Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

Zhenxiang Wang,Yongxin Zhang,Monica Zimmer,Ying Wang,Farhang Farhangfar,Giovambattista Pani

doi:10.1371/journal.pone.0040951

Zhenxiang Wang, Yongxin Zhang + Show 4 more

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https://doi.org/10.1371/journal.pone.0040951

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Abstract

Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.

Highlights

Wound repair and scar formation are complex and important processes in clinical care
And B, we found that (1) in all keratinocyte/fibroblast co-cultures without transwells (K/F), the keratinocyte concentrations correlated with heparin-binding EGF-like growth factor (HB-EGF) levels (r = 0.815; P,0.01) better than IL-1a and TGFb1 levels (P.0.05), (2) the keratinocytes in the co-culture without transwells proliferated significantly faster (P,0.01) than those in transwells (K//F), and (3) the inhibitory effects of siRNA transfection on IL-1a and TGFb1 productions in fibroblasts led to a reduction (P,0.05)in HB-EGF production and keratinocyte proliferation, but more significant reductions were observed in the antibody neutralization tests (P,0.01), especially with the anti-HB-EGF antibody. These results suggested that the keratinocyte proliferation that was enhanced by co-culturing the cells with fibroblasts was mainly mediated by the cytokines produced by both cells, in which HB-EGF might play a central role in stimulating keratinocyte growth and could be up-regulated by IL-1a and TGFb1 produced by fibroblasts
The migration of fibroblasts was unaffected by any experimental factor. These results suggest that fibroblast-produced cytokines can enhance keratinocyte migration when the two cell types are in contact, possibly due to the effects of IL-1a and TGF-b1 produced by fibroblasts at cell contact points [29], where the fibroblasts may have accessibility for the delivery of cytokines to keratinocytes

Summary

Introduction

Wound repair and scar formation are complex and important processes in clinical care. Scar formation characterized by hypertrophy, contracture, and scar instability significantly impairs late functional and aesthetic outcomes [1]. Conventional splitthickness skin grafts, which are often widely meshed and expanded, are utilized to close large wound deficits. The consequences of scarring and contracture often require lengthy courses of revisional surgery [1]. Tissue repair is divided into an inflammatory phase, a granulation phase with synthesis of new connective tissue and epithelial wound closure, and a scar remodeling phase once the epidermal barrier has been reestablished. In the mid- and late phases of wound healing, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts, which gradually shift the microenvironment away from an inflammatory to a synthesis-driven granulation phase [2]

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