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

Abstract

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.

Fibroblasts cocultured with keloid keratinocytes: normal fibroblasts secrete collagen in a keloidlike manner

“…Using keratinocyte and fibroblast cell strains from these samples, we have demonstrated, for the first time, differences between normal and keloid fibroblast collagen response to coculture with normal or keloid keratinocytes. Normal and keloid keratinocytes, like normal and keloid fibroblasts, also appear to be distinct subtypes from these observed differences, which reinforces earlier work (23) suggesting the role of epithelial-mesenchymal interactions in keloid pathogenesis.…”

“…Recently, using an in vitro two-chamber coculture model, we showed that normal dermal fibroblast proliferation was significantly increased when cocultured with keloid-derived keratinocytes (which we propose to be called "keloid keratinocytes") in a serum-free medium (23). These data suggested that soluble factors promoting fibroblast proliferation were elaborated by keloid keratinocytes in a manner different from normal skin-derived keratinocytes (termed "normal keratinocytes").…”

Hypertrophic Scarring and Keloids: Pathomechanisms and Current and Emerging Treatment Strategies

“…Greater thickness of neodermis production was seen in fibroblast-seeded skin models when keratinocytes from hypertrophic scars were added in culture (32). Similar results were detected with coculturing of keloidderived keratinocytes and fibroblasts (62), suggesting that keratinocytes might have an important role in keloid and hypertrophic scar pathogenesis by producing signals that stimulate the fibroblasts in the underlying dermis to proliferate or produce more ECM. The basis for an inherent abnormality among hypertrophic scar and keloid-derived keratinocytes remains elusive, however.…”

Role of IGF system of mitogens in the induction of fibroblast proliferation by keloid-derived keratinocytes in vitro

“…Passage 2 KK and fibroblasts were isolated from excised earlobe keloid samples as previously described (31). Normal keratinocytes (NK) and NF were randomly derived from a bank of foreskin circumcision specimens of healthy young children, again as previously described (31).…”

“…We extrapolated this concept of epithelial-mesenchymal interactions to keloids in two recent in vitro studies examining the possible effects of such interactions on normal fibroblasts (NF) in serum-free, two-chamber coculture with normal or keloid-derived keratinocytes (KK). We demonstrated significantly higher rates of proliferation (31), as well as a significant increase in soluble extracellular-matrix collagen production by NF cocultured with KK (30a).…”