Recently, we found that electrical stimulation can induce neuronal migration in neural networks cultured for more than 3 weeks on microelectrode arrays. Immunocytochemistry data showed that the aggregation of neurons was related to the emergence of astrocytes in culture. In this study, when neurons were cocultured with astrocytes, electrical stimulation could induce the migration of neuronal cell bodies after only 1 week in culture, while the same stimulation paradigm caused neural necrosis in neuron-only cultures. In addition, the stimulation-induced migration was inhibited by blocking action potentials in neural networks using the voltage-gated sodium channel blocker, tetrodotoxin. Immunocytochemistry was performed to monitor precisely the neuronal migration and count the number of neurons. These results indicate that neuronal migration of cell bodies is dependent on neuronal activity evoked by electrical stimulation and can be enhanced by coculturing with astrocytes. We believe this method can be employed as a means for modifying neural networks and improving the interface between electrodes and neurons.

Electrical stimulation (ES) can activate diverse biostimulatory responses in a range of tissues. Of various forms of ES, the application of biphasic electric current (BEC) is a new approach to bone formation. This study is to investigate the effects and mechanism of action of BEC in osteoblast differentiation and cytokine production in human mesenchymal stromal cells (hMSCs). Using an in vitro culture system with a modified version of the BEC stimulator chip used in our previous study, we exposed hMSCs to a 100 Hz ES with a magnitude of 1.5/15 muA/cm(2) for 250/25 mus. hMSCs showed increased proliferation during static BEC stimulation for 5 days. However, alkaline phosphatase activity and calcium deposition were enhanced in hMSCs 7 days after the stimulation, rather than during the period of ES. BEC induced vascular endothelial growth factor (VEGF) and BMP-2 production; the former can enhance the proliferation of human umbilical vein endothelial cells in culture using conditioned media from BEC cultures. Treatment with selective inhibitors of p38 MAPK (SB203580) or Erk (PD98059), as well as calcium channel blockers (verapamil and nifedipine), reduced the BEC-mediated increase of VEGF expression and cell proliferation. These findings reveal that BEC is involved in the osteoblast differentiation of hMSCs through enhancement of cell proliferation and modulation of the local endocrine environment through VEGF and BMP-2 induction through the activation of MAPK (Erk and p38) and the calcium channel. Thus, local stimulation using BEC might be most beneficial in promoting osteogenic differentiation of hMSCs, resulting in enhanced bone formation for bone tissue engineering.

This study investigated biphasic electric current (BEC) functions as a new type of electrical stimulation to induce rat calvarial osteoblasts to proliferate, differentiate and synthesize cytokines. The culture system was designed so that biphasic current flowed between upper and lower gold plates. BEC helps to minimize the net charge accumulation during cell exposure to the electrical stimulation. Osteoblasts were exposed to electrical stimulation of 1.5 microA/cm2 at 3000 Hz, and the effect of BEC was assessed in the interrupted mode (6 h daily) and in the continuous mode (24 h daily), depending on the interval of stimulation. Whereas proliferation increased by 31% after stimulation in the continuous mode for 2 days, it was unaffected in the interrupted mode. The transcriptional expression of osteogenesis-related genes such as alkaline phosphatase (ALP), osteopontin, and type I collagen was unchanged 4 days after stimulation in both modes, while cbfa1 was decreased under the same conditions. There was no detectable change in mRNA expression of growth factors (BMP-2, -4, IGF-2 and TGF-beta1) that promote osteoblast differentiation. However, real-time RT-PCR and ELISA demonstrated that vascular endothelial growth factor (VEGF) was markedly up-regulated by BEC. Induction of VEGF by BEC was not hypoxia driven. In conclusion, the present in vitro study demonstrates that BEC increases cell proliferation and induces the production of VEGF. The BEC was more effective with continuous stimulation than with interrupted stimulation. To confirm whether BEC can enhance osteogenesis, further in vivo studies are needed.

This work was supported by Korea Science and Engineering Foundation (KOSEF) through Nano Bioelectronics and Systems Research Center (NBSERC) in Seoul National University.

Planar microelectrode arrays (MEAs) are widely used to record electrical activity from neural networks. However, only a small number of functional recording sites frequently show electrical activity. One contributing factor may be that neurons in vitro receive insufficient synaptic input to develop into fully functional networks. In this study, electrical stimulation was applied to neurons mimicking synaptic input. Various stimulation paradigms were examined. Stimulation amplitude and frequency were tailored to prevent cell death. Two effects of stimulation were observed when 3-week-old cultures were stimulated: (1) clusters of neural cells were observed adjacent to stimulating electrodes and (2) an increase in spontaneous neuronal activity was recorded at stimulating electrodes. Immunocytochemical analysis indicates that stimulation may cause both new neuron process growth as well as astrocyte activation. These data indicate that electrical stimulation can be used as a tool to modify neural networks at specific electrode sites and promote electrical activity.

We developed a novel dental implant technology for early bone formation using electrical µ-current stimulator integrated in healing abutment of implant. Electrical stimulation makes a early bone formation possible for early loading of implants. In order to implement small sized stimulation system, we needed a small chip that could be operated on a tiny battery. Thus, we designed a current stimulator chip using 0.35µm fabrication process of SAMSUNG semiconductor through the 41st IDEC MPW program. The chip is composed of 5 parts, which is a clock generation block, duration setting block, pulse rate setting block, amplitude setting block and biphasic pulse control block. In our system, stimulation parameters are fixed with 120µs of duration, 100Hz of pulse rate and 20µA/㎠ of amplitude and power is delivered by a serial connected silver oxide battery(SR421SW, Sony) of which dimension is 1.65mm of thickness and 4.8mm of diameter. The chip consumed 142µW power and lasted 7 days in an implanted site. The fabricated chip is wire bonded with printed circuit board (PCB) and packaged in a healing abutment made with Poly-carbonate insulating materials. Reference electrode(screw) and channel electrode(cap) made with the material of pure titanium are soldered with the PCB using a fine pt wire. In order to give a tolerance against the external shock, the fast setting adhesive epoxy was injected inside the healing abutment with integrated system. We implanted the current stimulator system into the 10 dogs(Beagle). After pulling out premolar of maxillary and mandibular, wounded sites were stabilized for 45 days. Then, the system was implanted in mandibular first premolar with the control. Electrical stimulation was applied for 1 weeks and the test group was sacrificed at 2 weeks after stimulation. Histological specimens with H&E and Masson Trichrome staining were made and histomorphometrically analysed with image analyser. A new bone formation was observed around implants in the experimental group. The area of a new bone was increased from 13.77% to 43.93% compared to that of control. These results suggest that our newly developed system is effective in the early bone formation around the surface of implant. It can be a useful method in clinical application for the early loading of implant.

This work was supported by the MOCIE(Ministry of Commerce, Industry and Energy), KOREA under the IDEC Supported Program(MPW) and SAMSUNG Electronics Co. LTD.

Since the discovery of piezoelectric properties of natural bone, electrical stimulation has been widely used in the clinical treatment of orthopedic fracture. Nevertheless, in dental implant technologies, it is the methods of surface modification that has been recently developed to enhance early osteointegration between implant's surface and surrounding tissue. In this paper, in order to accelerate bone formation, we developed a battery powered biphasic electrical current stimulator integrated in healing abutment of dental implant. In vivo. animal test shows that the proposed electrical stimulation system expends the osteogenesis of the implant's surfaces by 2.15 times more than that of controls. Based on these results, we propose novel dental implant technology that can be applied to patient with osteoporosis.

This work was supported by the International Collaboration Program, NBS-ERC (Nano Bioelectronics and Systems Engineering Research Center)/ KOSEF (Korea Science and Engineering Foundation) and also supported in part by the Nanobiotechnology Center (NBTC), an STC Program of the National Science Foundation under Agreement No. ECS-9876771.