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Modulation of the Lipid Profile of Reconstructed Skin Substitutes After Essential Fatty Acid Supplementation Affects Testosterone Permeability

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Modulation of the Lipid Profile of Reconstructed Skin Substitutes After Essential Fatty Acid Supplementation Affects Testosterone Permeability

Mélissa Simard et al. Cells.

Abstract

Skin models with efficient skin barrier function are required for percutaneous absorption studies. The contribution of media supplementation with n-3 and n-6 polyunsaturated fatty acids (PUFAs) to the development of the skin barrier function of in vitro skin models remains incompletely understood. To investigate whether PUFAs, alpha-linolenic acid (ALA, n-3 PUFA) and linoleic acid (LA, n-6 PUFA), could enhance the impermeability of a three-dimensional reconstructed human skin model, skin substitutes were produced according to the self-assembly method using culture media supplemented with either 10 μM ALA or 10 μM LA. The impact of PUFAs on skin permeability was studied by using a Franz cell diffusion system to assess the percutaneous absorption of testosterone and benzoic acid. Our findings showed that ALA supplementation induced a decrease in the absorption of testosterone, while LA supplementation did not significantly influence the penetration of testosterone and benzoic acid under present experimental conditions. Both ALA and LA were incorporated into phospholipids of the skin substitutes, resulting in an increase in n-3 total PUFAs or n-6 total PUFAs. Collectively, these results revealed the under-estimated impact of n-3 PUFA supplementation as well as the importance of the n-6 to n-3 ratio on the formation of the skin barrier of in vitro reconstructed human skin models.

Keywords: lipidomics; polyunsaturated fatty acids; skin barrier function; skin substitutes; tissue engineering.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Impact of EFA supplementation on skin substitute cutaneous morphology. (a–c) Macroscopic aspect and (df) histological cross-section after Masson’s trichrome staining of the skin substitutes. Vertical bars indicate epidermal layers. b: basal layer, s: spinous layer, g: granular layer and sc: compact layers of the stratum corneum. Scale bars: (a–c) 1 cm, (d–f) 100 μm. (g) Epidermal and dermal thickness quantified from Masson’s trichrome staining. N = 18 (3 donors, 2 skin substitutes per donor, 3 measurements per skin substitute). One-way ANOVA followed by Tukey’s post-hoc test. p < 0.05 was considered statistically significant.
Figure 2
Figure 2
Skin substitute permeability to testosterone and benzoic acid. Influence of (a,c) ALA and (b,d) LA supplementation on the cumulative dose of (a,b) testosterone and (c,d) benzoic acid absorbed through the skin substitutes. Percutaneous absorption studies were performed on a Franz cells diffusion system. The dosing solutions were freshly prepared in ethanol/water (1:1), yielding a concentration of 4.0 mg/mL for each compound. Compounds were quantified with a Waters Acquity UPLC. Values are mean +/− standard error of the mean (SEM) (3 donors, 6 skin substitutes per donor), p-values were derived from Student’s t-tests. * p < 0.05.
Figure 3
Figure 3
EFA incorporation in the epidermal and dermal phospholipid fraction of the skin substitutes. (a,c,e,g) ALA supplementation impact on (a,e) n-3 PUFAs and (c,g) n-6 PUFAs and (b,d,f,h) LA supplementation impact on (b,f) n-3 PUFAs and (d,h) n-6 PUFAs. PUFAs were quantified following gas chromatography analysis. Results are expressed as μg per g of tissue. Two-way ANOVA followed by Sidak’s post-hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. For skin substitutes: n = 6 (3 donors, 2 skin substitutes per donor). Abbreviations: AA, arachidonic acid; ALA, α-linolenic acid; DGLA, dihomo-γ-linolenic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; DTA, docosatetraenoic acid; EPA, eicosapentaenoic acid; ETA, eicosatetraenoic acid; ETE, eicosatrienoic acid: GLA: γ-linolenic acid; LA, linoleic acid; OsA, osbond acid; PUFAs, polyunsaturated fatty acids.
Figure 4
Figure 4
Proportions of FAs in the skin substitutes quantified by gas chromatography. (a) FA classes are shown as a percentage of total FAs detected in phospholipids of the epidermis or the dermis. (b) Phospholipid FA ratios in the epidermis or the dermis of the skin substitutes. (c) UI of the phospholipid FAs in the epidermis and the dermis. For Substitute: n = 11 (3 donors, 3-4 skin substitutes per donor); for SubstituteALA+ and SubstituteLA+: n = 6 (3 donors, 2 skin substitutes per donor). (a) Two-way ANOVA followed by Tukey’s post-hoc test. (b,c) One-way ANOVA followed by Tukey’s post-hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Abbreviations: ALA, α-linolenic acid; FA, fatty acid; LA, linoleic acid; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; SFA, saturated fatty acid; UI, unsaturation index.
Figure 5
Figure 5
Schematic overview of n-3 and n-6 metabolism in the skin at the air-liquid interface. (a) The ALA and LA PUFAs added into the culture media are incorporated into the phospholipids of cells in contact with the media. During the air-liquid interface culture period, keratinocytes are no longer in contact with the culture media and PUFAs must be provided from the crosstalk with fibroblasts. (b) The PLA2 hydrolyzes PUFAs from the phospholipids. ALA and LA can be converted by a series of desaturation and elongation reactions in both fibroblasts and keratinocytes. (c) After phospholipid hydrolysis, PUFAs can also be brought to the endoplasmic reticulum where they are transformed into more complex lipids such as ceramides. Lipids are stored in lamellar bodies, which then merge with the cell membrane, releasing their content and thus forming the intercellular lipid matrix. (d) PUFAs such as EPA and DHA are activators of PPARs, which regulate keratinocyte differentiation. Abbreviations: AA, arachidonic acid; ALA, α-linolenic acid; Ap-1, activator protein 1; COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; DGLA, dihomo-γ-linolenic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; DTA, docosatetraenoic acid; EPA, eicosapentaenoic acid; ETA, eicosatetraenoic acid; GLA, γ-linolenic acid; LA, linoleic acid; LK, leukotriene; LOX, Lipoxygenase; OsA, osbond acid; PG, prostaglandin; PLA2, phospholipase A2; PPAR, peroxisome proliferator-activated receptor; PUFAs, polyunsaturated fatty acids; SDA, stearidonic acid.

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