organization and axonal outgrowth in neuron-like
PC12-Cells
Alexey Klymov, Charlotte T Rodrigues Neves, Joost te Riet, Martijn J
H Agterberg, Emmanuel A M Mylanus, Ad F M Snik, John A Jansen, X
Frank Walboomers
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Introduction
Congenital or acquired severe to profound sensorineural deafness, which results from defective loss or damage of sensory hair cells, is preferentially treated by placement of a cochlear implant (CI). More than 324000 patients worldwide have received such a CI to date.1 The implants are bypassing
the damaged hair cells, allowing direct stimulation of the spiral ganglion cells (SGCs). Although the quality of the implants and thereby the patient perception- and understanding-ability improved during the last decade, CI- based hearing still has substantial shortcomings. High fidelity hearing might be limited due to the poor spatial resolution on the interface between the electrode and SGCs. Sounds are perceived by separated stimulation of approximately 30000 neurones of the auditory nerve in a healthy situation, while only 4 – 8 broad range areas of SGCs can be triggered by up to 22 electrodes in the most recent devices.2 Transfer of temporal information
is difficult in this situation leading to limited possibilities for speech recognition-in-noise and music appreciation. A possible explanation for the rather crude stimulation of the SGCs in limited filter bands is the relatively large distance of about 150 µm between the electrode contacts, situated in the scala tympani of the cochlea, and the SGCs in Rosenthal’s canal. This results in an interaction of electrical stimuli and thereby in reduction of resolution. Approximation of the electrodes closer to the target cells has
shown an improvement in implant performance,3 allowing speculation
about a possible CI-optimization based on direct electrode-SGC contact. Furthermore, in vivo experiments and post mortem explantation studies, have shown the formation of fibrous tissue and occasionally even bone on the electrode surface, which can also affect the interaction between the electrode and SGCs.4-7 A direct electrode-SGC contact might also reduce
this irritating factor.
Manipulation of the electrode surface topography in combination with delivery of neurotrophins,8-10 may be a possible solution for the reduction
of fibrotic tissue formation and consequently a more direct electrode- auditory nerve contact.11 Moreover this combination can also be a method
for a spatially designed interaction between the implant and the SGCs. The last would allow (1) the achievement of a higher resolution in stimulation
and therefore higher fidelity of patient-experienced perception, and (2) an optimization of the electrode surface that could provide increased affinity for the outgrowing neurons towards the electrode. Studies in other biomedical device applications have shown that especially grooves with micro- and nanodimensions are able to control cell-body organization and alignment of different cell types,12 making such patterns interesting for
the utilization on CI electrode-surfaces. It should be taken into account that the introduced electrode length is highly limited (20 – 30 mm) and relative to damage that is induced in the patient ears.13 As in an ideal
situation up to 30000 cells have to be stimulated by the electrode surface, only nanosized patterns may deliver the necessary separation of signal transduction. Moreover, microgrooves may not be sufficient for control of orientation of outgrowing axons, which have width dimensions in the
submicron range14 and would need contact guidance by templates of
comparable size. The efforts of the semi-conductor industry have led to development of powerful techniques that allow design and structural manipulation of various material-surfaces effectively down to sizes of only few nanometers. Interestingly, several in vitro studies have shown already small-scale nanotopographical features to have enormous influence on cellular behaviour, such as proliferation, differentiation, gene expression and migration.12 However, the effect that a topography can have on the
cell is strongly cell-type dependent. Furthermore, the topography might interact with neurotrophins that stimulate neurite outgrow.
A mechanism by which microgrooves direct neurite organization strongly depends on the axonal localization inside the groove. Due to cellular dimensions these mechanisms may not be effective on nanogrooved surfaces, as cells including the neurites would always be located on top of the grooves. It is not known to which extent neuronal cells can sense and more importantly can be influenced in their behaviour by nanotopographies and what would be the smallest topography on which a recognizable effect could be observed. We hypothesize that a threshold exists for pattern recognition of neuronal cells and that the textures will have an effect on the outgrowth length of the axons. For the current study we made use of a polystyrene culturing surface that featured 5 different nanosized grooved surface topographies and a smooth control. The surfaces were used for
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cell-culture of PC12 neuron-like cells during a period of 7 days, in which the cells were treated with nerve growth factor allowing differentiation and axon formation. After culture the cells were evaluated in terms of overall pattern-size recognition and subsequent organization (measured as pattern-induced cell and axon alignment) and the affinity for the surface pattern (measured as axon outgrowth).