OH)2D3 leads to a similar morphogenesis andcomparable barrier formation in vitro.Drug Delivery Technolog

Recapitulation of Native Dermal Tissue in a Full-Thickness Human Skin Model Using Human Collagens

Dermatology-oncolog

Incorporation of chitosan in the dermal matrix improves epidermal barrier formation in full thickness human skin equivalents

Dermatology-oncolog