Bioengineered Pigmented Skin Equivalent for Wound Recovery

Drug Discovery Assay – reference number: B128

Overview

Testing Information

Assay Description

This platform can be used to assess the impact of wounding on skin health, and the potential ability of candidate actives to enhance the wound recovery process. A comparison can be drawn between standard model recovery data sets and those obtained with the candidate active-added test conditions. Wound generation can be discussed and tailored to the individual study specifications. Example wound types include scalpel cut to mimic acute trauma, tape-stripping to mimic diffuse mild trauma and temperature-controlled burns of a defined size. The specific results that will be provided upon request are the effects of test articles on: Transepidermal water loss (TEWL), gross morphology, histology and specific biomarker analysis. 

Test Article Requirements

Test article(s) to be provided by the Sponsor in storable aliquots at required test concentrations with information on the diluent vehicle used. Test compounds can either be applied to the model system via a systemic approach in the underlying media, or topically applied to the surface of the skin either in liquid or cream format. Stock solutions are prepared in distilled water unless otherwise requested. Sponsor to provide sufficient test article to run the entire study. 

Suggested Testing

A minimum of triplicate per condition with a maximum of 30 models per experiment. Several experiments can be run in a single study to increase model throughput.

Study Outline

Rationale and Experimental Design

To construct the bioengineered tissue representative of human skin, our researchers first generate a dermal foundation. The dermal foundation consists of fibroblasts seeded within Avletex® Scaffold, which populate the scaffold and neosynthesise endogenous extracellular matrix (ECM), along with providing paracrine support to the overlying epidermal tissue, in the form of soluble growth factors. Keratinocytes are then seeded upon the dermal foundation and following proliferation and differentiation phases, form mature stratified epidermal constructs at the air-liquid interface (ALI).  

Schematic representation of culture process. Downstream applications include both pigmentary studies through addition of single active to the culture medium or topical formulation application, and UV challenge. 

Once skin constructs have matured, wounding of models and application of the test actives can commence. This may be in the form of topical application of a complete formulation, or the soluble addition to culture medium of a candidate active as desired. End point analyses include gross morphology of wound site, recovery and repair markers and skin damage marker expression. 

Addition of Test Articles

Test articles can take the form of solubilized compounds added directly to the culture medium, or topically applied formulations to the surface of the skin. It is recommended a minimum of triplicates should be used for each condition. An example of the conditions assessed for three test agents would be as follows: 

1. Untouched Control
2. Sham Control 
3. Test Agent 1
4. Test Agent 2
5. Test Agent 3 
6. Positive Control 

End Point Analysis

There are a range of endpoints that can be used to assess effectiveness in this system including:  

• Barrier Integrity: Histological assessment, immunofluorescent staining, transepidermal water loss (TEWL) measurements 

• Cytotoxicity: Histological assessment, TUNEL assay to detect and quantify apoptotic cells 

• Biomarkers of Wound Repair: Histological assessment, immunofluorescent staining. 

• Morphology of Wound Site: Gross Images, Histological assessment, immunofluorescent staining. 

Example data

Diffuse Mild Trauma: Tape stripping induces mild trauma-like phenotype in-vitro which can be recovered. The application of a moisturizer post-insult enhances recovery. 

Schematic protocol depicting the adhesion and removal of tape strips from FT-HSEs in vitro (A). Tape strips were applied with controlled pressure using a spring-loaded applicator and removed from the surface of HSEs using forceps. Cellular material is visible on tape strips upon removal and models were either analysed immediately or returned to culture for 4 days prior to analysis. H&E staining (B) of HSEs immediately following insult (Ba-c) ranging from 0 -10 tape strip removal, or following a 4 day recovery period (Be-g). Transepidermal water loss (TEWL) increased with increasing number of tape strips immediately following insult (C) (data represent mean ± SEM, n=9). TEWL recovered over a 4 day period (D) (data represent ± SEM, n=9)  to a level comparable to 0 tape strip controls (dashed line). Scale Bar: 100 µm. ** = p < 0.01, **** = p < 0.0001. 

Schematic protocol depicting tape strip application and subsequent topical application (A). Two tape strips were removed from HSEs, a moisturising formulation was added topically to the surface and spread evenly using a glass rod, HSEs were then returned to culture for 4 days. H&E staining of HSEs immediately following removal of two strips (Ba) and 4-days post-insult treated with (Bc) and without moisturisation (Bb) or with no tape strips removed (Bd). Representative images of Ki67 staining (B e-h) (ki67 green, nuclei blue), demonstrating changes in proliferation marker expression with treatment conditions. Transepidermal water loss (TEWL) (C) (data represent mean ± SEM, n=3) as a percentage of day 0 measurements. TEWL increases with time in culture without tape stripping (dashed line, 0 TS) but to a greater degree with tape stripping, which is reduced with post-tape strip moisturisation. Epidermal thickness (D) (data represent mean ± SEM, n=3) and percentage ki67 positive cells (E) (data represent mean ± SEM, n=3) are both increased with tape strip insult and reduced by post-insult moisturisation. Concentration of pro-inflammatory cytokines: TNFα (F), IL-1β (G), IL-Ra (H) and IL-12p40 (I) were increased in the culture medium following tape strip insult, and subsequently reduced by moisturisation (data represent mean ± SEM, n=6). Scale Bar: 50 µm. * = p < 0.1, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. 

Development of a novel in vitro strategy to understand the impact of shaving on skin health: combining tape strip exfoliation and human skin equivalent technology. Costello L, Goncalves K, Maltman V, Barrett N, Shah K, Stephens A, Dicolandrea T, Ambrogio I, Hodgson E, Przyborski S. Frontiers in Medicine, section Dermatology, 2023, 2;10:1236790. DOI: 10.3389/fmed.2023.1236790. eCollection 2023.  

Acute Trauma Model Generation and Subsequent Recovery and Repair 

Demonstration of acute trauma model system. The cut is created in the epidermis alone, or in both the epidermis and dermis, in a controlled manner, and the wound healing process is monitored for up to 14 days. The wound has closed and a new stratum corneum is present after 3 days of culture, post-insult.