Kathy Q. Zhu

Review of the female Duroc/Yorkshire pig model of human fibroproliferative scarring

Hypertrophic scarring after burns is an unsolved problem and remains as devastating today as it was in the 40s and it may be that the main reason for this is the lack of an accepted, useful animal model. The female, red Duroc pig was described as a model of hypertrophic scarring nearly 30 years ago but then vanished from the literature. This seemed strange since the authors reported that 12 of 12 pigs developed thick scar. In the mid 90s we explored the model and found that, indeed, the red Duroc pig does make thick scar. Other authors have established that the Yorkshire pig does not heal in this fashion so there is the possibility of a same species control. We have continued to explore the Duroc/Yorkshire model and herein describe our experiences. Is it a perfect model of hypertrophic scarring? No. Is it a useful model of hypertrophic scarring? Time will tell. We have now obtained gene expression data from the Duroc/Yorkshire model and analysis is underway.

Spatial and Temporal Localization of the Melanocortin 1 Receptor and Its Ligand α–Melanocyte-Stimulating Hormone during Cutaneous Wound Repair

Growing evidence indicates that the melanocortin 1 receptor (MC1R) and its ligand α–melanocyte-stimulating hormone (α-MSH) have other functions in the skin in addition to pigment production. Activation of the MC1R/α-MSH signaling pathway has been implicated in the regulation of both inflammation and extracellular matrix homeostasis. However, little is known about the role of MC1R/α-MSH signaling in the regulation of inflammatory and fibroproliferative responses to cutaneous injury. Although MC1R and α-MSH localization has been described in uninjured skin, their spatial and temporal expression during cutaneous wound repair has not been investigated. In this study, the authors report the localization of MC1R and α-MSH in murine cutaneous wounds, human acute burns, and hypertrophic scars. During murine wound repair, MC1R and α-MSH were detected in inflammatory cells and suprabasal keratinocytes at the leading edge of the migrating epithelial tongue. MC1R and α-MSH protein levels were upregulated in human burn wounds and hypertrophic scars compared to uninjured human skin, where receptor and ligand were absent. In burn wounds and hypertrophic scars, MC1R and α-MSH localized to epidermal keratinocytes and dermal fibroblasts. This spatiotemporal localization of MC1R and α-MSH in cutaneous wounds warrants future investigation into the role of MC1R/α-MSH signaling in the inflammatory and fibroproliferative responses to cutaneous injury. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Expression of collagen genes in the cones of skin in the Duroc/Yorkshire porcine model of fibroproliferative scarring.

During the past decades there has been minimal improvement in prevention and treatment of hypertrophic scarring. Reasons include the lack of a validated animal model, imprecise techniques to dissect scar into the histologic components, and limited methodology for measurement of gene expression. These problems have been addressed with the Duroc/Yorkshire model of healing, laser capture microdissection, and the Affymetrix Porcine GeneChip®. Here we compared collagen gene expression in fibroproliferative healing in the Duroc breed to nonfibroproliferative healing in the Yorkshires. We made shallow and deep dorsal wounds, biopsied at 1, 2, 3, 12, and 20 weeks. We sampled the dermal cones by laser capture microdissection, extracted and amplified the RNA, and hybridized Affymetrix Porcine GeneChips®. We also obtained samples of human hypertrophic scar approximately 20 weeks postinjury. Data were normalized and statistical analysis performed with mixed linear regression using the Bioconductor R/maanova package. Genes for further analysis were also restricted with four biologic criteria, including that the 20-week deep Duroc expression match the human samples. Eleven ollagen genes and seven collagen types were differentially over expressed in deep Duroc wounds including 1a1, 1a2, 3a1, 4a1, 4a2, 5a1, 5a2, 5a3, 6a3 (transcript variant 5), 14a1 and 15a1. COL7a1 gene was differentially under expressed in deep Duroc wounds. The results suggest that collagens I, III, IV, V, VI, VII, XIV, and XV1 are involved in the process of fibroproliferative scarring. With these clues, we will attempt to construct the regulatory pathway(s) of fibroproliferative healing.

The microvasculature in cutaneous wound healing in the female red Duroc pig is similar to that in human hypertrophic scars and different from that in the female Yorkshire pig.

The female red Duroc pig has been found to be a promising model of hypertrophic scarring. The female Yorkshire pig has been demonstrated to heal in a very different manner, more resembling human normotrophic scarring. Given these observations, we studied microvessel density, an important aspect of wound healing, in human hypertrophic scars and the scars of the female Duroc and Yorkshire pigs. We studied microvessel density in uninjured skin; hypertrophic scars at 6 months or less, 7 to 12, and longer than 12 months; female Duroc tissues at 3 weeks and 3 and 5 months; and similar Yorkshire tissue, including uninjured skin and shallow and deep wounds. Antifactor VIII-related antigen was used to mark the endothelial cells. Computed assessment of microvessel density was used to quantify the microvasculature. In human hypertrophic scars, the microvessels were increased dramatically, and microvessel density and area were significantly elevated. We found similar results in the Duroc tissues at 5 months after deep wounding. In contrast, we found far less microvasculature and, at 5 months, the values had returned to normal in the Yorkshire tissues. This quantitative study of microvessel density further validates the female Duroc pig as an animal model of hypertrophic scarring and the female Yorkshire pig as a control.

Functional Genomics Unique to Week 20 Post Wounding in the Deep Cone/Fat Dome of the Duroc/Yorkshire Porcine Model of Fibroproliferative Scarring

Background Hypertrophic scar was first described over 100 years ago; PubMed has more than 1,000 references on the topic. Nevertheless prevention and treatment remains poor, because 1) there has been no validated animal model; 2) human scar tissue, which is impossible to obtain in a controlled manner, has been the only source for study; 3) tissues typically have been homogenized, mixing cell populations; and 4) gene-by-gene studies are incomplete. Methodology/Principal Findings We have assembled a system that overcomes these barriers and permits the study of genome-wide gene expression in microanatomical locations, in shallow and deep partial-thickness wounds, and pigmented and non-pigmented skin, using the Duroc(pigmented fibroproliferative)/Yorkshire(non-pigmented non-fibroproliferative) porcine model. We used this system to obtain the differential transcriptome at 1, 2, 3, 12 and 20 weeks post wounding. It is not clear when fibroproliferation begins, but it is fully developed in humans and the Duroc breed at 20 weeks. Therefore we obtained the derivative functional genomics unique to 20 weeks post wounding. We also obtained long-term, forty-six week follow-up with the model. Conclusions/Significance 1) The scars are still thick at forty-six weeks post wounding further validating the model. 2) The differential transcriptome provides new insights into the fibroproliferative process as several genes thought fundamental to fibroproliferation are absent and others differentially expressed are newly implicated. 3) The findings in the derivative functional genomics support old concepts, which further validates the model, and suggests new avenues for reductionist exploration. In the future, these findings will be searched for directed networks likely involved in cutaneous fibroproliferation. These clues may lead to a better understanding of the systems biology of cutaneous fibroproliferation, and ultimately prevention and treatment of hypertrophic scarring.