Human Probiotic Lactobacillus paracasei-Derived Extracellular Vesicles Improve Tumor Necrosis Factor-α-Induced Inflammatory Phenotypes in Human Skin.

Lactic acid bacteria (LAB), a probiotic, provide various health benefits. We recently isolated a new Lactobacillus paracasei strain with strong anti-inflammatory effects under lipopolysaccharide-induced conditions and proposed a new mode of action-augmenting the endoplasmic reticulum stress pathway for anti-inflammatory functions in host cells. The beneficial effects of the L. paracasei strains on the skin have been described; however, the effects of L. paracasei-derived extracellular vesicles (LpEVs) on the skin are poorly understood. Herein, we investigated whether LpEVs can improve inflammation-mediated skin phenotypes by determining their effects on primary human skin cells and a three-dimensional (3D) full-thickness human skin equivalent under tumor necrosis factor (TNF)-α-challenged inflammatory conditions. LpEVs were efficiently taken up by the human skin cells and were much less cytotoxic to host cells than bacterial lysates. Furthermore, low LpEV concentrations efficiently restored TNF-α-induced cellular phenotypes, resulting in increased cell proliferation and collagen synthesis, but decreased inflammatory factor levels (matrix metalloproteinase 1, interleukin 6, and interleukin 8) in the human dermal fibroblasts, which was comparable to that of retinoic acid, a representative antiaging compound. The beneficial effects of LpEVs were validated in a 3D full-thickness human skin equivalent model. LpEV treatment remarkably restored the TNF-α-induced epidermal malformation, abnormal proliferation of keratinocytes in the basal layer, and reduction in dermal collagen synthesis. Additionally, LpEVs penetrated and reached the deepest dermal layer within 24 h when overlaid on top of a 3D full-thickness human skin equivalent. Furthermore, they possessed superior antioxidant capacity compared with the human cell-derived EVs. Taken together, the anti-inflammatory probiotic LpEVs can be attractive antiaging and antioxidant substances for improving inflammation-induced skin phenotypes and disorders.

On-Site Construction of a Full-Thickness Skin Equivalent with Endothelial Tube Networks via Multilayer Electrospinning for Wound Coverage.

Novel full-thickness skin substitutes are of increasing interest due to the inherent limitations of current models lacking capillary networks. Herein, we developed a novel full-thickness skin tissue containing blood capillary networks through a layer-by-layer assembly approach using a handy electrospinning apparatus and evaluated its skin wound coverage potential in vivo. The average diameter and thickness of fabricated poly-ε-caprolactone-cellulose acetate scaffolds were easily tuned in the range of 474 ± 77-758 ± 113 nm and 9.43 ± 2.23-29.96 ± 5.78 µm by varying electrospinning distance and duration, as indicated by FE-SEM. Besides, keratinocytes exhibited homogeneous differentiation throughout the fibrous matrix prepared with electrospinning distance and duration of 9 cm and 1.5 min within five-layer (5L) epidermal tissues with thickness of 135-150 µm. Moreover, coculture of vascular endothelial cells, circulating fibrocytes, and fibroblasts within the 5L dermis displayed network formation in vitro, resulting in reduced inflammatory factor levels and enhanced integration with the host vasculature in vivo. Additionally, the skin equivalent grafts consisting of the epidermal layer, biomimetic basement membrane, and vascularized dermis layer with an elastic modulus of approximately 11.82 MPa exhibited accelerated wound closure effect indicative of re-epithelialization and neovascularization with long-term cell survival into the host, which was confirmed by wound-healing rate, bioluminescence imaging activity, and histological analysis. It is the first report of a full-thickness skin equivalent constructed using a battery-operated electrospinning apparatus, highlighting its tremendous potential in regenerative medicine.

Plant-Derived Extracts Plus Vitamin E and/or Aloe Vera Protect Against Intrinsic/Extrinsic Stressor in Human Skin: In Vitro and Clinical Evidence.

BACKGROUND: Humans are exposed to physical, biological, chemical, and psychological stressor throughout their life span. In recent years many medicinal plants have been shown to induce stress adapting and protective functions. Plant-derived extracts and vitamin E exhibit stress protection or resistance by normalizing cellular homeostasis and enhancing resistance to toxic stimuli to overcome cellular damage. Here we report the evaluation of a topical preparation (product test materials; PTM) containing an ingredient blend of Rhodiola Rosea, Eleutherococcus Senticosus (Siberian Ginseng), Rhaponticum Carthamoides, Inonotus Obliqus, and Slegainella Lepidophylla as the base formula and tested the addition of Lespedeza Capitata (leaf/stem) extract plus vitamin E and/or Aloe Vera to determine the induced protective functions in human skin when challenged with intrinsic and extrinsic stressors. METHODS: The base topical preparation plus Lespedeza Capitata extract plus vitamin E or the base topical preparation plus vitamin E and Aloe Vera were assayed in vitro on (a) intrinsically stressed excised abdominoplasty skin, (b) full thickness (FT) skin equivalent models post-treated with a combination of ultra-violet (UV) B light (250 mJ/cm2) and diesel particular matter (DPM) (75 µg/mL) skin, for their effect on antioxidant, inflammation, and stress biomarker geners. Additionally, the bioadaptive activity of the PTMs was confirmed in providing resilience and protection against UV-induced erythema. For example, in a clinical study, daily topical application of the PTMs on the buttocks of 20 woman (18-78 years old), average age of 51.1 years, median body mass index (BMI) of 26.5 for 8 weeks followed by 2 minimal erythema dose (MED) of UVB exposure was accessed 24 hours after irradiation. Statistical analysis was performed by t-test and ANOVA, repectively. RESULTS: Pretreatment with the topical PTMs on intrsinically stressed skin significantly reduced the expression of the stress gene biomarkers, p53, pro-inflammatory cytokines Interleukin-1ß (IL-1ß) and Tumor Necrosis Factor-α (TNFα) and the pro-apoptotic BCL2 associated X, apoptosis regulator (BAX) values compared to controls. Topical application of the PTMs on Full Thickness (FT) human skin treated with UVB light and DPM significantly enhanced the stress response by activating heat shock transcription factor 4 (HSF4) and heat shock protein family B (small) member 1 (HSPB1) gene levels belonging to the heat shock protein (HSP) family by significantly increasing the expression of heme oxygenase 1 (HMOX1). At the same time, significantly reducing IL-1ß levels were observed plus protection of skin cells from toxicity ocurred by significantly increasing the expression of B-cell lymphoma 2 (BCL2) (anti-apoptotic gene). In the clinical study, daily topical applications of the PTMs for 8 weeks followed by 2MED of UVB irradiation with clinical assessment 24 hours later revealed a significantly reduced intensity of erythema when compared to the buttock region treated with UVB alone. CONCLUSIONS: The PTMs containing adaptogen ingredients may confer stress resistance and induce stress protective responses against intrinsic as well as extrinsic stressors as demonstrated by the obtained in vitro and clinical evidence.

Advanced Online Monitoring of In Vitro Human 3D Full-Thickness Skin Equivalents.

Skin equivalents and skin explants are widely used for dermal penetration studies in the pharmacological development of drugs. Environmental parameters, such as the incubation and culture conditions affect cellular responses and thus the relevance of the experimental outcome. However, available systems such as the Franz diffusion chamber, only measure in the receiving culture medium, rather than assessing the actual conditions for cells in the tissue. We developed a sampling design that combines open flow microperfusion (OFM) sampling technology for continuous concentration measurements directly in the tissue with microfluidic biosensors for online monitoring of culture parameters. We tested our design with real-time measurements of oxygen, glucose, lactate, and pH in full-thickness skin equivalent and skin explants. Furthermore, we compared dermal penetration for acyclovir, lidocaine, and diclofenac in skin equivalents and skin explants. We observed differences in oxygen, glucose, and drug concentrations in skin equivalents compared to the respective culture medium and to skin explants.

Engineering a human skin equivalent to study dermis remodelling and epidermis senescence in vitro after UVA exposure.

Utra Violet type A (UVA) exposure strongly affects the ageing of human skin by modifying both epidermis and dermis and their cross talk as well. The possibility to get a deep understanding in vitro of such crucial mechanism would have a huge impact in the development of antiageing compounds. Here, we present a full thickness model of human skin equivalent formed by a millimeter-sized dermis completely composed of fibroblasts embedded in their own extracellular matrix. We show that such endogenous nature of the dermis compartment allows the replication of the complexity of the mutual interactions occurring between cellular and extracellular components of the skin under UVA exposure: (a) oxidative stress formation in the whole tissue (dermis and epidermis); (b) senescence of germinative layer of epidermal tissue in terms of p63, ki67, and activated caspase-3 regulation; (c) modification of the collagenous network architecture in the dermis compartment. By using this human skin model, it is possible to study a widely shared assumptions not yet proved in vitro such the effect of UVA on the self-renewal capability of skin stem cells.