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  • Characterization of a novel standardized human three-dimensional skin wound healing model using non-sequential fractional ultrapulsed CO2 laser treatments.

Characterization of a novel standardized human three-dimensional skin wound healing model using non-sequential fractional ultrapulsed CO2 laser treatments.

Lasers in surgery and medicine (2015-03-17)
Yvonne Marquardt, Philipp M Amann, Ruth Heise, Katharina Czaja, Timm Steiner, Hans F Merk, Claudia Skazik-Voogt, Jens M Baron
ABSTRACT

At present, there is no standardized in vitro human skin model for wound healing. Therefore, our aim was to establish and characterize an in vitro/ex vivo three-dimensional (3D) wound healing model, which we employed to analyze the effects of dexpanthenol on wound healing and gene regulation. The novel human 3D skin wound healing model using scaffold and collagen 3D organotypic skin equivalents was irradiated with a non-sequential fractional ultrapulsed CO2 laser. These standardized injured full-thickness skin equivalents enable qRT-PCR, microarray, and histological studies analyzing the effect of topically or systemically applied compounds on skin wound healing. These human laser-irradiated skin models were found to be appropriate for in vitro wound healing analysis. Topical treatment of skin wounds with a 5% dexpanthenol water-in-oil emulsion or two different 5% dexpanthenol oil-in-water emulsions clearly enhanced wound closure compared to laser-irradiated untreated control models. To find out whether this positive effect is caused by the active substance dexpanthenol, laser-irradiated skin models were cultured in calciumpantothenate containing medium (20 μg/ml) compared to skin equivalents cultured without calciumpantothenate. 3D models cultured in calciumpantothenate revealed considerably faster wound closure compared to the control models. Quantitative RT-PCR studies showed enhanced mRNA expression of MMP3, IL1α, keratin-associated protein 4-12 (KRTAP4-12), and decreased expression of S100A7 in laser-irradiated skin models cultured in medium containing calciumpantothenate. This novel standardized human 3D skin wound healing model proves useful for topical pharmacological studies on wound healing and reveals new insights into molecular mechanisms of dexpanthenol-mediated effects on wound healing. In addition, these novel 3D model systems can be used to monitor ex vivo effects of various laser systems on gene expression and morphology of human skin.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Ascorbic acid, FCC, FG
Supelco
Dexpanthenol, Pharmaceutical Secondary Standard; Certified Reference Material
Dexpanthenol, European Pharmacopoeia (EP) Reference Standard
USP
Dexpanthenol, United States Pharmacopeia (USP) Reference Standard
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L-Ascorbic acid, suitable for cell culture, suitable for plant cell culture, ≥98%
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L-Ascorbic acid, 99%
Sigma-Aldrich
L-Ascorbic acid, puriss. p.a., ≥99.0% (RT)
Supelco
L-Ascorbic acid, analytical standard
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L-Ascorbic acid, ACS reagent, ≥99%
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L-Ascorbic acid, powder, suitable for cell culture, γ-irradiated
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L-Ascorbic acid, reagent grade, crystalline
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L-Ascorbic acid, BioXtra, ≥99.0%, crystalline
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L-Ascorbic acid, meets USP testing specifications
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L-Ascorbic acid, puriss. p.a., ACS reagent, reag. ISO, Ph. Eur., 99.7-100.5% (oxidimetric)
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L-Ascorbic acid, BioUltra, ≥99.5% (RT)
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L-Ascorbic acid, tested according to Ph. Eur.
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L-Ascorbic acid, certified reference material, TraceCERT®, Manufactured by: Sigma-Aldrich Production GmbH, Switzerland
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Ascorbic Acid, Pharmaceutical Secondary Standard; Certified Reference Material
USP
Ascorbic acid, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
DAPI, for nucleic acid staining
Ascorbic acid, European Pharmacopoeia (EP) Reference Standard