Keratinocyte Electrotaxis Induced By Pulse Directed Current Electric Fields

BackgroundPeople on the electric field-induced cell migration and promote wound healing done a lot of research from cells to in vivo, since the 19 th century the discovery of the p Hysiology electric field on human. Some studies have found that there exist an ion flow between the epidermis and the epithelial which forming a potential longitudinal potential difference. When the skin wounded, the potential difference short-circuited rapidly and formed a transverse electric field from the normal area to the wound center. Directed migration of cells is a prerequisite for wound healing, and wound endogenous electric field is the key factors guiding cell directional migration. In vitro experiments have shown that cells in the DC electric field can produce sustained directional migration which called chemotaxis. Currently, a large number of experiments focused on constant directed current electric field-induced chemotaxis of cells. However, the constant directed current electric field will inevitably lead to thermal effects and electrolytic effect which limited application of DC electric field in vivo. We found that cell chemotaxis will not disappear immediately without electric field. And within a certain time cell still exhibit directional migration after removed the DC electric field which suggesting that inducing chemotaxis of cells, continuous electrical stimulation may not be required. So we suppose that pulse DC electric field which is intermittent stimulate cell may also guide cell chemotaxis.Pulsed DC electric field(PDCEF) is a low frequency, low voltage, intermittent DC electric field which making of three parameters: the field strength(EF), frequency(f), the duty cycle(D). The time of PDCEF on cell can be decreased due to the adjustable duty cycle. If the low duty cycle of pulsed DC can induce cell chemotaxis, it can significantly reduce the duration time of the electric field on cells, thereby reducing the electric field thermal effects and electrolytic effects. This can provide technical support to promote wound healing by electric field. And the experiment explored different parameters of PDCEFon cell chemotaxis, ultimately determine the best parameter for reference.Materials and Methods1. The isolation and culture of primary skin cells and the construction of electrotaxis modelThe full-thickness skin were removed from neonatal Balb/c mouse, the dermis soak in DispaseⅡ at 4°C overnight. After that separate epidermis and cut it into piece, with 0.25% trypsin for about 5min, then put it into the solution of medium and serum terminating digestion. And the solution was suspended, filtered, centrifuged. The suspension was divided into several dish after cell density adjustment, and those dishes were put into carbon dioxide incubator at 37°C with 5%CO2. The culture medium were replaced after cells achieve the desired density, and cultured for later use. The electrictaxis chamber model was soaked in 75% alcohol for 30 min and then placed in clean bench under UV light irradiation for 30 min. Put the glass seeded with epidermal cells into the electrictaxis chamber and adding culture medium on two opposite sides of chamber each 2ml. Building an applied electric field as usual and putting the entire electrictaxis model into a living cell workstation.2. The role and characteristics of pulsed DC electric field on cell electrictaxis1) In order to clear whether PDCEF can induce cell electrictaxis, we set the EF is 150 m V / mm, D is 60% and f is 0.1Hz.2) To study the variation of cell electrictaxis with EF of the electrical stimulation, we conducted the experiments on EF from 0 to 200 m V/mm with frequency 0.1Hz and duty cycle of 60%.3) The in order to study the variation of cell electrictaxis with the change of duty cycle, we set the experiments on duty cycle from 0 to 100% with frequency of 0.1Hz and EF of 150 m V/mm.4) Last, to study the variation of cell electrictaxis with frequency of the electrical stimulation, we performed the experiments at EF of 150 m V/mm and duty cycle of 60% with frequency from 0.001 Hz to 5000000 Hz.3. Comparative study of PDCEF and CDCEF on cell electrictaxis and electrochemical side reactions1) When the EF is the same(change from 50-250 m V/mm), compare PDCEF(frequency is 0.1Hz) with different duty cycle to CDCEF on cell electrictaxis.2) When the EF(change from 50-250 m V/mm) and the time which cell under electric filed is the same, compare PDCEF(frequency is 0.1Hz) with different duty cycle to CDCEF on cell electrictaxis.3) Setting the EF is 150 m V/mm, duty cycle is 60%, frequency is 0.1Hz for PDCEF and EF is 150mV/mm for CDCEF. Compare PDCEF with CDCEF on the change of p H and m V value in culture medium and buffer.Result1. The neonatal rat primary keratinocyte culture and the construction of electrictaxis modelPrimary keratinocyte start adherent around 4h, growing well, clear outline and good refraction, this laid a good foundation for the experiment next. The electrictaxis model can be put in living cell workstation for the observation of cell moving and the system having good stability.2. The analysis of PDCEF on cell electrictaxis1) Within 3 hours observation, cells showed random movement without electric field, but showed an obverse directional migration toward the cathode of electric field. And cell speed along X axis increased apparently. Time course analysis showed that cell began to move toward cathode under PDCEF for 30 minutes.2) Cell electrictaxis under PDCEF were positively correlated on EF, when the frequency is set at 0.1Hz and duty cycle is set at 60%. The cell directedness is 0.35 ± 0.05 under the EF of 50 m V/mm which means that the threshold EF for PDCEF is below 50 mV/mm.3) Cell electrictaxis under PDCEF were positively correlated on duty cycle, when the frequency is set at 0.1Hz and EF is set at 60%. The cell directedness is 0.26 ± 0.05 under the duty cycle of 20% which means that the threshold duty cycle for PDCEF is around 20% under this condition. When the duty cycle of PDCEF reaches or greater than 60%, cell directedness not increased apparently and there was no significant difference with CDCEF.4) The changing of frequency in 1000 Hz to 0.001 Hz could not affect PDCEF to induce cell electrictaxis. But it is not true for the frequency of 5000000 Hz.3. Comparative study of PDCEF and CDCEF on cell electrictaxis and electrochemical side reactions1) Under the same EF, PDCEF(frequency is 0.1Hz) reached a certain threshold can induce a directional response comparable with that induced by a CDCEF. The threshold duty cycle for a pulsed DCEF to induce a directional response comparable with that induced by a constant DCEF varied depending on the applied field strength: it was 60% at 150 m V/mm but 80% and 40% at 50 and 250 m V/mm, respectively.2) When EF and the actual time which cell under electric filed is the same, PDCEF can induce cell electrictaxis with shorter time compared to CDCEF. It is 30 min, 15 min and 15 min for PDCEF to induce cell electrictaxis but 45 min, 45 min and 30 min for CDCEF, when the EF is 50, 150 and 250 m V/mm. This result means that intermittent electric stimulation can enhance electrictaxis response of epidermal cells.3) The change of p H and m V value for culture medium or buffer is bigger under CDCEF than cell under PDCEF, and this trend is clearer as the time pass by.Conclusion1. PDCEF can induce keratinocyte electrictaxis in electric field, stimulation time as well as duty cycle dependent manner.2. PDCEF reaching threshold duty cycle is sufficient to trigger an electrotactic response comparable with that induced by a constant DCEF at the same field strength. The threshold duty cycle shifts toward lower duty cycles with increasing voltage: the higher the voltage is, the lower is the threshold duty cycle.3. Keratinocytes exhibit greater electrotactic sensitivity to pulsed DCEFs than to constant DCEFs given an identical duration of exposure to the electrical signal under the same EF.4. A pulsed DCEF induces fewer electrochemical reactions than does a constant DCEF.5. PDCEFs might be particularly suitable as a means of electrotherapy to guide the migration of keratinocytes in wound healing.