The linear excisional wound: an improved model for human ex vivo wound epithelialization studies

Abstract

Background/purpose: Wound healing is a complex process that involves multiple intercellular and intracellular processes and extracellular interactions. Explanted human skin has been used as a model for the re-epithelialization phase of human wound healing. The currently used standard technique uses a circular punch biopsy tool to make the initial wound. Despite its wide use, the geometry of round wounds makes it difficult to measure them reliably.

Methods: Our group has designed a linear wounding tool, and compared the variability in ex vivo human linear and circular wounds.

Results: An F test for differences in variances demonstrated that the linear wounds provided a population of wound size measurements that was 50% less variable than that obtained from a group of matched circular wounds. This reduction in variability would provide substantial advantages for the linear wound technique over the circular wound punch technique, by reducing the sample sizes required for comparative studies of factors that alter healing.

Conclusion: This linear wounding tool thus provides a method for wounding that is standardized, provides minimal error in wound gap measurements, and is easily reproducible. We demonstrate its utility in an ex vivo model for the controlled investigation of human skin wounds.

Figure 1. Linear wounds in explanted human skin

(a) Schematic representation of the linear excision tool. Schematic representation of the linear excision tool used for creating ex-vivo linear wounds examined in this study. Two scalpel blade handles were approximated and bonded to each other with cement, blades positioned at 45° angles, and blade-to-blade width set at 2 mm. (b) Human ex-vivo linear wounds in low serum conditions maintain cell viability and re-epithelialize after the replacement of low serum media with normal serum media. Both linear and circular wounds were incubated in low serum containing media for 4 days. No evidence of reepithelialization was found in day 4 samples. Samples were then split, one group continued to receive low serum media while the other group received normal serum media up until day 9. Wounds that received normal containing media were able to fully re-epithelialize while those in low serum media remained unhealed. The wound gap was determined as the measured distance between the originally cut epithelial borders of the wound (arrows, lower left image), as visualized with H&E staining under light microscopy. The new epithelial growth is easily differentiated from the original wound margin by the absence of fully stratified epithelium and absence of the stratum corneum (outlined and easily visualized) at the wound.

Figure 2. Linear ex-vivo wounds have less variability in wound gap measurements compared to circular punch wounds

Linear and circular wounds were compared by analyzing wound gap mean values, variance, and statistical analysis. (a) Box plot whiskers illustrate the range of wound gap measurements in millimeters. Cross bar in the boxes represents the mean wound gap values in the sample population. (b) Numerical values of wound gap minimum, maximum, mean, standard deviation of the two wounding techniques, and a two-sample t test with unequal variances. Statistical significance for the two-sample t test with unequal variances comparing wound gap means was at a p value = 0.0033. (c) Variance for each sample population (linear and circular wounds) was measured. An F test of equality of variances was employed for statistical analysis. Statistical significance for the F test was at a p value = 0.0072.

Figure 3. Potential for variation in circular vs linear wounds

. (a) Circular punch biopsy wounds can vary greatly in wound gap after histological sectioning. Based on a horizontal circular wound, vertical planes sectioned across the wound can generate different wound gaps values. (b) Linear excision wounds are consistently less variable in wound gap after histological sectioning compared to circular punch biopsies. The rectangular geometry of the linear excision wound creates very uniform sides and wound gaps along the entire length of all the vertical sections and results in more consistent and reproducible samples. Unlike a horizontal circle, a horizontal rectangular wound and resulting vertical planes will be equal in wound gap value.