Effect And Mechanism Interfacially Localized Electrical Stimulation On Osteogenic Differentiation Of Pre-osteoblasts

Electrical stimulation(ES)is an effective approach to modulate the growth behavior of cells and tissues(including bone tissue).And the bioactive coating on implants with ES functions can endow implants long-term and effective regulation of cell growth/remodeling of bone tissue as well as antibacterial ability.Considering the future goal of functional stimulating coatings,an interfacially localized electrical stimulation(ILES)model was proposed in this study by adopting planar microelectrodes,trying to understand the ES effect onto the cells adhered on the electrodes.The effect of ES on osteogenic differentiation of pre-osteoblast(MC3T3-E1 cells)was systematically studied under ILES model.Moreover,the mechanism of ILES model was also discussed.The main research achievement contains the following four aspects:1.Construction of ES cell culture devicesThe wire,wire/electrode connection,reproducibility and cell safety of cell culture devices were studied through the sticking contact-type or pressing contact-type method.For sticking contact-type method,the copper wire was connected to the planar microelectrode through the "silver paste/epoxy resin" sticking method.And the results showed that the appearance of copper element on the electrode surface at high voltages(1 to 2 V),indicated that the copper wire was electrochemically corroded during the stimulation.And it resulted in the obvious difference of protein adsorption on the positive,negative and intermediate regions.In addition,the wire/electrode connection was easily loosened after soaking in cell culture medium for 7 days.For pressing contact-type method,a tetrafluoroethylene cell culture device was designed to integrate a pressing contact to stably fix the wire onto the electrode surface and a wire within the tetrafluoroethylene to effectively avoid occurrence of electrochemical corrosion.The pressing contact-type device can reliably and safely guarantee the ES to cells under ILES model.2.Preparation of planar microelectrodes suitable for ILES modelThe effect of electrodes to cells is considered to depend on their injected charge(Qinj),which is closely related to the charge injection capacity(CQ)of the electrode material.CQ of the Au,ITO,and Ppy planar microelectrodes was measured,and the results showed that the CQ of Ppy electrode was 24 and 12 times larger than that of Au and ITO electrodes,respectively.The higher CQ endows the electrode higher Qinj at the same applied voltage.Therefore,the Ppy microelectrode was suitable to generate ILES to cells.During the preparation of Ppy planar microelectrodes by electropolymerization,the deposition current density was adjusted to regulate the Ppy polymerization/deposition kinetics for controlling electrode characteristic.The results showed that the Ppy polymerization/deposition process was intensified with an increase of deposition current density(3,5 and 8 mA/cm2),and the Ppy thickness was 20 nm,50 nm and 80 nm,respectively.Electrochemical measurements showed that the thickness could further adjust the CQ of the Ppy electrodes,and CQ increased with electrode thickness.Cell culture results showed that Ppy electrode had good biocompatibility.Therefore,a reasonable selection of Ppy deposition conditions could further maximize the stimulation effect to cells under ILES model.3.Effect of ILES on osteogenic differentiation of MC3T3-E1 cellsThe Ppy electrode with only 1 mV applied voltage have an effective promotion on osteogenic differentiation of the cells,while the ITO electrode needed to be applied 250 mV.This indicates that ES to cells on Ppy electrode was ILES model.Acoording to the stimulated results on different Ppy electrodes,the relationship between the stimulation intensity Qinj of all Ppy electrodes and osteogenic differentiation activity of MC3T3-E1 cells presented a parabola-shaped effect,and different Ppy electrodes showed a significant enhancement in cell osteogenic differentiation at Qinj ranged from 0.08 ?C to 0.15 ?C.For high CQ Ppy electrode(Ppy-III),only 1 mV was needed to generate Qinj of 0.08 ?C.It is found that the stimulated effect to cells under ILES model was independent to the applied voltage,and the Qinj value related to the electrode material characteristics is a determining parameter.Pulse frequency represents the number of ES of Qinj in one second and ES with frequency of I or 25 Hz showed an enhanced osteogenic differentiation of cells,while that with 50 Hz had no enhancement.Considering the stimulation time,the expression of ALP protein and osteogenesis-related genes was significantly up-regulated at 1 h/d rather than 0.5 and 24 h/d.The osteogenic differentiation potential induced by ILES was found to relate to the cell growth stage,the ES was performed at the early days(days 2-5)was more effective in promoting osteogenic differentiation than that at the later stage(days 6-8)of the overall cell culture of 8 days.4.The analysis of ILES mechanism to cellsCellular membrane voltage gate is considered to be a key point for ES to cells.According to type and characteristics of the gates,electrical field distribution characteristics of the planar microelectrode in this experiment,and the relationship between the ES intensity Qinj and the osteogenic differentiation response of cells,the mechanism of ILES to cells was revealed.The electrical field generated by the electrode Qinj mainly affects the cell membrane at the interface of the cell with the electrode,and the direction of the electric field is parallel to the cell membrane,which enhances the intracellular and extracellular ion migration variability.Therefore,the membrane potential is effectively changed at a very low applied voltage to open the L-type voltage gate of the cell membrane,and further activating calcium channels and osteogenic differentiation signal pathways.