Luận án Tiến sĩ: Tối ưu hóa tái tạo dây thần kinh ngoại vi bằng tín hiệu cấu trúc và sinh hóa

Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2011 Utilization of structural and biochemical cues to enhance peripheral nerve regeneration Balendu Shekhar Jha Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.edu/etd Part of the Nervous System Commons © The Author Downloaded from https://scholarscompass.edu/etd/2650 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact libcompass@vcu. © Balendu Shekhar Jha 2011 All Rights Reserved UTILIZATION OF STRUCTURAL & BIOCHEMICAL CUES TO ENHANCE PERIPHERAL NERVE REGENERATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University.

by BALENDU SHEKHAR JHA B.) Physical Therapy, Delhi University, 2003. Simpson, PhD Associate Professor Department of Anatomy & Neurobiology Virginia Commonwealth University Richmond, Virginia August, 2011. ii Acknowledgement Earning a PhD degree is truly a marathon event, and I would not have been able to complete this journey without the aid and support of countless people over these years. I must first express my gratitude towards my advisor, Dr.

David Simpson for his help and guidance. With his enthusiasm, inspiration, and great new ideas, he helped to make research work fun for me. I always considered him as Mr. He has a solution to each and every problem, and can make sense out anything (literally any data).

His way of seeing things and handling situations have set an example I hope to match someday. I would like to express my appreciation to my committee members: Dr. Raymond Colello, Dr. Scott Henderson, Dr.

Babette Fuss, Dr. Bob Diegelmann, and Dr. Gary Bowlin for their guidance towards completion of my bench work, and for the taking time for careful reading and commenting of my dissertation. Your expectations and concerns have always been right to the point.

This work would not have been possible without the constant assistance, guidance, and inputs provided by Dr. John Bigbee and Dr. Both of them have been my regular consultants, training me how to interpret science. I would like to thank the past and present Simpson lab fellows.

Rusty Bowman has always been a second mentor after my advisor. I am sure he has a big brain with more than 50% hippocampus where he has a huge knowledge database stored. He has an answer to any question with statistical and demographic figures. I huge thanks goes out to Thomas Turner for being the fun guy in the lab, keeping the lab alive with his jokes and funny online videos; you kept things light and smiling.

I would also like to thank Chantal Ayres for making me realize every now and then, that I should work in an organized fashion, keep the lab clean (glutaraldehyde-free), eat healthy and exercise regularly. A special thanks to Casey Grey for dealing with me every day now, and who has been always there for editing and proof-reading my work. Thank you for your encouragement, support, and most of all your humor. I would like to thank all my friends; thank you for being the surrogate family during my years at the VCU and for your continued moral support.

Most importantly, I am forever indebted to my parents and my wife, Vandana for their understanding, endless patience and encouragement when it was most required. I would also like to thank my younger sister, Pragya for being there with my parents, and taking care of them when needed, in my absence during the course of my PhD training. iii Table of Contents Page Acknowledgement……………………………………………………………………. ii List of tables……………………………………………………………………….

v List of figures……………………………………………………………………….… vi List of abbreviations…………………………………………………………………. Introduction to electrospinning…………. Regulating electrospinning – tweaking its variables……………… 12 3. Electrospun collagen: A tissue engineering scaffold with unique functional properties in a wide variety of applications……….

Materials and methods……. Two pole air gap electrospinning: Fabrication of highly aligned, three- dimensional scaffolds for nerve reconstruction…. Designing of a drug delivery platform for sustained release of gradients of growth factors at precise locations…. Electrospun 3D nerve guides: A comparative study….

Conclusions and future research directions …. Future research directions ………………………………………. 235 v List of Tables Page Table 4.1: Summary of specific electrospinning conditions for PCL in two pole electrospinning system…………………………………………………….1: Statistical analysis for sciatic functional index (SFI) assay……………….2: Statistical analysis for withdrawal reflex assay…………………………… 176 vi List of Figures Page Figure 2.1: Schematic of the process of electrospinning………………………….2A: Effect of Coulombic repulsion forces………………………….2B: Coiling of the electrospun jet………………………….1: Endothelial interactions with electrospun collagen and gelatin……….2: Osteoblast interactions with electrospun collagen & electrospun gelatin. Rates of wound closure in lesions treated with electrospun collagen or electrospun gelatin………………………….

Healing response to electrospun collagen and electrospun gelatin as a function of fiber diameter and pore dimension….5: Muscle fabrication: 3 weeks………………………….6: Muscle fabrication: 8 weeks………………………….7: Analysis of Type I collagen α chain content: Analysis of Type I collagen α chain content………………………….8: Ultrastructural and functional characteristics of collagen.1: Schematic representation of the mechanism of two pole air gap electrospinning. Schematic of the ground target used in a two pole air gap electrospinning system. Representative scanning electron micrographs (SEM).4: Average fiber diameter………………………….5: Analysis of fiber alignment by 2D FFT………………………….7: Cell culture experimentation………………………….8: Nerve reconstruction – frozen sections………………………….9: Nerve reconstruction – semi-thin sections. 107 vii Page Figure 4.10: Transmission electron microscopy………………………….1: Structure of alginic acid residues.2: Schematic of the characteristic egg-box structure……………………….3: Schematic of the electrospraying apparatus for preparing alginate microbeads………………………….4: Fabrication of alginate thread with concentration gradients…………….5: SEM images of alginaate microbeads, macrobeads, threads…………….6: NGF capture efficiency of different forms of alginate delivery platforms.7A: NGF capture efficiency of alginate threads and total NGF release in 7 days from different concentration alginate threads ….7B: NGF release profile from varying concentration alginate threads…….8 (A,B): NGF capture efficiency of alginate threads loaded with varying concentration of NGF.9: % NGF loss in the calcium chloride bath during the process of alginate thread polymerization………………………….10: NGF release profile from alginate threads………………………….11: NGF release and capture from alginate thread inside the electrospun 3D nerve guide………………………….12: DRG culture in scaffold with NGF in alginate delivery platform…….13: NGF gradient in the alginate thread…………………………………….1: Sciatic Functional Index……………………………………………….2: Gastrocnemius muscle atrophy comparison……………………………… 173 Figure 6.3: Sensory testing using the withdrawal reflex……………………………… 176 Figure 6.4: Lumbrical motor end plates…………………………………………….

178 viii Page Figure 6.5: Signal amplitudes across the implants at post-operative day 45………….6: Tangential semi-thin sections 45 days post-surgery ….8: Electron microscopy……………………………………………………… 191 ix List of Abbreviations 3D Three-dimensional ANOVA Analysis of variance BDNF Brain-derived neurotrophic factor BSA Bovine serum albumin CNTF Ciliary neurotrophic factor DRG Dorsal root ganglion ECM Extracellular matrix ELISA Enzyme-linked immunosorbent assay esC Electrospun collagen esG Electrospun gelatin FBS Fetal bovine serum FFT Fast fourier transform GAPDH Glyceraldehyde 3-phosphate dehydrogenase GDNF Glial cell line derived neurotrophic factor HDF Human dermal fibroblasts HFP 1,1,1,3,3,3-hexafluoro-2-propanol MEM Minimum essential media N-CAM Neural cell adhesion molecule NGF Nerve growth factor PBS Phosphate buffered saline PCL Poly-ε-caprolactone PGA/PLA Polylactic acid / Polyglycolic acid PNS Peripheral nervous system rEC Recovered electrospun collagen rEG Recovered electrospun gelatin RGD Arginine-glycine-aspartate SDS Sodium dodecyl sulfate SEM Scanning electron microscopy SFI Sciatic functional index TEM Transmission electron microscopy TFE 1,1,1-trifluoroethanol TGF Transforming growth factor x Abstract UTILIZATION OF STRUCTURAL & BIOCHEMICAL CUES TO ENHANCE PERIPHERAL NERVE REGENERATION. By Balendu Shekhar Jha, PMP, PT A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2011 Major Director: David G. Associate Professor, Department of Anatomy and Neurobiology This study examines the prospects of using the electrospinning process to fabricate tissue engineering scaffolds targeting a variety of regenerative applications, with a primary focus on the production of nerve guides for the treatment of long-defect nerve injuries in the peripheral nervous system.

A basic overview of the conventional electrospinning process is provided, and the utility of this fabrication scheme in the production of collagen-based tissue engineering scaffolds is demonstrated. Next, a novel modification of the basic electrospinning process is xi presented. This process, called two pole air gap electrospinning, was developed to produce nerve guides that exhibit an anisotropic structure that mimics the extracellular matrix of native peripheral nerve tissue. This electrospinning process makes it possible to produce macroscopic nerve guides that are cylindrical in shape and composed of dense arrays of nano- to micron-scale diameter fibers.

Unlike, conventional hollow core nerve guides, these electrospun constructs lack a central lumen, hence the designation 3D (for three-dimensional) nerve guide. The fibers are nearly exclusively arrayed in parallel with the long axis of the construct. This architectural feature provides thousands of individual channels, and aligned fibers that provide guidance cues that are designed to drive regenerating axons to grow in a highly directed fashion down the longitudinal axis of the guide. To supplement the structural cues provided by the fibrillar arrays of the electrospun 3D nerve guides, an alginate-based platform designed to deliver therapeutic reagents was developed and characterized.

This platform makes it possible to fabricate gradients of therapeutic reagents within the fibrillar arrays of an electrospun nerve guide. Functional and structural analyses of these constructs supplemented with or without a gradient of NGF, in a long-defect nerve injury in the rodent sciatic nerve indicate that the 3D design is superior to the gold standard treatment, the autologous nerve graft. Animals treated with the 3D grafts recovered motor and sensory function faster and exhibited far higher nerve-to-nerve and nerve- to-muscle signal amplitudes in electrophysiological studies than animals treated with autologous grafts or conventional hollow core cylindrical grafts. Overview The central hypothesis of this study states that tissue regeneration after injury can be maximized by identifying and recapitulating key features of the native extracellular matrix (ECM) [1].

In this study the central role that scaffold structure and composition play in the tissue engineering paradigm is explored. Tissue engineering is an evolving multidisciplinary field that has the potential to revolutionize medical practice and improve the health and quality of life for millions of people worldwide by restoring the structure and function to diseased or damaged tissues and organs. As a science, tissue engineering encompasses a broad range of potential applications including the repair, augmentation, or replacement of body tissues such as bone, muscle, skin, blood vessels, nerve, cartilage, and other connective tissues such as ligaments and tendons. Fundamental to nearly all tissue engineering processes is the scaffold used to establish the three-dimensional space necessary for cell attachment and growth at the injury site [1].

Typically, these scaffolds biodegrade or integrate themselves into the host tissue as the nascent ECM regenerates at the injury site. In effect, the scaffolds represent a template that act to guide the regenerative process and in most applications these structures are designed to be remodeled and completely replaced by native tissues. These scaffolds may or may not be supplemented with various types of cells designed to promote the reconstitution of functional tissue. A primary assumption of the tissue engineering paradigm is the notion that functional tissue will develop if the proper biological, guidance and or positional cues are provided by the tissue engineering scaffold [1].

It is becoming increasingly clear that each specific tissue requires its own unique set of these signals. The cues to be used in any specific application may be driven by biological, clinical, commercial and / or regulatory considerations. In the example of 1 peripheral nervous tissue, it may be guidance and / or positional cues that are paramount in design of the regenerative template. Superimposed on these basic considerations are the processing limitations that limit the ability to fabricate different materials into scaffolds with the features suitable to function as a regenerative template for the reconstruction of organs and tissue.