Graduate Student University of Cincinnati Lima, Ohio, United States
Introduction:: Introduction: Electrical stimulation of the cell can have numerous effects on cell phenotype. Many of the benefits observed present as improvements to the inflammatory, proliferative, and remodeling phases of wound healing, but many modern therapeutics utilizing these therapies require invasive, percutaneous devices or electrodes to effectively apply this type of stimulus. Electrospun polyvinylidene fluoride-trifluroethylene (PVDF-TrFE) is used here as an exciting platform for non-invasive, on demand electric stimulation of cells for nerve regeneration. This is due to its piezoelectric nature, or inherent ability to produce an electrical current upon mechanical deformation. Here, we have coupled electrospun PVDF-TrFE with ultrasound (US) treatments to noninvasively stimulate the scaffold to produce electric potentials. Slight distortion of the scaffold surface elicits electrical activity, thereby altering cell phenotype and morphology in a way that can potentially improve peripheral nerve regeneration.
Materials and Methods:: Materials/Methods: PVDF-TrFE scaffolds were fabricated via electrospinning for 3 hours per lab protocols1 to create aligned nanofibrous, piezoelectric scaffolds. Electrical activity was confirmed by measuring current produced by scaffold surface deformations and comparing to a nonpiezoelectric, PCL control (Fig 1A). Scaffolds were cut into 4 x 4 cm pieces to cover a 60 mm tissue culture plate. To secure the scaffold, each corner was anchored to the culture plate using microscopy mounting medium. Following 24 hours post-seeding, US was applied for a duration of 3 to 5 min via a transducer attached to the bottom of the culture plate with a thin layer of coupling gel. US parameters consisted of a modulation frequency at 1.0 MHz, pulse repetition frequency at 1.0 kHz, and an intensity of 0.08 W cm-2. 24 hours after stimulation, fibroblasts and Schwann cells were assayed and stained for phenotypic changes (Fig 1B, C).
Results, Conclusions, and Discussions:: Results and Discussion: Following a single US treatment, fibroblasts cultured on PVDF-TrFE showed greater metabolic activity when compared to cells subjected to US stimulation on coverslips (p < 0.005) and non-US-treated cells on both types of substrates (Fig 1D). As electrical activity is known to increase cell proliferation, the increase seen here suggests electrically activated PVDF-TrFE could be the result of this rise in metabolic activity. Furthermore, after one treatment of US, cells on activated PVDF-TrFE presented with heightened morphologic aspect ratios compared to cells on non-activated scaffolds (p < 0.05) (Fig 1E). While Schwann cell alignment and migration can be electrically driven, these results suggest Schwann cell morphology can be further transformed. This can lead to beneficial effects downstream, such as the promotion of key regenerative markers in regeneration of nerves, such as c-Jun expression.
Conclusions: US therapy applied to PVDF-TrFE holds incredible promise as a method for noninvasively generating the electrical activity needed for full nerve regeneration. This work demonstrates US therapeutics used to trigger electrical activity from PVDF-TrFE scaffolds can alter the metabolic activity of fibroblasts in a manner that benefits the proliferative stage of wound healing, showing statistically significant rates of increase. Furthermore, this work indicates the piezoelectric effect of PVDF-TrFE instigated by US-mediated surface deformations, causes Schwann cells to elongate, a morphological change desired in the remodeling phase of nerve regeneration.
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References (Optional): : References: 1. J. A. Orkwis, Macromol Biosci2020, Vol 20, Issue 9
