Final Report Summary - DREAMS (Diamond to retina artificial micro-interface structures) Electrical stimulation of neurons is a recognised therapeutic approach for the treatment of several neurodegenerative pathologies (Parkinson's disease, audio prosthesis, etc). These techniques could have a high impact on the treatment of other pathologies like epilepsy, or to restore function as in blindness. For these applications, the long-term stability of the device is mandatory and a closer neuro-electronic interface is required to lower the threshold of neuronal activation. Currently available commercial devices are based on metallised electrodes which are degraded in a physiological environment and induce reactive gliosis leading to insulating interfaces. The high resolution required for vision and the stimulation of graded potential neurons requires the use of complex and very precise stimulators capable of generating signals of varying intensity. Recent progress in nanotechnologies and their application for biology have motivated the emergence of new interfacing concepts for direct communication between biological and electronics. DREAMS proposed to study and fabricate novel types of nanotransducers, which are based on artificial nanocrystalline diamond (NCD). NCD is a semiconductor that exhibits extreme biocompatibility and stability in physiological media. From the NCD surface functionalisation, selective physiological applications can be fabricated to high extend. The use of NCD diamond films for coating of metallic electrodes or non-metallised CMOS devices will produce novel active hybrid structures on which neural cells can be grown to fabricate novel biocompatible implants to restore a useful vision. Survival of neurons and particularly retinal cells will be evaluated on NCD diamond surfaces. Then NCD neuro-compatible interfaces will be fabricated and tested in vitro for their ability to activate neurones that will be recorded individually with the patch clamp technique. Finally, the project will lead to the fabrication of matrix of field effect transistor structures that can be validated for stimulation of the retina as well as for readout of the retinal signals in-vivo. The project objectives were directed towards the development of novel approaches for electrical stimulation of neurons aiming at the treatment of neurodegenerative pathologies for restoring visual sight functions. by building up artificial retinal cell interfaces using stimulation devices and by tuning the biocompatibility of the stimulation electrodes and their surface interactions with neurons, we proposed to fabricate microelectrodes and microelectrode arrays (MEAs) as well as ion sensitive field effect transistors (ISFETs) for medical applications in vivo. For these applications long-term stability of the implant device is required and a closer proximity neuro-electronic interface is required to lower the threshold of neuronal activation. DREAMS enabled to study and fabricate these novel types of nanotransducers, which are based on synthetic NCD and epitaxial single crystal CVD diamond thin films. The key objectives of the project were to take advantage of both the biocompatibility and the semiconducting properties of diamond in order to explore the feasibility of fabrication of novel artificial retina implants. In the course of the DREAMS project, we have shown that mixed retinal cells can grow on nanocrystalline diamond. In these mixed retinal cells, we did find glial cells and retinal neurones, especially bipolar cells postsynaptic to photoreceptors. A specific staining of these different cells indicated that retinal neurones were directly adhering on the diamond surface with retinal glial cells present at a certain distance. Such features were present with or without polypeptide (polylysine and laminine) coating on the diamond material. Cell survival was confirmed by incubating the cultured cells in the live / dead assay showing calcein accumulation in the viable cells leading to a strong green fluorescence. To investigate if retinal neurones could survive on diamond independently of glial cells, adult retinal ganglion cells were purified and seeded on diamond. Again, these cells were found to survive and grow extended neurites on diamond. In the final quantification showed similar abilities to grow on diamond and glass. Finally, the preference between polypeptide coating and pure nanocrystalline diamond was compared by applying a polypeptide pattern on the diamond during this final semester period. All these results indicated that nanocrystalline diamond is a remarkably biocompatible material for retinal cells and more precisely for retinal neurones. They therefore suggest that this diamond material could provide an excellent biocompatible material for interfacing retinal prostheses and retinal tissue or more precisely retinal neurones. These data are currently compiled in a large paper for publication in an international journal. Using the diamond ISFETs arrays developed in this project, we have demonstrated for the first time the recording of the electrical activity of electrogenic cells which were cultured onto the devices. Electrical recordings from two different cell lines, both showing healthy growth and good adhesion to the substrate, have been successfully acquired. The diamond transistors are used to detect electric signals from both types of cells by recording the extracellular potential. The electrical activity from a three-days culture of HL-1 cells (cardiomyocytes, heart muscle cells) has been recorded, revealing the shape and the propagation of the spontaneously generated action potentials throughout the dense cell layer. In order to investigate the activity of individual cells, a better-controlled system has been investigated with patch-clamped human embryonic kidney cells (HEK 293), which stably expressed voltage-gated K+ channels. In our cell-sensor coupling experiments, the cell's membrane potential is controlled by the patch pipette, which is used to induce the opening and closing of the potassium channels in the cell membrane, thus changing the transductive extracellular potential. The induced change of the extracellular potential is successfully recorded by the diamond transistor. Furthermore, the ion sensitivity of the diamond surface enables the detection of released potassium ions accumulated in the cleft between transistor and cell. The results from our diamond solution-gated field effect transistors (SGFETs) attest to the promising capabilities of diamond for bioelectronics. Retinal implants with a diamond layer were produced by CEA with similar dimensions as those used in a previous study (Salzmann et al., 2006). They were implanted following a similar procedure in rats. During the in vivo period, an endoscope was used to visualise the retinal implant and control its position. Finally, animals were sacrificed one month after the operation, perfused with fixatives to maintain the retinal implant in the same position as in vivo. Retinal sections made with a cryostat enabled us to label the tissue with antibodies specific for reacting glial cells. These staining indicated that no major reactive gliosis was induced by diamond. However, in some instance, the diamond layer dissociated from polyimide, although this is likely to have occurred after this animal was sacrificed during the sample preparation. These studies are consistent with the in vitro studies showing a good biocompatibility for diamond in contact to the retinal tissue. It further supports the potential use of diamond in neuroprostheses and more specifically in retinal prostheses. Documenti correlati Final Report - DREAMS (Diamond to retina artificial micro-interface structures)
