Characterization of a novel standardized human three‐dimensional skin wound healing model using non‐sequential fractional ultrapulsed CO ₂ laser treatments

Abstract

Background and Objective: 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. Materials and Methods: The novel human 3D skin wound healing model using scaffold and collagen 3D organotypic skin equivalents was irradiated with a non-sequential fractional ultrapulsed CO 2 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. Results: 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 oilin-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 mg/ 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, IL1a, keratinassociated protein 4-12 (KRTAP4-12), and decreased expression of S100A7 in laser-irradiated skin models cultured in medium containing calciumpantothenate. Conclusion: 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.

Macrophages significantly enhance wound healing in a vascularized skin model

“…The cultivation period for our model needed to balance the requirements for angiogenesis and epithelium formation, and preliminary tests revealed the formation of vascular structures and stratified epithelium following cultivation under air–liquid interface conditions for 7–14 days. The selected cultivation period of 10 days at an air–liquid interface was chosen to prepare the samples for treatment with the CO 2 laser based on a previous report, as were the sampling points 0, 2, and 3 days after laser treatment …”

Topical use of dexpanthenol: a 70th anniversary article

“…Topical treatment of skin wounds with different dexpanthenol containing preparations clearly enhanced wound closure compared to untreated or vaseline-treated controls. Gene expression analysis showed increased mRNA expression of genes involved in wound healing (16).…”

In vitro wound healing assays – state of the art

“…As a result, there have been attempts to perform wounding in an automated manner, using devices (e.g. a rotating drill) [28] or lasers already used in dermatology [27] which inflict damage to the layer in a reproducible manner.…”

“…Several groups have made attempts to standardize the 3D wound healing assay procedure [27,28]. After the construct has matured, some form of wounding is carried out.…”