Use of organotypic coculture to study keloid biology

Abstract 

Background

Keloids are pathologic scars afflicting a large segment of our population and for which there is no definitive therapy. The lack of an animal model for keloid formation has hampered study. We developed an in vitro organotypic skin model to simulate normal keloid biology, which may allow us to study keloid formation without an animal model.

Methods

Normal (NFs) and keloid (KFs) human fibroblasts were cultured in a collagen matrix to create a 3-dimensional dermal structure. Normal human keratinocytes (NKs) were cultured as a second layer on top and exposed to an air–fluid interface to allow differentiation into a mature keratinocyte layer. The organotypic skin was maintained for 28 days in Dulbecco’s modified eagle medium with 10% fetal calf serum. Samples were collected, processed, sectioned, stained with hematoxylin and eosin, and then measured for qualitative analysis. α–smooth-muscle actin was also evaluated by immunoblotting.

Results

KF/NK organotypic skin showed increased collagen deposition, based on significantly denser collagen staining, with increased dermal thickness compared with NF/NK organotypic skin. We saw increased contracture in the KF/NK construct, and this correlated with increased organization of α–smooth-muscle actin fibers in the dermal layer of KF/NK organotypic skin compared with NF/NK skin.

Conclusions

We have shown that coculture of KFs with keloid keratinocytes leads to an increased collagen production and dermal contracture compared with NFs and NKs, consistent with known keloid behavior. Given the lack of an animal model, we believe that organotypic skin culture can serve as a surrogate to study keloid formation.

Keywords: Fibroblast, Keratinocyte, Keloid, Organotypic coculture, Scar, Wound healing