Long‐term organ culture of keloid disease tissue

Abstract

Keloid disease (KD) is a common fibroproliferative disorder of unknown aetiopathogenesis, with highly unsatisfactory treatment. Therefore, it is crucial to have a robust and clinically relevant model for studying KD pathobiology as well as preclinical testing of potential KD therapeutics. However, the unique occurrence of KD in human skin and the corresponding lack of animal models pose a major challenge in KD research. Therefore, we developed a simplified assay for the serum-free, long-term organ culture of KD tissue that facilitates quantitative analyses of major KD read-out parameters. Four millimetre KD punches embedded in a collagen matrix and organ-cultured at the epidermis air-liquid interphase (ALI) in supplemented William's E medium showed optimal tissue, cell and RNA preservation for up to 6 weeks (as measured by H & E and Pyronin Y histochemistry as well as by MTT assay, lactate dehydrogenase release and quantitative Ki67 ⁄ TUNEL immunohistomorphometry). The keloid phenotype persisted well during this period, as shown by collagen-I and -III synthesis (Herovici's histochemistry staining and ELISA), and analysis of the expression of significant KD markers (CD3, CD20, CD31, CD34, CD56, tryptase, Langerin, vimentin, neutrophil elastase, CTGF and Collagen). To functionally evaluate whether this assay can test the response to candidate therapeutics, dexamethasone, a glucocorticosteroid often used in KD therapy, was administered. Indeed, dexamethasone significantly reduced the keloid volume and cellularity plus induced epidermal shrinkage. Therefore, this novel assay provides a quantitative, clinically relevant model system for studying KD pathobiology and response to treatment.Key words: Dulbecco's modified Eagle's medium (DMEM) -enzyme linked immunosorbent assay (ELISA) -keloid disease -organ cultureterminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) -William's E medium (WE)

Altered expression of hyaluronan synthase and hyaluronidase mRNA may affect hyaluronic acid distribution in keloid disease compared with normal skin

“…A number of ECM molecules are involved in tissue remodelling, the majority of these such as collagen I, fibronectin and other glycosaminoglycans are increased in KD (1); however, the reasons for dysregulated HAS and HYAL expression, their role in lower HA concentrations in KD tissue and any subsequent effect in vivo remain elusive. The study of KD in whole tissue is more relevant than cell culture, as the interaction between different cell types and the ECM may be investigated (15), expression patterns also vary between the margin and the middle of the KD lesion (16).…”

Where we stand with human hypertrophic and keloid scar models

“…Despite our growing knowledge of the immune system, the inflammatory response, and proteomic and genomic changes after injury, we have neither created a model that demonstrates the complex nature of human scar formation, nor have we pinpointed ways to prevent hypertrophic scars and keloids (1,2). Our current models, both in cell cultures and in animals, do not reproduce the complexity of scar formation (3). These models simply demonstrate the fibrotic nature of scars but do not elucidate how and why adverse scar formation occurs in certain populations or why they develop in response to certain injuries (1-3).…”

“…They have led to a better understanding of biomarker expression, fibroblast activity and the role of keratinocytes in scar development. Traditionally, the investigation of hypertrophic scars and/or keloids has been based upon research of isolated respective scar cells in two-dimensional cell culture conditions (3,(15)(16)(17). This, however, fails to mirror the conglomerate of molecular and cellular synergies of hypertrophic scars or keloids in human skin.…”

“…A severe burn predetermines hypertrophic scarring -what is, however, the pathophysiological background for hypertrophic scarring and keloid formation after simple incisions? Conjectures remain that keloids and hypertrophic scars are immune-modulated or driven by an inflammatory response (both of which are time dependent), not just genetic predisposition (1)(2)(3)(4)(5).…”

“…More recent efforts have been made to better mimic the conditions upon which these scars form. Researchers developed three-dimensional models using a extracellular matrix, human skin constructed from keloid-derived tissue and mesenchymal cells (3). The evolution of the model has led to improvements in the study of the in vitro inflammatory response to scar formation to better mimic the in vivo response to scar (1).…”