Abstract
Polyunsaturated fatty acids (PUFAs) play an important role in the establishment and the maintenance of the skin barrier function. However, the impact of their derived lipid mediators remains unclear. Skin substitutes were engineered according to the self-assembly method with a culture medium supplemented with 10 μM of both α-linolenic acid (ALA) and linoleic acid (LA). The supplementation with ALA and LA decreased testosterone absorption through a tissue-engineered reconstructed skin model, thus indicating an improved skin barrier function following supplementation. The exogenously provided fatty acids were incorporated into the phospholipid and triglyceride fractions of the skin substitutes. Indeed, the dual supplementation increased the levels of eicosapentaenoic acid (EPA) (15-fold), docosapentaenoic acid (DPA) (3-fold), and LA (1.5-fold) in the epidermal phospholipids while it increased the levels of ALA (>20-fold), DPA (3-fold) and LA (1.5-fold) in the epidermal triglycerides. The bioactive lipid mediator profile of the skin substitutes, including prostaglandins, hydroxy-fatty acids, N-acylethanolamines and monoacylglycerols, was next analyzed using liquid chromatography-tandem mass spectrometry. The lipid supplementation further modulated bioactive lipid mediator levels of the reconstructed skin substitutes, leading to a lipid mediator profile more representative of the one found in normal human skin. These findings show that an optimized supply of PUFAs via culture media is essential for the establishment of improved barrier function in vitro.
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Key Points
Skin barrier function is provided by the outermost layer of the epidermis, the stratum corneum (SC), which is composed of corneocytes embedded in a lipid matrix. The SC is produced during a multistep process of differentiation of the epidermal keratinocytes in which lipid metabolism plays an essential role, both at a structural and a homeostatic level. First, fatty acids in the skin can be synthesized de novo or can be taken up from the diet or extracutaneous sites, with the exception of short-chain PUFAs such as α-linolenic acid (ALA) and LA, which cannot be synthesized in the skin and need to be obtained exclusively through dietary sources. The contribution of LA to the skin barrier is crucial, since it is incorporated into ω-hydroxylated ceramides. These ceramides are covalently bound to the corneocytes and serve as a scaffold for other lipids, allowing an optimal organization of the lipid matrix. The epidermal differentiation is highly regulated by different signaling systems, involving many different actors such as kinases and also a large number of bioactive lipid mediators derived from PUFAs.
The complex structure of the SC still represents a challenge to replicate in vitro. The tissue-engineered bilayered human skin model is the model that currently best represents normal human skin morphology. It is produced using both primary fibroblasts and keratinocytes and includes culture steps at the air-liquid interface, which allows complete differentiation of the reconstructed epidermis. Many factors can influence the formation of the barrier function in the reconstructed skin model, one of them being the fatty acids of the media used during long-term culture. Because of their essential nature, PUFAs must therefore be added to the culture media. ALA has never been documented as influencing skin permeability. Additionally, ALA is a preferred substrate for beta-oxidation and carbon recycling, and it possesses potential anti-inflammatory action in diverse diseases. Therefore, these previous results highlight the importance of the n-3 to n-6 PUFA ratio in cutaneous homeostasis. In the present study, the impact of a combined supplementation with ALA and LA on the permeability of the skin substitutes was assessed. In addition, the impact of such supplementation on the levels of bioactive lipid mediators in the skin substitutes was also analyzed in order to better understand the metabolism of PUFAs within the skin.
The present report is the first to study the lipidome of a tissue-engineered human skin model and its impact on the skin barrier function. As expected, the reconstructed skin substitutes displayed lower proportions of n-3 and n-6 PUFA. These results are consistent with other studies, in which a deficiency of n-6 PUFAs was found in tissue-engineered human skin produced with culture media that were not supplemented with PUFAs. The exogenous provision of ALA and LA had a similar impact on the barrier function of the reconstructed skin substitutes. These results showed that the metabolism of the n-3 and n-6 polyunsaturated fatty acids remains functional in the skin model produced in vitro.
The findings described in the present study show that an optimized supply of EFA via culture media is essential for the establishment of the barrier function in vitro, as it largely affects the lipidome of the reconstructed skin. The present study shows that n-6 PUFAs, and more specifically LA, play a crucial role in the skin barrier function and that they need to be used in combination with n-3 PUFAs for optimal incorporation and metabolization by the reconstructed tissue.
Supplementation of the culture medium with 10 μM of both α-linolenic acid (ALA) and linoleic acid (LA) improved the skin barrier function of a tissue-engineered skin model. The exogenously provided fatty acids were incorporated into the phospholipid and triglyceride fractions of the skin substitutes and further modulated bioactive lipid mediator levels, including prostaglandins, hydroxy-fatty acids, N-acylethanolamines and monoacylglycerols. These findings highlight the important role of ALA and LA in skin homeostasis and show that an optimized supply of polyunsaturated fatty acids via culture media is essential for the establishment of improved barrier function in vitro.