The Effect of Pulsed Electric Field on Expression of ECM proteins: Collagen, Elastin, and MMP1 in Human Dermal Fibroblasts.
Journal of electroanalytical chemistry (Lausanne, Switzerland)
Electrical stimulation of tissues has many uses in pain management, antibacterial treatment, and wound healing. The electric field stimulates epidermal migration and increases fibroblast cell proliferation. Here we show the effects of electrical field (EF) stimulation of human dermal fibroblasts (HDF) on the expression of collagen, elastin, and collagenase (MMP1; matrix metalloproteinase 1). The effects of EF stimulation are evaluated in terms of changes in cell morphology and extracellular matrix (ECM) protein expression, defined as intracellular concentration of collagen, elastin, and MMP1. HDF are stimulated in a bioreactor using square wave voltage pulses for up to 24 h. The pulse voltage (0-10V), pulse bias (0, +), pulse time (10-1000 ms), and rest time (0.1-10 s) were varied. We show that expression of collagen, elastin, and MMP1 increases in response to applied EF. The intracellular concentration of ECM proteins more than doubles depending on stimulation conditions with a threshold of effective stimulation above 3V/cm. The short time voltage pulses used for EF stimulation are more effective, while the rest time between pulses has a small effect on intracellular concentration of collagen, MMP1 and elastin. The previously studied HDF stimulation with chemical factors (i.e. TNF-α, TGF-β) shows negative correlation between concentration of collagen and MMP1. Contrary to that observation, we show that EF stimulation causes increase in the intracellular concentration of both collagen and MMP1. We also demonstrate that the transdermal stimulation of HDF in subcutaneous tissue is possible, thus it might be utilized in the future to improve the wound healing and tissue regeneration process.
10.1016/j.jelechem.2018.01.050
[Effects of direct current electric field on directional migration and arrangement of dermal fibroblasts in neonatal BALB/c mice and the mechanisms].
Liu Jie,Ren Xi,Guo Xiaowei,Sun Huanbo,Tang Yong,Luo Zhenghui,Zhang Qiong,Zhang Dongxia,Huang Yuesheng,Zhang Jiaping
Zhonghua shao shang za zhi = Zhonghua shaoshang zazhi = Chinese journal of burns
OBJECTIVE:To explore the effects of direct current electric fields on directional migration and arrangement of dermal fibroblasts in neonatal BALB/c mice and the related mechanisms. METHODS:Twelve neonatal BALB/c mice were divided into 4 batches. The skin on the back of 3 neonatal mice in each batch was obtained to culture fibroblasts. Fibroblasts of the second passage were inoculated in 27 square cover slips with the concentration of 5 × 10(4) cells per mL. (1) Experiment 1. Six square cover slips inoculated with fibroblasts of the second passage were divided into electric field group (EF) and sham electric field group (SEF), with 3 cover slips in each group. The cover slips were put in live cell imaging workstation. The cells in group EF was treated with electric power with EF intensity of 200 mV/mm, while simulating process without actual power was given to SEF group (the same below) for 6 h. Cell proliferation rate was subsequently counted. (2) Experiment 2. Six cover slips were divided and underwent the same processes as in experiment 1. Cell movement locus within EF hour (EFH) 6, direction change of cell migration at EFH 0 (immediately), 1, 2, 3, 4, 5, and 6 which was denoted as cos(α), cell migration velocity within EFH 6, direction change of long axis of cell within EFH 6, and direction change of cell arrangement at EFH 0, 1, 2, 3, 4, 5, and 6 which was denoted as polarity value cos[2(θ-90)] were observed under live cell imaging workstation. After EFH 6, the morphological changes in microtubules and microfilaments were observed with immunofluorescent staining. (3) Experiment 3. Six cover slips were divided into cytochalasin D group (treated with 1 μmol/L cytochalasin D for 10 min) and colchicine group (treated with 5 μmol/L colchicine for 10 min), with 3 cover slips in each group. The morphological changes in microfilaments and microtubules were observed with the same method as in experiment 2. (4) Experiment 4. Nine cover slips were divided into control group (no reagent was added), cytochalasin D group and colchicine group (added with the same reagents as in experiment 3), with 3 cover slips in each group. Cells in the 3 groups were exposed to an EF of 200 mV/mm for 6 h. Cell movement locus within EFH 6, cell migration velocity within EFH 6, cell polarity values at EFH 0, 3, and 6, and morphological changes of cells at EFH 0 and 6 were observed. Data were processed with independent samples t-test, one-way analysis of variance, and LSD test. RESULTS:(1) There was no statistically significant difference in cell proliferation rate in group EF and group SEF (t=-0.24, P﹥0.05). (2) Within EFH 6, cells in group EF migrated towards the anode of EF, while cells in group SEF moved randomly. At EFH 0, the values of cos(α) of cells in the 2 groups were both 0. The absolute value of cos(α) of cells in group EF (-0.57 ± 0.06) was significantly higher than that in group SEF (0.13 ± 0.09, t=6.68, P<0.01) at EFH 1, and it was still higher than that in group SEF from EFH 2 to 6 (with t values from 5.33 to 6.83, P values below 0.01). Within EFH 6, migration velocity of cells in group EF was (0.308 ± 0.019) μm/min, which was significantly higher than that in group SEF [(0.228 ± 0.021) μm/min, t=-2.76, P<0.01]. Within EFH 6, long axis of cells in group EF was perpendicular to the direction of EF, while arrangement of cells in group SEF was irregular. Cell polarity values in group EF were significantly higher than that in group SEF from EFH 2 to 6 (with t values from -7.52 to -0.90, P values below 0.01). At EFH 6, the morphology of microfilaments and microtubules of cells in EF group was similar to that in SEF group. (3) The fluorescent intensity of microfilaments of cells in cytochalasin D group became weakened, and the filamentary structure became fuzzy. The microtubules of cells in colchicine group became fuzzy with low fluorescent intensity. (4) Within EFH 6, cells in control group migrated towards the anode of EF, while cells in cytochalasin D group and colchicine group moved randomly. Within EFH 6, there was statistically significant difference in migration velocity of cells in the 3 groups (F=6.36, P<0.01). Migration velocity of cells in cytochalasin D group and colchicine group was significantly slower than that in control group (P<0.05 or P<0.01). At EFH 0, 3, and 6, cell polarity values in the 3 groups were close (with F values from 0.99 to 1.51, P values above 0.05). At EFH 0, cells in control group were spindle; cells in cytochalasin D group were polygonal or in irregular shapes; cells in colchicine group were serrated circle or oval. At EFH 6, no morphological change was observed in cells in control group; cells in cytochalasin D group were spindle with split ends on both ends; cells in colchicine group were serrated oval. CONCLUSIONS:The physiologic strength of exogenous direct current EF can induce directional migration and alignment of dermal fibroblasts in neonatal BALB/c mice. Microfilaments and microtubules are necessary skeleton structure for cell directional migration induced by EF, while they are not necessary for cell directional arrangement induced by EF.
10.3760/cma.j.issn.1009-2587.2016.04.007
[Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts].
Zhonghua shao shang yu chuang mian xiu fu za zhi
To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney test, one-way analysis of variance, independent sample test, and least significant difference test. After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with values of -5.33 and -5.41, respectively, <0.01), and the directionality was significantly enhanced (=-4.39, <0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (=-9.81, <0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased 0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (<0.05 or <0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (<0.05 or <0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (<0.05 or <0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (<0.01). The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.
10.3760/cma.j.cn501120-20210112-00017
Electrostimulation induces cardiomyocyte predifferentiation of fibroblasts.
Genovese Jorge A,Spadaccio Cristiano,Langer Jason,Habe Jaclyn,Jackson Johnna,Patel Amit N
Biochemical and biophysical research communications
Stem-cell therapy has become a promising therapeutic tool for myocardial repair. Cardiac pre-committed cells, which complete their differentiation in the myocardium, may reduce fibrosis and restore muscle function. However, many questions concerning a precise, functional integration of injected cells remain unanswered. Fibroblasts regulate the cardiac extracellular matrix and are the most abundant cell population in an infarcted area. Electrostimulation is a well-known trophic factor and can induce phenotypic changes in myoblasts. The objective of this study was to evaluate the effectiveness of electrical stimulation to induce pre-commitment of fibroblasts into cardiomyocytes in vitro. Using short-time electrostimulation in a cytokine-free culture system, we induced pre-commitment of two fibroblast cell lines to a cardiomyocyte phenotype. This partial differentiation in vitro may facilitate further differentiation within the cardiac environment and result in better electro-mechanical integration of the therapeutically introduced cells.
10.1016/j.bbrc.2008.03.115
In vitro stimulation with radiofrequency currents promotes proliferation and migration in human keratinocytes and fibroblasts.
Hernández-Bule María Luisa,Toledano-Macías Elena,Naranjo Aida,de Andrés-Zamora Marina,Úbeda Alejandro
Electromagnetic biology and medicine
Capacitive-resistive electric transfer (CRET) therapies have been proposed as strategies for regeneration of cutaneous tissue lesions. Previous studies by our group have shown that intermittent stimulation with 448 kHz CRET currents at subthermal densities promotes in vitro proliferation of human stem cells involved in tissue regeneration. The present study investigates the effects of the in vitro exposure to these radiofrequency (RF) currents on the proliferation and migration of keratinocytes and fibroblasts, the main cell types involved in skin regeneration. The effects of the electric stimulation on cell proliferation and migration were studied through XTT and wound closure assays, respectively. The CRET effects on the expression and location of proteins involved in proliferation and migration were assessed by immunoblot and immunofluorescence. The obtained results reveal that electrostimulation promotes proliferation and/or migration in keratinocytes and fibroblasts. These effects would be mediated by changes observed in the expression and location of intercellular adhesion proteins such as β-catenin and E-cadherin, of proteins involved in cell-to-substrate adhesion such as vinculin, -FAK and the metalloproteinase MMP-9, and of other proteins that control both processes: MAP kinases -p38, -JUNK and -ERK1/2. These responses could represent a mechanism underlying the promotion of normotrophic wound regeneration induced by CRET. Indeed, electric stimulation would favor completion of granulation tissue formation prior to the closure of the outer tissue layers, thus preventing abnormal wound cicatrization or chronification.
10.1080/15368378.2021.1938113
Directly Induced Neural Differentiation of Human Adipose-Derived Stem Cells Using Three-Dimensional Culture System of Conductive Microwell with Electrical Stimulation.
Heo Dong Nyoung,Acquah Nana,Kim Junghoon,Lee Se-Jun,Castro Nathan J,Zhang Lijie Grace
Tissue engineering. Part A
Adipose-derived stem cells (ADSCs) have the capacity to differentiate into neural precursor cells which can be used for nerve regeneration. However, their inherently low neurogenic differentiation efficiency limits further clinical applications. This study was designed to promote neurogenic differentiation efficacy of ADSCs by integrating conductive hydrogel-based microwells with electrical stimulation (ES). We hypothesize that ADSCs will differentiate more efficiently into neural precursor cells when electrically stimulated in conductive hydrogel microwells. To make the conductive hydrogel-based microwell, polyethylene glycol (PEG) diacrylate aqueous solution mixed with poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) was patterned with the polydimethylsiloxane mold and exposed to UV light to induce photo-cross-linking of the conductive hydrogel. After seeding the ADSCs in the microwells, the cells formed distinct cell spheres in PEG microwells and wide disks in the PEG/PEDOT:PSS microwells. Although the microwells yielded varying three-dimensional (3D) cell aggregate structure, cell viability was not affected. After neurogenic differentiation with ES, the ADSC aggregates in PEG/PEDOT:PSS microwells with ES expressed greater positive neuronal differentiation markers compared to nonstimulated PEG/PEDOT:PSS microwells. Although all neuronal gene expression levels were greater in PEG microwells with ES, the increased rates of gene expression levels between treated and untreated PEG/PEDOT:PSS microwells were much higher compared to PEG microwells. This would mean that electrically stimulating ADSC aggregates in conductive microwells is an effective method in increasing neurogenic differentiation. Therefore, we propose a most effective strategy taking advantage of a 3D conductive culture system which can be useful in a wide variety of electrical application.
10.1089/ten.TEA.2017.0150
Electrical stimulation promotes the wound-healing properties of diabetic human skin fibroblasts.
Journal of tissue engineering and regenerative medicine
This study evaluated the effect of low (20 and 40 mV/mm) intensities of electrical stimulation on the proliferation and migration of skin fibroblasts from diabetic donors. We also examined the effect of electrical stimulation on modulating the capacity of fibroblasts to contract collagen gel, express alpha-smooth muscle actin, and secrete proteolytic enzymes involved in regulating extracellular matrix synthesis and degradation. Our study shows that 20 and 40 mV/mm of stimulation increased the growth of fibroblasts extracted from diabetic patients but not from non-diabetic donors. Electrical stimulation increased the migration of diabetic fibroblasts, their capacity to contract collagen gel, and the expression of alpha-smooth muscle actin and promoted different proteolytic enzymes involved in accelerating wound healing. Overall results confirm the effectiveness of electrical stimulation in modulating the wound healing activities of fibroblasts extracted from diabetic skin donors. This study, therefore, suggests the possible use of electrical stimulation to promote diabetic foot ulcer healing by stimulating the wound healing properties of skin fibroblasts.
10.1002/term.3305