Development of Neuronal Guidance Fibers for Stimulating Electrodes: Basic Construction and Delivery of a Growth Factor

Abstract

State-of-the-art treatment for sensorineural hearing loss is based on electrical stimulation of residual spiral ganglion neurons (SGNs) with cochlear implants (CIs). Due to the anatomical gap between the electrode contacts of the CI and the residual afferent fibers of the SGNs, spatial spreading of the stimulation signal hampers focused neuronal stimulation. Also, the efficiency of a CI is limited because SGNs degenerate over time due to loss of trophic support. A promising option to close the anatomical gap is to install fibers as artificial nerve guidance structures on the surface of the implant and install on these fibers drug delivery systems releasing neuroprotective agents. Here, we describe the first steps in this direction. In the present study, suture yarns made of biodegradable polymers (polyglycolide/poly-ε-caprolactone) serve as the basic fiber material. In addition to the unmodified fiber, also fibers modified with amine groups were employed. Cell culture investigations with NIH 3T3 fibroblasts attested good cytocompatibility to both types of fibers. The fibers were then coated with the extracellular matrix component heparan sulfate (HS) as a biomimetic of the extracellular matrix. HS is known to bind, stabilize, modulate, and sustainably release growth factors. Here, we loaded the HS-carrying fibers with the brain-derived neurotrophic factor (BDNF) which is known to act neuroprotectively. Release of this neurotrophic factor from the fibers was followed over a period of 110 days. Cell culture investigations with spiral ganglion cells, using the supernatants from the release studies, showed that the BDNF delivered from the fibers drastically increased the survival rate of SGNs in vitro. Thus, biodegradable polymer fibers with attached HS and loaded with BDNF are suitable for the protection and support of SGNs. Moreover, they present a promising base material for the further development towards a future neuronal guiding scaffold.

Keywords: BDNF; cochlea implant; heparan sulfate; implant-associated drug delivery; polycaprolactone; polyglycolide.

FIGURE 1

Scheme of surface functionalization of PGA/PCL polymer fiber (grey) including amino-modification (green) of the surface and subsequent attachment of heparan sulfate (blue) and loading of the growth factor BDNF (red).

FIGURE 2

Scheme of the chemical operations used in surface functionalization of PGA/PCL polymer fibers (grey). Aminolysis with ethylenediamine (green); covalent attachment of HS (blue). Different functional groups of HS (amino (left)/carboxy (right)) lead to the coupling via amide bonds. The immobilization of BDNF (red) follows in both cases. IPA stands for isopropanol, DCC for N,N′-dicyclohexylcarbodiimide, NHS for N-hydroxysuccinimide, HS for heparan sulfate, MES for (2-N-morpholino)ethanesulfonic acid, EDC for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, BDNF for brain derived neurotrophic factor, PBS for phosphate buffered saline and BSA for bovine serum albumin.

FIGURE 3

Infrared spectra of unmodified and aminolyzed fibers as well as of ethylenediamine. In the spectrum of the aminolyzed fibers the bands of the two amide vibrations I and II are marked.

FIGURE 4

(A) Fluorescence microscopy images of aminolyzed (left) and control fibers (right) after treatment with FITC. (B) SEM images of unmodified (top) and amino-modified fibers (bottom) at two different magnifications.

FIGURE 5

pH-dependent zeta potential titration curves of unmodified (dashed grey line) and amino-modified (dashed green line) fibers as well as for those fibers coated with heparan sulfate: unmodified fibers (continuous grey line) and amino-modified fibers (continuous green line).

FIGURE 6

Results of TB assay: Absorption of TB solutions after contact with different types of fibers: Unmodified fibers without HS (grey stripes)/with HS (grey) as well as amino-modified fibers without HS (green stripes)/with HS (green) (right). Values are given as mean ± standard error of the mean (N = 3).

FIGURE 7

Release of HS from unmodified and amino-modified (PBS with 0.1% BSA, 110 days, 37°C). The left axis represents the cumulative HS release referred to 1 ml release medium and the right axis shows the cumulative release of HS with regard to 1 cm fiber length.

FIGURE 8

pH-dependent zeta potential titration curves of unmodified (dashed grey line) and amino-modified (dashed green line) fibers as well as for unmodified (continuous grey line) and amino-modified (continuous green line) fibers covered with heparan sulfate after a release of 21 days in PBS (with 0.1% BSA, 37°C).

FIGURE 9

Release of BDNF from unmodified and amino-modified fibers pre-coated with heparan sulfate (HS) (PBS with 0.1% BSA, 110 days, 37°C). The left axis represents the cumulative BDNF release referred to 1 ml release medium and the right axis shows the cumulative release of BDNF with regard to 1 cm fiber length.

FIGURE 10

Relative BDNF release curves for unmodified and amino-modified fibers, carrying heparan sulfate or not. The total amount released was set to 100% for each curve.

FIGURE 11

Relative cell viabilities of NIH 3T3 fibroblasts in the presence of unmodified and amino-modified fibers as determined by the NRU assay after an incubation of 4 days. Values are given as mean ± standard error of the mean (N = 3).

FIGURE 12

Representative microscopic images of spiral ganglion cell cultures using the supernatants from the two different fiber types, derived from the release experiment of day 1. SGNs are counted as survived neurons when a neurite with a length of at least three soma sizes has grown out of the cell soma. Scale bar 100 µm.

FIGURE 13

Results from spiral ganglion cell culture studies after cultivation for 2 days. Cells were incubated with the supernatants from the release experiments of either unmodified (grey columns) or amino-modified (green columns) fibers. Fibers carrying BDNF and HS are depicted in the darkest colour shades, those carrying only HS are depicted in medium colour shades, and those not further modified are depicted in the lightest colour shade. Values for controls are shown as white columns (PBS and medium as negative controls, medium with added BDNF as positive control). (A) Survival rates of spiral ganglion neurons. (B) Neurite lengths of survived SGNs. Values are given as mean ± standard error of the mean (N = 3, n = 3). Statistical assessment was performed using one-way ANOVA with Tukey’s Multiple comparison test (n.s. = not significant, *p < 0.05; **p < 0.01; ***p < 0.001). Asterisks over the bars indicate the significance of the survival rates of different conditions compared to the medium (serum-free SGN medium) and PBS control (as the results obtained for both cases are very similar to each other) as well as the significance of the neurite lengths of different conditions compared to the medium control.