Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1193 Strategies in Cochlear Nerve Regeneration, Guidance and Protection 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, © Fredrik Edin 2016 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 vestibu- lar ganglion shows large neural cell bodies and neural extensions stain- ing 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 spi- ral 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 .....................................................................................................