TISSUE ENGINEERING OF THE INNER EAR

Our knowledge of the regenerative ability of the auditory system is still inadequate. Moreover, new treatment techniques for hearing impairment using cochlear implant and tissue engineering, call for further investigations. Tissue engineering and regenerative strategies have many applications ranging from studies of cell behavior to tissue replacement and recently there have been significant advances in the biotechnological tools followed by development of new interventions, including molecules, cells, and even biodegradable biomaterials. This thesis presents results of tissue engineering approaches used in vitro with the long-term aim of facilitating auditory nerve and spiral ganglion regeneration. The first part describes the use of neurotrophic factors and neurosteroids for promoting survival and growth of nerve cells and the second part describes the effective usage of a biotechnology method, microcontact imprinting technique, to control key cellular parameters modifying chemical cues on the surface. The failure of the spiral ganglion neurons to regenerate was postulated to be due to the limited capacity of neurons to re-grow axons to their target. In paper I, we focused our studies on the role of GDNF in promoting spiral ganglion neuron outgrowth. The effect of three neurotrophins, among them GDNF, on spiral ganglion neurons in vitro was evaluated. The neuronal outgrowth was characterized by light microscopy and immunohistochemistry. The results speak in favor of GDNF, which promoted neuronal growth and branching, and Schwann cell alignment along the neurons in culture. The study support the role of GDNF as a potent factor, exerted neurogenic effects on cochlear cells in a degree dependent on the concentration used, confirming the hypothesis of GDNF being an oto-protector for chemicaland noiseinduced hearing loss and potential drug candidate for the inner ear. This might be relevant for future regenerative therapies and could have implications for tissue engineering techniques. In the second study, paper II, the objective was similarly to evaluate the efficacy of dendrogenin, a neurosteroid analogue, which can be applied to the cochlea. Dendrogenin was also tested in the presence and absence of other growth factors and the effect on adult neural stem cells was investigated. The study showed that neural stem cells exhibited proliferation/differentiation responses. Based on fluorescent labeling and a sphere-formation assay, we observed that adult neural stem cells induced proliferation. We asked whether the stem cells would differentiate into the major cell types of the nervous system and mainly neurons. Thus, neurotrophic supplement was added to the culture medium and was shown to have a selective effect on outgrowth of neuronal population. β3-tubulin positive neurons with BrdU positive nuclei were found and similar to other studies, we observed that the rate of differentiation increased with declining of BrdU expression. We found that despite the ongoing neuronal differentiation, there was an apparent difference of the neuronal outgrowth among the spheres treated with dendrogenin. The newly formed neurons were not found to send long projections into the local circuitry and the total cell number and length remained limited. Taken together, the protocols described inhere provide a robust tool to expand the biological role of dendrogenin that was in favor of differentiation when added to neuronal cell lines. The results of this study add new knowledge and better understanding of the possible action of dendrogenin in regenerative therapy. In paper III a strategy to guide spiral ganglion neurons was developed using a microcontact technique. The surface for neuronal guidance was designed with favorable extracellular proteins to promote the neurite outgrowth. Micro-contact imprinting provided a versatile and useful technique for patterning the guidance surface. Imprinting generated a patterned surface in a controllable, predictable, and quantifiable manner. A range of events followed the patterning including alignment, polarity and directionality was reported and observed by microscopic description. The dynamic microenvironment that resulted from the synergistic combination of extracellular guidance cues and Schwann cells selectively instructed and directed the terminal extension of neurons into unior bi-polar fate. In summary, applying new factors such as molecules, cells and surfaces provides unique possibilities to recruit spiral ganglion neurons into their regenerative ability. Additionally, creating an environment that incorporates multiple molecular and cellular cues will offer exciting opportunities for elucidating the mechanisms behind nerve regeneration and highlight specific considerations for the future tissue engineering. LIST OF PUBLICATIONS This thesis is based on the following original papers, which will be referred to in the text by their Roman numerals. I. Marja Bostrom, Shaden Khalifa, Henrik Bostrom, Wei Liu, Ulla Friberg, Helge Rask-Andersen. Effects of neurotrophic factors on growth and glial cell alignment of cultured adult spiral ganglion cells. Audiology Neurootology 2010; 15(3): 175-186. II. Shaden AM Khalifa, de Medina Philippe, Sandrine Silvente-Poirot, Anna Erlandsson, Hesham ElSeedi and Marc Poirot. The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells. Under revision in Biochemical and Biophysical Research Communications. III. Shaden AM Khalifa, Per Bjork, Christian Vieider, Mats Ulfendahl, and Eric Scarfone. Neuronal Polarity Mediated by Micro-scale Protein Patterns and Schwann Cells in vitro. Tissue Engineering and Regenerative Medicine 2013; 10(5): 266-272. CONTENTS Abstract List of publications Abbreviations Chapter 1: Introduction 1 1.1 Ear anatomy 1 1.2 Hearing physiology 6 1.3 Hearing Loss 8 1.4 Cochlear implant 9 1.5 Tissue engineering strategies 11 Aims 18 Chapter 2: Materials and methods 19 2.1 Ethical permission and animal care 19 2.2 Tissue dissection 19 2.3 Micro-pattern fabrication 20 2.4 Culture procedure 22 2.5 Immunohistochemistry 24 2.6 Imaging 25 2.7 Time Lapse Video 26 2.8 Imaging analysis 26 2.9 Axon analyzer software 27 2.10 Statistical Analysis 27 Chapter 3: Results 28 3.1 Neurotrophins stimulate neuronal regeneration in vitro 28 3.2 Dendrogenin activity on adult neural stem cells 29 3.3 Protein patterning 30 3.4 Neuronal polarity 30 3.5 Cells in culture 31 Chapter 4: Discussion 33 4.1 GDNF effects on spiral ganglion cells in vitro 33 4.2 Dendrogenin effects on adult neural stem cells in vitro 34 4.3 Patterning proteins 35 4.4 Neuronal guidance and polarity 35 Chapter 5: Conclusions and future prospective 37 5.1 Conclusions 37 5.2 Prospective 38 Acknowledgments 40 References 43 LIST OF ABBREVIATIONS