Photodynamic therapy on keloid fibroblasts in engineered keratinocyte-fibroblast co-culture

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Objectives: Keloids are disfiguring proliferative scars that are a pathologic response to cutaneous trauma. An organotypic tissue culture system (the RAFT model) was used to investigate the feasibility of treating keloids using photodynamic therapy (PDT). The RAFT co-culture recreates skin by layering keratinocytes on top of fibroblasts embedded in a collagen matrix. PDT uses drugs that produce singlet oxygen in situ when irradiated by light and may lead to a number of effects in living tissues varying from the modulation of growth to apoptosis. Methods: Normal adult, neonatal, and keloid fibroblasts and keratinocytes were used to construct the RAFTs. Mature RAFTS were incubated with 5-ALA, a photosensitizer, and were laser-irradiated (635 nm) for energy delivery of 5 J/cm2, 10 J/cm2, or 20 J/cm2. RAFTS were examined 24 hours and 14 days later. Cell viability was determined using confocal imaging combined with live-dead fluorescent dyes. Multiphoton microscopy (MPM) imaged collagen structure and density. As RAFTs contracted over time, surface area was measured using optical micrometry daily. Results: At 20 J/cm2, near-total cell death was observed in all constructs. At 10 J/cm2 some cell viability was maintained, while at 5 J/cm2 cell viability was comparable to controls. After 14 days, cell viability in keloid and neonatal RAFTs was greater than that observed in normal adult RAFTs. Treated RAFTs contracted less over the 14-day period compared to controls. Contraction and collagen density were greatest in keloid and neonatal RAFTS. Conclusions: A PDT dosimetry range has been established that reduces tissue contraction and collagen synthesis while preserving cell viability.