University of Central Florida STARS Electronic Theses and Dissertations, 2004-2019 2009 Tissue Engineered Myelination And The Stretch Reflex Arc Sensory Circuit: Defined Medium ormulation,F Interface Design And Microfabrication John Rumsey University of Central Florida Part of the Biology Commons Find similar works at: https://stars.library.ucf.edu/etd University of Central Florida Libraries http://library.ucf.edu This Doctoral Dissertation (Open Access) is brought to you for free and open access by STARS. It has been accepted for inclusion in Electronic Theses and Dissertations, 2004-2019 by an authorized administrator of STARS. For more information, please contact [email protected]. STARS Citation Rumsey, John, "Tissue Engineered Myelination And The Stretch Reflex Arc Sensory Circuit: Defined Medium Formulation, Interface Design And Microfabrication" (2009). Electronic Theses and Dissertations, 2004-2019. 3826. https://stars.library.ucf.edu/etd/3826 TISSUE ENGINEERED MYELINATION AND THE STRETCH REFLEX ARC SENSORY CIRCUIT: DEFINED MEDIUM FORMULATION, INTERFACE DESIGN AND MICROFABRICATION by JOHN WAYNE RUMSEY B.S. University of Florida, 2001 M.S. University of Central Florida, 2004 A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Burnett School of Biomedical Sciences in the College of Medicine at the University of Central Florida Orlando, Florida Fall Term 2009 Major Professor: James J. Hickman ABSTRACT The overall focus of this research project was to develop an in vitro tissue- engineered system that accurately reproduced the physiology of the sensory elements of the stretch reflex arc as well as engineer the myelination of neurons in the systems. In order to achieve this goal we hypothesized that myelinating culture systems, intrafusal muscle fibers and the sensory circuit of the stretch reflex arc could be bioengineered using serum-free medium formulations, growth substrate interface design and microfabrication technology. The monosynaptic stretch reflex arc is formed by a direct synapse between motoneurons and sensory neurons and is one of the fundamental circuits involved in motor control. The circuit serves as a proprioceptive feedback system, relaying information about muscle length and stretch to the central nervous system (CNS). It is composed of four elements, which are split into two circuits. The efferent or motor circuit is composed of an α-motoneuron and the extrafusal skeletal muscle fibers it innervates, while the afferent or sensory circuit is composed of a Ia sensory neuron and a muscle spindle. Structurally, the two muscular units are aligned in parallel, which plays a critical role modulating the system’s performance. Functionally, the circuit acts to maintain appropriate muscle length during activities as diverse as eye movement, respiration, locomotion, fine motor control and posture maintenance. Myelination of the axons of the neuronal system is a vertebrate adaptation that enables rapid conduction of action potentials without a commensurate increase in axon diameter. In vitro neuronal systems that reproduce these effects would provide a unique modality to study ii factors influencing sensory neuronal deficits, neuropathic pain, myelination and diseases associated with myelination. In this dissertation, results for defined in vitro culture conditions resulting in myelination of motoneurons by Schwann cells, pattern controlled myelination of sensory neurons, intrafusal fiber formation, patterned assembly of the mechanosensory complex and integration of the complex on bio-MEMS cantilever devices. Using these systems the stretch sensitive sodium channel BNaC1 and the structural protein PICK1 localized at the sensory neuron terminals associated with the intrafusal fibers was identified as well as the Ca2+ waves associated with sensory neuron electrical activity upon intrafusal fiber stretch on MEMS cantilevers. The knowledge gained through these multi- disciplinary approaches could lead to insights for spasticity inducing diseases like Parkinson’s, demyelinating diseases and spinal cord injury repair. These engineered systems also have application in high-throughput drug discovery. Furthermore, the use of biomechanical systems could lead to improved fine motor control for tissue- engineered prosthetic devices. iii “It is not the critic who counts; not the man who points out how the strong man stumbles, or where the doer of deeds could have done them better. The credit belongs to the man who is actually in the arena, whose face is marred by dust and sweat and blood, who strives valiantly; who errs and comes short again and again; because there is not effort without error and shortcomings; but who does actually strive to do the deed; who knows the great enthusiasm, the great devotion, who spends himself in a worthy cause, who at the best knows in the end the triumph of high achievement and who at the worst, if he fails, at least he fails while daring greatly. So that his place shall never be with those cold and timid souls who know neither victory nor defeat.” ~ Theodore Roosevelt iv ACKNOWLEDGMENTS I would like to thank Dr. James J. Hickman for his support, guidance and teaching during the course of this program; without his leadership this work would not have been possible. Additionally, I would like to thank the members of the Hybrid Systems Lab without whom many of these projects would have taken significantly more time. I would like to extend a special thanks to Dr. Mainak Das whose journey with me through this program resulted in good collaboration and a great friendship. His mentoring during the duration of this project has been invaluable. I would like to thank the members of my committee for feedback concerning the work documented here. Finally, I want to thank my family, friends and specifically Cindy Blecha for supporting me during this endeavor. v TABLE OF CONTENTS LIST OF FIGURES .......................................................................................................... x LIST OF TABLES ...........................................................................................................xx CHAPTER 1: GENERAL INTRODUCTION ..................................................................... 1 References ................................................................................................................ 16 CHAPTER 2: SUBSTRATE DIRECTED MYELINATION AND NODE OF RANVIER FORMATION ON SENSORY NEURONS IN VITRO..................................................... 21 Introduction ................................................................................................................ 21 Materials and Methods .............................................................................................. 24 Results ....................................................................................................................... 28 Discussion ................................................................................................................. 31 References ................................................................................................................ 41 CHAPTER 3: NODE OF RANVIER FORMATION ON MOTONEURONS IN VITRO .... 44 Introduction ................................................................................................................ 44 Materials and Methods .............................................................................................. 47 Results ....................................................................................................................... 51 Discussion ................................................................................................................. 54 References ................................................................................................................ 63 CHAPTER 4: DEVELOPING A NOVEL SERUM FREE CELL CULTURE MODEL OF SKELETAL MUSCLE DIFFERENTIATION BY SYSTEMATICALLY STUDYING THE ROLE OF DIFFERENT GROWTH FACTORS IN MYOTUBE FORMATION ................ 66 Introduction ................................................................................................................ 66 vi Materials and Methods .............................................................................................. 68 Results ....................................................................................................................... 72 Discussion ................................................................................................................. 76 References ................................................................................................................ 87 CHAPTER 5: TISSUE ENGINEERING INTRAFUSAL FIBERS: DOSE AND TIME DEPENDENT DIFFERENTIATION OF NUCLEAR BAG FIBERS IN A DEFINED IN VITRO SYSTEM USING NEUREGULIN 1-β-1 ............................................................. 92 Introduction ................................................................................................................ 92 Materials and Methods .............................................................................................. 96 Results ..................................................................................................................... 102 Discussion ............................................................................................................... 109 References .............................................................................................................