Electrical stimulation through conductive PPy-PLLA material promoted fibroblast interacting with keratinocytes generating better organised human skin equivalent

Hyunjin Park (Groupe de Recherche en écologie Buccale, Centre de recherche du CHU de Québec Université Laval)
Dounia Rouabhia (Groupe de Recherche en écologie Buccale, Faculté de Médecine Dentaire, Université Laval)
Ze Zhang (Centre de recherche du CHU de Québec, Département de chirurgie, Faculté de médecine, Université)
Denis Lavertu (Centre de recherche du CHU de Québec, Département de chirurgie, Faculté de médecine, Université)
Mahmoud Rouabhia (Groupe de Recherche en écologie Buccale, Faculté de Médecine Dentaire, Université Laval)

Introduction

Human body generates trans-epithelial electrical potential (TEP) ranging between 10 and 60 mV in various locations [1] contributing to wound healing [2]. Wound current corresponds to a relatively steady local EF between 40 and 200 mV/mm. The EF persists until complete wound re-epithelialisation is achieved [3,4]. This EF was suggested to guide cell migration including fibroblasts and keratinocytes directly toward the wound edge; this healing process is compromised if the EF is inhibited [4,5]. Thus ES offers a rational and potentially highly efficient approach to promote wound healing by activating fibroblast activities. We previously developed a biodegradable conductor made of 5% polypyrrole (PPy) and 95% polylactide (PLA) [6], as well as an electronic system that allows cells to be cultured on the surface of the conductors and then electrically stimulated [7]. Using this system we were able to demonstrate that ES promoted human skin fibroblast growth, and increased the secretion of fibroblast growth factors (FGF1 and FGF2) [8]. The effect of ES on fibroblasts can also promote the interaction between fibroblasts and keratinocytes in skin wound healing. The objective of the present study was to investigate the interactions between human keratinocytes and the ES-exposed fibroblasts using an engineered human skin equivalent (EHSE).

Materials and Methods

Normal human skin fibroblasts and keratinocytes were used in this study. The fibroblasts were cultured in a Dulbecco's modified Eagle's medium, and then used at passages 4 and 5 in the present work. Cells were cultured on the heparin-bioactivated conductive PPy/PLLA membranes which were connected to a DC constant potential source through external electrodes to form a complete circuit (Fig .1a). Two potential intensities, 50 and 200 mV/mm, were tested. The cells were exposed to ES for 6 h, and were further cultured for 24 h prior the analyses. Sham ES-exposed control groups followed the same conditions except exposure to ES. Fibroblasts were then collected and mixed with collagen type I to produce dermis. After 2h incubation, human keratinocytes extracted from normal human skin tissue were seeded on top of the engineered dermis and incubated for 3 days to complete the epidermis (Fig 1 b). After that, air-liquid interface procedure was performed for another 3 days to allow keratinocyte stratification. Finally, the SEs were fixed in a 4% paraformaldehyde solution. The morphology of the SEs was analyzed by histology. The secretion of growth factors, such as keratinocytes growth factor (KGF), by the ES-exposed fibroblasts was also analyzed by ELISA.

Results

The histology results showed nice keratinocyte stratification giving more cell layers in the ES groups than that in the controls. Furthermore the cohesion between the dermis and epidermis seems better in the skin containing ES-exposed fibroblasts. Interestingly the epidermis was also thicker in the SE prepared with the ES-exposed fibroblasts. Additionally, ELISA measurement showed that the level of KGF was higher in the ES-exposed fibroblasts. KGF stimulates keratinocytes proliferation and also enhances differentiation [9]. Thus, ELISA results support the histological observations.

Discussion and Conclusion

The structural analyses demonstrated morphological differences between the SEs prepared with the ES-exposed and control fibroblasts. The better stratified layers in the ES-exposed SEs could be linked to the higher level of KGF secretion in human skin fibroblast cells exposed to ES.

Figure 1: Fibroblasts exposure to ES through conductive PPy-PLLA membrane, and their use to engineer the skin equivalent.

References

[1] Foulds IS, Barker AT. Br J Dermatol 1983;109: 515–522. [2] Zhao M. Semin Cell Dev Biol 2009;20:674–82. [3] Chiang M, Robinson KR, Vanable JW Jr. Exp Eye Res 1992;54:999–1003. [4] Sta Iglesia DD, Vanable JW Jr. Wound Repair Regen 1998;6:531–42. [5] Song B, Zhao M, Forrester JV, McCaig CD. Proc Natl Acad Sci USA 2000;99:13577–82. [6] Shi GX, Rouabhia M, Wang Z, Dao LH, Zhang Z. Biomaterials 2004;25:2477–88. [7] Shi GX, Zhang Z, Rouabhia M. 2008 Oct;29(28):3792-8. [8] Rouabhia M, Park H, Meng S, Derbali H, Zhang Z. 2013. Plos One. 19. e71660. -11. [9] Fatimah SS, Chua K, Tan GC, Azmi TI, Tan AE, Rahman HA. Burns. 2013. 39. 905-915.

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