Electrical Stimulation and Cellular Behaviors in Electric Field in Biomedical Research.

Research on the cellular response to electrical stimulation (ES) and its mechanisms focusing on potential clinic applications has been quietly intensified recently. However, the unconventional nature of this methodology has fertilized a great variety of techniques that make the interpretation and comparison of experimental outcomes complicated. This work reviews more than a hundred publications identified mostly from Medline, categorizes the techniques, and comments on their merits and weaknesses. Electrode-based ES, conductive substrate-mediated ES, and noninvasive stimulation are the three principal categories used in biomedical research and clinic. ES has been found to enhance cell proliferation, growth, migration, and stem cell differentiation, showing an important potential in manipulating cellular activities in both normal and pathological conditions. However, inappropriate parameters or setup can have negative effects. The complexity of the delivered electric signals depends on how they are generated and in what form. It is also difficult to equate one set of parameters with another. Mechanistic studies are rare and badly needed. Even so, ES in combination with advanced materials and nanotechnology is developing a strong footing in biomedical research and regenerative medicine.

Growth and follow-up of primary cortical neuron cells on nonfunctionalized graphene nanosheet film.

BACKGROUND: Conductive biomaterials are an ideal biosubstrate for modifying cellular behaviors by conducting either internal or external electrical signals. In this study, based on a simple-preparation graphite exfoliation method in organic reagent, a nonfunctionalized graphene nanosheet film (NGNF) with high conductivity and large size was simply fabricated through spraying coating. The biocompatibility of the NGNF was carefully tested with primary cortical neuron cells, and its biocompatibility properties were compared with a chemical vapor deposition (CVD) graphene film. METHODS: Nonfunctionalized graphene nanosheet (NGN) was first exfoliated from graphite with a flat-tip ultrasonicator probe, and then spray-coated onto glass slide substrate to form the film. The morphology of NGNF was observed with light microscopy and SEM. The morphology and neuronal network formation of primary cortical neuron cells onto NGNF, as shown by DAPI and Alexa Fluor® 488 staining, were observed with fluorescent microscopy. Cell viability and proliferation were measured with MTT. RESULTS: NGNF had better cell biocompatibility than CVD graphene film. MTT test showed that NGNF exhibited no cytotoxicity. According to neuronal network formation at 7 days of cell culture, primary neuron cells aggregated into 50-µm "nuclei"; the average neurite number and length were 3 and 100 µm, respectively. However, these values were almost doubled after 14 days of cell culture. CONCLUSIONS: These results may improve the use of NGNF as a conductive scaffold for nerve regeneration.

Electrical stimulation promotes wound healing by enhancing dermal fibroblast activity and promoting myofibroblast transdifferentiation.

Electrical stimulation (ES) has long been used as an alternative clinical treatment and an effective approach to modulate cellular behaviours. In this work we investigated the effects of ES on human skin fibroblast activity, myofibroblast transdifferentiation and the consequence on wound healing. Normal human fibroblasts were seeded on heparin-bioactivated PPy/PLLA conductive membranes, cultured for 24 h, and then exposed to ES of 50 or 200 mV/mm for 2, 4, or 6 h. Following ES, the cells were either subjected to various analyses or re-seeded to investigate their healing capacity. Our findings show that ES had no cytotoxic effect on the fibroblasts, as demonstrated by the similar LDH activity levels in the ES-exposed and non-exposed cultures, and by the comparable cell viability under both conditions. Furthermore, the number of viable fibroblasts was higher following exposure to 6 h of ES than in the non-exposed culture. This enhanced cell growth was likely due to the ES up-regulated secretion of FGF-1 and FGF-2. In an in vitro scratch-wound assay where cell monolayer was used as a healing model, the electrically stimulated dermal fibroblasts migrated faster following exposure to ES and recorded a high contractile behaviour toward the collagen gel matrix. This enhanced contraction was supported by the high level of α-smooth muscle actin expressed by the fibroblasts following exposure to ES, indicating the characteristics of myofibroblasts. Remarkably, the modulation of fibroblast growth continued long after ES. In conclusion, this work demonstrates for the first time that exposure to ES promoted skin fibroblast growth and migration, increased growth factor secretion, and promoted fibroblast to myofibroblast transdifferentiation, thus promoting wound healing.

Biocompatibility of pristine graphene for neuronal interface.

OBJECT: Graphene possesses unique electrical, physical, and chemical properties that may offer significant potential as a bioscaffold for neuronal regeneration after spinal cord injury. The purpose of this investigation was to establish the in vitro biocompatibility of pristine graphene for interface with primary rat cortical neurons. METHODS: Graphene films were prepared by chemical vapor deposition on a copper foil catalytic substrate and subsequent apposition on bare Permanox plastic polymer dishes. Rat neuronal cell culture was grown on graphene-coated surfaces, and cell growth and attachment were compared with those on uncoated and poly-d-lysine (PDL)-coated controls; the latter surface is highly favorable for neuronal attachment and growth. Live/dead cell analysis was conducted with flow cytometry using ethidium homodimer-1 and calcein AM dyes. Lactate dehydrogenase (LDH) levels-indicative of cytotoxicity-were measured as markers of cell death. Phase contrast microscopy of active cell culture was conducted to assess neuronal attachment and morphology. RESULTS: Statistically significant differences in the percentage of live or dead neurons were noted between graphene and PDL surfaces, as well as between the PDL-coated and bare surfaces, but there was little difference in cell viability between graphene-coated and bare surfaces. There were significantly lower LDH levels in the graphene-coated samples compared with the uncoated ones, indicating that graphene was not more cytotoxic than the bare control surface. According to phase contrast microscopy, neurons attached to the graphene-coated surface and were able to elaborate long, neuritic processes suggestive of normal neuronal metabolism and morphology. CONCLUSIONS: Further use of graphene as a bioscaffold will require surface modification that enhances hydrophilicity to increase cellular attachment and growth. Graphene is a nanomaterial that is biocompatible with neurons and may have significant biomedical applications.

Electrical stimulation modulates osteoblast proliferation and bone protein production through heparin-bioactivated conductive scaffolds.

Electrical fields are known to interact with human cells. This principle has been explored to regulate cellular activities for bone tissue regeneration. In this work, Saos-2 cells were cultured on conductive scaffolds made of biodegradable poly(L-lactide) and the heparin-containing, electrically conducting polypyrrole (PPy/HE) to study their reaction to electrical stimulation (ES) mediated through such scaffolds. Both the duration and intensity of ES enhanced cell proliferation, generating a unique electrical intensity and temporal "window" within which osteoblast proliferation was upmodulated in contrast to the downmodulation or ineffectiveness in other ES regions. The favourable ES intensity (200 mV/mm) was further investigated in terms of the gene activation and protein production of two important osteoblast markers characterised by extracellular matrix maturation and mineralisation, that is alkaline phosphatase (ALP) and osteocalcin (OC). Both genes were found activated and the relevant protein production increased significantly following ES. In contrast, ES in the down-modulation region (400 mV/mm) suppressed the production of both ALP and OC. This work demonstrated that important osteoblast markers can be modulated with specific ES parameters mediated through conductive polymer substrates, providing a unique strategy for bone tissue engineering.

Accelerated osteoblast mineralization on a conductive substrate by multiple electrical stimulation.

One of the major benefits of a conductive PPy-based substrate is that the mediated electrical stimulation (ES) can be a stimulating factor to promote tissue regeneration. We cultured osteoblast-like Saos-2 cells on a conductive substrate made of biodegradable polylactide (95 wt%) and electrically conducting polypyrrole bioactivated with heparin (PPy/HE) (5 wt%). Using multi-well electrical cell culture plates, the effect of multiple ESs on osteoblast mineralization was investigated at various culture times. As ascertained by ARS, CPC and XPS analyses, the ES was able to promote osteoblast adhesion and growth, resulting in significantly higher calcium and phosphate content in the mineral deposition of the electrically stimulated membranes. Morphology, Ca/P ratio and crystalline structure demonstrated that the minerals on the conductive substrate surface were similar to those found on typical hydroxyapatite. ES also significantly upregulated the expression of the osteoblast-specific markers ALP, BMP2, Runx2 and OC. ES through a synthetic conductive polymer substrate therefore represents a vital option to promote bone regeneration.

Electroactivity and stability of polylactide/polypyrrole composites.

Electrically conductive biodegradable composites are useful in biomedical engineering to mediate electrical interactions between cells and electrical components. Because conductive polymers are often subjected to progressive oxidative deterioration and dedoping, their electrical stability in aqueous environment is, therefore, critical. We used cyclic voltammetry (CV) to investigate the electroactivity and stability of conductive membranes made of only 5% conductive polypyrrole (PPy) particles blended with 95% insulating polylactide (PLLA). During 1000 CV scans, the electroactivity of the composite membranes revealed a period of initial activation, followed by a relatively stable period and subsequent slow deterioration process. PPy membranes doped with heparin, a cell-adhesive polysaccharide and polyanion, displayed electrical stability superior to that of PPy membranes doped with chlorine anions. The latter, however, recorded a higher initial electroactivity during the first ca. 150 scans. XPS analysis showed that the deterioration of the electroactivity was likely due to the oxidation of the PPy. ATR-FT-IR and SEM were also used to characterize the materials. The PLLA/PPy membranes were thus electroactive and maximum electroactivity was achieved through an activation process. The heparin-doped PPy composite was electrically stable and may, thus, be used for multiple long-term electrical stimulations.

Heparin dopant increases the electrical stability, cell adhesion, and growth of conducting polypyrrole/poly(L,L-lactide) composites.

Polypyrrole (PPy) is a promising conductive polymer for tissue engineering and bioelectrical applications. However, its electrical conductivity deteriorates easily in aqueous conditions. Cell adhesion to PPy is also relatively poor. The goal of this study was to simultaneously improve the electrical stability of and cell adhesion to PPy by using heparin (HE) as dopant, for HE is both a polyanion and an important glycosaminoglycan in cell membranes and extracellular matrix. PPy particles doped with HE were synthesized through emulsion polymerization using Fenton's reagent as an oxidant. X-ray photoelectron spectroscopy (XPS), infrared and scanning electron microscopy (SEM) were used to investigate the PPy particles. Conductive biodegradable membranes of 10(2) to 10(3) Omega/square were prepared from 5% (w) PPy with various amounts of HE and 95% (w) poly(L,L-lactide) (PPy/PLLA). Azure A staining was employed to quantify the HE exposed on the surface of the PPy particles and PPy/PLLA membranes. The distribution of HE on membranes was demonstrated by DAPI staining. Results showed that HE was incorporated into the PPy particles as counterions and presented on particle surface. A unique "filament"-like morphology of the PPy preparation was observed at high-HE content. The electrical stability of the PPy/PLLA membranes was tested in saline at 37 degrees C for 500 h. Human skin fibroblasts were used to test the cell adhesion capacity. The conductive membranes containing HE-doped PPy particles recorded significantly increased electrical stability, cell adhesion, and growth. The electrically more stable and cell adhesive conductive biodegradable membrane may act as a platform for various biomedical applications.

Electrical stimulation enhances viability of human cutaneous fibroblasts on conductive biodegradable substrates.

Electrically conductive biodegradable polymer membranes were prepared by mixing conductive polypyrrole particles with poly(L-lactic acid) solution followed by solution casting and solvent evaporation. Multi-well electrical cell culture plates were fabricated to host electrically stimulated cell culture and monitor parameters. Human cutaneous fibroblasts were cultured on conductive membranes with or without electrical stimulation (ES). Cell count, MTT, Hoechst staining, and SEM were performed to characterize the cells. The membranes supported the adhesion and proliferation of the fibroblasts in both the presence and absence of ES. In the presence of direct electrical field strength of 100 mV/mm, cell viability on the PPy/PLLA membranes at 2 and 24 h was 2.2- and 4.0-fold (p < 0.05) respectively of that on the same membranes without ES. Direct electrical current ranging from 2.5 to 250 microA/mm had no effect on the viability of cells cultured on the gold-coated Petri dish. Electrical field applied to conductive biodegradable polymer surfaces is therefore an effective approach to upregulate the mitochondrial activity of human skin fibroblasts.