The active role of electrically stimulated fibroblasts in skin wound healing

摘要

Background: Electrical stimulation (ES) in its various forms has been shown to promote wound healing by increasing the migration of keratinocytes and macrophages, enhancing angiogenesis, and stimulating dermal fibroblasts. Delivery of ES to wound can be through biocompatible conductive biomaterials such as conductive membranes made of 5% polypyrrole (PPy) and 95% polylactide (PLA), as well as an electronic system that enables cells to be cultured on the surface of the conductors and then electrically stimulated. One of the key cells in wound healing is fibroblast secreting extracellular matrix and adopting myofibroblast phenotype to promote wound closure. The role of fibroblasts during wound healing can be promoted by ES. However, the underline molecular mechanisms are not clear and the approach to apply electrically activated fibroblasts to assist skin wound healing needs to be explored. Objectives: (ⅰ) To investigate the signaling pathway when cells were exposed to ES; (ⅱ) To demonstrate the advantages of ES on in vitro and in vivo skin tissue regeneration. Experimental Protocol: Conductive membranes were designed either by by combining polylactide (PLLA) and a heparin (HE)-doped polypyrrole (PPy) or by coating PPy to polyester fabrics. Membrane morphology was observed with scanning electron microscope (SEM) and conductivity assessed using four point probe. Primary human dermal fibroblasts were seeded onto the conductive membranes and allowed to adapt themselves to the new environment for 24 h prior ES. Cells were subjected to two different ES regimes, i.e., a continuous ES regime for 6 h at 50 or 200 mV/mm, or a pulsed ES regime consisting of 10 s ES followed by 1190 s rest or 300 s ES followed by 300 s rest for 24 h. The stimulated cells were then used to perform in vitro and in vivo analyses. These include cell signaling mechanisms (TGFβ1/ERK/NF-κB), interactions with human primary keratinocytes, tissue structure, vascularization, etc., and the grafting of EHS to nude mice. Results and discussion: We demonstrated that both continuous and pulsed ES induced fibroblast adhesion and proliferation. Furthermore, ES promoted fibroblasts to myofibroblast differentiation. Interestingly, the myofibroblast phenotype acquired following ES can be transferred to daughter cells. The effects of ES on human fibroblasts lead to an increased production of TGFβ1 by activating ERK and NF-κB signaling pathway. The ES-modulated fibroblasts adequately interacted with keratinocytes leading to a well-structured EHS tissue expressing basement membrane (BM) glycoproteins, including laminin and type Ⅳ collagen. Tissue organization was superior under 200 mV/mm of ES than under 50 mV/mm. This confirms the previously reported study suggesting that exogenous ES maintains the ex vivo epidermal integrity and cell proliferation of a human skin explant. Following 20 and 30 days of grafting, the newly regenerated skin was well vascularized, showing BM formation through laminin and type Ⅳ collagen secretion, and was totally formed by the implanted human cells. Conclusion: We demonstrated that electrically activated fibroblasts interacted with keratinocytes leading to in vitro/in vivo well-structured engineered skin. This study thus provides an innovative way to use electrically activated cells for skin wound repair applications.