Investigation of the Influence of Keloid-derived Keratinocytes on Fibroblast Growth and Proliferation in Vitro

Keloids are disfiguring, proliferative scars that represent a pathological response to cutaneous injury. The overabundant extracellular matrix formation, largely from collagen deposition, is characteristic of these lesions and has led to investigations into the role of the fibroblast in its pathogenesis. Curiously, the role of the epidermis in extracellular matrix collagen deposition of normal skin has been established, but a similar hypothesis in keloids has not been investigated. The aim of this study was to investigate the influence of keloid epithelial keratinocytes on the growth and proliferation of normal fibroblasts in an in vitro serum-free co-culture system. A permeable membrane separated two chambers; the upper chamber contained a fully differentiated stratified epithelium derived from the skin of excised earlobe keloid specimens, whereas the lower chamber contained a monolayer of normal or keloid fibroblasts. Both cell types were nourished by serum-free medium from the lower chamber. Epithelial keratinocytes from five separate earlobe keloid specimens were investigated. Four sets of quadruplicates were performed for each specimen co-cultured with normal fibroblasts or keloid-derived fibroblasts. Controls consisted of (1) normal keratinocytes co-cultured with normal fibroblasts, and (2) fibroblasts grown in serum-free media in the absence of keratinocytes in the upper chamber. Fibroblasts were indirectly quantified by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay, with results confirmed by DNA content measurement, at days 1 and 5 after the co- culture initiation.Significantly, increased proliferation was seen in fibroblasts co-cultured with keloid keratinocytes, as compared with the normal keratinocyte controls at day 5 (analysis of variance, p < 0.001). These results strongly suggest that the overlying epidermal keratinocytes of the keloid may have an important, previously unappreciated role in keloid pathogenesis using paracrine or epithelial-mesenchymal signaling.