Strategies in Cochlear Nerve Regeneration, Guidance and Protection

Home , Cochlear nerve, Spiral ganglion, Vestibular ganglion, Vestibular nerve

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1193

Prospects for Future Cochlear Implants

FREDRIK EDIN

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-9503-9 UPPSALA urn:nbn:se:uu:diva-276336 2016 Dissertation presented at Uppsala University to be publicly examined in Skoogsalen, Akademiska Sjukhuset, Ingång 78/79, Uppsala, Thursday, 28 April 2016 at 09:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Docent Maoli Duan (Karolinska Institutet, Department of Clinical Science, Intervention and Technology).

Abstract Edin, F. 2016. Strategies in Cochlear Nerve Regeneration, Guidance and Protection. Prospects for Future Cochlear Implants. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1193. 56 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9503-9.

Today, it is possible to restore hearing in congenitally deaf children and severely hearing- impaired adults through cochlear implants (CIs). A CI consists of an external sound processor that provides acoustically induced signals to an internal receiver. The receiver feeds information to an electrode array inserted into the fluid-filled cochlea, where it provides direct electrical stimulation to the auditory nerve. Despite its great success, there is still room for improvement, so as to provide the patient with better frequency resolution, pitch information for music and speech perception and overall improved quality of sound. A better stimulation mode for the auditory nerves by increasing the number of stimulation points is believed to be a part of the solution. Current technology depends on strong electrical pulses to overcome the anatomical gap between neurons and the CI. The spreading of currents limits the number of stimulation points due to signal overlap and crosstalk. Closing the anatomical gap between spiral ganglion neurons and the CI could lower the stimulation thresholds, reduce current spread, and generate a more discrete stimulation of individual neurons. This strategy may depend on the regenerative capacity of auditory neurons, and the ability to attract and guide them to the electrode and bridge the gap. Here, we investigated the potential of cultured human and murine neurons from primary inner ear tissue and human neural progenitor cells to traverse this gap through an extracellular matrix gel. Furthermore, nanoparticles were used as reservoirs for neural attractants and applied to CI electrode surfaces. The nanoparticles retained growth factors, and inner ear neurons showed affinity for the reservoirs in vitro. The potential to obtain a more ordered neural growth on a patterned, electrically conducting nanocrystalline diamond surface was also examined. Successful growth of auditory neurons that attached and grew on the patterned substrate was observed. By combining the patterned diamond surfaces with nanoparticle-based reservoirs and nerve- stimulating gels, a novel, high resolution CI may be created. This strategy could potentially enable the use of hundreds of stimulation points compared to the 12 – 22 used today. This could greatly improve the hearing sensation for many CI recipients.

Keywords: Human vestibular nerve, Scarpa's ganglion, Stem cells, Nanoparticles, Nanocrystalline diamond

Fredrik Edin,

ISSN 1651-6206 ISBN 978-91-554-9503-9 urn:nbn:se:uu:diva-276336 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-276336)

Till Morfar

Front cover: Double immunofluorescence staining of a human vestibular ganglion shows large neural cell bodies and neural extensions staining positive for Tuj1 (red) and TrkB (green). Nuclei are stained with DAPI (blue). Picture adapted from Paper II.

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Edin F, Liu W, Boström M, Magnusson PU, Rask-Andersen H. Differentiation of human neural progenitor cell-derived spiral ganglion-like neurons: a time-lapse video study. Acta Oto- laryngol. 2014 May;134(5):441-7. II Edin F, Liu W, Li H, Atturo F, Magnusson PU, Rask- Andersen H. 3-D gel Culture and Time Lapse Video Micros- copy of the Human Vestibular Nerve. Acta Otolaryngol. 2014 Dec;134(12):1211-8. III Li H, Edin F, Hayashi H, Gudjonsson O, Engqvist H, Rask- Andersen H and Xia W. Guided Growth of Auditory Neurons: Bioactive Particles Towards Gapless Neural - Electrode Inter- face. Submitted Manuscript. IV Cai Y, Edin F, Jin Z, Alexsson A, Gudjonsson O, Liu W, Rask-Andersen H, Karlsson M, and Li H. Strategy towards In- dependent Electrical Stimulation from Cochlear Implants: Guided Auditory Neuron Growth on Topographically Modi- fied Nanocrystalline Diamond. Acta Biomater. 2016 Feb;31:211-220.

Reprints were made with permission from the respective publishers.

Papers not included in thesis

I Brown TD, Edin F, Detta N, Skelton AD, Hutmacher DW, Dalton PD. Melt electrospinning of poly(ε-caprolactone) scaf- folds: phenomenological observations associated with collec- tion and direct writing. Mater Sci Eng C Mater Biol Appl. 2014 Dec;45:698-708. II Nordling S, Hong J, Fromell K, Edin F, Brännström J, Lars- son R, Nilsson B, Magnusson PU. Vascular repair utilising immobilised heparin conjugate for protection against early ac- tivation of inflammation and coagulation. Thromb Haemost. 2015 Jun;113(6):1312-22. III Liu W, Edin F, Atturo F, Rieger G, Lowenheim H, et al. The Pre- and Post-somatic segments of the Human Type I Spiral Ganglion Neurons - Structural and Functional Considerations Related to Cochlear Implantation. Neuroscience. 2015 Jan 22;284:470-82. IV Liu W, Edin F, Blom H, Magnusson PU, Schrott-Fischer A, Glueckert R, Santi P, Li H, Laurell G and Rask-Andersen G. Super-Resolution Structured Illumination Fluorescence Mi- croscopy of the Lateral Wall of the Cochlea –the Connex- in26/30 Proteins are Separately Expressed in Man. Cell Tissue Res 2016. Early online. V Natan M, Edin F, Perkas N, Yacobi G, Perelshtein I, Segal E, Homsy A, Laux E, Keppner H, Rask-Andersen H, Gedanken A and Banin E. Two Are Better than One: Combining ZnO and MgF2 Nanoparticles Reduces Streptococcus pneumoniae Biofilm Formation on Cochlear Implants. Accepted in Adv. Func. Mat. 2016.

VI Liu W, Edin F, Brännström J, Glueckert R, Santi P, Salven- moser W, Laurell G, Blom H, Schrott-Fischer A and Rask- Andersen H. The Epithelial Gap Junction Network in the Hu- man Cochlea. An Ultrastructural, Laser Confocal and Super- resolution Structured Illumination Microscopy Study. Submit- ted Manuscript.

Contents

Introduction ...... 13 The inner ear, hearing, and hearing loss ...... 13 Cochlear implants ...... 15 Auditory brainstem implants ...... 17 Approaches to improve CI outcome ...... 17 Electrode-based strategies ...... 17 Cell- and gene- based therapies against hearing loss ...... 18 Stem cells ...... 18 Restoring the auditory nerve ...... 19 Hair cell regeneration ...... 20 The NanoCI project ...... 21 Auditory nerve regeneration ...... 22 Electrode surface modifications ...... 23 Aims ...... 24 Paper I ...... 24 Paper II ...... 24 Paper III ...... 24 Paper VI ...... 24 Material & Methods ...... 25 Cell culture media ...... 25 Stem cell culture (Paper I, II, IV) ...... 26 Primary cultures (Paper II – IV) ...... 26 Time-lapse video microscopy (Paper I – III) ...... 27 Immunofluorescence (Paper I – IV) ...... 27 3-D culture with coated electrodes (Paper III) ...... 27 Cultures on diamond substrates (Paper IV) ...... 28 LigandTracer (Paper III) ...... 28 Results ...... 29 Paper I ...... 29 Paper II ...... 31 Paper III ...... 32 Paper IV ...... 34

Discussion & Future perspectives ...... 36 Paper I ...... 36 Paper II ...... 37 Paper III ...... 40 Paper IV ...... 42 General discussion ...... 44 Conclusions ...... 46 Sammanfattning på svenska ...... 47 Acknowledgements ...... 48 References ...... 50

Abbreviations

ABI Auditory brainstem implant ABR Auditory brainstem response BDNF Brain-derived neurotrophic factor bFGF Basic fibroblast growth factor BM Basilar membrane CI Cochlear implant CNT Carbon nanotubes CPHS Calcium phosphate hollow nanospheres DMEM Dulbecco’s modified eagles medium EGF Epidermal growth factor ECM Extracellular matrix ES Embryonic stem (cells) FGF Fibroblast growth factor GDNF Glial cell line-derived neurotrophic factor HEI House Ear Institute hNPC Human neural progenitor cell IHC Inner hair cell IPS Induced pluripotent stem cell LIF Leukemia inhibitory factor NCD Nanocrystalline diamond NT-3 Neurotrophin-3 OC Organ of Corti OHC Outer hair cell PFA Paraformaldehyde PI Platinum-iridium SG Spiral ganglion SNHL Sensory neural hearing loss TLVM Time-lapse video microscopy TM Tectorial membrane TrkB Tropomyosin related kinase B, BDNF receptor (a.k.a. NTRK2) Tuj1 Neuron-specific class III beta-tubulin VG Vestibular ganglion

Introduction

The inner ear, hearing, and hearing loss The human inner ear is one of the most intriguing structures of the human body. It relies on different highly specialized cell types set in a precise order to execute its functions. Landmark discoveries about the ear were made by Antonio Scarpa (1752 – 1832) and Alfonso Corti (1822 – 1876), who also lent their names the structures they discovered. At the end of the 19th century, the anatomy of the inner ear in humans and several animals was described in remarkable detail by the Swedish anatomist Gustaf Retzius (Retzius, 1884). The inner ear consists of two parts, with separate functions, that both develop from the otocyst (Fekete, 1999). The cochlea is responsible for hearing, while the vestibular organ provides the senses of balance and motion. Innervation of the two organs is separated. The spiral ganglion (SG) innervates the cochlea and is connected to the brainstem via the auditory nerve. The vestibular nerve relays information from the vestibular ganglion (VG, or Scarpa’s ganglion) which innervates the balance organ. Together, the auditory and vestibular nerves form the VIIIth cranial nerve.

Sound consists of vibrations. When sound reaches the external ear, which consists of the pinna and external ear canal, it generates minute vibrations in the tympanic membrane. The tympanic membrane is connected to the ossicular chain, consisting of the maleus, incus and stapes. This is a highly tuned system, capable of amplifying sound in a frequency-dependent man- ner, both in the external ear canal and the ossicles. The ossicular chain trans- fers mechanical vibrations via the middle ear to the inner ear. The stapes emits vibrations to the oval window of the cochlea, which causes pressure changes in the cochlear fluids. Mechanosensory receptors, called inner hair cells (IHC), inside the cochlea then transform the mechanical vibrations into electrical impulses, transferred to the brainstem via the auditory nerve. When the pressure changes reach the cochlear fluids, they give rise to a traveling wave in the basilar membrane (BM), which separates the scala tympani from the scala media (Von Békésy & Wever, 1960). The BM con- tributes to the cochlear frequency resolution as, from base to apex, its width linearly increases four times whilst the thickness decreases six times (Khanna & Leonard, 1982; Patuzzi et al., 1982; Liu et al., 2015).

Figure 1. Cross section showing the sensory organ (Organ of Corti) and the three fluid filled spaces of the cochlea. The scala vestibuli is connected to the oval window where the stapes attaches. Cochlear implants are inserted into the scala tympani. Picture adapted from work by Oarih Ropshkow, available according to CC BY-SA.

The organ of Corti (OC) sits on the BM, beneath the tectorial membrane (TM) and contains the outer and inner hair cells (Figure 1). The BM move- ments result in shearing forces between the stereociliary bundles of the outer hair cells (OHCs) and the TM. The highly specialized OHCs can act as local amplifiers and their mechanical response increases not only sound sensitivity but also frequency resolution. The TM is also believed to contribute to cochlear tuning, but the exact mechanisms remain to be elucidated (Ghaffari et al., 2007; Hayashi et al., 2015). The 3,400 IHC then convert the vibrations into electrical signals. The IHC are connected to approximately 30,000 Type I neurons that mediate afferent signals to the brain via the VIIIth cranial nerve (Otte et al., 1978). Consequently, the BM and OHCs may act as primary and secondary fil- ters partly responsible for the highly precise frequency resolution in the mammalian cochlea. Together they shape a finely tuned, tonotopically ar- ranged inner ear map (Greenwood, 1961). The highest frequency sounds are filtered near the round window, whereas the lowest frequencies are filtered near the apex (Liberman, 1982). According to the World Health Organization (WHO, 2013), more than 5 % of the world’s population suffers from disabling hearing loss. This is often caused by infections, ototoxic drugs, noise exposure, or age-related hearing loss (also known as presbyacusis) (Niihori et al., 2015). Fragmented OHC loss as seen in presbyacusis reduces hearing sensitivity and capacity in noisy environments. It can often be amended through sound amplification using hearing aids. When IHCs are lost, on the other hand, the cochlea progressively loses its afferents, resulting in gradual deafness. In severe cases of inner ear associated hearing loss, also known as sensory- neural hearing loss (SNHL), the most common treatment is cochlear implants (CI) (Shepherd et al., 1993).

14 Cochlear implants The first electrically evoked sensation of hearing was reported by Ales- sandro Volta in the late 18th century. He connected a series of batteries to two rods which were inserted into fluid-filled external ear canals (Volta, 1800). Apart from being extremely unpleasant, the effect was described as the sound of boiling thick soup. Groundbreaking results in the 1930s showed that, at certain frequencies, the original sound could be reconstructed from signals recorded from the auditory nerve of a cat while providing sound stimulus. This was the first evidence of a correlation between perceived sound and the signaling to the brain (Wever & Bray, 1930). In 1957, the first human trial using direct electrical stimulation of the auditory nerve was performed by French clinicians. A sense of sound was evoked by electrically stimulating part of the VIIIth cranial nerve in a patient who had lost both inner ears during surgery (Djourno & Eyries, 1957). Alt- hough the implant was later removed, the patient received some pitch perception.

Current CI technology stems from the 1970s and the first single channel electrode that was inserted into the cochlea of a deaf patient by the pioneer William House (Mudry & Mills, 2013). He worked at the House Ear Insti- tute (HEI) in Los Angeles, US. This institute was for a long time a leader in auditory implant innovation and development, being one of the first CI cen- ters, and the first center to implant a child with a CI in the US. Several research laboratories worked in parallel, and by the end of the 1970s the first multichannel CI was implanted in France (Chouard, 2015), followed by their commercial launch in the 1980s (Wilson, 2013).

CI electrodes are inserted into the scala tympani, either through a cochleostomy or the less traumatic round window approach (Adunka et al., 2004b). A CI acts through a direct electrical stimulation of the auditory nerve, by- passing dysfunctional IHCs (Michelson & Schindler, 1983). The exact site of electrical stimulation of the auditory nerve remains elusive and it is cur- rently unknown if the stimulation activates voltage-gated ion channels on the SG soma, the hillock region, or at the Ranvier´s node of the peripheral or proximal afferents.

Figure 2. Cochlear implants. The external processor and transmitter sit behind the external ear and connect wirelessly to the subcutaneous receiver. The receiver is connected to the electrodes placed inside the cochlea. Picture by MED-EL.

Sound is collected by a microphone situated on the external processor. The acoustic signal is transformed into several band-passed electrical signals that are wirelessly transmitted through the skin to a subcutaneous receiver. The receiver decodes the signals into electrical pulses that stimulate the SG through multiple stimulation points on the electrode placed inside the cochlea (Figure 2 and Figure 3 C). Speech can be perceived from temporal cues, but the fine structure and spectral features are difficult to convey through the implant (Hochmair et al., 2015). The implants are generally made from flexible silicone and wrap around the helical, central structure of the cochlea, also known as the modiolus. Typically a CI has 12 – 22 electrodes (stimulation points) composed of an inert platinum-iridium (PI) alloy. Electrical stimulation of a certain region of the modiolus will produce a hearing sensation in a particular frequency region as neural responses in the SG are place-mapped, roughly following the aforementioned tonotopic map (Stakhovskaya et al., 2007). This means that a pulse near the round window is perceived as a high-pitched sound whereas an apical pulse will provide a lower frequency sound. However, the electrical pulse characteristics, such as pulse rate and amplitude, influence the psy- chophysical outcome and perception which is taken advantage of in modern CI processors. Through hearing preservation, surgery patients with residual hearing can receive so-called hybrid hearing or electro-acoustic stimulation, which relies on both acoustic and electrical stimulation (Kiefer et al., 2004). The patient can perceive low frequency sounds through their natural hearing while high frequency sounds are mediated through the CI electrical stimulation. This strategy often gives the patient a softer and more natural hearing sensation, including better speech perception in noisy surroundings (Erixon et al., 2012).

16 In recent years, development of more atraumatic surgical techniques that allow for structural preservation has been considered essential. As it is im- possible to foresee how future SNHL therapies may act, any structural damage caused during implantation may prevent a patient from receiving novel therapies when the previous implant has to be replaced. Life expectancy in many industrialized countries today is around 80 years (U.N., 2012). This means that implanted children will, most likely, go through at least one re- implantation during their lifetime (Hochmair et al., 2015).

Thus far, more than 450,000 people have received a CI and the number of implant recipients is increasing exponentially, suggesting that more than one million people will receive a CI by 2020. Thanks to the cochlear implant technology, deaf children can today be taught in regular schools, and adults can access job opportunities and maintain connections with their families, friends, and society.

Auditory brainstem implants The use of CI is limited to patients with an innervated cochlea. If the auditory nerve is lacking or dysfunctional, an auditory brainstem implant (ABI) electrode can be placed directly on the brain stem and provide hearing sensa- tions through direct electrical stimulation of the cochlear nucleus (Vincent, 2012). A patient group that might receive an ABI is those suffering from bilateral acoustic tumors as they often lose both of their auditory nerves during surgery. An ABI can then be implanted to provide some sense of hearing (Siegbahn et al., 2014). ABI results are generally poorer in terms of speech perception and quality of hearing quality compared to a CI, most likely due to anatomical variations in frequency mapping (Lundin et al., 2015).

Approaches to improve CI outcome Electrode-based strategies Various strategies have been developed to improve CI performance. For instance, it would be desirable to increase the number of stimulation points to provide a more discrete stimulation in each frequency region, as this is thought to be beneficial for speech perception and the ability to enjoy music (Wilson, 2013). This has proven difficult since tissue in the anatomical gap between CI and SG acts as resistance while the perilymph is highly conductive, causing wide current spread in the cochlea. By limiting or eliminating the gap, the electrodes could employ less powerful pulses to evoke a SG

17 neuron response (Shepherd et al., 1993) which has also been demonstrated in vitro (Hahnewald et al., 2015). This in turn would limit current spread and allow the stimulation points to be positioned more closely without risking stimulation overlap, allowing for higher resolution CI (O'Leary et al., 2009; Wilson, 2013) as well as lower power consumption. The modiolus hugging electrode, also known as the perimodiolar electrode, is pre-shaped to wrap around the modiolus as it is inserted. These electrodes are already in use and were found to improve speech understanding in clinical studies (Bacciu et al., 2004). They have also exhibited decreased power consumption and stimulation thresholds (Wackym et al., 2004) compared to straight electrodes. One downside of this electrode type is that the increased size often necessitates insertion through a cochleostomy (Doshi et al., 2015) which could cause unnecessary trauma (Adunka et al., 2004a), particularly in patients with residual hearing. To further decrease the distance, attempts have been made to combine perimodiolar implants with micro-needles to penetrate the wall of the modiolus, providing intra-modiolar stimulation (Badi et al., 2002). Other layouts for intra-modiolar electrodes have been designed with 194 stimulation points (Volckaerts et al., 2007). Neither of the two strategies has reached the clinic.

Signal focusing can also be achieved by grounding, or providing negative currents through electrodes neighboring the stimulating electrode. Studies employing such strategies have shown that neural responses are more limited and focused, which indicates a more selective stimulation (George et al., 2014). Focusing of the signal comes at a cost, since stimulation thresholds increase compared to monopolar stimulation, causing higher power consumption (George et al., 2015). As the strategy has already been tested clini- cally and regular CI electrodes can be used, this may very well be the next step in electrode improvement (van den Honert & Kelsall, 2007).

Cell- and gene- based therapies against hearing loss Stem cells For many years, embryonic stem (ES) cells have been the main focus of stem cell research. These cells are capable of proliferating indefinitely and are pluripotent, which means that they are capable of differentiating into cells of all three germ layers (Thomson et al., 1998). Stem cell populations have been found also in adult tissue; these are so-called progenitor cells, which slowly divide and provide new cells, e.g. for skin or intestinal villi. Innate, adult stem cell populations have also been found in the nervous system and even the adult cochlea (Rask-Andersen et al., 2005). These progenitor cells generally have a more limited potential for differentiation.

18 In recent years, the induced pluripotent stem (IPS) cells have come into focus. Any somatic cell can, in theory, be transformed into an IPS cell by transfection with the genes Oct3/4, Sox2 and Nanog (Takahashi & Yamanaka, 2006). This technique was awarded the Nobel Prize in 2012 and is considered to have great promise in future therapies, as the patient’s own cells can be reprogrammed to supplement damaged tissue. Both IPS and ES cells are considered as important in vitro supplements to animal models, and may have potential in the treatment of neurodegenerative diseases such as Parkinson’s and Alzheimer’s.

Restoring the auditory nerve The performance of a CI may depend on the status of the auditory nerve, but a clear correlation between implantation outcome and the remaining number of neurons does not seem to exist (Fayad & Linthicum, 2006). It has been suggested that as little as 10 % of the SG population is sufficient for a CI to function and give a satisfactory outcome (Linthicum et al., 1991). Supplementation of neurons to the auditory nerve, either by stem cells or nerve grafts, has been considered as a strategy to improve CI outcome. This treatment could benefit cases where the auditory nerve has been severely degenerated by disease or lost during surgery. Such treatments could also be a prerequisite for future generations of CI, which may require a larger neural population to function (Olivius et al., 2003; Olivius et al., 2004; Palmgren et al., 2011; Chen et al., 2012; Palmgren et al., 2012). The success of the trans- plant would depend on the successful integration of the newly formed cells into the auditory nerve and the formation of functional connections to the brainstem. Animal studies have shown that transplanted neurons can re-innervate IHCs in a denervated OC (Martinez-Monedero et al., 2006; Martinez- Monedero & Edge, 2007; Chen et al., 2012). Improved hearing thresholds in deafened animals after stem cell injections have also been reported, suggesting that functional connections to the brainstem can be achieved (Chen et al., 2012). The anatomy of the cochlea makes intra-cochlear administration of stem cells difficult without causing irreversible structural damage. By injecting the stem cells into the auditory nerve, these risks could be reduced, since the cochlea would not be opened (Corrales et al., 2006; Palmgren et al., 2012). The risk of tumor formations, both benign and malignant, must be taken into account when evaluating the potential benefits of stem cell transplantation (Amariglio et al., 2009).

19 Hair cell regeneration Hair cell regeneration would be a giant leap in hearing restoration and make the CI obsolete. Unfortunately, the complex and highly ordered structures would easily be damaged by a stem cell injection, and it would be difficult to target stem cells towards the OC. Regenerating hair cells and integrating them into the OC would most likely require differentiation of the adjacent supporting cells, normally surrounding the hair cells (Mizutari et al., 2013; Bramhall et al., 2014). As the supporting cells degenerate after hair cell loss, leaving a flat epithelium, treatments stimulating differentiation would need to be administered before the OC degenerates for this to be a realistic option (Izumikawa et al., 2008).

The stem cell source is also an important issue. Unless IPS or innate stem cells isolated from the tissue of the patient are used, chemical immunosup- pressive treatments would be needed to avoid rejection (Nussbaum et al., 2007). Presumably, we will see further improvements of the CI technology before stem cell-based therapies will become a reality.

20 The NanoCI project The work in this thesis was largely funded and conceptualized within the project “Nanotechnology based cochlear implant with gapless interface to auditory neurons”, with the acronym “NanoCI”, within the 7th framework program of the EU. The project started September 2012 and ended August 2015 and aimed at closing the anatomical gap between CI and SG. The idea of the project was based on results from the aforementioned animal studies, which showed that SG can re-sprout peripheral extensions towards the scala tympani after hair cell loss (Glueckert et al., 2008). To achieve this, factors providing stimulation and guidance to neurons were developed and produced within the consortium, as well as matrices to replace the perilymph of the scala tympani (Figure 3). Uppsala University, along with collaborators at the Universities of Tübingen, Germany and Bern, Switzerland, were involved in the in vitro testing of these factors. The group in Tübingen also performed in vivo studies based on the most promising in vitro results at the end of the project. All work is summarized into reports available to the public through the NanoCI consortium home page (www.nanoci.org).

Figure 3. The NanoCI project. Images show scanning electron microscopy images of half of a human cochlea cut through the modiolus to reveal internal structures. A) Showing approximate innervation (yellow) of SG in one turn of an ear with normal hearing. B) After hair cell loss, peripheral extensions degenerate leaving monopolar SG neurons. C) A regular CI placed in the scala tympani to stimulate the monopolar SG neurons. D) NanoCI approach, with a gel replacing the perilymph, and an implant with neurotrophin reservoirs stimulating regeneration of the peripheral extensions of the SG and guiding them towards a modified implant. Scale bars 1 mm. Adapted from Rask-Andersen et al. (2012).

21 Auditory nerve regeneration The presence of stem cells in rodents and even in the adult human cochlea has been previously described, indicating that there is a certain innate regenerative capacity in the mammalian inner ear (Rask-Andersen et al., 2005; Oshima et al., 2009). Murine SG and vestibular ganglion (VG) can also re- sprout neural extensions in a Matrigel-like matrix in vitro (Gaboyard et al., 2005), and paper II shows the potential for human VG do the same. Human SG neurons can remain electrically excitable in a monopolar form for decades after IHC loss thus enabling the use of CI (Linthicum & Fayad, 2009). As they are still viable, they might be susceptible to neurotrophic stimulation. In contrast, some mammals show a dramatic decrease in SG neuron numbers that can be observed within months after deafening (Dodson & Mohuiddin, 2000). In deafened guinea pigs, only around 5 % of the SG neuron population remains after long term deafness (Dodson, 1997). The discrepancy between SG survival in humans and animals may not be entirely physiological. It could to some extent be explained by the harsh deafening conditions used in animal experiments (Kong et al., 2010).

Various approaches have been used to deliver neurotrophins into the cochlea in animal studies. The aim of the studies is often to maintain the SG population after deafening, as well as to encourage re-sprouting of peripheral extensions (Glueckert et al., 2008; Wise et al., 2010; Pinyon et al., 2014). One approach employs chronic infusion of growth factors through a cath- eter in the round window. Using this method, brain-derived neurotrophic factor (BDNF) and fibroblast growth factor (FGF) was delivered to the cochlea of guinea pigs, which stimulated peripheral extensions to invade the scala tympani (Glueckert et al., 2008). Using an external supply permits long-term administration at high concentrations, but the risk of infections may out- weigh the advantages. Another approach is to deliver an intra-cochlear neurotrophin supply. One method that has been suggested is to place neurotrophin-producing cells on the surface of the CI (Rejali et al., 2007). Techniques to genetically modify cochlear cells in situ into producing the desired neurotrophins have also been considered (Wise et al., 2010; Pinyon et al., 2014). Receiving clinical ap- proval for these approaches could prove difficult, due to fears of tumor formation and immunological reactions. Providing factors for an extended time period merely to maintain SG neuron populations may also prove unnecessary in humans, as previously mentioned (Linthicum & Fayad, 2009). If the therapies aim at re-sprouting and guiding dendrites towards a CI, the neurotrophin reservoirs may only need to last for a limited amount of time. Adding biodegradable reservoirs into the scala tympani (Wang et al., 2014) or directly onto the CI could provide a sufficiently long release of the guidance cue. To further assist the bridging of the gap between modiolus and

22 CI, the distance could first be decreased using a perimodiolar implant. A gel could be used to replace the perilymph and facilitate neural outgrowth (Xie et al., 2013). Conditioning the gel with growth factors would provide an additional neurotrophin source (Hutten et al., 2014). A potential carrier of growth factors that could be applied directly to the CI is calcium phosphate hollow nanospheres (CPHS). Calcium phosphates are the major constituent of bone and teeth and are both biocompatible and biodegradable. They can also bind proteins and drugs (Ginebra et al., 2012). By introducing pores and making the structure hollow, the surface area for binding growth factors is increased; this also makes the nanoparticles easier to degrade.

Electrode surface modifications The interest in using nano-patterned diamond substrates made from nanocrystalline diamond (NCD) has increased greatly in the past years. NCD surfaces have been found to be biocompatible, durable (Amaral et al., 2008), have anti-bacterial properties (Jakubowski et al., 2004), and promote formation of neural networks. Neurons cultured on the NCD surfaces can also form electrically active networks (Thalhammer et al., 2010). The field of artificial retinal replacements is a research area where the need for an increased number of stimulation points was identified early. It has been estimated that such an implant will require at least 600 functional pixels to distinguish between faces and for reading (Bendali et al., 2015). This is a vast number considering the 12 – 22 stimulation points of the current CI. To reach 600 stimulation points the retinal implants and CI will have to overcome similar technical challenges, albeit with a more accessible neural population in the eye. 64-channel, flexible implant prototypes have already been produced with NCD and in vivo studies have shown that they can be implanted to the retina with minimal glial scarring (Bergonzo et al., 2011). Diamond surfaces structured with wells to limit current spread stimulated retinal glia and bipolar cells to integrate with the implant (Djilas et al., 2011). This approach could potentially be suitable for an ABI rather than a CI as it is positioned in direct contact with the neural tissue in the brainstem. By replacing the PI electrodes of the CI with nanostructured NCD, a gapless CI could potentially have thousands of stimulation points to provide an improved frequency discrimination.

23 Aims

The overall aim of this thesis is to study the regenerative capacity of the human VIIIth cranial nerve and the implications for development of a novel cochlear implant.

Paper I To evaluate the human neural progenitor cell (hNPC) line ENstem-A as a potential source for SG-like cells.

Paper II To establish a 3-D culture model for human vestibular ganglions collected during vestibular schwannoma surgery.

Paper III To investigate the efficacy of a nanoparticle-based slow release system and establish a model for neural guidance in vitro.

Paper IV To investigate the potential of patterned diamond substrates to act as a new surface for an interfaceable cochlear implant.

24 Material & Methods

Cell culture media All cultures were maintained using the following media compositions:

Table 1. Neurobasal-based culture media (Papers I – IV). ”Neurobasal media” Concentration Supplier Neurobasal – A Gibco B27 supplement 1:50 Gibco L-glutamin 1 mM Gibco Gentamicine 0.04 % Gibco Concentrations of growth factors added in ”Expansion media” bFGF 20 ng/mL Millipore LIF 10 ng/mL Millipore Concentrations of growth factors added in “Complete media” BDNF 20 ng/mL R&D Systems GDNF 20 ng/mL R&D Systems NT-3 20 ng/mL R&D Systems

Table 2. DMEM/F12 –based culture media (Papers II, IV). ”DMEM/F12 media” Concentration Supplier DMEM/F12 Gibco B27 supplement 1:50 Gibco L-glutamin 1 mM Gibco N2 supplement 1:10 Gibco Gemtamicine 0.04 % Gibco EGF 20 ng/mL Austral Biologicals bFGF 10 ng/mL Millipore Heparan sulfate 50 µg/mL Sigma-Aldrich

25 Stem cell culture (Paper I, II, IV) Two commercially available neural progenitor lines were used. In paper I the non-immortalized, h9-derived, hNPCs called ENstem-A (Millipore) were used. They were proliferated in “Expansion media” containing leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) which promote expansion and limits differentiation. They were typically passaged 1:3 Accutase (Life Technologies) or Try- plEx (Life Technologies) as Accutase gradually became ineffective at disso- ciating cells. For experiments, cells were counted in a TC20 Automated cell counter (Bio-Rad) and typically seeded at a density of approximately 3,000 cells/cm2. For differentiation “Complete media” was used. The trio of growth factors, BDNF, GDNF and neurotrophin 3 (NT-3) at a 20 ng/mL concentration has previously been used to differentiate and maintain SG neurons in primary culture, where they were found to be most effective when administered together (Rask-Andersen et al., 2005; Boström et al., 2010). In paper IV the immortalized ReNcell line (Millipore) was used. These cells are also neural progenitors but are, in our hands, easier to culture than ENStem-A. These cells were only used in a non-differentiated state in DMEM/F12 media. Passaging was performed using a suspension of 0.1 wt% Trypsin and 0.002 wt% EDTA (Life technologies). Cells were counted manually using in a Bürker chamber.

Primary cultures (Paper II – IV) Human tissues were harvested in accordance with ethical permits (no. 99398, 22/9 1999, cont., 2003 and Dnr. 2013/190), with patient consent, and in accordance with the Helsinki declaration, during surgery to remove vestibular schwannomas or petroclival meningiomas using a translabyrinthine approach. In the operating theater, the tissue was immediately placed in cooled Leibovitz’s L15 media. It was then dissected under dissection microscope into pieces suitable for culture, between 0.5 – 1 mm in diameter. After washing in PBS, explants were embedded in Matrigel-gel, containing 1:1 of Matrigel (8 mg/mL) and DMEM/F12 media further supplemented with 50 ng/mL of IGF (Sigma-Aldrich). The gel was allowed to set for 30 min at 37 °C, 5 % CO2, after which it was covered with the supplemented DMEM/F12 media. After 24 hours the media was replaced with “Complete media” to stimulate neural outgrowth.

26 As the human material is rare, SG tissue from guinea pigs was also used, in accordance with ethical permits (C98/12). SG tissue from P9 neonatal mice was also used and was harvested from residual tissue in accordance with supplemented ethical approvals (C346/11 and C1/15). After dissecting out the cochlea and isolating the SG, this tissue was treated in the same way as the human material.

Time-lapse video microscopy (Paper I – III) Time-lapse video microscopy (TLVM) material was mainly captured using a Nikon TE2000-E fitted with a CO2 incubator and temperature control as well as a perfect focus system. Pictures were taken every 1 – 3 minutes. Playback rate was typically 14 – 15 fps.

Immunofluorescence (Paper I – IV) Cells and tissues were fixated for 20 – 30 minutes in 4 % fresh paraformaldehyde (PFA) at room temperature. After rinsing, specimens were permea- bilized with 0.4 % Triton-X followed by rinsing and blocking with 2 % BSA solution. Primary antibodies were diluted in 2 % BSA and bound overnight at 4 °C. After rinsing, secondary antibodies dissolved in 2 % BSA were applied for two hours at room temperature. This was followed by rinsing and mounting in Vectashield mounting media with DAPI. After PFA fixation, tissues in Matrigel were cryopreserved and then sec- tioned into 8 µm slices and captured on SuperFrost plus slides (Menzel- Gläser). Sections were post-fixated in cold acetone for 10 minutes before staining. Sections were then air-dried and blocked with 2 % BSA. This was followed by overnight incubation at 4 °C with primary antibodies. After rinsing, sections were covered with secondary antibodies and incubated for two hours at room temperature. Specimen were then rinsed and mounted in Vectashield mounting media with DAPI.

3-D culture with coated electrodes (Paper III) A 2 % CPHS and 1 % cellulose solution was selectively applied to the PI surface of a single PI electrode on a 2 mm CI fragment (MED-EL). The cellulose acts as a glue, binding nanoparticles to the CI. Once dry, a 0.2 µL drop of neurotrophins was applied to the CPHS surface. Protocol for testing the coated CI surfaces is based on the protocol from paper II. Explants were encapsulated in Matrigel as described in Primary

27 Cultures and a coated electrode was then placed in close proximity to the explant (< 700 µm), which was cultured for up to two weeks.

Cultures on diamond substrates (Paper IV) Each NCD surface had four different inter pillar spaces, 4, 5, 9 and 14 µm. Human and murine material collected as described in Primary cultures were placed on uncoated, washed, and dried, diamond surfaces and a small drop of “Complete media” was applied, just covering the surface of the diamond and the explant. After 24 – 48 hours when the explant was fully attached, 2 mL media was added, filling the Petri dish. As the diamond was opaque it was not possible to follow and assess outgrowth during the culture period; the only indication of health was whether the explant anchored. After 14 days the explants were fixated and stained.

ReNCells were maintained as described. After passaging and counting, the cells were seeded onto three different substrates: either uncoated silicon, silicon coated with 20 µg/mL laminin, or the NCD diamond substrate.

LigandTracer (Paper III) To investigate the uptake and release of GDNF in the CPHS a 125I labeled GDNF was used. The 125I labeling was done using a chloramine-T reaction as previously described (Hunter & Greenwood, 1962). All in vitro slow release characteristics were measured using the LigandTracer Grey Instrument (Ridgeview), detecting gamma radiation (Figure 4).

Duplicate nanoparticle-containing gels were placed inside near the rim of a 10 cm dish along with pure drop of Matrigel. After background measurements in 3 mL of buffer, 200 µL of 1 µg/mL 125I- labeled GDNF was added and the uptake was measured once per minute for 23 hours. Background measurements were Figure 4. Illustration of the Ligand- made on untreated plastic. Tracer experimental setup.

For the retention experiments the 125I-buffer was first exchanged to fresh buffer and retention was measured for seven hours at room temperature. The buffer was then exchanged again and replaced with 5 mL of fresh buffer. Retention rate measurements then continued at 37 °C. Data was analyzed using the TraceDrawer software.

28 Results

Paper I The hNPC were investigated after 0 – 14 days of differentiation. We found that cells stained positive for nerve markers that are also found in the inner ear spiral ganglion, such as BDNF/NT-3 growth factor receptor (TrkB) and Neuron-specific class III beta-tubulin (Tuj1) (Figure 5, Table 3). A GlutR1 B TrkB DAPI DAPI

100 µm

C Neurog1 D Tuj 1 DAPI Olig1 DAPI

E Tuj 1 F S-100 DAPI DAPI

100 µm

Figure 5. Immunohistochemically stained hNPC after 14 days of differentiation against A) Glutamate receptor 1 (green). B) Tropomyosin receptor kinase B (green). C) Neurogenin 1. D) Tuj1 (red) and oligodendrocyte marker Olig1 (green). E) Tuj1 (green). F) Calcium binding protein S-100 (green). G) Phase contrast image of F.

29 Table 3. List of antibodies used in the study. The table also shows if cells stained positive or negative for each antibody and at what stage of differentiation staining was performed. Scoring was based on intensity and background, +++ = excellent, ++ = strong, + = positive, (+) = faint, - = negative.

Day 0 Day 7 Day 14 Antigen Source Dilution Pos/neg Pos/neg Pos/neg Tubulin (Tuj 1) Rabbit 1-300 + +++ +++ Tubulin (Tuj 1) Mouse 1-300 (+) +++ +++ Olig1 Rabbit 1-600 ++ ++ ++ Olig1 Mouse 1-40 ++ Brn3a Mouse 1-400 + + + Brn3a Rabbit 1-50 + GATA-3 Goat 2-125 - - - GATA-3 Mouse 1-50 - NTRK2 (TrkB) Goat 1-50 + NTRK2 (TrkB) Mouse 1-50 (+)/-+ + Neurogenin 1 Rabbit 1-50 + ++ ++ Calreg Mouse 1-50 - - - S100 Rabbit 1-100 + GFAP Rabbit 1-500 - GFAP Mouse 1-500 (+) Cx30 Rabbit 1-50 + Glutamatreceptor Rabbit 1-100 ++ Neurofilament 160 Mouse 1-100 ++

The differentiation process was followed through TLVM analysis, which revealed the morphological stages of cell differentiation the cells underwent. At the start, cells are clustered and fairly uniform in shape and size. Only minor morphological changes occur until day five, when colonies start con- necting to each other through thin axon-like structures. This becomes more visible at day seven when the cytoplasm also appears tighter around the nuclei and colonies are less densely packed. At day nine, morphologies vary and include nerve-like cells. At 14 days few cells remain and clusters of heterogeneous cells are connected by long axon-like structures.

30 Paper II We found that human VG could be cultured as explants in Matrigel and migration was followed by TLVM. The culture protocol also proved successful in promoting 3-D axonal outgrowth from murine and guinea pig explants as well as differentiated hNPC.

Figure 6. Cultured human VG. A) Phase contrast image of VG neurites extending into the 3-D matrix. B) Cryosectioned VG explant showing large neural cell bodies positive for Tuj1 (red) and TrkB (green). Scale bars 100 µm.

Various cell types migrated into the surrounding gel starting within 24 hours. Using TLVM, it was possible to follow the long axon-like structures migrat- ing out into the gel, with growth cones finding their way through gel (Figure 6 A). Cryosectioning human explants revealed internal structures and large Tuj1 positive cell bodies could be identified (Figure 6 B). This was also done for mouse spiral ganglion where type I SG neurons were found. Guinea pig SG neurons could also be visualized on the border between gel and explant (Figure 7 A). In rodent cultures neural outgrowth was ob- scured by cells aligning along the axonal processes (Figure 7 B).

Figure 7. Cultured guinea pig SG. A) Phase contrast image merged with immuno- fluorescent image showing Tuj1 positive neural cell bodies bordering the explant and sending extensions out into the surrounding gel (*). B) Image from TLVM. Supporting cells align with and migrate along the nerve-like extensions. Cell division can also be observed (arrow). Scale bars 100 µm.

31 Paper III CPHS nanoparticles were successfully anchored onto the CI electrodes using cellulose. The extent of the coating could be visualized by scanning electron microscopy. When encapsulated in a gel, the GDNF uptake and release properties of the CPHS could be clearly distinguished from both the empty gel and background using LigandTracer. Results showed that the CPHS had a large loading capacity at room temperature and initially a rapid release rate at room temperature. The uptake and release was fitted to a Langmuir binding model and the theoretical association (ka), and dissociation (kd) could be calculated (Table 4).

Table 4. Summary of uptake and release experiments showing calculated maximum capacity and actual uptake as well as calculated association and dissociation con- stants.

Uptake Maximum ka (1/(M*s)) kd (1/s) Affinity 23 h (ng) capacity (ng) KD (nM) Average 10.4 20.1 6.3*103 2.6*10-5 4.5 Stdev 2.1 6.7 1.7*103 4.3*10-6 1.9

When continued at 37 °C, the release was biphasic. Initially, the release rate was high, but after 4 – 8 hours it leveled out and stabilized at a lower rate (Figure 9 A-B). The uptake and release patterns of Matrigel and cellulose indicated that within 1 hour after GDNF addition or removal they had equilibrated with the surrounding buffer.

When the CPHS were loaded with GDNF and placed in 3-D cultures, neurites of both human and murine origin showed affinity for the coated regions and Tuj1-positive extensions could be seen reaching for these areas. 3-D confocal scans indicated that these extensions interacted with the coated surfaces (Figure 8 C).

Figure 8. In vitro 3-D cultures with CPHS coated CI. GDNF was loaded into CPHS and selectively coated on the metal electrode. (A) The CPHS were barely visible in stereo microscope. (B-C) Tuj1-positive extensions (green) sprouting from human VG were attracted to the GDNF coated electrode 14 days after co-culture. Scale bars 100 µm.

32 A B

Figure 9. GDNF uptake and release curves from LigandTracer experiments. (A) A representative curve of the uptake and release of GDNF at room temperature. (B) The continued release was monitored after the dish was moved to 37 °C. The data was normalized, and the curves show the release properties of both the measuring points at 37 °C during 16 hours for the three duplicate experiments (n=6).

A nuclear staining showed that cells growing out from a human VG explant in the 3-D gel also had affinity for the neurotrophin-loaded coating and pref- erentially followed the coated surface (Figure 10 A, B, D & E) rather than distributing randomly in the gel. Empty CPHS did not trigger such organization (Figure 10 A-F). A B C

D E F

Figure 10. A 3-D reconstruction of Z-stacked images visualized the distribution of cells in the gel surrounding the coated electrodes. Cells were stained with DAPI (green). (A, D) The control-coated surface did not attract the growth of cells onto the electrode and the cells grew randomly near the implant surface. Both BDNF- (B, E) and GDNF-coated (C, F) CI electrode fragments attracted cells onto the coated surfaces. The co-cultures were observed vertically (A, B and C) and horizontally (D, E and F) in the gel. Metal wires are also visible as they reflect light.

33 Paper IV NCD surfaces were successfully produced by the Department of Engineering Sciences, Uppsala University, as described in paper IV. The pillars were interspaced at 4, 5, 9 and 14 µm with a thin diamond head. Primary neurons of both human and murine origin were capable of attaching to the NCD surface without extracellular matrix coating and following the patterned diamond structures. Immunostaining revealed Tuj1 positive extensions growing in an orderly fashion (Figure 11 A, C). When studied with 3-D confocal microscopy, the neurons appear to stay at the top of the pillars when inter-pillar distance was below 9 µm, while nuclei from non-neural cells were located between the pillars (Figure 11 B). At the 14 µm spacing neural outgrowth still followed the pillars but was instead located on the silicon surface between the pillars.

Figure 11. Mouse neurons showed high affinity for NCD pillared surface. (A–C) Confocal laser microscopy showing murine SG outgrowth (Tuj1, green) on the NCD pillars with a spacing of 4 µm. (B) 3-D rendering of Z-stacked images revealing axon growth on the top of the NCD pillars, with the cell nuclei in between. (A, C) Most sprouting neurites remained on the structured NCD surface. Dotted line indicates the border between the structured and flat NCD surfaces. Scale bars 100 µm.

VG cultures showed that human tissue also attached and projected Tuj1 positive extensions along the surface (Figure 12 A-C). Patch-clamping of cells from the VG revealed that neurons were alive but not spontaneously firing (Figure 12 D). Neurite length was assessed to find the most nerve-stimulating inter-pillar distance. In murine cultures the longest axons were observed at 4 and 14 µm. In the human cultures only the 4 and 9 µm spacing were studied, with no discernible difference (Figure 13 A-B).

34 The ReNcell cultures showed that stem cells could attach to the uncoated patterned NCD surface equally or better than to a laminin-coated surface. The optimal distance for ReNcell adhesion was 9 µm.

Figure 12. Human neurites growing on the NCD pillars expressed Tuj1 but not parvalbumin. (A) Immunofluorescence staining with Tuj1 (green) show regenerating human VG neurite growth following the NCD pillars with 4 µm spacing. (B) 3-D reconstruction of the Z-stacks revealed Tuj1 and parvalbumin (red) co-expressed in sprouted neurites. (C) The expression of parvalbumin (red) was weaker in neurites reaching the NCD surface. (D) The neuron was held at -60 mV by constant current injection. In order to evoke action potentials, a series of increasing depolarizing currents (from 50 to 300 pA in 50 pA increments, 500 ms) were injected.

Figure 13. Longest axons were observed on NCD pillared surface with 4 and 14 µm spacings. (A) Axonal length was measured using the simple neurite tracer plugin in ImageJ. Purple indicates measured axons, and the longest axon is shown in green. (B) Diagram summarizing data of the longest axons measured from each explant. NCD pillars with a spacing of 4 and 14 µm show a tendency towards longer axons; however, the difference was not statistically significant. Human VG explants showed no difference in axonal length between the compared surfaces. n = 3.

35 Discussion & Future perspectives

Paper I As human material is difficult to obtain, it is vital to find alternative sources. In this study we differentiated the hNPC in media previously used to differentiate primary spheres derived from human SG tissue. During differentiation, they started expressing markers found in the SG, such as Tuj1 and the BDNF receptor TrkB.

The TrkB receptor is the primary target of stimulation in the NanoCI project because of its successful stimulation of neural re-sprouting, as described by Glueckert et al. (2008). A number of small molecule BDNF-mimetics were tested within the project and TrkB-expression in the differentiated cells indicate that they could respond to a mimetic that binds this receptor.

In the TLVM videos we saw that the differentiation times vary within the cell population, with some cells sprouting axon-like projections within days while most require longer differentiation times. Cell density also declines steadily as many cells die during the process. The heterogeneity of the population at 14 days could indicate that a longer differentiation time might be necessary for terminal differentiation. One drawback of using hNPC in studies is that they are very sensitive to seeding density and external cues such as growth factors. Most likely, other additives with batch-to-batch variation also affect their behavior. It can be difficult to control them and to get reproducible results. One of the main errors leading to the lack of statistics in this study was that it was conducted for a prolonged period, in the end making it difficult to compare old results to new, as suppliers and batches as well as environment and handling changed. The B27 supplement used in our cultures has been identified as a potential culprit responsible for variations in serum free nerve cell cultures. The two animal derived components of B27, albumin and transferrin, are sus- pected to cause this variability, and new suggestions for optional supplements with exclusively defined components are available (Chen et al., 2008). The laminin, derived from mouse tail, could of course also suffer from batch-to-batch variations.

36 In the future it would be interesting, but challenging, to perform in vivo experiments using the hNPC, since previous studies have shown engraftment into the modiolus (Corrales et al., 2006; Palmgren et al., 2012) and even improved hearing thresholds after IHC re-innervation (Chen et al., 2012). The ENstem-A neural progenitor cells have already been used in vivo in a rat stroke model, resulting in improved healing and recovery (Chang et al., 2013). The influence of the interaction between the newly forming neurons and existing neuronal network and connections with the brainstem on the tonotopic map of the cochlea remains an open question. Of particular interest is to see if hNPC may establish functional links between novel, interfaceable electrode surfaces and the innate SG population to improve the nerve- electrode-interface. The plasticity of the undifferentiated cells may also be advantageous. An advantage of applying neural supplementation strategies in combina- tion with a CI, rather than for reconnecting IHCs, is that signals from the CI can be reprogrammed to match the actual frequency distribution in the newly innervated cochlea in case the tonotopical map was shifted. IHCs would probably only respond to a certain frequency that might not match the new nerve.

Future work will focus on the further characterization of these cells and their capacity to supplement primary cultured material. Their capacity to form electrically active networks should also be investigated.

Paper II As the regenerative capacity of the VIIIth cranial nerve is paramount for the new strategies in cochlear implantation, we analyzed the capacity of human neurons to re-sprout and penetrate through a gel. Primary human SG tissue is rare and difficult to obtain as it is located inside the cochlea. The VG is located in the internal acoustic meatus, outside the labyrinthine structures, and this makes it more available during translabyrinthine surgery. Both SG and VG derive from the otocyst (Fekete, 1999) but differ in the response to some neurotrophins (Ernfors et al., 1995).

We show, for the first time, that a human VG can sprout neurites into a surrounding 3-D matrix in vitro. This indicates that there is an innate regenerative capacity in the adult auditory nerve. As expected, Matrigel had a highly stimulating effect on both neurons and non-neural cells. TLVM showed that growth cones moved seemingly unimpaired in several focal planes. Due to the heterogeneity of the VG samples we could not quantify them with statistical certainty. This is a limitation with primary human material, as each patient history is unique. As vestibular schwannomas originate from the

37 auditory nerve, the successful retrieval of material requires favorable anatomy. The tumor also has to be small enough not to invade the internal acoustic meatus, destroying the ganglion. Recent studies have also shown that vestibular schwannomas can have a degenerative effect on the neural population of the VG (Moller et al., 2015). This could increase the variability between samples as not only tumor size, but also duration becomes a factor.

Previous work with dissociated single cell cultures of both human and guinea pig SG showed low yields, and few of the approximately 30,000 type I neurons survived and sprouted (Rask-Andersen et al., 2005; Boström et al., 2010). As a VG contains fewer nerves, we chose to culture it as explants. Cells in explant cultures may shield neurons from external stimuli as the surrounding cells can e.g. absorb growth factors from the media. In this way, explant cultures may better resemble the in vivo environment. In 3-D cultures, a dissociated neuron would also be exposed to sheer stress and potential pH variations as the matrix solidifies.

When culturing dissociated primary inner ear-derived cells the supplemented DMEM/F12 media gave better cell morphology, compared to supplemented Neurobasal media (unpublished data). We therefore initiated cultures in DMEM/F12 media to maintain the whole cell population in the explant. By then switching to Neurobasal media containing BDNF, GDNF and NT-3 we aimed mainly at promoting neural outgrowth from the explant. This approach was found to be successful and we did not initially change this protocol. In later experiments (i.e. Paper III and IV) the use of DMEM/F12 media was abandoned as we gained more experience from animal tissue cultures. All cultures thereafter only relied on the supplemented Neurobasal.

Fully encapsulating the tissue inside the gel made it more difficult to observe outgrowth as the cells grew in several planes. This approach was still pur- sued as it is already known that Matrigel can serve as a coating for stem cell cultures (Ishikawa et al., 2015) and we were not interested in 2-D outgrowth. Secondly, 3-D outgrowth of murine vestibular neurites had been previously demonstrated (Travo et al., 2012) and our results using the hNPC showed that 3-D networks could form inside the Matrigel. The extra gel material was also added to ensure attachment as, in our experience, sheer stress from the culture media easily dislodges explants before they are able to attach to a culture dish. Experience from partners in the NanoCI consortium showed that although neurites protruding from explants cultured adjacent to a gel could reach and interact with the gel, they followed the path of least resistance and grew on top of it (unpublished results). Encapsulation then seemed like the only option to force neurites to grow inside the gel and achieve true 3-D outgrowth.

38 In the in vivo application the transition from tissue into gel could be an obstacle. If the gel composition was tuned to mimic the ECM of the surrounding tissue, the transit would be facilitated. Studies of the composition of ECM in the cochlea could therefore help in formulating an optimal matrix composition (Liu et al., 2015). This tuning might, however, risk increasing the invasion of fibroblasts and inflammatory cells which could interfere with the neural regeneration and guidance. The optimal gel composition remains to be determined.

Despite the promising in vitro results, Matrigel is unfortunately inappropri- ate for clinical use, as it is produced by an Engelbreth-Holm-Swarm mouse sarcoma cell line. It has an undefined composition, batch-to-batch variations, and contains trace growth factors (Hughes et al., 2010). It is therefore necessary to find an alternate, chemically defined matrix that would be approvable by the regulatory authorities. Within the NanoCI consortium the synthetic Purmatrix was modified with the laminin-derived IKVAV-motif in an attempt to create this kind of matrix (EMC microcollections, Tübingen, Germany). Initially, our attempts to culture explants and stem cells in this gel using direct encapsulation as with Matrigel were unsuccessful. By adding 10 % fetal bovine serum and allowing the gel to set before explant insertion, neural outgrowth was finally achieved (Figure 14, unpublished results). Still, cells would rather grow on the surface of the gel rather than penetrating it.

Figure 14. 3-D rendered confocal image of murine SG explant cultured in modified Puramatrix with IKVAV motif. (A) Cultured murine neurites staining positive for the beta-III-tubulin epitope TU-20 centrally and Tuj1 peripherally. (B) Side view of the cultured explant in A. Dotted outline shows approximate outline of gel.

The need to supplement the defined gel with animal-derived, undefined additives ultimately defeats its purpose. It also shows that our knowledge of what is truly required to stimulate outgrowth is incomplete.

Although rare and heterogeneous, human VG cultures can provide a valuable tool in the gap between animal models and human in vivo trials.

39 Paper III Creating direct contacts between SG neurons and the CI will require a supply of neurotrophins providing cues to stimulate outgrowth and guidance for weeks, or perhaps months. In this paper we investigated the use of a nanoparticle-based delivery system applied to the surface of the CI. We found that these nanoparticles could bind neurotrophic factors and provide a slow release mechanism. 3-D cultured primary auditory nerve material showed affinity for the neurotrophin-filled nanoparticles.

With the LigandTracer technique, we showed that GDNF is absorbed and slowly released from the CPHS. The CPHS have a large uptake capacity and affinity. Results also showed that adding GDNF to a gel, like Matrigel, will only provide short-term bulk release as it is rapidly saturated during loading and swiftly equilibrated with the rinsing solution. Adding neurotrophic substances to the gel could still be useful to boost the initial signal, but chemical linkage would most likely be necessary to achieve any slow release. Cellu- lose was used as a glue to attach nanoparticles to the surface of the electrodes. Measurements on gels containing cellulose showed a similar limited uptake pattern and maximum capacity as pure Matrigel. This indicated the contribution of growth factors from the cellulose was negligible in the in vitro experiments. As the cellulose becomes brittle when dry and may de- laminate from the CI during insertion, new methods of attaching the CPHS to the CI surface will be necessary in the future. The bulk of the GDNF was released from the CPHS fairly rapidly at room temperature, before leveling out after a few hours at 37 °C. This bulk might be bound to the outside of the particles with lower affinity, and the slower release could be from inside the pores of the particles. The binding and release characteristics may be modified by altering the surface chemistry of the calcium phosphate. An early bulk release could be advantageous for neurotrophins to initiate neurite re-sprouting. The initial signal may also need to be robust to penetrate the tissue surrounding the SG soma. Once neurites reach the gel in the scala tympani, a weaker signal may be sufficient for guidance. The cochlear volume is also substantially smaller than the volume used in the in vitro setting. This may lead to a considerably lower total release rate in vivo since it would increase the growth factor concentration near the surface. The higher concentration in turn would shift the equilibrium of the continuous uptake and release from the CPHS. The GDNF remaining once the release curve had stabilized was not per- manently bound to the CPHS. When a second rinse was performed, the signal showed a second decline, indicating that a new level of equilibrium was reached (results not shown).

40 Cell- and gene-based therapies, as previously mentioned, could provide a long term delivery of neurotrophins (Wise et al., 2010; Pinyon et al., 2014) but safety concerns could prevent or delay clinical applications. The long- term effects of continuous growth factors administration are also difficult to predict. A benefit often shown in animal experiments is the increased SG neuron survival following IHC loss, where SG neuron degeneration is oth- erwise fairly rapid. This may, however, be a problem of small clinical signif- icance as human SG neurons survive long term after IHC loss and as only 10 % of the neural population is required for a successful CI (Linthicum & Fayad, 2009; Kong et al., 2010). Prolonged growth factor administration has also been found to maintain the peripheral dendrites after IHC loss, which could be beneficial as the reduced distance may lower stimulation thresholds (Pinyon et al., 2014). Pro- moting neurite outgrowth without providing guidance or a modified implant could however ultimately obscure the tonotopic mapping by rearranging the re-sprouting dendrites, reducing the potential benefits.

Compared to the cell- and gene-based delivery methods, treatments with biologically degradable, clinical grade polymer reservoirs might more rapidly be approved for clinical use by the regulatory authorities. Such systems could also more easily regulate the amount of growth factors delivered. The amount of growth factors administered from transfecting cells in situ might be highly variable, both in amount and duration. Freeze-dried neurotrophins also have a long shelf life when stored appro- priately, although the influence of the CPHS on their biological activity should be investigated. Cell-based delivery methods, on the other hand, would require rapid delivery and specialized laboratories near the hospital.

One clinical trial using viral therapies to regenerate hair cells was initiated in 2014 (GenVec, 2015). This approach could be particularly useful in cases where the hair cell population is only partially degraded, and the OC is fairly intact. The potential benefits of such treatments motivate further studies, since natural hearing outperforms any of today’s implants.

The binding capacity of the CPHS was evaluated using only GDNF. Since the binding capacity of CPHS is unspecific, the uptake and release patterns of additional growth factors, such as BDNF, could also be assessed. The capacity of the CPHS to act as a stand-alone intra-cochlear drug carrier should also be evaluated in the future. The CPHS may then be used to deliver anti-inflammatory drugs which may ameliorate healing after implantation.

Promoting outgrowth from human monopolar neurons could prove a great challenge. Certain antibiotics such as gentamicin destroy hair cells and are ototoxic (Niihori et al., 2015). Guiding SG dendrites in these patients might

41 be more plausible as the connections to the IHCs were recently lost and their peripheral extensions may remain.

In the NanoCI project, a functional prototype CI electrode with slow release reservoirs on the surface was developed. The mechanism of these reservoirs is not fully disclosed. In vivo results produced within the consortium applying the prototype CI in a guinea pig model show that it is possible to regenerate axons using a slow release mechanism and a 3-D matrix (unpublished results). This confirms the viability of this regeneration approach. The results are available in the final publishable summaries on the NanoCI consortium home page (NanoCI-Consortium, 2016).

Paper IV In this study we investigated the use of patterned NCD-based surfaces as a new electrode surface for the interfaceable CI. NCD-surfaces were produced with four different inter-pillar distances 4, 5, 9 and 14 µm. Results using both human VG and murine SG tissue indicate that it is possible for auditory neurons to attach to and follow the patterned NCD surfaces. Neurons seemed to prefer the structured surface compared to unstructured areas. The inter-pillar distance also influenced outgrowth and cell attachment.

Compared to the 3,400 IHC the 12 – 22 PI electrodes of the CI provide a poor pitch perception. It is believed that increasing the number of stimulation points to stimulate neurons more discretely at a specific frequency region would be beneficial. The helical and compressed structure of the SG, combined with the spreading of currents, makes it difficult to increase the number of stimulation points without overlap. Most likely, it is necessary to construct a gapless interface to achieve a more selective stimulation. Each of the pillars on the NCD surface could, in theory, be a stimulus generator. If the PI electrodes could be replaced by patterned NCD surfaces, or completely redesigned around the NCD, the potential number of stimulation points could be dramatically increased. By adopting techniques for multipolar stimulation (George et al., 2014) into the programming for the NCD surface, further improvements in selectivity could be achieved and allow discrete stimulation even between closely related stimulation points. The successful implementation of these patterned surfaces would depend on the regeneration and guidance of peripheral dendrites towards the surface, as discussed for Paper III. A direct contact between the stimulus generator and the nerve would additionally, more closely resemble the in vivo situa- tion. The ability of cells attaching to the uncoated material is clearly advan-

42 tageous, as any surface modification using extracellular matrix-derived materials would greatly increase the risk of an adverse immune response. Creating an environment of gels and neurotrophins suitable for neural outgrowth could increase the risk for infections and biofilm formation. NCD has been shown to have good antibacterial properties, making it a suitable surface in such settings (Jakubowski et al., 2004).

Steering neurites to the proper position and stopping them from elongating once they have reached the target area also remains a challenge. We observed that the extension of neural outgrowth varied with the pillar spacing. This might indicate that inter-pillar distance could be used as a modifier of growth to create optimal neural networks and improve guidance and targeting. By using the spacing with less cell attachment, as indicated by the ReN- cell experiments, and better neural outgrowth, the risk of glial scarring could perhaps be reduced while maintaining neural attachment. The ability of the nerve-electrode interface to persist over time will be critical. Once the neurotrophin supply has been expended some cue to maintain the connection may be needed. As the connection ideally would be per- manent, any biological additives to the surface would eventually be degraded or depleted over time. It was previously shown that electrical stimulation may promote neural survival (Leake et al., 1999) and the electrical stimulation could perhaps help maintaining the neurite-CI link over time. Additional structural modifications, such as the addition of carbon nanotubes (CNTs), could potentially also be applied to regions of interest to increase the surface area and provide mechanical anchoring sites. The excellent conductive properties of CNTs might also improve the connection (Mattson et al., 2000; Lovat et al., 2005).

The lack of an electrical response in patch-clamp experiments was unfortu- nate since this would reveal characteristics of the firing patterns of the human VG. Electrically active neuronal cultures are needed to investigate the functional connection between the NCD pillars and neurites. Optimizations of the culture protocol may be essential for such studies. The limited amount of human material unfortunately makes optimization difficult. Culture time may e.g. be a critical factor for the electrical activity of the re-sprouting neurites as well as the composition of the extracellular solutions used in the experiments (Hahnewald et al., 2015). Further experience with animal tissues may help in resolving these issues.

Future work will focus on functionalizing NCD surfaces to elicit and record electrical signals. This would allow us to further investigate the connection between nerve and diamond.

43 General discussion

Cochlear implants have provided, and continue to provide, tremendous help for many hearing-impaired and deaf children and adults. This treatment, though initiated to some degree after benchmarked works, was essentially developed empirically. Therefore, we need to better understand how and why it really works. For this, we need to further analyze the human inner ear. With studies on cochlear sections from patients with a known history of hearing, we could gain further insights into potentially critical interspecies differences that might affect the way results from animal models translate to the clinic.

To improve the CI we need more insights into the firing patterns and syn- chronized nerve activity of the auditory nerve. Several types of Type I SG neurons were described with varying electrical properties (Chen et al., 2011; Liu et al., 2014a; Davis & Crozier, 2015). As approximately ten Type I SG neurons innervate each IHC, the pre- processing of the auditory signal could be considerable at the level of the SG somas. Connexins found in the glia network surrounding the human Type I SG neurons should permit exchange of signaling molecules within the clusters of Type I neurons (Liu et al., 2014c). This could potentially allow the neurons to further modulate signals from the IHCs before conveying information to the brainstem. These cell associations and the fact that human SG neurons lack a compact myelin layer may also improve cell survival following IHC loss (Liu et al., 2014b). The structural differences between human and animal SG motivate further studies. However, characterizing the human SG neuron population electrophysiologically and tonotopically provides a major challenge. Electrophysiological studies of cultured human auditory neurons could, hopefully, provide further insights. The functional outcome after cochlear implantation may also depend on the electrical properties of the remaining Type I neurons. Variances in excitability and physiological responses may influence the strategies needed to regenerate and guide the neurites.

There are several issues to consider concerning auditory neurophysiology, nerve restoration and cochlear implantation. The current CI can successfully provide speech perception for many recipients; would it be worth potentially upsetting the neural network through regeneration? Searching for means to

44 improve the nerve potential seems rational, as it may be crucial for new CI therapies with an increased number of stimulation points. Re-sprouting of SG neurons could also modify the electrophysiological responses. Amazing- ly, the neurons remain electrically excitable after dendrite atrophy, but, the normal electrophysiology might be imbalanced in the remaining monopolar neurons. Re-sprouting could theoretically restore some of these imbalances, which could further improve CI results.

For future experiments it will be necessary to functionalize the NCD surface and make it electrically active. Ideally, the system should be able to both stimulate neurons and record their electrical responses. By combining the 3- D culture techniques with the CPHS reservoirs and active NCD surfaces, a complex in vitro model could be created where the efficacy of the system can be tested. The connection between neurites and the NCD surface could be confirmed by stimulating and recording the responses from the neural population. An external stimulator may also be placed near the explant, using the NCD only to monitor the neural response (Hahnewald et al., 2015). It would be critical to verify a functional connection in vitro before any in vivo studies are considered. In vitro studies can also reveal if further surface modifications, such as the addition of CNTs, are necessary to improve the connection. Differentiating hNPC on the functionalized surface may determine whether they are electrically active. Their capacity to act as a bridge between the primary ganglion and electrode could also be investigated.

Developing coding strategies that could take advantage of hundreds or even thousands of stimulation points would be an entirely new challenge for the CI manufacturers. As neurites reach the surface CI it is likely that even frequency mapping of each channel could be problematic. This, however, lies beyond the scope of this study.

Considering the CI as treatment of patients with profound hearing loss and deafness, progress in hearing rehabilitation has been remarkable in the past 30 years. Hopefully, some results from the present study can form some basis for the future efforts in this field.

45 Conclusions

The regenerative capacity of the human VIIIth cranial nerve was studied and models for in vitro studies were suggested. The studies showed that neural progenitor cells expressed spiral ganglion-associated markers after differentiation, indicating that they could be used as an in vitro model.

Human adult auditory neurons retain a regenerative capacity and can be stimulated to re-sprout neural extensions in vitro that grow 3-dimensionally inside a 3-D matrix.

Biodegradable calcium phosphate hollow nanoparticles can bind growth factors and provide a controlled release over time. These nanoparticles could also be applied to the cochlear implant surface to act as neurotrophin reservoirs. Re-sprouted neurites from the auditory nerve were attracted by the nanoparticle-coated electrodes.

Neurites from auditory nerve explants can adhere and grow on patterned nanocrystalline diamond (NCD) surfaces without extracellular matrix coating. The pattern of pillars structured the outgrowth of neural networks. Pat- terned NCD surfaces could increase the number of stimulation points in a gapless nerve-implant interface.

These studies show for the first time that the human VIIIth cranial nerve has a regenerative capacity and can re-sprout severed peripheral extensions. Human auditory neurons can grow in a gel and interact with modified surfaces. These findings could give prospects for future cochlear implants, aim- ing to develop novel strategies for cochlear nerve regeneration, guidance, and protection.

46 Sammanfattning på svenska

Hörselnedsättning drabbar ca 340 miljoner människor globalt varav ca 34 miljoner är barn (WHO, 2012). Orsaken är oftast en avsaknad av fungerande hårceller i innerörat men även nervpåverkan kan förekomma. Ca 15 000 hårceller tar emot ljudinducerade vibrationer som omvandlas till elektriska impulser i hörselnerven. Vid allvarlig hörselnedsättning kan hörsel delvis återskapas genom s.k. cochleaimplantat (CI). Ett CI består av en elektrod som förs in i hörselsnäckan och kringgår skadade hårceller genom direkt elektrisk stimulering av hörselnerven. Avståndet mellan elektrod och nerv leder till en spridning av den elektriska signalen till ett större antal nervceller än önskat. Detta begränsar antalet stimuleringspunkter. Vi har ingått i ett större EU-konsortium (NanoCI) som syftar till att ta fram ett nytt CI där nerver kan växa fram och koppla till elektroden. Detta skulle kunna möjliggöra ett större antal stimuleringspunkter och förbättra patientens hörselupplevelse. I denna avhandling har jag fokuserat på att odla nerver från innerörat från människa och mus. Vi har försökt styra de utväxande neuriterna mot elek- troderna. Till vår hjälp har vi haft olika geler bestående av extracellulär gel- matrix samt reservoarer av nervattraherande ämnen laddade i nanopartiklar. Vi har även använt en stamcellspopulation som utdifferentierades till nerver som experimentell modell. Studier av kirurgiskt material från innerörat visar att en human nerv kan odlas och stimuleras till återväxt i en gel och har potentiell regenerationsförmåga. Nervmigration och riktning kunde påverkas med hjälp av nanopartikelre- servoirer av tillväxtfaktorer som placerats på ytan av ett CI. För att skapa en yta med ökat antal stimuleringspunkter användes nano- kristalin diamant deponerad på kiselyta. Ytan består av mikrometer stora pelare. Odlade nerver kunde växa ut på ytan och följde pelarna i ett ordnat mönster. Diamantytan kan i framtiden modifieras så att individuella pelare blir strömförande. Därigenom kan man skapa förutsättningar att framställa en begränsad elektrodyta med ett mycket stort antal stimuleringspunkter.

47 Acknowledgements

This work was performed at the inner ear research lab at Uppsala University. I would like to thank the following in particular:

My always enthusiastic supervisor Helge Rask-Andersen – thanks for taking me in and guiding me through my PhD. You’ve literally sent me half way around the world and given me a chance to grow in my role as a scientist, and meet a lot of interesting people in the process.

My co-supervisor Peetra Magnusson - thanks for all the advice and discussions. I truly appreciate the way you have welcomed me into your group and that you always had your door open for me.

Hao Li for helping me through my studies and for all the good discussions and advice. Also, thanks for initiating an unknowing Viking into the secrets of the Underground Chinese Research Network!

Marja, I need another 40 years of experience before I can come close to your ability to culture cells. Wei Liu, thanks for all the brilliant sections and insights into the secrets of the cochlea.

The guys at Rudbeck, Johan Brännström who introduced me to the various and marvelous microscopes. Sofia Nordling, good luck with the Vampire diaries! Moa Fransson, who got a new job but never really left.

Our visiting researchers Francesca Atturo and Hisamitsu Hayashi. You both helped set a good mood in the lab and did a lot of good research while at it.

Our old roommates at the plastic surgery department, Åsa, Alexandra and Cassandra.

The Oto- and Neurosurgical teams at Uppsala University Hospital - your skills made these studies possible.

I also wish to acknowledge the whole NanoCI consortium, and in particular our coordinator Pascal Senn for making it such a success. This project is the ruler against which all my future collaborations will be measured.

48 Börje Runögård for his kind donations to the lab which kept the lab running and allowed me do my PhD.

Paul Dalton who first told me I should meet a Dr. Rask-Andersen if I wanted to work with something cool in Uppsala.

Thanks to all my co-authors, coworkers and collaborators.

Tomas, Erik and Anders, I can’t believe that 11 years have passed since we first met. Then again I can’t believe that it has only been 11 years…

My parents and JoJ who have always supported me. Even when you haven’t been able to understand, or even pronounce what I’ve been doing you have always cheered me on!

Ett speciellt tack till Mormor för att du alltid finns där. Tack också till hela klanen Sundling och alla mina vänner!

My darling Lina, I couldn’t have done it without you. Thanks for putting up with me for the past five years; I can’t wait to see what our future has in store.

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