Residual rubber shielded multi walled carbon nanotube electrodes for neural interfacing in active medical implants

Advanced neuroprostheses need high density, mechanically flexible contacts with superior electrophysiological performance. Carbon nanotubes have shown interweaving with neurites are well suited but are opposed by ongoing nanoparticle biocompatibility discussions. We present a route circumventing those issues by immersing multiwalled carbon nanotubes (MWCNT) in silicone rubber and re-etch the surface yielding a MWCNT-lawn electrically contacted towards the percolative bulk. The use of tetra-n-butylammonium fluoride (TBAF) and sodium hydroxide solution (NaOH) leads to desired freestanding CNT strands still covered by residual rubber of approximately 13 nm thickness. The biocompatibility of such interfaces has been proven by WST-1-Assays for cell metabolism of 3T3NIH fibroblasts and SH-SY5Y neuroblastoma cells in terms of growth and morphology. Neural cell adhesion is proven with biomolecular markers. The electrical performance reaches percolation conductivities of up to 1.6 × 102 S/m. The lowest impedance was 1.3 × 102 Ωcm2 at 1 kHz, which is similar to gold reference electrodes whilst their capacitive roll off is lowered in electrophysiological arrangements. When compared to pure MWCNTs the performance is decreased due to the insulating residual rubber encasement. However, this is seen to be a reasonable loss in the light of the increased biosafety of rubber shielded MWCNT neural interfaces. Keywords: Carbon nanotubes, PDMS, Active neural implant, Biocompatibilit

Magnetic beads enhance adhesion of NIH 3T3 fibroblasts: A proof-of-principle in vitro study for implant-mediated long-term drug delivery to the inner ear

Introduction Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated. Materials and Methods Murine NIH 3T3 fibroblasts-genetically modified to produce BDNF-were labelled with MB. Results Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release. Discussion Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest. © 2016 Aliuos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Dissociated Neurons and Glial Cells Derived from Rat Inferior Colliculi after Digestion with Papain

<div><p>The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode–tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue–material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3<b>_</b>5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2<b>×</b>10⁴ cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.</p></div

Synoptic representation of the proteolytic enzymes used for dissociation.

<p><i>Each assay includes at least two independent preparations with 2–10 wells;</i></p><p><i>w/ = with DNase I.</i></p><p><i>w/o = without DNase I.</i></p

In Vitro Evaluation of PCL and P(3HB) as Coating Materials for Selective Laser Melted Porous Titanium Implants

Titanium is widely used as a bone implant material due to its biocompatibility and high resilience. Since its Young’s modulus differs from bone tissue, the resulting “stress shielding” could lead to scaffold loosening. However, by using a scaffold-shaped geometry, the Young’s modulus can be adjusted. Also, a porous geometry enables vascularisation and bone ingrowth inside the implant itself. Additionally, growth factors can improve these effects. In order to create a deposit and release system for these factors, the titanium scaffolds could be coated with degradable polymers. Therefore, in the present study, synthetic poly-ε-caprolactone (PCL) and the biopolymer poly(3-hydroxybutyrate) (P(3HB)) were tested for coating efficiency, cell adhesion, and biocompatibility to find a suitable coating material. The underlying scaffold was created from titanium by Selective Laser Melting (SLM) and coated with PCL or P(3HB) via dip coating. To test the biocompatibility, Live Cell Imaging (LCI) as well as vitality and proliferation assays were performed. In addition, cell adhesion forces were detected via Single Cell Force Spectroscopy, while the coating efficiency was observed using environmental scanning electron microscopy (ESEM) and energy-dispersive X-ray (EDX) analyses. Regarding the coating efficiency, PCL showed higher values in comparison to P(3HB). Vitality assays revealed decent vitality values for both polymers, while values for PCL were significantly lower than those for blank titanium. No significant differences could be observed between PCL and P(3HB) in proliferation and cell adhesion studies. Although LCI observations revealed decreasing values in cell number and populated area over time on both polymer-coated scaffolds, these outcomes could be explained by the possibility of coating diluent residues accumulating in the culture medium. Overall, both polymers fulfill the requirements regarding biocompatibility. Nonetheless, since only PCL coating ensured the maintenance of the porous implant structure, it is preferable to be used as a coating material for creating a deposit and release system for growth factors

Fluorescence images of different antibodies for glial cell characterization.

<p>Papain digested cells were labelled with GFAP (red) and MAG (green) and are depicted in the first row. Positive staining for GFAP as well as MAG discriminate astrocytes as well as oligodendrocytes, respectively. A second astrocytic marker (S100, red) was also tested in combination with TUJ1 (green) and presented in the second row. Positive staining for S100 confirmed previously obtained results with GFAP (second row). Two different magnifications are presented in the left (20×; scale bar: 100 µm) and right column (40×; scale bar: 50 µm).</p

Synoptic representation of dissociation kits.

<p><i>NTDK = Neural Tissue Dissociation Kit (from Miltenyi Biotech);</i></p><p><i>Each assay includes at least two independent preparations with 2–10 wells;</i></p>$<p><i> = supplemented Panserin 401;</i></p>*<p><i> = supplemented MACS® Neuro Medium.</i></p

Fluorescent images from the testing of proteolytic enzymes.

<p>Cells digested either with trypsin (15 min: first row) or papain (30 min: second row and 90 min: third row) were triturated without (left column) and with DNase I (right column). Merged pictures of cells labelled with TUJ1 antibody (green) and GFAP antibody (red) were depicted. When comparing all depicted conditions, an improved cytoarchitecture with high neuronal (and glial) yield was obtained in cultures dissociated with papain for 30 min without DNase I. Scale bar: 100 µm.</p

Immunocytochemical results of different dissociation protocols and media.

<p>Improved neuronal yield and branching was observed in cells cultivated for 5 days with MACS® Neuro Medium (right column). By contrast, cells cultivated with Panserin 401 (left column) showed less branching with prominent neurite fragmentation (A, E). IC tissue was dissociated with different protocols: NTDK T (A, B), NTDK P (C, D) and NTDK PN (E, F) as well as SGN-protocol (G, H). After fixation and labelling with TUJ1 (green), poor neuronal yield was obtained after dissociation with NTDK T independent from the cultivation medium. Scale bar: 100 µm.</p