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Evaluation of biomimetic polymer nerve guidance conduit for peripheral nerve regeneration

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Title:
Evaluation of biomimetic polymer nerve guidance conduit for peripheral nerve regeneration
Creator:
Lee, David Jay ( author )
Language:
English
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1 electronic file (92 pages) : ;

Subjects

Subjects / Keywords:
Nervous system -- Regeneration ( lcsh )
Nervous system -- Wounds and injuries -- Treatment ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Review:
Surgical treatment of peripheral nerve injury clinically remains an unmet medical need as the current gold standard autograft is associated with many drawbacks, including a second surgical procedure, donor site morbidity, mismatch of donor nerve size, and limited donor nerve length. Nerve guidance conduits are a promising alternative to the autograft that promote neuronal growth and guide axonal extension. A nerve guidance conduit was designed using a blend of arginylglycylaspartic acid conjugated polyurea and polycaprolactone containing intraluminal microchannels with aligned nanofibers. The nerve guidance conduit was evaluated in a 10 mm sciatic nerve transection rat model. Functional, electrophysiological, and histological assessments were used to evaluate nerve regeneration of the conduit. Although generally no statistically significant improvement in nerve regeneration was observed for the nerve guidance conduit compared the autograft, the conduit consistently demonstrated comparable, if not improved, recovery characteristics.
Thesis:
Thesis (M.S.) University of Colorado Denver.
Bibliography:
Includes bibliographic references,
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System requirements: Adobe Reader.
General Note:
Department of Bioengineering
Statement of Responsibility:
by David Jay Lee.

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|University of Colorado Denver
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Auraria Library
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925843627 ( OCLC )
ocn925843627
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LD1193.E56 2015m L44 ( lcc )

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Full Text
EVALUATION OF A BIOMIMETIC POLYMER NERVE GUIDANCE
CONDUIT FOR PERIPHERAL NERVE REGENERATION
by
DAVID JAY LEE
B.S., Colorado School of Mines, 2013
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Master of Science
Bioengineering
2015


This thesis for the Master of Science degree by
David Jay Lee
has been approved for the
Bioengineering Program
by
Daewon Park, Chair
John H. Caldwell
Karin A. Payne
July 23,2015
11


Lee, David Jay (M.S. Bioengineering)
Evaluation of a Biomimetic Polymer Nerve Guidance Conduit for Peripheral Nerve
Regeneration
Thesis directed by Assistant Professor Daewon Park
ABSTRACT
Surgical treatment of peripheral nerve injury clinically remains an unmet medical need as
the current gold standard autograft is associated with many drawbacks, including a
second surgical procedure, donor site morbidity, mismatch of donor nerve size, and
limited donor nerve length. Nerve guidance conduits are a promising alternative to the
autograft that promote neuronal growth and guide axonal extension. A nerve guidance
conduit was designed using a blend of arginylglycylaspartic acid conjugated polyurea and
polycaprolactone containing intraluminal microchannels with aligned nanofibers. The
nerve guidance conduit was evaluated in a 10 mm sciatic nerve transection rat model.
Functional, electrophysiological, and histological assessments were used to evaluate
nerve regeneration of the conduit. Although generally no statistically significant
improvement in nerve regeneration was observed for the nerve guidance conduit
compared the autograft, the conduit consistently demonstrated comparable, if not
improved, recovery characteristics.
The form and content of this abstract are approved. I recommend its publication.
m
Approved: Daewon Park


ACKNOWLEDGEMENTS
I would like to express my gratitude to the many individuals that have contributed to the
research project and to those that have been involved in my personal and professional
development. None of this work would have been possible without them.
I would like to thank my advisor, Dr. Daewon Park, for his guidance, patience, and the
opportunity to work in his lab. Under his instruction, I have learned invaluable skills that
will be essential to my future success. Recognition must also be given to Dr. John
Caldwell and Dr. Karin Payne for their insight and teachings throughout the project.
My graduate experience would be far from complete without the encouragement and
assistance from my labmates in the Translational Biomaterials Research Laboratory,
especially Melissa Laugher, James Bardill, and Anna Laura Nelson. I am very fortunate
to have worked with them and I will cherish our continued friendship.
Several individuals have also contributed to this project at various points.
Electrophysiological assessment would not have been possible without the help of Arjun
Fontaine. Melissa Card dedicated much of her time on animal care training and Dr. Chris
Manuel provided training for the surgical procedure.
I would like to express my appreciation to my family. My dad has been supportive
throughout my life and has led me to be the person I am today. My mom has been a
constant source of inspiration and is the example of how I want to live my life. Finally,
my brother, who has grown up right alongside me, continuously challenged me and
pushed me to become a better person. I am truly blessed to have such a family.
IV


Declaration of original work
by
David Jay Lee
This masters thesis was independently composed and authored by myself, using the
support from my advisor, committee members, lab members, fellow students, and the
Department of Bioengineering. The research and ideas presented in this document
originated from the Translational Biomaterials Research Laboratory under the guidance
of Dr. Daewon Park. The electrophysiological setup was arranged by Dr. John Caldwell.
All other resources and funds were provided by Dr. Daewon Park and the Department of
Bioengineering.
David Jay Lee
v


TABLE OF CONTENTS
List of Figures...................................................................xi
List of Abbreviations............................................................xiv
Chapter
1 Introduction..................................................................1
1.1 Overview..................................................................1
1.2 Anatomy and physiology....................................................1
1.3 Injury classification.....................................................5
1.4 Pathophysiology...........................................................6
1.5 PNI diagnosis and treatment...............................................8
1.6 Study obj ective..........................................................8
2 Background....................................................................9
2.1 PNI treatment with surgical intervention..................................9
2.1.1 Neurorrhaphy..........................................................9
2.1.2 Autografts............................................................9
2.1.3 Allografts...........................................................10
2.1.4 Hollow tubes.........................................................11
2.2 NGC design considerations................................................12
2.2.1 Biocompatibility.....................................................12
2.2.2 Mechanical stability.................................................13
vi


2.2.3 Semipermeability......................................................13
2.2.4 Biodegradability......................................................13
2.2.5 Surface functionalization.............................................14
2.2.6 Intraluminal channels.................................................15
2.2.7 Lumen fillers.........................................................15
2.2.8 Growth factors........................................................16
2.2.9 Support cells.........................................................17
2.2.10 Electric conductivity.................................................18
2.3 Electrospinning of polymer solutions......................................18
2.3.1 Setup and process.....................................................19
2.3.2 Fiber arrangement.....................................................19
2.3.3 Parameters............................................................20
2.4 Rat SNI model.............................................................20
2.4.1 Functional assessment.................................................21
2.4.2 Electrophysiological assessment.......................................23
2.4.3 Histological assessment...............................................24
2.4.4 Limitations...........................................................25
3 Previous Work.................................................................26
3.1 Biocompatibility..........................................................26
3.2 PSHU-RGD and PC 12 cell culture.......................................27
vii


3.3 Fiber alignment and PC 12 cell culture.....................................30
3.4 NGC with aligned nanofibers along intraluminal channels....................31
3.5 NGC and hNSC culture.......................................................32
4 Hypothesis and Specific Aims..................................................35
4.1 Hypothesis.................................................................35
4.2 Specific aims..............................................................35
5 Materials and Methods.........................................................36
5.1 Materials..................................................................36
5.2 Equipment..................................................................37
5.3 N-BOC serinol synthesis....................................................37
5.4 PSHU synthesis.............................................................37
5.5 PSHU deprotection..........................................................38
5.6 RGD conjugation to dPSHU...................................................38
5.7 NGC fabrication by electrospinning.........................................39
5.8 Sciatic nerve transection and graft implantation...........................41
5.9 Walking track analysis.....................................................42
5.10 Ankle motion analysis....................................................42
5.11 Euthanasia and tissue harvest............................................43
5.12 CAP recordings...........................................................44
5.13 Gastrocnemius muscle mass................................................45
viii


5.14 Histology...............................................................45
5.14.1 IHC of nerve grafts...................................................45
5.14.2 Massons trichrome staining of gastrocnemius muscle...................46
5.15 Statistical analysis....................................................47
6 Results and Discussion........................................................48
6.1 PSHU, dPSHU, and PSHU-RGD reaction sequence.........................48
6.2 PSHU and dPSHU characterization using *H NMR..............................48
6.3 PSHU, dPSHU, and PSHU-RGD characterization using FT-IR....................50
6.4 Sciatic nerve transection and graft implantation.........................51
6.5 Walking track analysis....................................................53
6.6 Ankle motion analysis.....................................................54
6.7 CAP recordings............................................................56
6.8 Gastrocnemius muscle mass.................................................59
6.9 Histology.................................................................60
6.9.1 IHC of nerve grafts...................................................60
6.9.2 Massons trichrome stain................................63
7 Conclusion....................................................................66
8 Future Work...................................................................68
8.1 Increase sample size......................................................68
8.2 Additional negative control...............................................68
IX


8.3 Additional time points
68
8.4 Electrophysiological assessment using muscle action potentials.............69
8.5 Larger animal model.........................................................69
8.6 Growth factor and Schwann cell integration..................................69
References...........................................................................70
x


LIST OF FIGURES
Figure
1.1 The structural organization of the nervous system. [4].............................1
1.2 The structure of a neuron. [6].....................................................2
1.3 Peripheral distribution of spinal nerves. (A) the process of sending afferent
sensory signals to the CNS, (B) the process of relaying efferent motor signals
from the CNS to peripheral tissues. [7].................................................3
1.4 Cross-sectional anatomy of a peripheral nerve. [9].................................5
1.5 Peripheral nerve regeneration. (A) nerve transection, (B) degeneration, (C)
growth cone regeneration, (D) Schwann cell alignment. [9]...............................7
2.1 Electrospinning setup. Fiber arrangement is dependent on the collector. Flat
collectors result in random fiber arrangement and split electrode collectors result
in aligned fiber arrangement. [71]......................................................19
2.2 Measured variables used to calculate SFI in walking track analysis. [75]...........22
3.1 Biocompatibility assessment using PC12 cells with cell viability assessed by
MTT assay. PSHU extract was prepared by incubating PSHU in cell culture
media for 24 h in standard conditions and filtered to remove particulates. Error
bars represent one standard deviation...................................................27
3.2 Fluorescence microscopy of the neuronal response of PC12 cells seeded on
either surfaces coated with either laminin or PSHU-RGD in cell culture media
supplemented with and without NGF. PC 12 cells were stained with (3111-tubulin
and Alexa 488. Scale bars apply to respective columns...................................28
3.3 Neuronal response of PC12 cells by cell differentiation. Differentiated cells
were classified as cells with at least one neurite. Error bars represent one standard
deviation. indicates p < 0.0005.......................................................29
3.4 Neuronal response of PC12 cells by neurite length. Error bars represent one
standard deviation. indicates p < 0.001, ** indicates p < 0.0005......................29
3.5 Neuronal response of PC12 cells to fiber arrangement. (A) random fiber
arrangement, (B) aligned fiber arrangement, (C) neurite extension on random
fiber arrangement, (D) neurite extension on aligned fiber arrangement. Scale bars
apply to respective columns.............................................................31
xi


3.6 SEM images of a PSHU-RGD/PCL conduit. (A) transverse section of conduit
with embedded sucrose fibers, (B) transverse section after dissolving sucrose
fibers, (C-D) longitudinal sections after dissolving sucrose fibers...........32
3.7 Fluorescence microscopy images of hNSC behavior on PCL conduit after 14 d.
(A) DAPI stain in blue, (B) P-III tubulin stain in green. Arrow indicates the
direction of nanofiber alignment..............................................33
3.8 Confocal microscopy images of hNSC behavior on PSHU-RGD/PCL conduit
after 14 d. (A-C) along center of microchannel, (E-G) along inner wall of
microchannel, (A, D) DAPI stain in blue, (B, E) P-III tubulin stain in green, (C, F)
combined DAPI and P-III tubulin stains. Arrows indicate the direction of
nanofiber alignment..............................................................34
5.1 Two electrode electrospinning setup. Parameters: distance between needle and
collector at 10 cm, flow rate at 1 ml/h, voltage at 7.5 kV (PSHU-RGD/PCL and
PSHU/PCL) or 9 kV (PCL), room temperature, relative humidity at 30 %.............39
5.2 Rolling of flat sheet into a tube..........................................40
5.3 Different phases of walking in which ankle angle was measured for ankle
motion analysis..................................................................43
5.4 Platform of parallel conducting wires used to measure CAP..................44
6.1 Reaction sequence of PSHU-RGD synthesis......................................48
6.2 *H NMR spectrum of PSHU confirming molecular structure.....................49
6.3 *H NMR spectrum of PSHU and dPSHU confirming the removal of the BOC
protecting group with the disappearance of the b peak...........................49
6.4 FT-IR spectrum of PSHU, dPSHU, and PSHU-RGD.................................50
6.5 FT-IR spectrum of PSHU, dPSHU, and PSHU-RGD. The presence of free
amine groups on dPSHU after deprotection is confirmed from region a. The
conjugation of RGD to dPSHU is confirmed with the shift in wavelength with
carbonyl absorbance shown in region b.........................................50
6.6 Sciatic nerve transection and graft implantation. (A) longitudinal skin incision
from the knee to the hip exposing underlying muscles, (B) location of connective
tissue separating two muscles, (C) blunt dissection through connective tissue, (D)
isolation of sciatic nerve, (E) sutured autograft, (F) sutured PSHU-RGD/PCL
graft.................................................................................52
6.7 Calculated SFI values. Error bars represent standard error of the mean. *
indicates p < 0.05....................................................................53
Xll


6.8 The difference in the ankle angle between the grafted and healthy contralateral
sides during the initial contact phase of walking. Error bars represent standard
error of the mean. indicates p < 0.05...............................................54
6.9 The difference in the ankle angle between the grafted and healthy contralateral
side during the mid-swing phase of walking. Error bars represent standard error of
the mean. indicates p < 0.05........................................................55
6.10 The difference in the ankle angle between the grafted and healthy
contralateral side during the toe off phase of walking. Error bars represent
standard error of the mean. indicates p < 0.05......................................55
6.11 Representative CAP recordings 8 w after nerve transection and graft
implantation...........................................................................57
6.12 CAP amplitude ratio of the grafted to healthy contralateral nerves.
Amplitude was determined as the maximum peak value of the CAP. Error bars
represent standard error of the mean. indicates p < 0.05.............................58
6.13 CAP AUC ratio of the grafted to healthy contralateral nerves. AUC was
calculated by subtracting the area under the stimulus artifact modeled as a
Gaussian from the total area under both the stimulus artifact and CAP. Error bars
represent standard error of the mean...................................................58
6.14 Gastrocnemius muscle mass ratios of the grafted to healthy contralateral
sides. Error bars represent standard error of the mean................................60
6.15 Representative images of double immunostaining for axonal regeneration
and Schwann cell activity. (A-B) autograft, (C-D) PCL conduit, and (E-F) PSHU-
RGD/PCL conduit. Longitudinal sections from left to right represent proximal to
distal ends. Axons were stained with NF-M and Alexa Fluor 594 and appear in
red. Schwann cells were stained with SI00b and Alexa Fluor 488 and appear in
green. Scale bar represents 500 pm...................................................62
6.16 Representative images of gastrocnemius muscle stained with Massons
trichrome stain. (A) autograft, (B) PCL conduit, (C) PSHU-RGD/PCL conduit.
Muscle fibers are stained in red, while collagen is stained in blue. Scale bars
represent 100 pm.......................................................................64
6.17 The ratio of collagen to muscle fiber by area. The presence of collagen
implies the formation of fibrotic tissue. Error bars represent standard error of the
mean. indicates p < 0.05.............................................................64
xiii


LIST OF ABBREVIATIONS
'h nmr proton nuclear magnetic resonance
aFGF acidic fibroblast growth factor
ANOVA analysis of variance
AUC area under the curve
BDNF brain-derived neurotrophic factor
bFGF basic fibroblast growth factor
CAP compound action potential
CMAP compound muscle action potential
CNS central nervous system
CNTF ciliary neurotrophic factor
DCM methylene chloride
DMF N,N-dimethylformamide
DMSO dimethyl sulfoxide
dPSHU deprotected poly(serinol hexamethylene urea)
ECM extracellular matrix
EDC N-(3-dimethylamino-propyl)-N'-ethylcarbodiimide hydrochloride
EITS experimental intermediate toe spread
EPL experimental print length
ESC embryonic stem cell
ETS experimental toe spread
FT-IR fourier transform infrared spectroscopy
GDNF glial cell line-derived neurotrophic factor
GRGDS Gly-Arg-Gly-Asp-Ser
HDI hexamethylene diisocyanate
HFP 1,1,1,3,3,3 -hexafluoro-2-propanol
hNSC human neural stem cell
IACUC institutional animal car and use committee
me immunohi stochemi stry
IR infrared
ISO international organization for standardization
ITS intermediate toe spread
MRI magnetic resonance imaging
MSC mesenchymal stem cell
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NF-M neurofilament-medium
NGC nerve guidance conduit
NGF nerve growth factor
nh2 amine group
NHS N-hydroxysuccinimide
NITS normal intermediate toe spread
NPL normal print length
NSC neural stem cell
NT-3 neurotrophin-3
NT-4/5 neuroprophin-4/5
xiv


NTS OCT PBS PCL PGA PL PLGA PNI PNS PSHU PSHU-RGD RGD SEM SFI SNI TEM TFA TFE TS normal toe spread optimal cutting temperature phosphate buffered saline polycaprolactone polyglycolic acid print length poly(lactic-co-glycolic acid) peripheral nerve injury peripheral nervous system poly(serinol hexamethylene urea) arginylglycylaspartic acid conjugated poly(serinol hexamethylene urea) arginylglycylaspartic acid, Arg-Gly-Asp scanning electron microscope sciatic function index sciatic nerve injury transmission electron microscopy trifluoroacetic acid 2,2,2-trifluoroethanol toe spread
XV


1 Introduction
1.1 Overview
Peripheral nerve injury (PNI) is reported in 3 % of all trauma patients [1], Yearly in the
United States, $150 billon is spent on nerve injuries and over 200,000 peripheral nerve
repair procedures are performed [2,3], These injuries often occur from physical distress
including traumatic injury, surgery, or compression. PNI can also result from underlying
conditions such as autoimmunity, vasculitis, systemic illness (e.g. diabetes), cancer (e.g.
paraneoplastic neuropathy), infection, dysproteinemia (e.g. myeloma), drug toxicity, and
congenital disease. Nerve damage often leads to loss of function, pain, sensory loss, and
motor deficits. Despite advances in the reconstruction of segmented nerves following PNI,
functional recovery remains inadequate.
1.2 Anatomy and physiology
The nervous system is divided into the central nervous system (CNS) and the peripheral
nervous system (PNS) (Figure 1.1). The CNS consists of the spinal cord and brain while
the PNS comprises of all the neural tissue outside the CNS. The peripheral nervous
system consists of sensory neurons and motor neurons contributing to the sensory
division and motor division, respectively.
Peripheral nervous system (PNS)
________________II
Sensory (afferent)
division
Motor (efferent) division
1
1 1
Somatic nervous Autonomic nervous
system system (ANS)
i
1
Sympathetic Parasympathetic
division division
Figure 1.1 The structural organization of the nervous system. [4]
1


Peripheral nerves are comprised of a cell body, dendrites, and axons that are capable of
propagating electrical impulses called action potentials (Figure 1.2). These axons are
myelinated by Schwann cells. As the primary glial cells of the PNS, Schwann cells form
insulating myelin sheaths around axons, decreasing membrane capacitance and increasing
conduction velocity [5], Along myelinated axons are nodes of Ranvier. These nodes are
gaps in myelin sheath between adjacent Schwann cells and play a key role in generating
action potentials.
Cell body
(soma)
\
Initial segment
of axon
/
Node of Ranvier /Schwann cell
7
Axon
. iNOue ui nanvier


Terminal buttons
Figure 1.2 The structure of a neuron. [6]
The PNS sends afferent sensory signals to the CNS (Figure 1.3 A). Sensory receptors are
activated by stimuli that generate action potentials which trigger nerve impulses along the
length of the axons. These impulses travel across the cell bodies located in the dorsal root
ganglia and deliver the sensory information to the sensory nuclei located in the spinal
cord. The PNS can also relay efferent motor signals to peripheral tissues and systems
(Figure 1.3B). The cell bodies of motor neurons are located in the anterior horn of the
spinal cord. Once motor commands originate from the motor nuclei in the spinal cord,
motor information is conveyed to skeletal and smooth muscle fibers.
2


From interoceptors From exteroceptors.
of back proprioceptors of back Dorsal Somatic Visceral
B
Postganglionic fibers T
to smooth muscles. >
glands, etc of back
Dorsal ramus '
Ventral ramus
To skeletal
muscles of body
wal. limbs
Postganglionic fibers to
smooth muscles, glands
etc, of body wal. kmbs
To skeletal
muscles of back
Dorsal
Dorsal root ganglion root
Visceral Somatic
motor motor
Ramr
communicantes
KEY
Gray ramus
(postganglionic)
Whito ramus
(preganglionic)
Sympathetic nerve
Somatic motor
command*
Visceral motor
command*
Spinal nerve
Sympathetic ganglion
Postganglionic fibers
to smooth muscles,
glands, visceral organs
in thoracic cavity
Preganglionic fibers to
sympathetic ganglia
innervating abdomino-
pe tv ic viscera
Figure 1.3 Peripheral distribution of spinal nerves. (A) the process of
sending afferent sensory signals to the CNS, (B) the process of relaying
efferent motor signals from the CNS to peripheral tissues. [7]
3


Nerve fibers in the PNS can be classified by function, nerve fiber diameter, and
conduction velocity. Aa and Ap fibers are larger (5-20 pm) and are primarily responsible
for proprioception and mechanoreception, respectively. A8 and C fibers are smaller (0.3-
5 pm) and are associated with nociception and thermoreception. The extent of
myelination varies with nerve fibers. Aa and Ap fibers have higher degrees of
myelination, while AS fibers are thinly myelinated and C fibers are unmyelinated. Due to
the difference in the degrees of myelination, conduction velocities of nerve fibers vary as
conduction velocity increases with increasing myelin thickness. [6,8]
The peripheral nerve is composed of the endoneurium, perineurium, and epineurium that
constitute the nerve tissue (Figure 1.4). Individual axons are surrounded by the
endoneurium. The endoneurium consists of a loose collagenous matrix and provides
nutrients and protection for axons. Axons are bundled into fascicles held together by the
perineurium. The perineurium is comprised of tighter connective tissue that surrounds
fascicles and contributes to tensile strength. All the fascicles are enclosed by the
epineurium. The epineurium contains a tough fibrous sheath providing mechanical
support for fascicles and blood vessels. [9,10]
4


Figure 1.4 Cross-sectional anatomy of a peripheral nerve. [9]
1.3 Injury classification
PNI can be classified into three pathological descriptions. Neurapraxia occurs from
compression, lack of blood flow, or mild physical distress to the nerve and is
characterized by myelin breakdown and dysfunction. No physical disruption of nerve
tissue or axons is presented and no surgical treatment is necessary as nerve function is
eventually restored, although this may take several months. Axonotmesis is a more severe
degree of PNI when motor, sensory, and autonomic function is impaired due to traumatic
crush or stretch of the nerve. Axonal damage is apparent, but some form of the nerve
tissue remains intact, including the perineurium and epineurium. Surgical intervention is
usually not required, but may be considered to remove any scar tissue that may have
formed. Neurotmesis is the most severe class of PNI and is classified by the complete or
partial transection of nerve tissue and axonal disruption. [9,10]
5


Neurotmesis can further be categorized depending on the extent of injury. Less severe
neurotmesis involves a lesion of the endoneurium, but the perineurium and epineurium
are preserved. Surgical intervention may be considered. In more severe cases of
neurotmesis in which only the epineurium remains intact or complete transection occurs,
surgical repair is necessary to regain nerve function. [9,10]
1.4 Pathophysiology
When a peripheral nerve is transected, the nerve undergoes a period of degeneration and
subsequent regeneration (Figure 1.5). After injury, degeneration occurs both proximally
and distally by chromatolysis. Traumatic degeneration is characterized by the
deterioration of the nerve proximally from the transection site to the next node of Ranvier.
24-48 h after injury, Wallerian degeneration occurs in which axons and myelin distal to
the injury site breakdown. During nerve degeneration, proliferating Schwann cells,
macrophages, and monocytes cooperate to remove myelin and axonal debris, release
neurotrophins, and guide axons toward adjacent synapses. After Wallerian degeneration,
growth cones form at the distal ends of regenerating axons that consist of filopodia,
allowing for axonal contraction and elongation. As growth cones are formed, Schwann
cells align longitudinally and form Biingner bands which provide a growth permissive
environment for regenerating axons. [9,11-14]
6


B
Traumatic
degeneration
\
Wallerian
degeneration
/ V

Nervex Ca* K*
Vj^ ________ Ma* o,.
Microglial cell
'7/vr sprout Na* Protein
D
Figure 1.5 Peripheral nerve regeneration. (A) nerve transection, (B)
degeneration, (C) growth cone regeneration, (D) Schwann cell alignment.
[9]
7


1.5 PNI diagnosis and treatment
PNI diagnosis is based on neurological and physical examinations, patient history, and
medical screening (e.g. blood test). Nerve damage can be assessed using nerve
conduction velocity tests, electromyography, magnetic resonance imaging (MRI), and
biopsies (e.g. nerve, skin). Current non-surgical treatments of PNI are limited to symptom
management. Neuropathic pain can be controlled using over-the-counter medication for
mild cases, while prescription medications are used for more severe and chronic cases.
Muscle weakness is often addressed using mechanical aids (e.g. braces).
Surgical intervention is considered for more severe cases of PNI, characterized by nerve
transection. For shorter nerve gaps, neurorrhaphy is performed which directly reconnects
the two nerve stumps through suturing. Autografts, allografts, and hollow tubes are used
to span longer nerve gaps. However, current techniques in surgical intervention have
clinical limitations and often lead to poor functional recovery.
1.6 Study objective
PNI repair is clinically an unmet medical need. Due to the limitations and drawbacks
associated with current surgical intervention in the treatment of PNI, a synthetic nerve
guidance conduit (NGC) has been developed to promote nerve regeneration. The
objective of this study is to evaluate a arginylglycylaspartic acid conjugated poly(serinol
hexamethylene urea) (PSHU-RGD) and polycaprolactone (PCL) blended nanofiber
scaffold that has previously been reported [15,16], The NGC has successfully been
characterized and shown to promote cell attachment and neurite extension in vitro. The
focus of this study was to evaluate the NGC for peripheral nerve regeneration in a rat
sciatic nerve injury (SNI) model.
8


2 Background
2.1 PNI treatment with surgical intervention
Transected peripheral nerves are approached differently depending on the severity and
the length of the gap induced by injury when considering treatment. Although advances
in microsurgical instrumentation and technique have been made, all surgical treatment
options have major drawbacks in their efficacy of PNI repair.
2.1.1 Neurorrhaphy
Neurorrhaphy is one surgical repair technique of PNI which involves the direct suturing
of discontinued nerve stumps. There are two different approaches to neurorrhaphy,
epineurial repair and grouped fascicular repair. Epineurial repair involves suturing of the
epineurium of the two ligated ends while grouped fascicular repair involves matching and
suturing fascicular groups. Although realigning axons by grouping fascicles leads to
improved functional recovery, suturing of the fascicles leads to increased scarring and
damage to blood vessels, preventing optimal recovery [17], Although relatively
successful at recovering nerve function, neurorhaphy is limited to shorter nerve gaps, no
greater than 5 mm in length [18], Excessive tension on the nerve damages the nerve
tissue layers as it disrupts connective tissue matrices and reduces blood flow, inducing
necrosis and chronic ischemia [19],
2.1.2 Autografts
For longer nerve gaps, autografts are considered the gold standard of PNI repair [20],
Autografts currently offer the best results in terms of nerve regeneration, but are also
associated with many drawbacks including a second surgical procedure, donor site
9


morbidity, mismatch of donor nerve size, and limited donor nerve length. Common
sources of donor nerve are the sural, medial antebrachial cutaneous, and lateral femoral
cutaneous nerves as they are readily accessible and relatively expendable. There are
several types of autografts including single, cable, trunk, and vascularized grafts.
A single graft is a segment of a donor nerve that is of similar diameter to the two ends of
the transected nerve. The single graft is restricted to nerves of a certain diameter due to
the limited number of expendable nerves that would be similar in diameter. Cable grafts
are multiple segments of donor nerve of smaller diameter aligned in parallel to span gaps
of larger diameter. The multiple segments are held together by suture or fibrin glue [21],
Trunk grafts are segments from a large nerve used to repair a gap in a proximal nerve.
However, truck grafts are associated with poor recovery due to fibrosis and poor
vascularity attributable to the thickness of the graft [22], Vascularized grafts are donor
nerves that are used to span gaps without disturbing the blood vessels supplying the nerve.
Although these grafts offer superior recovery in areas that are poorly vascularized, donor
site morbidity is of major concern [23],
2.1.3 Allografts
Allografts are obtained from human cadaveric nerves. The use of allografts avoids
several of the limitations of autografts, but the complexity and cost of producing
allografts remains a challenge [24], Also, allografts are not as effective in restoring nerve
function compared to autografts due to their tendency to elicit an immune response. An
increase in T-cell response has been linked to donor Schwann cells and requires the use
of immunosuppressants [25],
10


To avoid the use of immunosuppressants, allografts can be decellularized using freeze-
thawing, chemical detergents, enzyme degradation, or irradiation. However,
decellularization causes cell debris formation and impairs neurite outgrowth. Acellular
allografts were shown to regenerate axons across gaps only up to 3 cm [26],
2.1.4 Hollow tubes
Several biological and synthetic hollow tubes have been developed and are approved by
the FDA for clinical use. Although many of the drawbacks of autografts and allografts
can be avoided through the use of hollow tubes, neurite outgrowth has been limited to
gaps less than 3 cm in length and is associated with poor functional recovery [27], The
random dispersion of regenerating axons through hollow tubes leads to inappropriate
target reinnervation.
These implantable devices are fabricated using either non-absorbable or biodegradable
materials. Non-absorbable tubes can be fabricated from various materials, such as
polyvinyl alcohol and silicon. These tubes are mechanically stable and easily sterilized,
but are associated with nerve compression and tension at the sutured areas of the nerve
[27], Biodegradable tubes can be produced using natural or synthetic materials. Natural
materials that are produced in the body include collagen, chitosan, and fibrin. Although
these materials are easily obtainable and biocompatible, complete biodegradation of the
tubes may take up to a year and batch-to-batch variations prevents consistent nerve
regeneration properties [27,28], Synthetic materials such as polyglycolic acid (PGA),
poly-lactic-co-glycolic acid (PLGA), and PCL can also be used to fabricate
biodegradable tubes. These tubes have excellent biodegradability, but are often not
11


mechanically stable, have low solubility, and produce undesired products from
degradation (e.g. acidic products) [27,29,30],
2.2 NGC design considerations
Due to the limitations associated with current nerve grafts used for PNI repair, much of
the research surrounding nerve regeneration has focused on NGCs. In order for a NGC to
be a clinically relevant alternative to current grafting techniques, functional recovery
needs to be comparable to that of the autograft. NGCs are relatively successful at
recovering nerve function across shorter nerve gaps, but are incapable of selectively
guiding axons toward appropriate end tissue for longer gaps [31],
When designing NGC for use in PNI repair, several considerations may be implemented
to promote nerve regeneration. Although not all of the following conduit characteristics
necessarily need to be incorporated into the final design, some are often required due to
the physiology of axonal extension. The following NGC characteristics have shown to
promote nerve regeneration.
2.2.1 Biocompatibility
Biocompatibility of a material refers to the tendency of the material to support and
sustain appropriate cellular behavior. For nerve conduits, this will include allowing for
molecular and mechanical signaling systems during regeneration, without presenting
cytotoxic effects or eliciting an immune response [32,33], The formation of Biingner
bands relies heavily on cellular and axonal migration to promote axonal extension, and in
order for a NGC to be considered biocompatible for neural tissue, it must not interfere
with this healing process [34],
12


2.2.2 Mechanical stability
NGCs must provide the mechanical strength necessary during sterilization, implantation,
and nerve regeneration processes. To prevent infection, sterilization is required for all
implants and NGCs must retain their mechanical strength throughout this process. During
implantation, the NGCs must resist tear from sutures and have the structural integrity
when handled during the procedure. Mechanical strength must also be considered during
nerve regeneration as NGCs should have comparable mechanical strength to native nerve
tissue. This is usually a balance between flexibility and rigidity. NGCs must not collapse
or break down until the nerve regenerates and can provide ample mechanical stability,
and they must not be too stiff causing compression or dislocation. [33]
2.2.3 Semipermeability
The porosity of the NGC is another design consideration. The exchange of fluids between
the regenerating nerve and the surrounding fluid is essential to optimal regeneration.
Semipermeable conduit walls allow for the diffusion of gas exchange (e.g. oxygen,
carbon dioxide) and other nutrients vital for nerve regeneration into the conduit while
restricting the influx of infiltrating inflammatory cells into the conduit and outflow of
neurotrophic factors out of the conduit [35], Pore sizes of 10-38 pm have shown to yield
optimal permeability [36],
2.2.4 Biodegradability
Nerve regeneration is usually limited due to toxicity and long term complications that are
associated with NGCs fabricated from materials that are not biodegradable. The use of
non-absorbable materials requires a second surgical procedure after initial implantation to
13


remove the NGC after some nerve regeneration is observed. Therefore, interest in
biodegradable materials has increased. [37]
Mechanical stability is of major concern for NGCs made from biodegradable materials,
as the conduits need to withstand the mechanical stress from neighboring tissues without
collapsing while the conduit is degrading. Materials that degrade too rapidly will
compromise the structural integrity of the regenerating nerve, while materials that
degrade too slowly will lead to compression and dislocation. Ideally, the NGC should
remain intact for the time needed for axons to regenerate across the nerve gap and then
degrade gradually to minimize undesired affects. The degradation products must also be
considered. Biodegradable materials often have undesirable byproducts (e.g. acidic
products).
2.2.5 Surface functionalization
Surface functionalization of the lumen enhances the interactions between the conduit and
nerve cells. Cell adhesion molecules and short peptide motifs commonly found in
extracellular matrix (ECM) proteins can be incorporated into NGC design to enhance
these interactions between the cells and conduits. Arginylglycylaspartic acid (RGD, Arg-
Gly-Asp) is a cell-binding motif found in fibronectin, laminin, collagen, and vitronectin
that is involved in several cellular processes including cell differentiation, embryogenesis,
proliferation, and gene expression [38,39], The mechanism by which RGD promotes cell
attachment is by increasing microfilament and focal adhesion formation [40], RGD
coated surfaces were shown to promote neurite outgrowth and increase biocompatibility
in implants [41,42], RGD surface density above 4.0 pmol/cm has been found to induce
cell adhesion, spreading, focal contact formation, and cytoskeletal organization [43],
14


NGC surfaces can also be functionalized to provide topographical signals for directing
cellular function in regenerating axons. Axonal growth generally occurs more randomly
in direction, making it difficult for reinnervation in tissues distant to the transection site.
With the introduction of topical cues, axons can be guided to extend more linearly,
improving neurite extension across large distances [44], One strategy is to use
longitudinally aligned fibers to induce linear outgrowth of axons [45], Longitudinally
aligned fibers also contribute to high surface area which is favorable for cell attachment
and growth [46],
2.2.6 Intraluminal channels
Intraluminal channels can also be utilized to allow for more surface functionalization and
to mimic the structural orientation of native nerve fascicles. NGCs with intraluminal
channels allow for improved nerve regeneration as the channels provide more surface
area for cell attachment and migration [47], These channels also reduce the random
dispersion of regenerating axons as axonal extension will be confined to a restricted
surface area once a regenerating axon enters a single intraluminal channel [48],
2.2.7 Lumen fillers
The alignment of Schwann cells and the formation of Biingner bands are reduced during
the regenerating process across larger nerve gaps. Lumen fillers provide a cell supporting
matrix that promotes axonal outgrowth as they provide topological cues to promote
attachment, proliferation, and migration of Schwann cells [32], Lumen fillers can be
made from biological materials found in the body (e.g. collagen) or synthetic biomaterials
(e.g. PLGA, PCL) and processed into various fibers, gels, or sponges.
15


2.2.8 Growth factors
Growth factors regulate cellular proliferation and differentiation of various cell types
during nerve regeneration. During nerve regeneration, the growth cone reacts favorably
to several different growth factors that promote axonal outgrowth, but as cellular
production of these growth factors diminish after injury, so does regeneration [49],
One major class of growth factors that can be implemented to stimulate axonal outgrowth
and nerve regeneration are neurotrophins. Neurotrophins consist of nerve growth factor
(NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and
neurotrophin-4/5 (NT-4/5) that promote various neural responses involved in nerve
regeneration. NGF has been shown to enhance nerve regeneration by promoting survival
and differentiation of sensory and sympathetic neurons [50], BDNF is associated with
increasing motor neuron survival, enhancing axonal growth, and facilitating in the
myelination of regenerating axons [51], NT-3 and NT-4/5 support the survival, growth,
and differentiation of motor and sensory neurons and their use in NGCs has been shown
to increase the number of regenerated axons, axonal diameter, and myelin thickness [52],
Glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF),
acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) are
growth factors outside the neurotrophin family, but are associated with various
neurotrophic functions. GDNF is associated with neuron survival and has shown to
enhance sensory and motor axon regeneration [53], CNTF has been correlated to increase
motor nerve conduction velocity, muscle action potential amplitudes, axon diameter,
number of axons, neurite outgrowth, and myelin thickness [54], aFGF has been shown to
16


increase the number of myelinated regenerating axons [55], bFGF has been linked to an
increase in axonal sprouting and proliferation of Schwann cells [56],
2.2.9 Support cells
Schwann cells play a critical role in peripheral nerve regeneration and can be integrated
into NGCs through processes including injections and cell seeding. Schwann cells
produce essential ECM molecules (e.g. laminin, collagen) that provide structural and
adhesive support for elongating axons. Schwann cells also produce neurotrophic
molecules that provide biochemical cues for neurite extension. Although incorporating
Schwann cells into NGCs is beneficial for axonal extension, autologous Schwann cells
are difficult to obtain in large quantities and allogeneic Schwann cells induce
immunogenic responses and their use requires immunosuppressants. [57-59]
Peripheral nerve injury severely decreases the number of healthy neurons and one
strategy to replenish neuronal activity has been to use stem cells as support cells
including embryonic stem cells (ESCs), neural stem cells (NSCs), and mesenchymal stem
cells (MSCs) that can proliferate and undergo rapid cellular expansion in response to
nerve injuries. ESCs are pluripotent cells that can differentiate into neuronal cells.
Retinoic acid and NGF stimulate neuronal differentiation, extensive outgrowth, and leads
to the expression of neuron-specific molecules [60], Implanted ESC derived neural
progenitor cells were found to increase axonal regrowth and nerve repair as they
differentiated into myelinating cells resembling a phenotype similar to Schwann cells
[61], NSCs are multipotent cells that can differentiate into neurons. The use of NSCs has
been shown to facilitate in increased electrophysiological activity and increased nerve
regeneration observed through histological examination [62], MSCs are pluripotent stem
17


cells and can differentiate into neurons and myelinating cells [63,64], The use of MSCs in
conduits has shown to support nerve regeneration as the MSCs were shown to behave
similarly to myelinating Schwann cells [65],
2.2.10 Electric conductivity
Neural communication relies on action potentials generated at the synapse of neurons
which is dependent on electric fields. This implies that electrical conductivity is
important to promote neurite outgrowth and enhance nerve regeneration [66], NGCs
produced from electrically conductive materials were shown to improve nerve cell
migration as they promote microtubule disassembly and creates a charged neuronal
cytoskeleton [67], Polymer systems based on biodegradable and conducting polymers
have been shown to provide local stimulation of desired tissue, time controlled drug
release, and stimulation of proliferation and differentiation of cells [68],
2.3 Electrospinning of polymer solutions
Electrospinning has become a common technique for producing nanofiber polymer
scaffolds for biomedical applications. Electrospun scaffolds have an overall porous
structure, high surface area due to the small fiber diameters, and resemble the three-
dimensional networks of the ECM of biological tissues and organs [69], This
environmental similarity is beneficial for cell attachment and migration, signal
transduction, and nutrient transport. By electrospinning polymer solutions, flexible and
fibrous structures can be fabricated that have ideal semipermeable properties that can
interact well in the biological system [70],
18


2.3.1 Setup and process
The process of electrospinning requires a needle, high-voltage power supply, and a
grounded collector (Figure 2.1). A syringe pump is used to flow a viscoelastic polymer
solution through a needle at a constant and controlled rate. As a high voltage is applied to
the needle, the polymer solution is highly electrified and induces charges over the surface
of the viscoelastic solution. The viscoelastic solution at the tip of the needle forms a
Taylor cone and when the electrostatic forces overcome the surface tension of the
viscoelastic solution, a liquid jet is ejected from the tip of the needle. Electrostatic
repulsion from the surface charges and evaporating solvent stretches the liquid jet into a
long fiber. Due to the electrical gradient between the positively charged liquid jet and the
grounded collector, fibers are deposited onto the collector. [71]
Figure 2.1 Electrospinning setup. Fiber arrangement is dependent on the
collector. Flat collectors result in random fiber arrangement and split
electrode collectors result in aligned fiber arrangement. [71]
2.3.2 Fiber arrangement
The arrangement of electrospun fibers is dependent on the distribution of the electric field
of the collector. Although many different collectors exist to control fiber arrangement,
19


random or aligned fibers are usually desired. Random fiber arrangement can be obtained
using a simple flat collector and aligned fibers can be obtained using a split electrode
collector (Figure 2.1). With the use of a flat collector, random bending is associated with
the charges on the liquid jet that are quickly dissipated and deposited on the collector.
When a split electrode collector is used, the charges on the liquid jet induces opposite
charges between the collecting electrodes and the electrostatic attractions between the
electrodes stretch and align the nanofibers across the gap [72],
2.3.3 Parameters
The morphology and diameter of electrospun fibers is dependent on various
electrospinning parameters that involve the intrinsic properties of the polymer solution
and operating conditions. The intrinsic properties of the polymer solution include
viscosity, concentration, conductivity, surface tension, and polymer molecular weight.
Operating conditions include electric field strength, voltage, flow rate, distance between
needle tip and collector, temperature, and humidity.
2.4 Rat SNI model
Peripheral nerve regeneration is most often investigated using the SNI model to evaluate
sensory and motor nerve function. The sciatic nerve is the largest nerve trunk in
mammals and the large nerve allows for easy surgical access and facilitates in the
surgical repair of nerve injury. The sciatic nerve divides into the tibial, sural, and
common peroneal nerves and the site of terminal branching needs to be carefully
considered. The evaluation of NGCs in SNI models have been carried out in a number of
different animal species, but most commonly in rats. The extensive use of rats is due to
their small size and large availability, in addition to being relatively easy to work with.
20


Since the majority of studies involving PNI have been modeled using SNI models, data
from previous studies is readily available for comparison. As most clinically and
surgically relevant nerve injuries are characterized by at least partial transection of the
nerve, the SNI model consisting of complete nerve transection is ideal for evaluation of
clinically relevant peripheral nerve regeneration [73],
2.4.1 Functional assessment
One method of evaluating nerve regeneration is performing a functional assessment,
often investigating motor function. Functional recovery of nerves is based on axon
regeneration and selective target reinnervation. As the clinical objective in the use of
NGCs is to restore function to denervated tissue following PNI, functional assessment is
the most direct method of evaluation. However, functional tests are highly variable.
One of the most commonly used tests to assess motor function recovery is the walking
track analysis. Through the walking track analysis, motor function is quantified through
several variables that are measured after recording hind feet footprints. The quantified
value, the sciatic function index (SFI), is calculated using several measured variables
consisting of print length (PL), toe spread (TS), and intermediate toe spread (ITS) of both
the experimental or grafted side (EPL, ETS, EITS) and the normal contralateral side
(NPL, NTS, NITS) (Figure 2.2, Equation 2.1) [74], The walking track analysis utilizes
the increased PL, decreased TS, and decreased ITS characteristics of the intrinsic loss of
muscle function [75], SFI ranges from values of 0 to -100, where 0 indicates normal
nerve function and -100 indicates total impairment.
21


ITS
TS
PL
4_________________________.1
Figure 2.2 Measured variables used to calculate SFI in walking track
analysis. [75]
SFI = -38.3
(EPL NPL}
V nFl )
+ 109.5
. (ETS NTS}
NTS J
+ 13.3
fEITS NITS}
V NITS )
-8.8
(2.1)
Another method of evaluating motor function recovery is utilizing video recording for a
gait analysis. There are several different techniques and variables that can be measured to
quantify motor function. One technique is to measure the toe out angle, the angle between
the direction of progression during walking and the tip of the third digit. The toe out
angle accounts for the biomechanical differences of the external rotation of the hind foot
in the stance phase of walking [76], Another technique is to measure the ankle motion
during walking. With injury to the sciatic nerve, both the angles of maximum ankle
22


plantar flexion and dorsiflexion decrease. Ankle motion analysis is a reliable and
sensitive technique that can be used to measure even subtle differences in both ankle
plantar flexion and dorsiflexion angles during nerve regeneration [77],
Standing or static methods in assessing behavioral deficits have also been developed. A
static footprint video analysis utilizes TS and ITS while the animal under observation is
static to calculate a quantified value, the static sciatic index [78],
Both dynamic and static parameters of behavior and motor deficits can also be assessed
simultaneously. The CatWalk gait analysis is a behavioral test for detecting both dynamic
and static parameters after nerve transection by using several variables including print
area, print intensity, stance duration, and swing duration to evaluate nerve function [79],
Functional assessments of nociceptive function can also be used to evaluate nerve
function. One test that is often used is the withdrawal reflex latency test. This test
measures the withdrawal time after placing a hind foot on a heat stimulus, such as a hot
plate [80],
2.4.2 Electrophysiological assessment
The sciatic nerve consists of both sensory and motor neurons, allowing for
electrophysiological assessment to be performed on both afferent and efferent
components. These tests study sensory and motor nerve conduction utilizing
electromyography and spinal reflex tests, often inducing motor and sensory potentials.
However, when considering clinical relevance, the recovery of motor function is often
considered more significant over sensory function. [81]
23


The most common approach to electrophysiological assessment is inducing compound
muscle action potentials (CMAPs). CMAPs can be evoked by electrical stimulation
proximal and distal to the transection site and action potentials can be recorded in muscle
distal to the stimulation site, such as the gastrocnemius muscle [82],
2.4.3 Histological assessment
Although functional and electrophysiological assessments can be used to evaluate
functional recovery of nerve regeneration, histological examination is essential to
complement these assessments. By histological assessment, the presence of regenerated
axons can be directly observed and any incidence of inflammation and fibrosis can be
examined.
Histological assessment is most commonly done on transverse sections of the regenerated
nerve. This can be done by toluidine blue staining on sections from osmium post-fixed
and resin-embedded blocks or by transmission electron microscopy (TEM). The number
and size of regenerating axons and myelin sheath thickness are often quantified. Axon
regeneration can also be observed by immunohistochemistry (IHC). IHC on longitudinal
sections can be used to show axon regeneration across the length of the transection site.
Histological assessment can be used to observe morphological changes in denervated
tissue and tissue during reinnervation. Massons trichrome stain is often used to stain
gastrocnemius muscle. Muscle fiber diameter and the presence of collagenous and
fibrotic tissue can be quantified.
24


2.4.4 Limitations
Although the rat SNI model is an appropriate model to evaluate NGC efficacy in vivo,
there are limitations that must be considered. Due to the smaller size of rats, the
maximum length of a sciatic nerve transection is limited to 1.5 cm for an adult rat. For
studies investigating larger nerve gaps, rabbits and dogs are commonly used for their
larger body size and sciatic nerve length [83], Another limitation of using rats to model
SNI is the shorter life span of rats, making more chronic nerve injuries difficult to model.
Another limitation in using rats in a SNI model is autotomy. Autotomy is one of the most
frequent postoperative complications that arises in rats and ranges in severity. Loss of
toenails is observed in minor cases, while loss of toes can occur in more severe cases.
Autotomy can affect functional assessment and often raises concerns regarding animal
welfare.
25


3 Previous Work
The potential use of PSHU-RGD/PCL for nerve regeneration has previously been
examined. Cytotoxicity, cell viability, cell proliferation, cell differentiation, neurite
outgrowth, and guided neurite sprouting in PC 12 cell culture has been investigated for
PSHU-RGD [15], A method of introducing intraluminal microchannels with aligned
nanofibers was developed and was found to promote neuronal growth and guide axonal
extension, in addition to enhancing cell attachment, survival, and migration in human
neural stem cell (hNSC) culture [16],
3.1 Biocompatibility
Biocompatibility assessment was conducted according to the guidelines developed by the
International Organization for Standardization (ISO) 10993-5 which describes test
methods to assess in vitro cytotoxicity of medical devices. Cytotoxicity was evaluated
using the PC12 cell line which is derived from rat pheochromocytoma and has an
embryological origin. PC12 cells differentiae into cells that behave similarly to neurons,
making them a suitable as a model system for neuronal differentiation.
PSHU extract was prepared by incubating PSHU in cell culture media for 24 h in
standard conditions and filtered to remove particulates. PC 12 cells were exposed to fresh
media, PSHU extract, or diluted PSHU extract and cell viability was measured using an
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure 3.1).
No statistical difference was observed between cell culture media and PSHU extract
suggesting biocompatibility.
26


120
Cell PSHU 1:2 1:4 1:8 1:16,
Culture Extract Diluted PSHU Extract in Media
Media
Figure 3.1 Biocompatibility assessment using PC 12 cells with cell
viability assessed by MTT assay. PSHU extract was prepared by
incubating PSHU in cell culture media for 24 h in standard conditions and
filtered to remove particulates. Error bars represent one standard deviation.
3.2 PSHU-RGD and PC12 cell culture
The neuronal response to PSHU-RGD was examined by seeding PC 12 cells onto coatings
of either laminin or PSHU-RGD in cell culture media supplemented with and without
NGF. NGF was used as a positive control as the growth factor induces cell differentiation,
microtubule assembly, and neurite outgrowth. An increase in neuronal activity was
observed for surfaces coated with PSHU-RGD compared to surfaces coated with laminin
(Figure 3.2).
27


Laminin Laminin NGF PSHU-RGD PSHU-RGD NGF
100 pm____________| 100 pm_____________| 100 pm____________| 100 pm
Figure 3.2 Fluorescence microscopy of the neuronal response of PC12
cells seeded on either surfaces coated with either laminin or PSHU-RGD
in cell culture media supplemented with and without NGF. PC12 cells
were stained with pill-tubulin and Alexa 488. Scale bars apply to
respective columns.
Cell differentiation and neurite length were compared between the different surface
coatings, with and without NGF (Figure 3.3, Figure 3.4). Differentiated cells were
classified as cells with at least one neurite. PC12 cells on laminin coated surfaces showed
minimal neurite growth through 14 d, while PC 12 cells on PSHU-RGD coated surfaces
showed significantly improved cell differentiation and neurite outgrowth, demonstrating
the potential beneficial impact of using PSHU-RGD. When NGF was introduced to PC 12
cells on laminin and PSHU-RGD surfaces by supplementation in media, extensive neurite
networks developed over 14 d.
28


Day 1 Day 7 Day 14
Figure 3.3 Neuronal response of PC12 cells by cell differentiation.
Differentiated cells were classified as cells with at least one neurite. Error
bars represent one standard deviation. indicates p < 0.0005.
Day 1 Day 7 Day 14
Figure 3.4 Neuronal response of PC12 cells by neurite length. Error bars
represent one standard deviation. indicates p < 0.001, ** indicates p <
0.0005.
Although the supplemented use of NGF on surfaces coated with PSHU-RGD showed the
highest level of cell differentiation and neurite length, the delivery of NGF in clinical
29


settings remains a challenge. NGF concentration at the injury site must be sustained
during the nerve regenerative process that may often take long periods of time [84],
Therefore, it is ideal if sufficient neuronal activity can be supported by material alone, as
seen with PC12 cells on surfaces coated with PSHU-RGD without the supplementation of
NGF.
3.3 Fiber alignment and PC12 cell culture
The importance of fiber alignment and guided neurite extension has been investigated by
seeding PC12 cells on surfaces with varying fiber alignment (Figure 3.5). Scaffolds with
random and aligned fiber arrangements were fabricated by electrospinning with fiber
arrangements confirmed by scanning electron microscope (SEM). PC 12 cells seeded onto
random fiber arrangements presented high branching and randomly extending neurites,
while PC 12 cells seeded onto aligned fiber arrangements exhibited low branching and
extensions further from cell bodies along the direction of the aligned fibers.
30


Figure 3.5 Neuronal response of PC12 cells to fiber arrangement. (A)
random fiber arrangement, (B) aligned fiber arrangement, (C) neurite
extension on random fiber arrangement, (D) neurite extension on aligned
fiber arrangement. Scale bars apply to respective columns.
3.4 NGC with aligned nanofibers along intraluminal channels
A method of electrospinning was developed to fabricate NGCs with aligned nanofibers
along intraluminal channels using a split electrode collector from a blended polymer
solution of PSHU-RGD and PCL (Section 5.7, Figure 3.6). The structure of the PSHU-
RGD/PCL conduit closely resembles the native nerve structure of the perineurium and
epineurium.
31


Figure 3.6 SEM images of a PSHU-RGD/PCL conduit. (A) transverse
section of conduit with embedded sucrose fibers, (B) transverse section
after dissolving sucrose fibers, (C-D) longitudinal sections after dissolving
sucrose fibers.
3.5 NGC and hNSC culture
PSHU-RGD/PCL conduits were fabricated and hNSCs were seeded onto one end of each
NGC and neuronal activity was investigated. PCL conduits served as a negative control,
of which very limited neuronal activity was expected when seeded with hNSCs. The PCL
conduits were constructed similarly to PSHU-RGD/PCL conduits, but PSHU-RGD was
not blended with the PCL solution during electrospinning. The hNSCs seeded on PCL
conduits were sparsely distributed and showed no directional preference in extension
after 14 d (Figure 3.7). Although the PCL conduits contained intraluminal microchannels
with aligned nanofibers, the absence of RGD prevented ample cell attachment, survival,
and migration. No neuronal growth or axonal extension was observed.
32


Figure 3.7 Fluorescence microscopy images of hNSC behavior on PCL
conduit after 14 d. (A) DAPI stain in blue, (B) P-III tubulin stain in green.
Arrow indicates the direction of nanofiber alignment.
The hNSCs seeded on PSHU-RGD/PCL conduits were higher in cell density and the cells
migrated into the microchannels with considerable neurite extension after 14 d (Figure
3.8). In contrast to the PCL conduits, PSHU-RGD/PCL conduits allowed for cell
attachment, survival, and migration. Significant neuronal growth and axonal extension
were observed.
33


Figure 3.8 Confocal microscopy images of hNSC behavior on PSHU-
RGD/PCL conduit after 14 d. (A-C) along center of microchannel, (E-G)
along inner wall of microchannel, (A, D) DAPI stain in blue, (B, E) P-III
tubulin stain in green, (C, F) combined DAPI and P-III tubulin stains.
Arrows indicate the direction of nanofiber alignment.
34


4 Hypothesis and Specific Aims
4.1 Hypothesis
Based on preliminary data that suggests that an NGC containing intraluminal
microchannels with aligned nanofibers promote neuronal growth and guide axonal
extension, it was hypothesized that the electrospun PSHU-RGD/PCL conduit was
functionally comparable to the gold standard autograft when used to surgically treat a
peripheral nerve transection.
4.2 Specific aims
The first specific aim was to synthesize and characterize PSHU-RGD. As the major
functional material component of the NGC, it was important to confirm the conjugation
of RGD and the overall molecular structure of the polymer. The second specific aim was
to evaluate the NGC for nerve regeneration in a rat SNI model. Evaluation included
functional, electrophysiological, and histological assessments after sciatic nerve
transection and graft implantation. Functional recovery was investigated using a walking
track analysis and ankle motion analysis. Electrophysiological activity was measured by
recording the compound action potential (CAP) of the grafted nerves. Gastrocnemius
muscle mass was used to assess muscle reinnervation. Regenerating axons and Schwann
cells were observed using IHC with stains specific for neurofilament of regenerating
axons and calcium-binding proteins in Schwann cells. The morphology associated with
the reinnervation of the gastrocnemius muscle was observed using Massons tri chrome
stain.
35


5 Materials and Methods
5.1 Materials
Serinol, urea, hexamethylene diisocyanate (HDI), anhydrous N,N-dimethylformamide
(DMF), PCL (Mn 80,000 g/mol), dimethyl sulfoxide (DMSO)-d6, sucrose,
paraformaldehyde, Massons trichrome stain kit, and xylene were purchased from Sigma-
Aldrich (St. Louis, MO, USA). Di-tert-butyl dicarbonate, ethyl acetate, trifluoroacetic
acid (TFA), 2,2,2-trifluoroethanol (TFE), N-(3-Dimethylamino- propyl)-N'-
ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 1,1,1,3,3,3-
Hexafluoro-2-propanol (HFP) were purchased from Alfa Aesar (Ward Hill, MA, USA).
Hexane and anhydrous diethyl ether were purchased from Fisher Scientific (Pittsburgh,
PA, USA). Anhydrous methylene chloride (DCM) and 10 % formalin were purchased
from JT Baker (Phillipsburg, NJ, USA). Gly-Arg-Gly-Asp-Ser (GRGDS) was
purchased from Biomatik (Wilmington, DE, USA). Sprague Dawley rats were purchased
from Charles River Laboratories (Wilmington, MA, USA). Isoflurane, ketoprofen, and
bupivacaine (0.5 % Marcaine) were purchased from MWI Veterinary Supply (Boise, ID,
USA). Prolene polypropylene 7-0 and coated Vicryl 4-0 sutures were purchased from
Ethicon (Somerville, NJ, USA). Optimal cutting temperature (OCT) compound was
purchased from Sakura (Torrance, CA, USA). Phosphate Buffered Saline (PBS) and
Cytoseal 60 were purchased from Thermo Scientific. Goat serum, neurofilament-medium
(NF-M, rabbit IgG), Alexa Fluor 594 (goat anti-rabbit IgG), SlOOb (mouse IgGl), Alexa
Fluor 488 (goat anti-mouse IgG), and SlowFade Diamond antifade mountant with DAPI
were purchased from Life Technologies (Carlsbad, CA, USA). Triton X-100 was
purchased from MP Biomedicals.
36


5.2 Equipment
Proton nuclear magnetic resonance (*H NMR) was performed on a Varian Inova 500
NMR Spectrometer and samples were run in DMSO-d6 at room temperature. Fourier
transform infrared spectroscopy (FT-IR) was performed on a Nicolet 6700 FT-IR
Spectrometer and samples were run on polyethylene infrared (IR) sample cards.
Electrophysiological assessment was performed using Axon CNS MultiClamp 700B,
Axon Digidata 1440A, and Grass SD9 Stimulator. Tissue was sectioned using a CryoStar
NX70 Cryostat. Confocal images were taken using an Olympus FVI000. Brightfield
images were taken using a Nikon Eclipse 80i. ImageJ was used to quantify variables for
image analysis.
5.3 N-BOC serinol synthesis
Serinol (1.96 g, 21.5 mmol) was dissolved in ethanol (20.0 ml) and cooled to 4 C. A
mixture of di-tert-butyl dicarbonate (5.97 ml, 26.0 mmol) and ethanol (10.0 ml) was
added dropwise. The solution was heated at 37 C for 1 h and subsequently
rotoevaporated at 50 C and 20 mbar to remove ethanol, yielding a white powder. The
white powder was dissolved in a 1:1 volume mixture of ethyl acetate and hexane at 60 C.
Hexane was added dropwise until crystalline structures were formed and excess hexane
was added to ensure complete precipitation. The precipitate was allowed to settle at 4 C
and filtered to remove hexane, yielding a crystalline white powder.
5.4 PSHU synthesis
N-BOC serinol (1.15 g, 6.00 mmol) and urea (0.360 g, 6.00 mmol) were dissolved in
DMF (6.00 ml). Then, HDI (1.93 ml, 12.0 mmol) was added and the solution was heated
at 90 C for 7 d under a nitrogenous atmosphere. The solution was then cooled to room
37


temperature and rotoevaporated at 80 C and 20 mbar to remove DMF. The product was
precipitated using cooled diethyl ether. To purify the product, a Milli-Q water wash and
diethyl ether wash was performed. After the last wash, the product was dried by
rotoevaporation at 50 C and 20 mbar, yielding the polyurea as a white powder.
5.5 PSHU deprotection
Deprotection functionalizes the polyurea by removing the BOC protecting groups and
exposing free amine groups (NH2), producing deprotected PSHU (dPSHU). PSHU (1 g,
1.96 mmol) was dissolved in DCM (15.0 ml), and TFA (15.0 ml) was subsequently added
dropwise. Deprotection occurred by hydrogenation at room temperature for 45 min.
DCM and TFA were rotoevaporated at 50 C and 20 mbar and the product was dissolved
in DMF (1.00 ml). The product was precipitated using cooled diethyl ether. To purify the
product, the precipitate was dissolved in TFE and precipitated in ether. The product was
dried by rotoevaporation at 50 C and 20 mbar, yielding a white powder.
5.6 RGD conjugation to dPSHU
GRGDS pentapeptide was used rather than RGD tripeptide to preserve the integrity of the
entire RGD binding motif. It was determined that the number of free amine groups in
dPSHU is 2.05 mmol NH2 /g PSHU after confirming the structure (Sections 6.1, 6.2).
GRGDS (151 mg, 0.308 mmol), EDC (70.9 mg, 0.370 mmol), and NHS (53.2 mg, 0.462
mmol) was dissolved in DMF (1.00 ml) and NHS-ester activation was allowed for 2 h.
dPSHU (150 mg, 0.308 mmol NH2) was separately dissolved in DMF (1.0 ml) and added
dropwise to the NHS-ester activated solution. The solution was allowed to react for 24 h
at room temperature. The product was rotoevaporated at 40 C and 20 mbar to remove
DMF and subsequently precipitated in diethyl ether. To purify the product, the precipitate
38


was washed in Milli-Q water. The product was dried by lyophilization, yielding PSHU-
RGD as a white powder. PSHU-RGD was stored away from light at 4 C.
5.7 NGC fabrication by electrospinning
Prior to electrospinning, sucrose fibers with diameters between 150-200 pm were formed
using a fiber drawing method. Sucrose was heated to 75 C until it melted with a thick
consistency and fibers were drawn. The collector was constructed using two copper wire
electrodes spaced 3.5 cm apart and the sucrose fibers were fit to span the gap between the
copper wire electrodes. 8 w/w % polymer solutions in HFP were prepared for PSHU-
RGD/PCL (30/70) blend, PSHU/PCL (30/70) blend, and pure PCL. A two electrode
electrospinning setup was used to fabricate the NGC (Figure 5.1).
Figure 5.1 Two electrode electrospinning setup. Parameters: distance
between needle and collector at 10 cm, flow rate at 1 ml/h, voltage at 7.5
kV (PSHU-RGD/PCL and PSHU/PCL) or 9 kV (PCL), room temperature,
relative humidity at 30 %.
39


The collector was placed 10 cm from the needle of the syringe filled with polymer
solution. The polymer solution was ejected at 1.0 ml/h through a 21 gauge stainless steel
flat-tip needle at room temperature and relative humidity at 30 %. A positive 7.5 kV
electrostatic potential was applied to the needle for the two blended solutions, and a 9 kV
potential for the pure PCL solution. The collector was rotated every 5 min to evenly
distribute the nanofibers across both sides of the collector. For PSHU-RGD/PCL conduits,
the PSHU-RGD/PCL blend was electrospun for the initial 10-15 min, and then the
PSHU/PCL blend was used to deposit the remainder of the nanofibers. The pure PCL
conduits were electrospun using its respective solution during the entire electrospinning
process. The flat sheet was then removed from the collector and rolled into a tube (Figure
5.2). Using the same electrospinning setup, the rolled tube was held in front of a flat
collector and manually rotated until a thin layer of polymer was coated onto the surface,
sealing the outer layer of the tube and preventing it from unraveling. The tubes were then
cut into 10 mm long conduits.
<----->
Sucrose Fiber
Aligned Fiber
Direction of Fiber Alignment
<-----------
Figure 5.2 Rolling of flat sheet into a tube.
40


5.8 Sciatic nerve transection and graft implantation
The sciatic nerve transection and NGC implantation procedure was approved by the
Institutional Animal Care and Use Committee (IACUC). A total of 24 Sprague Dawley
rats were used for the study, 4 rats per each graft (autograft, PCL, PSHU-RGD/PCL) for
2 time points (4, 8 w). The rats weighing 250-275 g were allowed 7 d to acclimate prior
to implantations and maintained on a 14/10-hour light/dark cycle with access to food and
water ad libitum.
The rats were anaesthetized using continuous isoflurane and oxygen inhalation. Initial
induction was at 5 % isoflurane in oxygen and then maintained at 2 % isoflurane in
oxygen. To minimize post-operative pain, preoperative doses of ketoprofen at 5 mg/kg
and bupivacaine (0.5 % Marcaine) at 2 mg/kg were administered via subcutaneous
injection. Artificial tears ointment was administered to prevent dry eyes during the
procedure. The sciatic nerve that underwent transection and implantation was determined
randomly, either the left or right sciatic nerve. The sciatic nerve that was not selected for
transection did not undergo any experimental manipulation and was used as a control for
normal nerve function. The rats were placed on their side and the skin around the gluteal
region on the randomly selected side was shaved and disinfected with chlorhexidine and
isopropyl alcohol. A longitudinal skin incision from the knee to the hip was made to
expose the underlying muscles that were retracted to isolate the sciatic nerve. An incision
of the sciatic nerve was made 5 mm in each direction from mid-thigh for a total of a 10
mm gap. In the case of the autograft, the ends of the 10 mm transected nerve were
reversed and sutured to the proximal and distal nerve stumps using Prolene
polypropylene 7-0 sutures. For PCL and PSHU-RGD/PCL conduits, the ends of the
41


conduit were sutured to the proximal and distal nerve stumps using Prolene
polypropylene 7-0. After graft implantation, the muscle layer was closed with coated
Vicryl 4-0 sutures using a continuous suturing pattern. The skin incision was closed using
coated Vicryl 4-0 sutures using a continuous subcuticular suture technique. Postoperative
doses of ketoprofen at 5 mg/kg were administered daily for 3 d.
5.9 Walking track analysis
4 or 8 w after implantation, a walking track analysis was performed. A walking alley with
a darkened goal box at one end was used to assess functional recovery after implantation.
The floor of the walking alley with dimensions 45 cm x 8 cm x 5 cm was covered with
white paper. The hind feet of the rats were smeared with finger paint and the rats were
allowed to walk down the track leaving footprints on the paper. The hind feet were
cleaned and disinfected with chlorhexidine and alcohol after obtaining footprints. SFI
was calculated with EPL, NPL, ETS, NTS, EITS, and NITS measurements for three sets
of prints that were averaged (Equation 2.1).
5.10 Ankle motion analysis
Immediately after obtaining footprints for the walking track analysis, ankle motion was
recorded by video on a standard recording device. The angle of the ankle was measured
during initial contact, mid-swing, and toe off phases of walking (Figure 5.3). The
difference between grafted and normal contralateral sides for each phase during walking
were calculated (Equation 5.1).
42


Initial Contact
Mid-Swing------
i
Figure 5.3 Different phases of walking in which ankle angle was
measured for ankle motion analysis.
Grafted Side Healthy Contralateral Side
Difference [%] = --------Healthy Contralateral Side------X 100%
5.11 Euthanasia and tissue harvest
One day following the recording of ankle movement for ankle motion analysis, the rats
were euthanized by carbon dioxide and bilateral thoracotomy. Both of the sciatic nerves
of the grafted and healthy contralateral sides were exposed similarly to the initial
43


implantation protocol (Section 0). The sciatic nerves from the spinal cord to the terminal
branching site of the tibial, sural, and common peroneal nerves were harvested. The
gastrocnemius muscle samples were harvested by making an incision of the skin just
above the heel of the hind foot and the skin surrounding the gastrocnemius muscle was
removed. Both the gastrocnemius muscles from the grafted and healthy contralateral
sides were harvested.
5.12 CAP recordings
Immediately after nerve tissue harvest, the CAPs of both the sciatic nerves of the grafted
and healthy contralateral sides were recorded. The harvested nerves were placed on a
platform with parallel conducting wires (Figure 5.4). The proximal end of the graft or
healthy nerve was stimulated for 0.15 ms at 10 V and the CAP was recorded at the distal
end. The grafted side to healthy contralateral side ratio of CAP amplitude and area under
the curve (AUC) were calculated (Equation 5.2).
Stimulating
Electrode
Recording
Electrode
Ratio =
Grafted Side
Healthy Contralateral Side
(5.2)
44


5.13 Gastrocnemius muscle mass
The gastrocnemius muscles of both the grafted and healthy contralateral sides were
weighed and the ratio of grafted side to healthy contralateral side was calculated
(Equation 5.2).
5.14 Histology
5.14.1 IHC of nerve grafts
After recording CAP measurements of the sciatic nerves, the grafted nerves were fixed
using 4 % PFA in PBS for 1 h, cryoprotected with 30 % sucrose in PBS for 2 d,
embedded in OCT compound, and frozen at -80 C. The nerves were sectioned
longitudinally with a thickness of 18 pm and placed on glass slides.
The sections were fixed in acetone for 10 min and washed 2 times in PBS for 3 min each.
Clear nail polish was used at the very ends of each nerve and allowed to dry to secure the
nerves onto the slides. The sections were blocked in 3 % hydrogen peroxide in PBS for
10 min to block endogenous peroxidase activity and washed 3 times in PBS for 3 min
each. Blocking Buffer (5% goat serum, 0.4% Triton X-100, PBS) was used to block the
sections for 30 min. All antibodies were diluted in Dilution Buffer (1% serum, 0.4%
Triton X-100, PBS). The sections were stained with NF-M (1:500) for 60 min, washed 3
times in PBS for 3 min each, stained with Alexa Fluor 594 (1:500) for 30 min, and
washed 3 times in PBS for 3 min each. The sections were double immunostained with
SlOOb (1:1000) for 60 min, washed 3 times in PBS for 3 min each, stained with Alexa
Fluor 488 (1:500) for 30 min, and washed 3 times in PBS for 3 min each. SlowFade
Diamond antifade mountant with DAPI was used to mount the slides.
45


5.14.2 Massons trichrome staining of gastrocnemius muscle
After measuring muscle mass, the gastrocnemius muscle of both the experimental or
grafted side and the healthy contralateral side were fixed in 10 % formalin for 24 h,
cryoprotected with 30% sucrose in PBS for 24 h, embedded in OCT compound, and
frozen at -80 C. The muscles were sectioned with a thickness of 5 pm and placed on
glass slides.
The sections were washed in running deionized water to remove OCT compound and
fixed in Bourns solution (71 % saturated aqueous picric acid, 24 % formaldehyde, 5 %
acetic acid) at room temperature overnight and washed in running deionized. The first
stain consisted of Biebrich Scarlet-Acid Fucshin solution (0.9 % Biebrich scarlet, 0.1 %
acid fuchsin, 1.0 % acetic acid) for 5 min and subsequent wash in running deionized
water. The second stain involved a mixed solution of 1 volume 10 % Phosphotungstic
acid, 1 volume 10 % Phosphomolybdic acid, and 2 volumes deionized water for 5 min.
The third and final stain comprised of Aniline Blue solution (2.4 % Aniline blue, 2 %
acetic acid) for 5 min. The sections were placed in 1 % acetic acid for 2 min and washed
in running deionized water. Subsequent washes consisted of 70 % ethanol for 1 min and
100 % ethanol for 1 min. The sections were cleared in xylene for 2 min and mounted
using Cytoseal 60.
The amount of collagen was quantified by calculating the area of collagen to muscle fiber
ratio for the grafted and healthy contralateral sides (Equation 5.3). Collagen to muscle
fiber ratio was then set in another ratio to determine the amount of collagen of the grafted
side to the amount of collagen on the healthy contralateral side (Equation 5.2)
46


Area of Collagen
Collagen to Muscle Fiber Ratio = -------------------- (5.3)
Area of Muscle Fibers ' 7
5.15 Statistical analysis
All results are expressed as means standard error of the mean. Analysis of variance
(ANOVA) was used to determine significant differences between groups and followed by
Tukeys post hoc test when applicable. Statistical significance was considered when p <
0.05.
47


6 Results and Discussion
6.1 PSHU, dPSHU, and PSHU-RGD reaction sequence
The synthesized PSHU-RGD was designed to be biodegradable and biocompatible. The
amide and ester groups are the sites of degradation and the hydrophobic alkyl groups
allow for slower degradation characteristics and allows for enhanced cell attachment
(Figure 6.1).
HDl
O
x
h2n nh2
U rea
HO
HO
NH
N-BOC Serinol
O
O^NH
90 C, 7 (I
rYc
H
O N
Y'
o
o o o
XJLX
X
Poly(serinol hexamethylene urea) (PSHU)
NH2
TFA/DCM
Room Tempearture. 45 min
T
Y
0
o o o
XJLX.
Unprotected Poly(serinol hexamethylene urea) (dPSHC)
GRGDS
GRGDS, EDCVNHS
Room l empearture. 24 h
rY T
o o o
X.X..X.
Arginylglycylaspartic Acid Conjugated Poly(serinol hexamethylene urea) (PSIIIJ-RGD)
Figure 6.1 Reaction sequence of PSHU-RGD synthesis.
6.2 PSHU and dPSHU characterization using NMR
The synthesis of PSHU from the N-BOC serinol, urea, and HDI was confirmed using 'h
NMR (Figure 6.2).
48


Chemical Shift [ppm]
Figure 6.2 'HNMR spectrum of PSHU confirming molecular structure.
*H NMR was used to confirm the removal of BOC protecting groups. The resulting free
amines were used to conjugate RGD. The disappearance of the b peak confirms the
removal of the BOC groups (Figure 6.3).
1.7 1.6 1.5 1.4 1.3 1.2 1.1 1
Chemical Shift [ppm]
Figure 6.3 'HNMR spectrum of PSHU and dPSHU confirming the
removal of the BOC protecting group with the disappearance of the b peak.
49


6.3 PSHU, dPSHU, and PSHU-RGD characterization using FT-IR
After confirmation of PSHU and dPSHU structures using NMR, FT-IR was used to
further confirm the reaction and to verify RGD conjugation (Figure 6.4, Figure 6.5).
4000 3500 3000 2500 2000 1500 1000 500
Wavelength [cm'1]
Figure 6.4 FT-IR spectrum of PSHU, dPSHU, and PSHU-RGD.
Wavelength [cm1]
Figure 6.5 FT-IR spectrum of PSHU, dPSHU, and PSHU-RGD. The
presence of free amine groups on dPSHU after deprotection is confirmed
from region a. The conjugation of RGD to dPSHU is confirmed with the
shift in wavelength with carbonyl absorbance shown in region b.
50


The presence of free amine groups on dPSHU was confirmed in region a (Figure 6.5).
The wavelength in this region corresponds to primary amines. The conjugation of RGD
can be confirmed from region b (Figure 6.5). This region is associated with carbonyl
groups found in PSHU and carbonyl groups of RGD. The wavelength correlated to
carbonyl groups of RGD is slightly lower than that of the carbonyl groups of PSHU.
Therefore, there is a shift in the carbonyl peak with the conjugation of RGD.
6.4 Sciatic nerve transection and graft implantation
One of three different grafts was implanted into each rat. The autograft was used as a
positive control in which the greatest amount of nerve regeneration was expected. The
PCL conduit was used as a negative control. As seen previously, the PCL conduit is
associated with poor neuronal activity due to the absence of RGD to promote cell
attachment, survival, and migration. The PSHU-RGD/PCL conduit served as the
experimental graft. As a result of promising outcomes of neuronal growth and guided
axonal extension observed in preceding studies, the PSHU-RGD/PCL conduit was
believed to be comparable to the gold standard autograft in terms of nerve regeneration.
[15,16]
The sciatic nerve transection and graft implantation procedures were successful for 23 of
the 24 rats used in the study. After making a longitudinal skin incision and retracting the
underlying muscles surrounding the sciatic nerve, a 10 mm long transection was made
and grafts were implanted at the transection site (Figure 6.6). Other than for one rat, no
complications arose during the suturing of the grafts to the proximal and distal nerve
stumps. The rat that did have complications was euthanized after a failed attempt at
suturing an autograft. During the surgical procedure, the suture needle was passed
51


repetitively though the autograft until which the autograft was too damaged for suturing.
The rat was euthanized before recovering from anesthesia. All data analysis was
performed without the inclusion of this rat, therefore, the autograft group for the 8 w time
point consisted of 3 rats rather than the typical 4 rats for all other groups.
Figure 6.6 Sciatic nerve transection and graft implantation. (A)
longitudinal skin incision from the knee to the hip exposing underlying
muscles, (B) location of connective tissue separating two muscles, (C)
blunt dissection through connective tissue, (D) isolation of sciatic nerve,
(E) sutured autograft, (F) sutured PSFTU-RGD/PCL graft.
Although autotomy is one of the most frequent postoperative complications that arise
with the rat SNI model, self-mutilation was limited to slight nibbling of toenails.
However, after obtaining footprints for the walking track analysis, severe autonomy was
observed for three rats. It is presumed that the finger paint used to record footprints on
paper triggered this behavior. Although the hind feet were cleaned and disinfected with
chlorhexidine and alcohol after the analysis, any remaining residue may have caused the
rats to excessively groom the hind feet leading to the loss of toes. However, severe cases
52


of autotomy were only observed after the walking track analysis and did not affect the
footprint recordings.
6.5 Walking track analysis
A walking track analysis was used to assess the functional recovery using a quantified
SFI value to measure sciatic nerve function (Figure 6.7). As nerve function improves, PL
decreases, TS increases, and ITS increases with increased muscle function. This
improvement in sciatic nerve function is reflected by an increase in SFI (Equation 2.1).
Time [w]
Figure 6.7 Calculated SFI values. Error bars represent standard error of
the mean. indicates p < 0.05.
No significant difference between grafts was observed, but a significant improvement
was observed in sciatic nerve function between 4 and 8 w for the PSHU-RGD/PCL
conduit. At 4 w, the SFI values for each graft were similar, but at 8 w, more of a
difference was observed. Although not statistically significantly, the PSHU-RGD/PCL
conduit seemed to outperform the autograft. This encouraging level of functional
regeneration can be attributed to RGD and intraluminal microchannels with aligned
nanofibers. However, the PCL conduit also seemed to attribute to equal, if not superior,
53


functional recovery compared to the autograft. Although the PCL conduit was expected
to see a very limited amount of functional recovery, the positive effect of intraluminal
microchannels with aligned nanofibers may have been underestimated. Axons in the PNS
are known to be able to regenerate on their own, but the topical cues associated with the
microchannels and aligned nanofibers may have accelerated this regeneration process.
6.6 Ankle motion analysis
Ankle motion analysis was performed by recording the movement of the ankle during
various phases of walking. The difference in the ankle angle between the grafted and
healthy contralateral sides during initial contact, mid-swing, and toe off were calculated
(Figure 6.8, Figure 6.9, and Figure 6.10).
Figure 6.8 The difference in the ankle angle between the grafted and
healthy contralateral sides during the initial contact phase of walking.
Error bars represent standard error of the mean. indicates p < 0.05.
54


0
Figure 6.9 The difference in the ankle angle between the grafted and
healthy contralateral side during the mid-swing phase of walking. Error
bars represent standard error of the mean. indicates p < 0.05.
Figure 6.10 The difference in the ankle angle between the grafted and
healthy contralateral side during the toe off phase of walking. Error bars
represent standard error of the mean. indicates p < 0.05.
No statistical difference was observed between the three grafts for initial contact and
mid-swing. However, significant recovery was seen with initial contact for the autograft
and with mid-swing for the PCL conduit between 4 and 8 w. When considering only toe
55


off angle, the PSHU-RGD/PCL conduit showed discernible improvement from 4 to 8 w
and was statistically different from the PCL conduit.
When considering all three phases of walking that were analyzed, no solid conclusion can
be made on functional recovery between each graft. No graft seemed to consistently show
improved function recovery with all phases. However, it appears that substantial recovery
is occurring between 4 and 8 w after nerve transection.
6.7 CAP recordings
CAP recordings of the nerve grafts were used to assess the recovery of
electrophysiological activity across the transection gap. CAP is dependent on axon
diameter and myelin sheath thickness, and therefore, recovering electrophysiological
activity after nerve transection is an important step towards achieving functional recovery.
After stimulating the nerve samples at the proximal end, the CAP was recorded at the
distal end (Figure 6.11). The CAP recordings consisted of two peaks. The first peak is a
stimulus artifact that is produced from electrical stimulation. The second peak is the CAP
of which the amplitude and AUC were calculated. The recorded CAPs for all the grafts
had low latency, characteristic of the short delay of the peak after the stimulus artifact.
This suggests that the majority of the axons that are contributing to the CAP have high
degrees of myelination. Within 4 w after implantation, a CAP was observed for all grafts.
This implies that a sufficient number of myelinated axons have regenerated across the
entire length of the nerve gap.
56


10
>
E
CL
<
u
8 -
6 -
4 -
2 -
0 -
-2 -
0.005 0.006 0.007 0.008 0.009 0.01
Time [s]
Figure 6.11 Representative CAP recordings 8 w after nerve transection
and graft implantation.
Immediately after sciatic nerve transection, CAPs are expected to be unmeasurable. Due
to the disconnection of axons, the nerve is incapable of propagating electrical impulses
across the nerve gap. As axons regenerate across the transection site during the
regeneration process, the CAP amplitude and AUC are expected to increase. Amplitude
will increase as the number of axons increases. However, the degree of myelin thickness
varies greatly for axons during regeneration and the amplitude may not always account
for more thinly myelinated axons. This is characteristic of a broader CAP. Therefore,
AUC was also considered for analysis.
Due to the variability of CAPs of the healthy contralateral sciatic nerves between each rat,
a ratio of CAP amplitude and AUC of the grafted nerve to the healthy contralateral nerve
was calculated (Figure 6.12, Figure 6.13). The amplitude was determined as the
maximum peak value. Calculating the AUC required several steps as the stimulus artifact
and the CAP overlapped with each other. AUC was calculated by initially measuring the
AUC of both the stimulus artifact and CAP. The stimulus artifact was then modeled as a
57


Gaussian by using the parts of the peak that did not overlap with the CAP. The AUC of
the Gaussian was calculated and subtracted from the area of both the stimulus artifact and
CAP. The resulting value was considered the AUC of the CAP.
Time [w]
Figure 6.12 CAP amplitude ratio of the grafted to healthy contralateral
nerves. Amplitude was determined as the maximum peak value of the
CAP. Error bars represent standard error of the mean. indicates p < 0.05.
Figure 6.13 CAP AUC ratio of the grafted to healthy contralateral nerves.
AUC was calculated by subtracting the area under the stimulus artifact
modeled as a Gaussian from the total area under both the stimulus artifact
and CAP. Error bars represent standard error of the mean.
58


The CAP amplitude was significantly higher for the PSHU-RGD/PCL conduit compared
to the autograft and PCL conduit after 4 w. However, no statistical difference was
observed at 8 w, presumably due to the high variability associated with taking recordings
and limited sample size. Even so, it appears that CAP amplitude increased with the use of
PSHU-RGD/PCL over the other grafts. No significant difference was observed for AUC.
Although it appears that the PSHU-RGD/PCL conduit has higher AUC attributed by
more axonal regeneration and myelination, high variability and limited sample size,
prevented a statistically significant result.
Although no statistical significant electrophysiological result was observed, the CAP
recordings seem to imply that PSHU-RGD/PCL may provide a comparable, if not
improved, environment for promoting axonal extension and myelination.
6.8 Gastrocnemius muscle mass
As a target muscle of the sciatic nerve, the gastrocnemius muscle is affected by the
transection of the nerve. Muscle mass and the degree of innervation are dependent on the
motor neurons located in the sciatic nerve. After nerve transection, the gastrocnemius
muscle undergoes a process of denervation, causing muscle atrophy and the overall loss
of muscle mass. Through the use of nerve grafts, it was expected that the gastrocnemius
muscle was gradually reinnervated during the regeneration process, causing muscle mass
to increase. Due to variability of muscle mass between animals that can arise (e.g. size),
the grafted to healthy contralateral side mass ratio was used for comparison between the
different grafts (Figure 6.14). However, no significant difference in muscle mass was
observed between any of the grafts.
59


0.5
re
0£
S 0.45
i/>
3
0.4
0.35
E
ID
C
u
O
0.3
re
0.25
3 4 5 6 7 8 9
Time [w]
Figure 6.14 Gastrocnemius muscle mass ratios of the grafted to healthy
contralateral sides. Error bars represent standard error of the mean.
6.9 Histology
6.9.1 IHC of nerve grafts
IHC was used to directly observe regenerated axons. Axons were stained with NF-M and
Alexa Fluor 594, while Schwann cells were stained with SI00b and Alexa Fluor 488
(Figure 6.15). As measurable CAPs were recorded for all the grafts at both 4 and 8 w
time points, it was expected to see axonal extension from the proximal end of the graft
across the entire length towards the distal end.
60


61


Figure 6.15 Representative images of double immunostaining for axonal
regeneration and Schwann cell activity after 8 w. (A-B) autograft, (C-D)
PCL conduit, and (E-F) PSHU-RGD/PCL conduit. Longitudinal sections
from left to right represent proximal to distal ends. Axons were stained
with NF-M and Alexa Fluor 594 and appear in red. Schwann cells were
stained with SI00b and Alexa Fluor 488 and appear in green. Scale bar
represents 500 pm.
In all grafts, axons and Schwann cells were observed to span the entire length of the graft.
Axonal extension was not always continuous through each section, but the presence of
axons at the distal end was used to infer that axons have spanned the entire length of the
transection gap. In the case of the PCL conduit, axonal extension was observed mostly
along the outer surface of the conduit with minimal axonal extension through the
intraluminal microchannels. For the PSHU-RGD/PCL conduit, extension of axons was
observed along the entire length of an intraluminal microchannel in a continuous linear
path. Schwann cell activity was observed much more prevalently throughout the grafts as
Schwann cells are involved in the removal of myelin and axonal debris following nerve
injury, as well as in the formation of Biingner bands. The myelination of axons is also
attributed to Schwann cells. Myelin sheaths are expected to be present in areas where
axonal activity and Schwann cells activity overlap.
It appears that axons and Schwann cells are much more prevalent in the autograft
compared to the two conduits. However, a direct comparison of axon and Schwann cell
62


density between the conduits cannot be made based on these images. The autograft
initially contained axons and Schwann cells with implantation and both of the stains are
not specific for only regenerating axons and Schwann cells involved in regeneration.
Also, the physical characteristics of the conduits prevent direct comparison. As the wall
thickness of the intraluminal channels is larger than the section thickness of 18 pm, axons
and Schwann cells within a specific microchannel may leave the field of view if sections
were not cut perfectly parallel to the microchannels. Therefore, it can only be inferred
that the PSHU-RGD/PCL conduit was more effective in supporting axonal growth
compared to the PCL conduit due to higher axonal density.
6.9.2 Massons trichrome stain
Massons trichrome stain was used to examine the morphology of the gastrocnemius
muscle. Muscle atrophy occurs with the denervation of the gastrocnemius muscle
following sciatic nerve transection and fibrotic tissue is formed. With the regeneration of
motor neurons and reinnervation of the muscles, the degree of muscle atrophy and
fibrotic tissue will gradually decrease. The quantity of collagen present in muscle was
used to assess the disease state of the muscle as collagen fibers indicate the formation of
fibrotic tissue (Figure 6.16, Figure 6.17).
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Figure 6.16 Representative images of gastrocnemius muscle stained with
Massons trichrome stain after 8 w. (A) autograft, (B) PCL conduit, (C)
PSHU-RGD/PCL conduit. Muscle fibers are stained in red, while collagen
is stained in blue. Scale bars represent 100 pm.
Figure 6.17 The ratio of collagen to muscle fiber by area. The presence of
collagen implies the formation of fibrotic tissue. Error bars represent
standard error of the mean. indicates p < 0.05.
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The area of collagen to muscle fibers was used to compare the amount of fibrotic tissue
formation between the different grafts. At 4 w after implantation, no statistical difference
was observed between any of the grafts, but after 8 w, the autograft and PSHU-RGD/PCL
conduit had a significantly lower presence of collagen. These results suggest that the
PSHU-RGD/PCL conduit had comparable motor neuron regeneration and muscle
reinnervation characteristics to the autograft.
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7 Conclusion
The study started with the synthesis and characterization of the functional polymer used
for the NGC. PSHU-RGD was successfully synthesized and the molecular structure was
verified along with the confirmation of RGD conjugation through *H NMR and FT-IR.
The NGC was then evaluated for nerve regeneration in a rat SNI model. After sciatic
nerve transection and graft implantation, several assessments were performed. The
PSHU-RGD/PCL conduit was compared to the gold standard autograft and a PCL
conduit associated with poor neuronal activity [16],
Functional recovery was investigated using a walking track analysis and ankle motion
analysis. Through the walking track analysis, SFI was calculated to quantitatively
evaluate functional recovery. Although no statistical difference was observed, the PSHU-
RGD/PCL conduit seemed to outperform the autograft. The ankle motion analysis
consisted of three measured ankle angles during initial contact, mid-swing, and toe off
phases of walking. When considering all three phases of walking, the PSHU-RGD/PCL
conduit showed similar functional recovery compared to the other grafts.
Electrophysiological activity was measured by recording the CAP of the grafted nerves
and calculating CAP amplitude and AUC. An increase in CAP amplitude and AUC was
observed for the PSHU-RGD/PCL graft compared to other grafts, but a statistically
significant improvement was only observed for CAP amplitude.
Gastrocnemius muscle mass was used to assess muscle reinnervation. No significant
difference in muscle mass was observed between any of the grafts.
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Histological assessment was used to directly observe regeneration and the presence of
fibrotic tissue formation. Axons and Schwann cells were observed using IHC with stains
specific for neurofilament and calcium-binding proteins, respectively. Axonal extension
from the proximal end of the graft across the entire length towards the distal end was
observed for the PSHU-RGD/PCL conduit. The morphology associated with the
reinnervation of the gastrocnemius muscle was observed using Massons tri chrome stain.
The quantity of collagen present in muscle was used to assess the disease state of the
muscle. The PSHU-RGD/PCL conduit had comparable motor neuron regeneration and
muscle reinnervation characteristics to the autograft and statistically reduced fibrotic
tissue formation compared to the PCL conduit.
Although nerve regeneration using the PSHU-RGD/PCL conduit did not have statistically
significant improvements for all assessments considered for evaluation, the NGC
consistently showed similar or improved nerve regeneration characteristics. These results
are encouraging as autografts are associated with many drawbacks and it was determined
that the PSHU-RGD/PCL conduit is a functionally comparable alternative for surgical
treatment of PNI.
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8 Future Work
8.1 Increase sample size
For several of the grafts, statistically significant differences in several of the assessments
after sciatic nerve transection and graft implantation were not observed even though some
of the trends implied that there may be significant difference. This is due to the high
variability in the functional, electrophysiological, and histological assessments, as well as
the low sample size. It is expected with the increase in sample size, statistical significant
differences will be observed between the various grafts.
8.2 Additional negative control
The PCL conduit was used as a negative control as it was associated with poor neuronal
activity [16], However, the results from several of the assessments show that the PCL
conduit has substantial nerve regeneration characteristics. The positive effect of
intraluminal microchannels with aligned nanofibers may have been underestimated as the
topical cues associated with the microchannels and fiber alignment may have accelerated
the natural regeneration process. Therefore, another negative control should be used to
assess conduits without the intraluminal microchannels with aligned nanofibers. This
control could be a hollow tube or simply leaving the nerve untreated after transection.
8.3 Additional time points
Although statistical significant differences between several of the assessments were not
observed, the trends associated with the nerve regeneration process showed that a
possible difference may be observed at time points longer than 8 weeks after implantation.
With additional time points, a difference between the grafts may be more apparent.
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8.4 Electrophysiological assessment using muscle action potentials
The electrophysiological assessment in this study was performed by taking CAP
recordings of just the nerve. This assessment only accounts for regenerated axons across
the transection site. However, a more comprehensive analysis will account for axons that
have reinnervated target muscle. Therefore, compound muscle action potential (CMAP)
recordings may be of more significance as stimulation occurs at the grafted nerve and the
CMAP is recorded at the target muscle.
8.5 Larger animal model
Current FDA approved hollow conduits have been shown to effectively regenerate axons
across gaps up to 3 cm in length [27], In order for the PSHU-RGD/PCL to become a
clinically applicable treatment option to PNI repair a study of the conduit must be
performed in a model with a longer transection nerve gap. Due to the smaller size of rats,
the operable nerve length is limited. Options for a larger animal model include rabbits
and dogs [83],
8.6 Growth factor and Schwann cell integration
Although ample nerve regeneration without the need to use growth factors or support
cells is ideal for clinical applications, rapid axonal regeneration is often desired. When
considering the integration of growth factors and Schwann cells, there are many options
that could potentially maximize the rate of nerve regeneration (Section 2.2.8, Section
2.2.9).
69


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Full Text

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EVALUATION OF A BIOMIMETIC POLYMER NERVE GUIDANCE CONDUIT FOR PERIPHERAL NERVE REGENERATION by DAVID JAY LEE B.S. Colorado School of Mines, 2013 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment o f the requirements for the degree of Master of Science Bioengineering 2015

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ii This thesis for the Master of Science degree by David Jay Lee has been approved for the Bioengineering Program by Daewon Park, Chair John H Caldwell Karin A Payne July 23, 2015

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iii Lee, David Jay (M.S. Bioengineering) Evaluation of a Biomimetic Polymer Nerve Guidan ce Conduit for Peripheral Nerve Regeneration Thesis directed by Assistant Professor Daewon Park ABSTRACT Surgical treatment of peripheral nerve injury clinically remains an unmet medical need as the current gold standard autograft is as sociated with many drawbacks including a second surgical procedure, donor site morbidity, mismatch of donor nerve size, and limited donor nerve length. Nerve guidance conduits are a promising alternative to the autograft that promote neuronal growth and g uide axonal extension A nerve guidance conduit was designed using a blend of arginylglycylaspartic acid conjugated polyurea and polycaprolactone containing intraluminal microchannels with aligned nanofibers The nerve guidance conduit was evaluated in a 1 0 mm sciatic nerve transection rat model Functional, electrophysiological, and histological assessments were used to evaluate nerve regeneration of the conduit. Although generally no statistically significant improvement in nerve regeneration was observed for the nerve guidance conduit compared the autograft, the conduit consistently demonstrated comparable, if not improved, recovery characteristics. The form and content of this abstract are approved. I recommend its publication. Approved: Daewon Park

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iv ACKNOWLEDGEMENTS I would like to express my gratitude to the many individuals that have contributed to the research project and to those that have been involved in my personal and professional development. None of this work would have been possible without them. I would like to thank m y advisor, Dr. Daewon Park for his guidance, patience, and the opportunity to work in his lab. U nder his instruction, I have learned in valuable skills that will be essential to my future success. Recognition must also be given to Dr. John Caldwell and Dr. Karin Payne for their i nsight and teachings throughout the project My graduate experience would be far from complete without the encou ragement and assistance from m y labmates in the Translat ional Biomaterials Research Lab oratory, especially Melissa Laugher, James Bardill, and Anna Laura Nelson I am very fortunate to have worked wi th t hem and I will cherish our continued friendship. Sev eral individuals have also contributed to this project at various points. E lectrophysiological assessment would not have been possible with out the help of Arjun Fontaine. Melissa Card dedicated much of her time o n animal care training and Dr. Chris Manuel provided training for the surgical procedure. I would like to express my appreciation to my family. M y dad has been supportive throughout my life and has le d me to be the person I am today. My mom has been a constant source of inspiration and is the example of how I want to live my life. Finally my brother, who has grown up right alongside me, continuously challenged me and pushed me to become a better person I am truly blessed to have such a fam ily.

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v Declaration of original work by David Jay Le e This was independently composed and authored by myself, using the support from my advisor, committee members, lab members, fellow students, and the Department of Bioengineering. The research and ideas presented in this document originate d from the Translational Biomaterials Research Laboratory under the guidance of Dr. Daewon Park. The e lectrophysiological setup was arranged by Dr. John Caldwell. All other resources and funds were provided by Dr. Daewon Park and the Department of Bioengin eering. David Jay Lee

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vi TABLE OF CONTENTS List of Figures ................................ ................................ ................................ .................... xi List of Abbreviations ................................ ................................ ................................ ....... xiv Chapter 1 Introduction ................................ ................................ ................................ ................. 1 1.1 Overview ................................ ................................ ................................ .............. 1 1.2 Anatomy and physiology ................................ ................................ ..................... 1 1.3 Injury classification ................................ ................................ .............................. 5 1.4 Pathophysiology ................................ ................................ ................................ ... 6 1.5 PNI diagnosis and treatment ................................ ................................ ................ 8 1.6 Study objective ................................ ................................ ................................ ..... 8 2 Background ................................ ................................ ................................ ................. 9 2.1 PNI treatment with surgical intervention ................................ ............................. 9 2.1.1 Neurorrhaphy ................................ ................................ ................................ 9 2.1.2 Autografts ................................ ................................ ................................ ..... 9 2.1.3 Allografts ................................ ................................ ................................ .... 10 2.1.4 Hollow tubes ................................ ................................ ............................... 11 2.2 NGC design considerations ................................ ................................ ................ 12 2.2.1 Biocompatibility ................................ ................................ ......................... 12 2.2.2 Mechanical stability ................................ ................................ .................... 13

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vii 2. 2.3 Semipermeability ................................ ................................ ........................ 13 2.2.4 Biodegradability ................................ ................................ .......................... 13 2.2.5 Surface functionalization ................................ ................................ ............ 14 2.2.6 Intraluminal channels ................................ ................................ .................. 15 2.2.7 Lumen fillers ................................ ................................ ............................... 15 2.2.8 Growth factors ................................ ................................ ............................ 16 2.2.9 Support cells ................................ ................................ ................................ 17 2.2.10 Electric conductivity ................................ ................................ ................... 18 2.3 Electrospinning of polymer solutions ................................ ................................ 18 2.3.1 Setup and process ................................ ................................ ........................ 19 2.3.2 Fiber arrangement ................................ ................................ ....................... 19 2.3.3 Parameters ................................ ................................ ................................ ... 20 2.4 Rat SNI model ................................ ................................ ................................ .... 20 2.4.1 Functional assessment ................................ ................................ ................. 21 2.4.2 Electrophysiological assessment ................................ ................................ 23 2.4.3 Histological assessment ................................ ................................ .............. 24 2.4.4 Limitations ................................ ................................ ................................ .. 25 3 Previous Work ................................ ................................ ................................ .......... 26 3.1 Biocompatibility ................................ ................................ ................................ 26 3.2 PSHU RGD and PC12 cell culture ................................ ................................ .... 27

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viii 3.3 Fiber alignment and PC12 cell culture ................................ ............................... 30 3.4 NGC with aligned nanofibers along intraluminal channels ............................... 31 3.5 NGC and hNSC culture ................................ ................................ ...................... 32 4 Hypothesis and Specific Aims ................................ ................................ .................. 35 4.1 Hypothesis ................................ ................................ ................................ .......... 35 4.2 Specific aims ................................ ................................ ................................ ...... 35 5 Materials and Methods ................................ ................................ .............................. 36 5.1 Materials ................................ ................................ ................................ ............. 36 5.2 Equipment ................................ ................................ ................................ .......... 37 5.3 N BOC serinol synthesis ................................ ................................ .................... 37 5.4 PSHU synthesis ................................ ................................ ................................ .. 37 5.5 PSHU deprotection ................................ ................................ ............................. 38 5.6 RGD conjugation to dPSHU ................................ ................................ .............. 38 5.7 NGC fabrication by electrospinning ................................ ................................ .. 39 5.8 Sciatic nerve transection and graft implantation ................................ ................ 41 5.9 Walking track analysis ................................ ................................ ....................... 42 5. 10 Ankle motion analysis ................................ ................................ .................... 42 5.11 Euthanasia and tissue harvest ................................ ................................ ......... 43 5.12 CAP recordings ................................ ................................ ............................... 44 5.13 Gastrocnemius muscle mass ................................ ................................ ........... 45

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ix 5.14 Histology ................................ ................................ ................................ ........ 45 5.14.1 IHC of nerve grafts ................................ ................................ ..................... 45 5.14.2 .............................. 46 5.15 Statistical analysis ................................ ................................ ........................... 47 6 Results and Discussion ................................ ................................ ............................. 48 6.1 PSHU, dPSHU, and PSHU RGD reaction sequence ................................ ......... 48 6.2 PSHU and dPSHU characterization using 1 H NMR ................................ .......... 48 6.3 PSHU, dPSHU, and PSHU RGD characterization using FT IR ....................... 50 6.4 Sciatic nerve transection and graft implantation ................................ ................ 51 6.5 Walking track analysis ................................ ................................ ....................... 53 6.6 Ankle motion analysis ................................ ................................ ........................ 54 6.7 CAP recordings ................................ ................................ ................................ .. 56 6.8 Gastrocnemius muscle mass ................................ ................................ ............... 59 6.9 Histology ................................ ................................ ................................ ............ 60 6.9.1 IHC of nerve grafts ................................ ................................ ..................... 60 6.9.2 ................................ ................................ ............ 63 7 Conclusion ................................ ................................ ................................ ................ 66 8 Future Work ................................ ................................ ................................ .............. 68 8.1 Increase sample size ................................ ................................ ........................... 68 8.2 Additional negative control ................................ ................................ ................ 68

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x 8.3 Additional time points ................................ ................................ ........................ 68 8.4 Electrophysiological assessment using muscle action potentials ....................... 69 8.5 Larger animal model ................................ ................................ .......................... 69 8.6 Growth factor and Schwann cell integration ................................ ...................... 69 References ................................ ................................ ................................ ........................ 70

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xi LIST O F FIGURES Figure 1.1 The structural organization of the nervous system. [4] ................................ .................. 1 1.2 The structure of a neuron. [6] ................................ ................................ ........................ 2 1.3 Peripheral distribution of spinal nerves. (A) the process of sending afferent sensory signals to the CNS, (B) the process of relaying efferent motor signals from the CNS to peripheral tiss ues. [7] ................................ ................................ ............... 3 1.4 Cross sectional anatomy of a peripheral nerve. [9] ................................ ....................... 5 1.5 Peripheral nerve regeneration. (A) nerve transection, (B) degeneration, (C) growth cone regeneration, (D) Schwann cell alignment. [9] ................................ ............... 7 2.1 Electrospinning setup. Fiber arrangement is dependent on the collector. Flat collectors result in random fiber arrangement and split electrode c ollectors result in aligned fiber arrangement. [71] ................................ ................................ ..................... 19 2.2 Measured variables used to calculate SFI in walking track analysis. [7 5] .................. 22 3.1 Biocompatibility assessment using PC12 cells with cell viability assessed by MTT assay. PSHU extract was prepared by inc ubating PSHU in cell culture media for 24 h in standard conditions and filtered to remove particulates. Error bars represent one standard deviation. ................................ ................................ ............... 27 3.2 Fluorescence microscopy of the neuronal response of PC12 cells seeded on either surfaces coated with either laminin or PSHU RGD in cell culture media supplemented with and without NGF. PC12 cells were stained with III tubulin and Alexa 488. Scale bars apply to respective columns. ................................ ................... 28 3.3 Neuronal response of PC12 cells by cell differentiation. Differentiate d cells were classified as cells with at least one neurite. Error bars represent one standard deviation. indicates p < 0.0005. ................................ ................................ ...................... 29 3.4 Neuronal response of PC12 cells by neurite length. Error bars represent one standard deviation. indicates p < 0.001, ** indicates p < 0.0005. ................................ .. 29 3.5 Neuronal response of PC12 cells to fiber arrangement. (A) random fiber arrangement, (B) aligned fiber arrangement, (C) neurite extension on random fiber arrangement, (D) neurite extension on aligned fiber arrangement. Scale bars apply to respective columns. ................................ ................................ .............................. 31

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xii 3.6 SEM images of a PSHU RGD/PCL conduit. (A) transverse section of conduit with embedded sucros e fibers, (B) transverse section after dissolving sucrose fibers, (C D) longitudinal sections after dissolving sucrose fibers. ................................ ... 32 3.7 Fluorescence microscopy images of hNSC behavior on PCL conduit after 14 d. (A) DAPI stain in blue (B) III tubulin stain in green Arrow indicates the direction of nanofiber alignment. ................................ ................................ ....................... 33 3.8 Confocal microscopy images of hNSC behavior on PSHU RGD/PCL conduit after 14 d. (A C) along center of microchannel, (E G) along inner wall of microchannel, (A, D) DAPI stain in blue (B, E ) III tubulin stain in green (C, F) combined DAPI and III tubulin stains. Arrows indicate the direction of nanofiber alignment. ................................ ................................ ................................ .......... 34 5.1 Two electrode electrospinning setup. Parameters: distance between needle and collector at 10 cm, flow rate at 1 ml/h, voltage at 7.5 kV (PSHU RGD/PCL and PSHU/PCL) or 9 kV (PCL), room temperature, relative humidity at 30 %. ..................... 39 5.2 Rolling of flat sheet into a tube. ................................ ................................ ................... 40 5.3 Different phases of walking in which ankle angle was measured for ankle motion analysis. ................................ ................................ ................................ ................. 43 5.4 Platform of parallel conducting wires used to measure CAP. ................................ ..... 44 6.1 Reaction sequence of PSHU RGD synthesis. ................................ .............................. 48 6.2 1 H NMR spectrum of PSHU confirming molecular structure. ................................ .... 49 6.3 1 H NMR spectrum of PSHU and dPSHU confirming the removal of the BOC protecting group with the disappearance of the b peak. ................................ ..................... 49 6.4 FT IR spectrum of PSHU, dPSHU, and PSHU RGD. ................................ ................ 50 6.5 FT IR spectrum of PSHU, dPSHU, and PSHU RGD. The presence of free amine groups on dPSHU after deprotection is confirmed from region a The conjugation of RGD to dPSHU is confirmed with the shift in wa velength with carbonyl absorbance shown in region b ................................ ................................ ............ 50 6.6 Sciatic nerve transection and graft implantation. (A) longitudinal ski n incision from the knee to the hip exposing underlying muscles, (B) location of connective tissue separating two muscles, (C) blunt dissection through connective tissue, (D) isolation of sciatic nerve, (E) sutured autograft, (F) sutured PSHU RGD/PCL graft ................................ ................................ ................................ ................................ ... 52 6.7 Calculated SFI values. Error bars represent standard error of the mean. indicates p < 0.05. ................................ ................................ ................................ .............. 53

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xiii 6.8 The difference in the ankle angle between the grafted and healthy contralateral sides during the initial contact phase of walking. Error bars represent standard error of the mean. indicates p < 0.05. ................................ ................................ ............. 54 6.9 The difference in the ankle angle between the grafted and healthy contralateral side during the mid swing phase of walking. Error bars represent standard error of the mean. indicates p < 0.05. ................................ ................................ .......................... 55 6.10 The difference in the ankle angle between the grafted and healthy contralateral side during the toe off phase of walking. Error bars represent standard error of the mean. indicates p < 0.05. ................................ ............................... 55 6.11 Representative CAP recordings 8 w after nerve transection and graft implantation. ................................ ................................ ................................ ...................... 57 6.12 CAP amplitude ratio of the grafted to healthy contralateral nerves. Amplitude was determined as the maximum peak value of the CAP. Error bars represent standard error of the mean. indicat es p < 0.05. ................................ ............... 58 6.13 CAP AUC ratio of the grafted to healthy contralateral nerves. AUC was calculated by subtracting the area under the stimulus artifact modeled as a Gaussian from the total area under both the stimulus artifact and CAP. Error bars represent standard error of the mean. ................................ ................................ ................. 58 6.14 Gastrocnemius muscle mass ratios of the grafted to healthy contralateral sides. Error bars represent standard error of the mean. ................................ ...................... 60 6.15 Representative images of double immunostaining for axonal regeneration and Schwann cell activity. (A B) autograft, (C D) PCL conduit, and (E F) PSHU RGD/PCL conduit. Longitudinal sections from left to right re present proximal to distal ends. Axons were stained with NF M and Alexa Fluor 594 and appear in red Schwann cells were stained with S100b and Alexa Fluor 488 and appear in green Scale bar represents 500 m. ................................ ................................ .................. 62 trichrome stain. (A) autograft, (B) PCL conduit, (C) PSHU RGD/PCL conduit. Muscle fibers are stained in red while collagen is stained in blue Scale bars represent 100 m. ................................ ................................ ................................ .............. 64 6.17 The ratio of collagen to muscle fiber by area. The presence of collagen implies the formation of fibrotic tissue. Error bars represent standard error of the mean. indicates p < 0.05. ................................ ................................ ................................ 64

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xiv LIS T O F ABBREVIATIONS 1 H NMR proton nuclear magnetic resonance aFGF acidic fibroblast growth factor ANOVA a nalysis of variance AUC area under the curve BDNF brain derived neurotrophic factor bFGF basic fibroblast growth factor CAP compound action potential CMAP compound muscle action potential CNS central nervous system CNTF ciliary neurotrophic factor DCM methylene chloride DMF N,N dimethylformamide DMSO dimethyl sulfoxide dPSHU d eprotected poly(serinol hexamethylene urea) ECM extracellular matrix EDC N (3 d imethylamino p ropyl) N e thylcarbodiimide h ydrochloride EITS experimental intermediate toe spread EPL experimental print length ESC embryonic stem cell ETS experimental toe spread FT IR fourier transform infrared spectroscopy GDNF glial cell line derived neurotrophic factor GRGDS Gly Arg Gly Asp Ser HDI hexamethylene diisocyanate HFP 1,1,1,3,3,3 h exafluoro 2 propanol hNSC human neural stem cell IACUC institutional animal car and use committee IHC immunohistochemistry IR infrared ISO international organization for standardization ITS intermediate toe spread MRI magnetic resonance imaging MSC mesenchymal stem cell MTT 3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide NF M neurofilament medium NGC nerve guidance conduit NGF nerve growth factor NH 2 amine group NHS N hydroxysuccinimide NITS normal intermediate toe spread NPL normal print length NSC neural stem cell NT 3 neurotrophin 3 NT 4/5 neuroprophin 4/5

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xv NTS normal toe spread OCT optimal cutting temperature PBS phosphate buffered saline PCL polycaprolactone PGA polyglycolic acid PL print length PLGA poly(lactic co glycolic acid) PNI peripheral nerve injury PNS peripheral nervous system PSHU poly(serinol hexamethylene urea) PSHU RGD arginylglycylaspartic acid c onjugated poly(serinol hexamethylene urea) RGD arginylglycylaspartic acid, Arg Gly Asp SEM scanning electron microscope SFI sciatic function index SNI sciatic nerve injury TEM transmission electron microscopy TFA trifluoroacetic acid TFE 2,2,2 trifluoroethanol TS toe spread

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1 1 Introduction 1.1 Overview Peripheral nerve injury ( PNI ) is reported in 3 % of all trauma patients [1] Yearly in the United States, $150 billon is spent on nerve injuries and o ver 200,000 peripheral nerve repair procedure s are performed [2,3] These injuries often occur from physical distress including traumatic injury, surgery, or compression PNI can also result from underlying conditions such as autoimmunity, vasculitis, systemic illness (e.g. diabetes), cancer (e.g. paraneoplastic neuropathy ), infection dysproteinemia (e.g. myeloma), drug t oxicity, and congenital disease Nerve damage often lead s to loss of function pain sensory loss and motor deficits. Despite advances in the reconstruction of segmented nerve s following PNI, functional recovery remains inadequate 1.2 Anatomy and p hysiology The nervous system is divided into the central nervous system (CNS) and the peripheral nervous system (PNS) ( Figure 1 1 ) The CNS consists of the spinal cord and brain while the PNS comprises of all the neural tissue outside th e CNS The peripheral nervous system consis ts of sensory neurons and motor neurons contributing to the sensory division and motor division, respectively. Figure 1 1 T he structural organization of the nervou s system. [4]

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2 Peripheral nerves are comprised of a cell body, dendrites, and axons that are capable of propagating electrical impulses called action potentials ( Figure 1 2 ). These axons are myelinated by Schwann cells. As the primary glial cells of the PNS Schwann cells form insulating myelin sheaths aro und axons, decreasing membrane capacitance and increas ing conduction velocity [5] Along myelinated axons are nodes of Ranvier. These nodes are gaps in myelin sheath between adjacent Schwann cells and pl ay a key role in generating action potentials Figure 1 2 The structure of a neuron. [6] The PNS sends afferent sensory signals to the CNS ( Figure 1 3 A) Sensory receptors are activated by stimuli that generate action potentials which trigger nerve impulses along the length of the axons These impulses travel across the cell bodies located in the dorsal root ganglia and deliver the sensory information to the sensory nuclei located in the spinal cord. The PNS can also relay efferent motor signals to peripheral tissues and systems ( Figure 1 3 B). The cell bodies of motor neurons are located in the anterior horn of the spinal cord. Once motor commands originate from the motor nuclei in the spinal cord, motor information is conveyed to skeletal and smooth muscle fibers.

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3 Figure 1 3 Peripheral d istribution of spinal nerves. ( A) the process of sending afferent sensory signals to the CNS ( B) the process of relaying efferent motor signals from the CNS to peripheral tissues [7]

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4 Nerve fibers in the PN S can be classified by function, nerve fiber diameter, and conduction velocity A are larger (5 and are primarily responsible for proprioception and mechanoreception, respectively A and C fibers are smaller ( 0.3 5 are associated with nociception and thermoreception The extent of myeli nation varies with nerve fibers A fibers have higher degrees of myelination while A fibers are thinly myelinated and C fibers are unmyelinated. Due to the difference in the degrees of myelination, conduction velocities of ner ve fibers vary as c onduction velocity increases with increasing myelin thickness [6,8] The peripheral nerve is composed of the endoneurium, perineurium, and epineurium that constitute the nerve tissue ( Figure 1 4 ) Individual a xons are surrounded by the endoneurium. The endoneurium consists of a loose collagenous matrix and provides nutrients and protection for axons. Axons are bundled into fascicles held together by the perineurium T he perineurium is comprised of tighter connective tissue that surrounds fascicles and contr ibutes to tensile strength All the fascicles are enclosed by the epineur ium The epineurium contains a tough fibrous sheath provi ding mechanical support for fascicles and blood vessels. [9,10]

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5 Figure 1 4 Cross section al anatomy of a peripheral nerve [9] 1.3 Injury classification PNI can be classified into three pathological descriptions. Neurapraxia occurs from compression, lack of b lood flow, or mild physical distress to the nerve and is characterized by myelin breakdown and dysfunction No physical disruption of nerve tissue or axons is presented and no surgical treatment is necessary as nerve function is eventually restored, althou gh this may take several months. Axonotmesis is a more severe degree of PNI when motor, sensory, and autonomic function is impaired due to traumatic crush or stretch of the nerve Axonal damage is apparent, but some form of the nerve tissue remains intact, including the perineurium and epineurium. S urgical intervention is usually not required, but may be considered to remove any scar tissue that may have formed Neurotmesis is the most severe class of PNI and is classified by the complete or partial transection of nerve tissue and axonal disruption. [9,10]

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6 Neurotmesis can further be categorized depending on the extent of injury. Less severe neurotmesis involve s a lesion of the endoneurium, but the perineurium and epineurium are preserved. Surgical inte rvention may be considered. In more severe cases of n eurotmesis in w hich only the epineurium remains intact or complete transection occurs s urgical repair is necessary to regain nerve function [9,10] 1.4 Pathophysiology When a peripheral nerve is transected, the nerve undergoes a period of degeneration and subsequent regeneration ( Figure 1 5 ) After injury, degeneration occurs both proximally and dista lly by chromatolysis T raumatic degeneration is characterized by the deterioration of the nerve proximally from the transection si te to the next node of Ranvier. 24 48 h after injury, Wallerian degeneration occurs in which axons and myelin distal to the injury site breakdown. During nerve degeneration, proliferating Schwann cells, macrophages, and monocytes cooperate to remove myelin and axonal debris, release neu ro tro phins, and guide axons toward adjacent synapses. After Wallerian degeneration, growth cones form at the distal ends of regenerating axons that consist of filopodia, allowing for axonal contraction a nd elongation As grow th cones are formed, Schwann cells align longitudinally and form B ngner bands which provide a growth permissive environment for regenerating axons. [9,11 14]

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7 Figure 1 5 Peripheral nerve regeneration. ( A) nerve transection, ( B) degeneration, ( C) growth cone regeneration, ( D) Schwann cell alignment. [9]

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8 1.5 PNI diagnosis and treatment PNI diagnosis is based on neurological and physical exa minations, patient history, and medical screening (e .g. blood test). Nerve damage can be assessed using nerve conduction velocity tests, electromyography, magnetic resonance imaging (MRI) and biopsies (e.g. nerve, skin). Current non surgical treatments of PNI are limited to symptom management. Neuropathic pain can be controlled using over the counter medication for mild cases while prescription medications are used for more severe and chronic cases. Muscle weakness is often addressed using mec hanical aids (e.g. braces). Surgical intervention is considered for more severe cases of PNI characterized by nerve transection. For shorter nerve gaps, neurorrhaphy is performed which directly reconnects the two nerve stum ps through suturing. Autografts, allografts, and hollow tube s are used to span longer nerve gaps. However, current techniques in surgical intervention have clinical limitations and often lead to poor functional recovery. 1.6 Study objective PNI repair is clinically an unmet medical need. Due to the lim itations and drawbacks associated with current surgical intervention in the treatment of PNI a synthetic nerve guidance conduit (NGC) has been developed to promote nerve regeneration The objective of this study is to evaluate a arginylglycylaspartic acid c onjugated poly(serinol hexamethylene u rea) (PSHU RGD ) and polycaprolactone ( PCL ) blended nanofiber scaffold that has previously been reported [15,16] The NGC has succe ssfully been characterized and shown to promote cell attachment and neurite extension in vitro The focus of this study was to evaluate the NGC for peripheral nerve rege neration in a r at sciatic nerve injury (SNI) model

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9 2 Background 2.1 PNI treatment with surgi cal intervention Trans ected peripheral nerves are approached differently depending on the severity and the length of the gap induced by injury when considering treatment Although advances in microsurgical instrumentation and technique have been made, all surgical treatment options have major drawbacks in their efficacy of PNI repair. 2.1.1 Neurorrhaphy N eurorrhaphy is one surgical repair technique of PNI which involves the direct suturing of discontinued nerve stumps. There are two different approaches to neurorrhaphy epineurial repair and grouped fascicular repair. Epineurial repair involves suturing of the epineurium of the two ligated ends while grouped fascicular repair involves matching and suturing fascicular groups. Although realigning axons by gro uping fascicles leads to improved functional recovery, suturing of the fascicles leads to increased scarring and da mage to blood vessels, preventing optimal recovery [17] Although relatively successful at recovering nerve function, neurorhaphy is limited to short er nerve gaps n o greater than 5 mm in length [18] E xcessive tension on the nerve damages the nerve tissue layers as it disrupts connective tissue matrices and reduces blood flow, inducing necrosis and chroni c ischemia [19] 2.1.2 Autografts For longer nerve gaps, autografts are consi dered the gold standard of PNI repair [20] Autogra fts currently offer the best results in terms of nerve regeneration but are also associated with many drawbacks including a second surgical procedure, donor site

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10 morbidity, mismatch of donor nerve size, and limited donor nerve length. Common sources of do nor nerve are the sural, medial antebrachial cutaneous, and late ral femoral cutaneous nerves as they are readily accessible and relatively expendable. There are several types of autografts including single, cable, trunk, and vascularized grafts A single graft is a segment of a donor nerve that is of similar diameter to the two ends of the t ransected nerve. The single graft is restricted to nerves of a certain diameter due to the limited number of expendable nerves that would be similar in diameter. Cable grafts are multiple segments of donor nerve of smaller diameter aligned in parallel to span gaps of larger diameter. The multiple segments are held together by suture or fibrin glue [21] Trunk grafts are segments from a large nerve used to repair a gap in a proximal nerve. However, truck grafts are associated with poor recovery due to fibrosis and poor vascularity attributable to the thi ckness of the graft [22] Vascularized grafts are donor nerves that are used to span gaps without disturbing the blood vessels supplying the nerve. Although these grafts offer superior recovery in area s that are poorly vascularized, donor site morbidity is of major concern [23] 2.1.3 Allografts Allografts are obtained from human cadaveric nerves. The use of allografts avoi ds several of the limita tions of autografts, but the c omplexity and cost of producing allografts remains a challenge [24] Also, a llografts are not as effective in restoring nerve function compared to autografts due to their tendency to elicit an immune response. An increase in T cell response has been linked to donor Schwann cell s and requires the use of immunosuppressants [25]

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11 To avoid the use of immunosuppressants, allografts can be decellularized using freeze thawing, chemical detergents, enzyme d egradation, or irradiation. However, decellularization causes cell debris formation and impairs neurite outgrowth. Acellular allografts were shown to regenerate axons across gaps only up to 3 cm [26] 2.1.4 Hollow tubes Several b iological and synthetic hollow tubes have been developed and are approved by the FDA for clinical use. Although many of the drawbacks of autografts and allografts can be avoided through the use of hollow tubes neurite outgrowth has been limited to gaps less than 3 cm in length and is associ ated with poor functional recovery [27] The random dispersion of regenerating axons through hollow tubes leads to inappropriate target reinnervation. These implantable devices ar e fabricated using either non ab sorbable or biodegradable materials. Non ab sorbable tubes can be fabricated from various materials such as polyvinyl alcohol and silicon. These tubes are mechanically stable and easily sterili zed, but are associated with nerve compression and tension at the sutured areas of the nerve [27] B iodegradable tubes can be produced using natural o r synthetic materials Natural materials that are produced in the body include collagen, chitosan, and fibrin. Although these materials are easily obtainable and biocompatible, complete biodegradation of the tube s may take up to a year and batch to batch v ariations prevents consistent nerve regeneration properties [27,28] S ynthetic materials s uch as polyglycolic acid (PGA), poly lactic co g lycolic acid (PLGA), and P CL can also be used to fabricate biodegradable tubes These tubes have excellent biodegradability, but are often not

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12 mechanically stable, have low solubility, and produce undesired products from degradation (e.g. acidic products ) [27,29,30] 2.2 NGC desi gn considerations Due to the limitations associated with current nerve grafts used for PNI repair, much of the research surrounding nerve regeneration has focused on NGCs. In order for a NGC to be a clinically relevant alternative to current grafting techn iques, functional recovery needs to be comparable to that of the autograft. NGCs are relatively successful at recovering nerve function across shorter nerve gaps, but are incapable of selectively guiding axons toward appropriate end tissue for longer gaps [31] When designing NGC for use in PNI repair, several considerations may be impleme nted to promote nerve regeneration. Although not all of the following conduit characteristics necessarily need to be incorporated into the final design, some are often required due to the physiology of axonal extension. The following NGC characteristics ha ve shown to promote nerve regeneration. 2.2.1 Biocompatibility Biocompatibility of a material refers to the tendency of the material to support and sustain appropriate cellular behavior. For nerve conduits, this will include allowing for molecular and mechanical signaling systems during regeneration without presen ting cytotoxic effects or eliciting an immune response [32,33] The formation of B ngner bands relies heav ily on cellular and axonal migration to promote axonal extension and in order for a NGC to be considered biocompatible for neural tissue it must not interfere with this healing process [34]

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13 2.2.2 Me chanical stability NGCs must provide the mechanical strength necessary during sterilization implantation, and nerve regeneration process es To prevent infection, sterilization is required for all implants and NGCs must retain their mechanical strength thr oughout this process During implantation, the NGCs must resist tear from sutures and have the structural integrity when handled during the procedure Mechanical strength must also be considered during nerve regeneration as NGCs should have comparable mechanical strength to native nerve tissue. This is usually a balance between flexibility and rigidity NGCs must not collapse or break down until the nerve regenerates and can provide ample mechanical stability an d they must not b e too stiff causing compression or dislocation. [33] 2.2.3 Semipermeability The porosity of the NGC i s another design consideration. The exchange of fluid s between the regenerating nerve and the surrounding fluid is essential to optimal regeneration. Semipermeable conduit walls allow for the diffusion of gas exchange ( e.g. oxygen carbon dioxide ) and other nutrien ts vital for nerve regeneration into the conduit while restricting the influx of infiltrating inflammatory cells into the conduit and outflow of neurotrophic factors out of the conduit [35] Pore sizes of 10 38 m have shown to yield optimal permeabilit y [36] 2.2.4 Biodegradability Nerve regeneration is usually limited due to toxicity and long term complica tions that are associated with NGCs fabricated from materials that are not biodegradable. The use of non absorbable materials requires a second surgical procedure after initial implantation to

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14 remove the NGC after some nerve regeneration is observed There fore, interest in biodeg radable materials has increased. [37] Mechanical stability is of major concern for NGCs made from biodegradable materials as the conduits need to withstand the mechanical stress from neighboring tissues without collapsing while the conduit is degrading. Materials that degrade too rapidly will compromise the structural integrity of the regenerating nerve while materials that degrade too slowly will lead to compression and dislocation. Ideally, the NGC should rema in intact for the time needed for axons to regenerate across the nerve gap and then degrade gradually to minimize undesired affects T he degradation products must also be considered. B iodegradable materials often have undesirable byproducts (e.g. acidic pr oducts). 2.2.5 S urface functionalization Surface functionalization of the lumen enhances the interactions between the conduit and nerve cells Cell adhesion molecules and s hort peptide motifs commonly found in extracellular matrix (ECM) proteins can be incorpora ted into NGC design to enhance these interactions between the cells and conduits Arginylglycylaspartic a cid ( RGD, Arg Gly Asp ) is a cell binding motif found in fibronectin, laminin, collagen, and vitronectin that is i nvolved in several cellular processes including cell differentiation, embryogenesis, pro liferation, and gene expression [38,39] The mechanism by which RGD promotes ce ll attachment is by increasing microfilament and focal adhesion formation [40] RGD coa ted surfaces were shown to promote neurite outgrowth and increase biocompatibility in implants [41,42] RGD surface density above 4.0 pmol/cm 2 has been found to induce cell adhesion, spreading, focal contact formation, and cytoskeletal organization [43]

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15 NGC surfaces can also be functiona lized to provide topographical signals for directing cellular functi on in regenerating axons. Axon al growth generally occurs more randomly in direction making it difficult for reinnervation in tissues distant to the transection site With the introduction of topical cues axon s can be guided to extend more linearly improving neurite extension across large distances [44] One strategy is to use l ongitudinally aligned fibers to induce linear outgrowth of axons [45] Longitudinally aligned fibers also contribu te to high surface area which is favorable for cell attachment and growth [46] 2.2.6 Intraluminal c hannels Intraluminal channels can also be utilized to allow for more surface functionalization and to mimic the structural orientation of native nerve fascicles. NGCs with intraluminal channels allow for improved nerve regeneration as the channels provide more surface area for cell attachment and migration [47] These channels also reduce the random d ispersion of regenerating axons as axonal extension will be confined to a restricted surface area once a regenerating axon enters a single intraluminal channel [48] 2.2.7 Lumen fillers The alignment of Schwann cell s and the formation of B ngner bands are reduced during the regenerating process across larger nerve gaps. Lumen fillers provide a cell supporting matrix that promotes axonal outgrowth as they provide topological cues to promote attachment, proliferation, and migration of Schwann cells [32] Lumen fillers can be made from biological materials found in the body (e.g. collagen) or synthetic biomaterials (e.g. PLGA, PCL) and processed into various fibers, gels, or sponges.

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16 2.2.8 Growth factors Growth factors regulate cellular proliferation and differentiation of various cell types during nerve regeneration. During nerve regeneration, t he growth cone reacts favorably to several diff erent growth factors that promote axonal outgrowth, but as cellular production of these growth factors diminish after injury, so does regeneration [49] One major class of growth factors that can be implemented to stimulate axonal outgrowth and nerve regeneration are neurotrophins. Neurotrophins consist of n erve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT 3), an d neurotrophin 4/5 (NT 4/5) that promote various neural responses involved in nerve regeneration. NGF has been shown to enhance nerve regeneration by promoting survival and differentiation of sensory and sympathetic neurons [50] BDNF is associated with increasing motor neuron survival enhancing axonal growth and facilitating in the myelination of regenerating axons [51] NT 3 and NT 4/5 support the survival, growth, and differentiatio n of motor and sensory neurons and t heir use in NGCs has been shown to increase the number of regenerated axons, axonal diameter, and myelin thickness [52] Glial cell line derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), acidic fibroblast growth facto r (aFGF) and basic fibroblast growth factor (bFGF) are g rowth factors outside the neur otrophin family but are associated with various neurotrophic functions. GDNF is associated with neuron survival and has shown to enhance sensory and motor axon regenerat ion [53] CNTF has been corre lated to increase motor nerve conduction velocity muscle acti on potential amplitudes, axon diameter, number of axons, neurite outgrowth, and myelin thickness [54] a FGF has been shown to

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17 increase the n umber of myelinated regenerating axons [55] bFGF has been linked to an increase in axonal sprouting and proliferation of Schwann cells [56] 2.2.9 Support cells Schwann cells play a critical role in peripheral nerve regeneration and can be integrated into NGCs through process es including injectio ns and cell seeding Schwann cells produce essential ECM molecules ( e.g. laminin, collagen) that provide structural and adhesive support for elongating axons. Schwann cells also produce neurotrophic molecu les that provide biochemical cu es for neurite exten sion. Although incorporating Schwann cells into NGCs is beneficial for axonal extension, autologous Schwann cells are difficult to obtain in large quantities and allogeneic Schwann cells induc e immunogenic responses and their use requires immunosuppressants. [57 59] Peripheral nerv e injury severel y decreases the number of healthy neurons and one strategy to replenish neuronal activity has been to use stem cells as support cells including embry onic stem cells (ESCs), neural stem cells (NSCs) and mesenchymal stem cells (MSCs) that ca n proliferate and undergo rapid cellular expansion in response to nerve injuries ESCs are pluripotent cells that can differen tiate into neuronal cells. Retinoic acid and NGF stimulate neuronal differentiation, extensive outgrowth, and leads to the express ion of neuron specific molecules [60] Implanted ESC derived neural progenitor cells were found to increase ax onal regrowth and nerve repair as they differentiated into myelinating cells resembling a phenotype similar to Schwann cells [61] NSCs are multipotent cells that can differentiate into neurons The use of NSCs has been shown to facilitate in increased electrophysiolog ical activity and increased nerve regeneration observed through histological examination [62] MSCs are pluripotent stem

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18 cells and can differentiate into neurons and myelinating cells [63,64] The use of MSCs in conduits has shown to support nerve r egeneration as the MS Cs were shown to behave similarly to myelinating Schwann cells [65] 2.2.10 Electric conductivity Neural communication relies on action potentials generated at the synapse of neurons which is dependent on electric fields. This implies that electrical conductivity is important to promote neurite outgrowth and enhance nerve regeneration [66] NGCs produced from electrically conductive materials were shown to improve nerve cell migration as the y promote mic rotubule disassembly and creates a charged neuronal cytoskeleton [67] Polymer system s based on biodegradable and conducting polymers have b een shown to provide local stimulation of desired tissue, time controlled drug release, and stimulation of proliferation and differentiation of cells [68] 2.3 Ele ctrospinning of polymer solutions Electrospinning has become a common technique for producing nanofiber polymer scaffolds for biomedical applications. Electrospun scaffolds h ave an overall porous structure, high surface area due to the small fiber diameter s and resemble the three dimensional networks of the ECM of biological tissues and organs [69] This environmental similarity is beneficial for cell attachment and migration, signal transduction, and nutrient transport. By electrospinning polymer solutions, flexible and fibrous structures can be fabricated that have ideal semipermeable p r operties that can interact well in the biological system [70]

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19 2.3.1 Setup and process T he process of electrospinning requires a needle, high voltage power supply, and a grounded collector ( Figure 2 1 ) A syringe pump is used to flow a viscoelastic polymer solution through a needle at a constant and controlled rate. As a high voltage is applied to the needle, the polymer solution is highly electrified and induces charges over the surface of the viscoelastic solution. T he viscoelastic sol ution at the tip of the needle form s a Taylor cone a nd when the electrostatic forces overcome the surface tension of the viscoelastic solution, a liquid jet is ejected from the tip of the needle. Electrostatic repulsion from the surface charges and evaporating solvent stretches the liquid jet into a long fi ber. Due to the electrical gradient between the positively charged liquid jet and the grounded collector, fibers are deposited onto the collector. [71] Figure 2 1 E lectrospinning setup. Fiber arrangement is dependent on the collector. F lat collector s result in random fiber arrangement and split electrode collector s result in aligned fiber arrangement [71] 2.3.2 F iber arrangement The arrangement of electrospun fibers is dependent on the distribution of the electric field of the collector Although many different collectors exist to control fiber arrangement

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20 random or aligned fibers are usually desired. R andom fiber arrangement can be obtained using a simple flat collector and aligned fibers can be obtained using a split electrode collector ( Figure 2 1 ) With the use of a flat collector, random bending is associated with the charges on the liquid jet that are quickly dissipated and d eposited on the collector. When a split electrode collector is used the charge s on the liquid jet induces opposite charges between the collecting electrodes and the electrostatic attractions between the electrodes str etch and align the nanofibers across the gap [72] 2.3.3 Parameters The morphology and diameter of electrospun fibers is dependent on various electrospinning parameters that involve the intrinsic properties of the polymer solution and operati ng conditions. The intrinsic properties of the polymer solution include viscosity, concentration, conductivity, surface tension, and polym er molecular weight. Operating co nditions include electric field strength, voltage, flow rate, distance between needle tip and collector, temperature, and humidity. 2.4 Rat SNI model Peripheral nerve regeneration is most often investigated using the SNI model to evaluate sensory and motor ne rve function The sciatic nerve is the largest nerve trunk in mammals and the large nerve allows for easy surgical access and facilitates in the surgical repair of nerve injury. The sciatic n erve divides into the tibial sural, and common peroneal nerve s a nd the site of terminal branching needs to be carefully considered. The evaluation of NGCs in SNI models have been carried out in a number of different animal species, but most commonly in rats. The extensive use of rats is due to their small size and larg e availability in addition to being relatively easy to work with

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21 Since the majority of studies involving PNI have been model ed using SNI models data from previous studies is readily available for comparison. As m ost clinically and surgically relevant ne rve injuries are characterized by at least pa rtial transection of the nerve, the SNI model consisting of complete nerve transection is ideal for evaluation of clinically relevan t peripheral nerve regeneration [73] 2.4.1 F unction al assessment One method of evaluating nerve regeneration is performing a functional assessment often investigating motor function. F unction al recovery of nerves is based on axon regeneration and selective target reinnervation. As the clinical objective in the use of NGCs is to restore function to denervated tissue followi ng PNI f unctional assessment is the most direct method of evaluation However, functional tests are highly variable. One of the most commonly used test s to ass ess mot or function recovery is the walking track analysis Through the walking tra ck analysis motor function is quantified through several variables that are measured after recording hind feet footprints The quantified value, the sciatic function index (SFI), is calculated using several measured variables consisting of print length (PL), toe spread (TS), and intermediate toe spread (ITS) of both the experimental or grafted side (EPL, ETS, EITS) and the normal contralateral side (NP L, NTS, NITS) ( Figure 2 2 Equation 2 1 ) [74] The walking track analysis utilizes the increased PL, decreased TS, and decreased ITS characteristics of the intrinsic loss of muscle function [75] SFI ranges from values of 0 to 100 where 0 i ndicat es normal nerve function and 100 indicates total impairment.

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22 Figure 2 2 Measured v ariables used to ca lculate S FI in walking track analysis. [75] 2 1 ( 2 1 ) Another method of evaluating motor function recovery is utilizing video recording for a gait analysis T here are several different techniques and variables that can be measured to quantify motor function. One technique is to measure the toe out angle the angle between the direction of progression during walking and the tip of the third digit. The toe out angle accounts for the biomechanical differences of the external rotation of the hind foot in the stance phase of walking [76] Another technique is to measure the ankle motion during walking. With injury to the sciatic nerve both the angles of maximum ankle

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23 plantar flexion and dorsiflexion decrease. A nkle motion analysis is a rel iable and sensitive technique that can be used to measure even subtle differences in both ankle plantar flexion and dorsiflexion angles during nerve regeneration [77] Standing or static methods in assessing behavioral deficits have also been developed. A static footprint video analysis utilizes TS a nd ITS while the animal under observation is static to calculate a quantified value, the static sciatic index [78] Both dynamic and static parameters of b ehavior and motor deficits can also be assessed simultaneou sly The CatWalk gait analysis is a b ehavioral test for detecting both dynamic and static par ameters after nerve transection by using several variables including print area, print intensity, stance duration, and swing duration to evaluate nerve function [79 ] Functional assessment s of nociceptive function can also be used to evaluate nerve function. One test that is often used is the w ithdrawal reflex latency test This test measures the withdrawal time after placing a hind foot on a heat stimulus such a s a hot plate [80] 2.4.2 Electrophysiological assessment T he sciatic nerve consists o f both sensory and motor neurons, allowing for electrophysiological assessment to be performe d on both afferent and efferent components. These tests study sensory and motor nerve conduction utilizing electromyography and spinal reflex tests, often inducin g motor and sensory potentials However, when considering clinical relevance, the recovery of motor function is often considered more significant over sensory function. [81]

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24 T he most common approach to electrophysiological assessment is inducing compound muscle action potentials (CMAPs) CMAPs can be evoked by electrical stimulation proximal and distal to the transection site and action potentials can be recorded in muscle distal to the stimulation site, such as the gastrocnemius muscle [82] 2.4.3 Histological assessment Although functional and electrophysiological assessments can be used to evaluate functional recovery of nerve regeneration, histological examination is essential to complement these assessments. By histological assessment, the presence of regenerated axons can be directly observ ed and any incidence of inflammation and fibrosis can be examined. Histological assessment is most commonly done on transverse sections of the regenerated nerve. This can be done by t oluidine blue staining on sections from osmium post fixed and resin embedded blocks or by transmission electron microscopy (TEM). The number and size of regenerating axons and myelin sheath thickness are often quantified. Axon regeneration can also be observed by immunohistochemistry (IHC). IHC on longitudinal sections can be used to show axon regeneration across the length of the transection site. Histological assessment can be used to observe morphological changes in denervated often used to stain gastrocnemius muscle. Muscle fiber diameter and the presence of collagenous and fibrotic tissue can be quantified.

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25 2.4.4 Limitations Althou gh the rat SNI model is an appropriate model to evaluate NGC efficacy in vivo, there are limitations that must be considered. D ue to the smaller size of rats t he maximum length of a sciatic nerve transection is limited to 1.5 cm for an adult rat. For studies investigating larger nerve gaps, rabbits and dogs are commonly used for their larger body size and sciatic nerve length [83] Another limitation of using rats to model SNI is the shorter life span of rat s mak ing more chronic nerve injuries difficult to model. Another limitation in using rats i n a SNI model is autotomy. Autotomy is one of the most frequent postoperative complications that arise s in rats and ranges in severity. Loss of toenails is observed in mi nor cases, while loss of toes can occur in more severe cases Autotomy can affect functional assessment and often raises concerns regarding animal welfare.

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26 3 Previous Work The potential use of PSHU RGD/PCL for nerve regeneration has previously been exami ned. C ytotoxicity, cell viability, cell proliferation, cell differentiation, neurite outgrowth, and guided neurite sprouting in PC12 cell culture has been investigated for PSHU RGD [15] A method of introducing intraluminal micro channels with aligned nanofibers was developed and was found to promote neuronal growth and guide axonal extension in addition to enhancing cell attachment, surviva l and migration in human neural stem cell (hNSC) culture [16] 3.1 Biocompatibility Biocompatibility assessment was conducted according to the guidelines developed by the International Organization for Standardization (ISO) 10993 5 which describes test methods to assess in vitro cyto toxicity of medical devices. C ytotoxicity was evaluated using the PC12 cell line which is derived from rat pheochromocytoma and has an embryological origin. PC12 cells differentiae into cells that behave similar ly to neurons, making them a suitable as a mo del system for neuronal differentiation. PSHU extract was prepared by incubating PSHU in cell culture media for 24 h in standard conditions and filtered to remove particulates. PC12 cells were exposed to fresh media, PSHU e xtract, or diluted PSHU extract a nd cell viability was measured using an 3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide (MTT) assay ( Figure 3 1 ) No statistical difference was observed between cell culture media and PSHU extract suggesting biocompatibility

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27 Figure 3 1 Biocompatibilit y assessment using PC12 cells with cell viability assessed by MTT assay PSHU extract was prepared by incubating PSHU in cell culture media f or 24 h in standard conditions and filtered to remove particulates. Error bars represent one standard deviation 3.2 PSHU RGD and PC12 cell culture The neuronal response to PSHU RGD was examined by seeding PC12 cells o nto coating s of either laminin or PSHU RGD in cell culture media supplemented with and without NGF. NGF was used as a positive control as the growth factor induce s cell differentiation, microtubule assembly, and neurite outgrowth. An increase in neuronal activity was observed for surfaces coated with PSHU RGD compared to surfaces coated with laminin ( Figure 3 2 )

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28 Figure 3 2 Fluorescence microscopy of the n euronal response of PC12 cells seeded on either surfaces coated with either laminin or PSHU RGD in cell culture media supplemented with and without NGF. PC12 cells were s tained with III tubulin and Alexa 488. Scale bars apply to respective columns. Cell differ entiation and neurite leng th were compared between the different surface coatings, with and without NGF ( Figure 3 3 Figure 3 4 ). Differentiated cells were classified as cells with at least one neurite. PC12 cells on laminin coated surfaces showed minimal neurite growth through 14 d, while PC12 cells on PSHU RGD coated surfac es showed significantly improved cell differentiation and neurite outgrowth demonstrating the potential beneficial impact of using PSHU RGD. When NGF was introduced to PC12 cells on laminin and PSHU RGD surfaces by supplementation in media extensive neur ite networks developed over 14 d.

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29 Figure 3 3 Neuronal response of PC12 cells by cell differentiation. Differentiated cells were classified as cells with at least one neurite. Error bars represent one standard deviation. indicates p < 0.0005 Figure 3 4 Neuronal response of PC12 cells by neurite length Error bars represent one standard deviation. indicates p < 0.001 ** indi cates p < 0.00 05 Although the supplemented use of NGF on surfaces coated with PSHU RGD showed the highest level of cell differentiation and neurite length the delivery of NGF in clinical

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30 settings remains a challenge NGF concentration at the injury site must be s ustained during the nerve regenerative process that may often take long periods of time [84] Therefore, it is ideal if sufficient neuronal activity can be supported by material alone as seen with PC12 cell s on surfaces coated with PSHU RGD wit hout the supplementation of NGF 3.3 F iber alignment and PC12 cell culture The importance of fiber alignment and guided neurite extension has been investigated by seeding PC12 cells on surfaces with varying fiber alignment ( Figure 3 5 ) Scaffolds with random and aligned fiber arrangements were fabricated by electrospinning with fiber arrangement s confirmed by scanning electron microscope (SEM) PC12 cells seeded onto random fiber arrangements presented high branching and randomly extending neurites, while PC12 cells seeded onto aligned fiber arrangements exhibited low branching and extensions further from cell bodies along the direction of the aligned fiber s.

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31 Figure 3 5 Neuronal response of PC12 cells to fiber arrangement. (A) random fiber arrangement, (B) aligned fiber arrangement, (C) neurite extension on random fiber arran gement, (D) neurite extension on a ligned fiber arrangement. Scale bars apply to respective columns 3.4 NGC with aligned nanofibers along intraluminal channels A method of electrospinning was developed to fabricate NGCs with aligned nanofibers along intraluminal channels using a split electro de collector from a blended polymer solution of PSHU RGD and PCL (Section 5.7 Figure 3 6 ) The structure of the PSHU RGD/PCL conduit closely resembles the native nerve structure of the perineurium and epineurium

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32 Figure 3 6 SEM images of a PSHU RGD/PCL conduit ( A) transverse section of conduit with embedded sucrose fibers, (B) transverse section after dissolving sucrose fibers (C D) longitudinal section s after dissolving sucrose fibers. 3.5 NGC and hNSC culture PSHU RGD/PCL conduits were fabricated and hNSCs were seeded onto one end of each NGC and neuronal activity was investigated. PCL conduits served as a negative control, of which very limited neuronal activity was expected when seeded with hNSCs. The PCL conduits were constructed similarly to PSHU RGD/PCL conduits but PSHU RGD was not blended with the PCL solution during electrospinning. The h NSCs seeded on PCL conduits were sparsely distributed and showed no directional preference in extension after 14 d ( Figure 3 7 ). Although the PCL conduits contained intra luminal microchannels with aligned nanofibers, the absence of RGD prevented ample cell attachment, survival, and migration No neuronal growth or axonal extension was observed.

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33 Figure 3 7 Fluorescence microscopy images of hNSC behavior on PCL conduit after 14 d (A) DAPI stain in blue (B) III tubulin stain in green Arrow indicates the direction of nanofiber alignment. The h NSCs seeded on PSHU RGD/ PCL conduits were higher in cell density and the cells migrated into the microchannels with considerable neurite extension after 14 d ( Figure 3 8 ) In contrast to the PCL conduits, PSHU RGD/PCL conduits allowed for cell attachment, survival, and migration. Significant neuronal growth and axonal extension were observed.

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34 Figure 3 8 Confocal microscopy images of hNS C behavior on PSHU RGD/PCL conduit after 14 d. (A C) along center of microchannel, (E G) along inner wall of microchannel, (A, D) DAPI stain in blue (B, E) III tubulin stain in green (C, F) combined DAPI and III tubulin stains. Arrow s indicate the di rection of nanofiber alignment.

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35 4 Hypothesis and Specific A ims 4.1 Hypothesis Based on preliminary data that suggests that an NGC containing intraluminal microchannel s with aligned nanofibers promote neuronal growth and guide axonal extension it was hypothesized that the electrospun PSHU RGD/PCL conduit was functionally comparable to the gold standard autograft when used to surgically treat a peripheral nerve transection. 4.2 Specific aims T he first specific aim was to s ynthesize and ch aracterize PSHU RGD A s the major functional material component of the NGC it was important to confirm the conjugation of RGD and the overall molecular structure of the polymer. The second specific aim was to evaluate the NGC for nerve regeneration in a r at SNI model. Evaluation included functional, electrophysiological, and histological assessments after sciatic nerve transection and graft implantation Functional recovery was investigated using a walking track analysis and ankle motion analysis Electrop hysiological activity was measured by recording the compound action potential (CAP) of the grafted nerves Gastrocnemius muscle mass was used to assess muscle reinnervation Regenerating axons and Schwann cells were observed using IHC with stains specific for neurofilament of regenerating axons and calcium binding protein s in Schwann cells. The morphology associated with the reinnervation stain

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36 5 Materials and Methods 5.1 Materials Serinol urea, hexamethylene diisocyanate (HDI), anhydrous N,N dimethylformamide (DMF ), PCL ( Mn 80,000 g/mol) dimethyl sulfoxide (DMSO) d6 sucrose paraformaldehyde , and xylene were purchased from Sig ma Aldrich (St. Louis, MO, USA). Di te rt butyl dicarbonate ethyl acetate trifluoroacetic acid (TFA) 2,2,2 trifluoroethanol (TFE) N (3 Dimethylamino propyl) N ethylcarbodiimide hydrochloride (EDC), N hydroxysuccinimide (NHS) and 1,1,1,3,3,3 Hexafluoro 2 propanol (HFP) were purchased from Alfa Aesar (Ward Hill, MA USA ). Hexane and a nhydrous diethyl ether w ere purchased from Fisher Scientific (Pit tsburgh, PA, USA). Anhydrous methylene chloride (DCM) and 10 % formalin were purchased from JT Baker (Phillipsburg, NJ, USA). Gly Arg Gly Asp Ser (GRGDS) was purchased from Biomatik (Wilmington, DE USA ). Sprague Dawley rats were purchased from Charles River Laboratories (Wilmington, MA USA). Isoflurane, k etoprofen and b upivacaine (0.5 % Marcaine) were purchased from MWI Veterinary Supply (Boise, ID, USA) Prolene polypropylene 7 0 and c oated Vicryl 4 0 sutures were purchased from Ethicon (Somerville, NJ, USA) Optimal cutting temperature ( OCT ) compound was purchased from Sakura (Torrance, CA, USA). Phosphate Buffered Sali ne (PBS) and Cytoseal 60 were purchased from Thermo Scientific. Goat serum, n eurofilament medium ( NF M rabbit IgG ), Alexa Fluor 594 ( goat anti rabbit IgG) S100 b (mouse IgG1) Alexa Fluor 488 (goat anti m ouse IgG ) and SlowFade Diamond antifade m ountant w ith DAPI were purchased from Life Technologies (Carlsbad, CA, USA) Triton X 100 was purchased from MP Biomedicals.

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37 5.2 Equipment Proton nuclear magnetic resonance ( 1 H NMR ) was performed on a Varian Inova 500 NMR Spectrometer and samples were run in DMSO d6 at room temperature. F ourier transform infrared s pectroscopy (FT IR) was performed on a Nicolet 6700 FT IR S pectrometer and samples were run on polyethylene infrared (IR) sample cards Electrophysiological assessment was performed using Axon CNS MultiClamp 7 00B, Axon Digidata 1440A and Grass SD9 Stimulator Tissue was sectioned using a CryoStar NX70 Cryostat Confocal images were taken using a n Olympus FV1000. Brightfield images were taken using a Nikon Eclipse 80i. ImageJ was used to quantify variables for image analysis. 5.3 N BOC serinol synthesis Serinol (1.96 g, 21.5 mmol) was dissolved in ethanol ( 20 .0 ml) and cooled to 4 C. A mixture of d i tert butyl dicarbonate (5.97 ml 26. 0 mmol) and ethanol (10 .0 ml) was added dropwise. The solution was heated at 37 C for 1 h and subsequently rotoevaporated at 50 C and 20 mbar to remove ethanol yielding a white powder. The white powder was dissolved in a 1:1 volume mixture of ethyl acetate and hexane at 60 C Hexane was added dropwise until cry stalline structures w ere formed and excess hexane was added to ensure complete preci pitation. The precipitate was allowed to settle at 4 C and filtered to remove hexane yielding a crystalline white powder 5.4 PSHU synthesis N BOC serinol (1. 15 g, 6 .0 0 mmol) and urea (0.360 g, 6 00 mmol) were dissolved in DMF (6 0 0 ml) Then, HDI (1.93 ml, 12 0 mmol) was added and the solution was heated at 90 C for 7 d under a nitrogen ous atmosphere The solution was then cooled to room

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38 temperature and rotoevaporated at 80 C and 20 mbar to re move DMF. T he product was precipitated using cooled diethyl ether. T o purify the product, a Milli Q water wash and diethyl ether wash was performed. After the last wash, the product was dried by rotoevaporation at 50 C and 20 mbar yielding the polyurea as a white powder. 5.5 PSHU deprotection D eprotection functionalizes the polyurea by removing the BOC protecting groups and e xposing free amine groups (NH 2 ) prod ucing deprotected PSHU (dPSHU). PSHU (1 g, 1.96 mmol) was dissolved in DCM (15 .0 ml) and TFA (15 .0 ml) was subsequently added dropwise. Deprotection occurred by hydrogenation at room temperature for 45 min. DCM and TFA were rotoevaporated at 50 C and 20 mbar and the product was dissolved in DMF (1 0 0 ml). The product was precipitated us ing cooled diethyl ether. To purify the product, the precipitate was dissolved in TFE and precipitated in ether. The product was dried by rotoevaporation at 50 C and 20 mbar, yielding a white powder. 5.6 RGD conjugation to d PSHU GRGDS pentapeptide was used rather than RGD tripeptide to preserve the integrity of the entire RGD binding motif. It was determined that the number of free amine groups in dPSHU is 2.05 mmol NH 2 / g PSHU after confirming the structure ( Sections 6.1 6.2 ) GRGDS ( 151 mg, 0.308 mmol ), EDC ( 70.9 mg, 0.370 mmol), and NHS ( 53.2 mg, 0.462 mmol) was dissolved in DMF (1.0 0 ml) and NHS ester activation was allowed for 2 h dPSHU ( 150 mg, 0.308 mmol NH 2 ) was separately dissolved in DMF (1.0 ml) and added dropwise to the NHS ester activated solution. T he solutio n was allowed to react for 24 h at room temperature. The product was rotoevaporated at 40 C and 20 mbar to remove DMF and subsequently precipitated in diethyl ether. To purify the product, the precipitate

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39 was washed in Milli Q water The product was dried by lyophiliza tion y ielding PSHU RGD as a white powder. PSHU RGD was stored away from light at 4 C. 5.7 NGC fabrication by electrospinning Prior to electrospinning, sucrose fib ers with diameters between 150 200 using a fiber drawing method. Sucrose was heated to 75 C until it m elted with a thick consistency and fibers were drawn. The collector was constructed using two copper wire electrodes spaced 3.5 cm apart and the sucrose fibers were fit to span the gap between the copper wire electrodes. 8 w/w % polymer solutions in HFP were prepared for PSHU RGD /PCL (30/70) blend, PSHU/PCL (30/ 70) blend, and pure PCL. A two electrode electrospinning setup was used to fabricate the NGC ( Figure 5 1 ) Figure 5 1 Two electrode electrospinning setup. Parameters: distance between needle and collector at 10 cm, flow rate at 1 ml/h, voltage at 7.5 kV (PSHU RGD/PCL and PSHU/PCL) or 9 kV (PCL), room temperature, relative humidity at 30 %.

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40 The collect or was placed 10 cm from the needle of the syringe filled with polymer solution. The polymer s olution was ejected at 1 .0 ml/h through a 21 gauge stainless steel flat tip needle at room temperature and relative humidity at 30 %. A positive 7.5 kV electrostatic potential was a pplied to the needle for the two blended solutions, and a 9 kV potential for the pure PCL solution. Th e collector was rotated every 5 min to evenly distribute the nanofibers across both sides of the collector. For PSHU RGD/PCL conduits, the PSHU RGD/PCL bl end was e lectrospun for the initial 10 15 min, and then the PSHU/PCL blend was used to deposit the remainder of the nanofibers. The pure PCL conduits were electrospun using its respective solution during the entire electrospinning proces s. The flat sheet w as then removed from the collector and rolled into a tu be ( Figure 5 2 ) Using the same electrospinning setup the rolled tube was held in front of a flat collector and manually rotated until a thin layer of polymer was coated onto the surface seal ing the outer layer of the tube and prevent ing it from unraveling. The tubes were then cut into 10 m m long conduits. Figure 5 2 Rolling of flat sheet into a tube.

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41 5.8 Sciatic nerve transection and graft implantation The sciatic nerve transection and NGC implantation procedure was approved by the Institutional Animal Car e and Use Committee (IACUC). A total of 24 Sprague Dawley rats were used for the study, 4 rats per each graft (autograft, PCL, PSHU RGD/PCL) for 2 time points (4, 8 w) The rats weighing 250 275 g were allowed 7 d to acclimate prior to implantations and maintained on a 14/10 hour light/dark cycle w ith acces s to food and water ad libitum. The rats were anaesthetized using continuous isoflurane and oxygen inhalation. Initial induction was at 5 % isoflurane in oxygen and then maintai ned at 2 % isoflurane in oxygen. To minimize post operativ e pain, pre operative doses of ketoprofen at 5 mg/kg and bupivacaine (0.5 % Marcaine) at 2 mg/kg were administer ed via sub cutaneous injection. A rtificial tears ointment was administered to prevent dry eyes during t he procedure. The sciatic nerve that underwent t ransec tion and implantation was determin ed randomly, either the left or right sciatic nerve. The sciatic nerve that was not selected for t ransection did not undergo any experimental manipulation and was used as a control for normal nerve function. The rats were placed on their side and the skin around the gluteal region on the randomly selected side was shaved and disinfected with chlorhexidine and isopropyl alcoho l. A longitudinal skin incision from the knee to the hip was made to expose the underlying muscles that were retracted to isolate the sciatic nerve. An incision of t he sciatic nerve was made 5 mm in each direction from mid thigh for a total of a 10 mm gap. In the case of the autograft, the ends of the 10 mm transect ed nerve were reversed and sutured to the proximal an d distal nerve stumps using Prolene polypropylene 7 0 sutures. For PCL and PSHU RGD/PCL conduits the ends of the

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42 conduit were sutured to the proximal and distal nerve stumps using Prolene polypropylene 7 0. After graft implantation, the mus cle layer was closed with coated Vicryl 4 0 sutures using a continuous suturing pattern. The sk in incision was closed using coated Vicryl 4 0 sutures using a continuous subcuticular suture technique. Postoperative doses of ketoprofen at 5 mg/kg were admini stered daily for 3 d. 5.9 Walking track analysis 4 or 8 w after implantat ion, a walking track analysis was performed A walking alley with a darkened goal box a t one end was used to assess function al recovery after implantation. The floor of the walking alley with dimensions 45 cm 8 cm 5 cm was covered with white paper. The hind feet of the rats were smeared with finger paint and the rat s were allowed to walk down the track leaving footprints on the paper. The hind feet were cleaned and disinfected with ch lorhexidine and alcohol after obtaining footprints SFI was calculated with EPL, NPL, ETS, NTS, EITS and NITS measurements for three sets of prints that were averaged ( Equation 2 1 ) 5.10 Ankle motion analysis Immediately after obtaining footprints for the walking track analysis, a nkle motion was recorded by video on a standard recording device. The angle of the ankle was meas ured during initial contact, mid swing, and toe off phases of walking ( Figure 5 3 ) The difference between grafted and normal contralateral side s for each phase during walking wer e calculated (Equation 5 1 )

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43 Figure 5 3 Different phases of walking in which ankle angle was measured for ankle motion analysis. 5 1 ( 5 1 ) 5.11 Euthanasia and tissue harvest One day following the recording of ankle movement for ankle motion analysis, the rats were euthanized by carbon dioxide and bilateral thoracotomy. Both of the sciatic nerves of the grafted and healthy contralateral sides were exposed similarly to the initial

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44 implantation protocol (Section 0 ). The sciatic nerve s from the spinal cord to the terminal branching site of the tibial, sural and common peroneal nerves were harvested The gastrocnemius muscle samples were harvested by ma king an incision of the skin just above the heel of the hind foot and the skin surrounding the gastrocnemius muscle was removed. Both the gastrocnemius muscle s from the grafted and healthy contralateral side s were harvested. 5.12 CAP recordings Immediately afte r nerve tissue harvest, t he CAP s of b oth the sciatic nerve s of the grafted and healthy contralateral side s were recorded The harvested nerves were placed on a platform with parallel conducting wire s ( Figure 5 4 ). The proximal end of the graft or healthy nerve was stimulated for 0.15 ms at 10 V and the CAP was recorded at the distal end. The grafted side to healthy contralateral side ratio of CAP amplitude and area under the curve (AUC) were calculated (Equation 5 2 ). Figure 5 4 Platform of parallel conducting wires used to measure CAP. 5 2 ( 5 2 )

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45 5.13 Gastrocnemius muscle mass The gastrocnemius muscles of both the grafted and healthy contralateral sides were weighed and the ratio of grafted side to healthy contralateral side was calculated (Equation 5 2 ). 5.14 Histology 5.14.1 IHC of nerve grafts After recording CAP measurements of the sciatic nerves, the grafted nerves were fixed using 4 % PFA in PBS for 1 h cryoprotected with 30 % sucrose in PBS for 2 d embedded in OC T compound and frozen at 80 C. The nerves were sectioned longitudinally with a thickness of 18 m and placed on glass slides The sections were fixed in acetone for 10 min and washed 2 times in PBS for 3 min each. Clear n ail polish wa s used at the very ends of each nerve and allowed to dry to secure the nerves onto the slides. The sections were blocked in 3 % hydrogen peroxide in PBS for 10 min to block endogenous peroxidase activity a nd washed 3 times in PBS for 3 min each. Blocking Buffer (5% goat serum 0.4% Triton X 100 PBS) was used to block the sections for 30 min. All antibodies were diluted in Dilution Buffer (1% serum, 0.4% Triton X 100 PBS ). The sections were stained with NF M (1: 500) for 60 min washed 3 times in PBS for 3 min each stained with Alexa Fluor 594 (1:500) for 30 min, and washed 3 times in PBS for 3 min each. The sections were d ouble immunostain ed with S100b (1:1000) for 60 min, washed 3 times in PBS for 3 min each, stained with Alexa Fluor 488 (1:500) for 30 m in, and washed 3 times in PBS for 3 min each. SlowFade Diamond antifade mountant with DAPI was used to mount the slides.

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46 5.14.2 ing of g astrocnemius muscle After measuring muscle mass the gastrocnemius muscle of both the experimental or grafted side and the healthy contralateral side were fixed in 10 % formalin for 24 h, cryoprotected with 30% sucrose in PBS for 24 h embedded in OCT compound, and frozen at 80 C. The muscles were sectioned with a thickness of 5 m and placed on glass sl ides. The s ections were washed in running deionized water to remove OCT compound and acetic acid) at room temperature overnight and washed in running deionized. The first stain consisted of Biebrich Scarlet Acid Fucshin solution (0.9 % Biebrich scarlet, 0.1 % acid fuchsin, 1.0 % acetic acid) for 5 min and subsequent w ash in running deionized water The second s tain involved a mixed solution of 1 volume 10 % Phosphotun gstic acid, 1 volume 10 % Phosphomolybdic acid, and 2 volumes deionized water for 5 min The third and final stain comprised of Aniline Blue solution (2.4 % Aniline blue, 2 % acetic acid) for 5 min The sections were p laced in 1 % acetic acid for 2 min and washed in running deionized water Subsequent washes consisted of 70 % ethanol for 1 min and 100 % ethanol for 1 min The sections were c leared in xylene for 2 min and m ounted using Cytoseal 60 The amount of collagen was quantified by calculating the a r ea of collagen to muscle fiber ratio for the grafted and healthy contralateral sides (Equation 5 3 ) Collagen to muscle fiber ratio was then set in another ratio to determine the amount of collagen of the grafted side to the amount of collagen on the healthy contralateral side ( Equation 5 2 )

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47 5 3 ( 5 3 ) 5.15 Statistical analysis All results are express ed as means standard error of the mean. Analysis of variance (ANOVA) was used to determine significant differences between groups and followed by post hoc test when applicable. Statist ical significance was considered when p < 0.05.

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48 6 Results and Discussion 6.1 PSHU, dPSHU, and P SHU RGD reaction sequence The synthesized PSHU RGD was designed to be biodegradable and biocompatible. The amide and ester groups are the sites of degradation and the h ydrophobic alkyl group s allow for slower degradation characteristics and allows for enhanced cell attachment ( Figure 6 1 ) Figure 6 1 Reaction sequence of PSHU RGD synthesis. 6.2 PSHU and dPSHU characterization using 1 H NMR The synthesis of PSHU from the N BOC serinol, urea, and HDI was confirmed using 1 H NMR ( Figure 6 2 ).

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49 Figure 6 2 1 H NMR spectrum of PSHU confirming molecular structure. 1 H NMR was us ed to confirm the removal of BOC protecting groups. The re sulting free amines were used to conjugate RGD. The disappearance of the b peak confirms the removal of the BOC group s ( Figure 6 3 ) Figure 6 3 1 H NMR spectrum of PSHU and dPSHU confir ming the removal of the BOC protecting group with the disappearance of the b peak.

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50 6.3 PS HU, dPSHU, and PSHU RGD characterization using FT IR After confirmation of PSHU and dPSHU structures using NMR, FT IR was used to further confir m the reaction and to verify RGD conjugation ( Figure 6 4 Figure 6 5 ) Figure 6 4 FT IR sp ectru m of PSHU, dPSHU, and PSHU RGD Figure 6 5 FT IR spectrum of PSHU, dPSHU, and PSHU RGD. The presence of free amine groups on dPSHU after deprotection is confirmed from region a The conjugation of RGD to dPSHU is confirmed with the shift in wavelength with carbonyl absorbance shown in region b

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51 The presence of free amine groups on dPSHU was confirmed in region a ( Figure 6 5 ) The wavelength in this region corresponds to primary amines. The conjugation of RGD can be confirmed from r egion b ( Figure 6 5 ). This region is associated with carbonyl groups found in PSHU and carbonyl groups of RGD. The wavelength correlat ed to carbonyl groups of RGD is slightly lower than that of the carbonyl groups of PSHU. Therefore, there is a shift in the carbonyl peak with the conjugation of RGD. 6.4 Sciatic nerve transection and graft implantation One of three different grafts was implanted into each rat. The a utograft was used as a positive control in which the greatest amount of nerve regeneration was expected. The PCL conduit was used as a negative control As seen previously, the PCL conduit is associated with poor neuronal activity due to the absence of RGD to promote cell attachment, survival, and migration. The PSHU RGD/PCL conduit served as the experimental graft. As a result of promising outcomes of neuronal growth and guide d axonal extension observed in preceding studies, the PSHU RGD/PCL conduit was be lieved to be comparable to the gold standard autograft in terms of nerve regeneration [15,16] The sciatic nerve transection and graft implantation procedures were successful for 23 of the 24 rats used in the study. After making a longitudinal skin incision and retracting the underlying muscles surrounding the sciatic nerve, a 10 mm long transection was made and grafts were implanted at the transection site ( Figure 6 6 ). Other than for one rat, no complications arose during the suturing of the gra fts to the proximal and distal nerve stumps. The rat that did have complications was euthanized after a failed attempt at suturing an autograft. During the surgical procedure, the suture needle was passed

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52 repet itively though the autograft until which the a utograft was too damaged for suturing. The rat was euthanized before recovering from anesthesia. All data analysis was performed without the inclusion of this rat, therefore, the autograft group for the 8 w time point consisted of 3 rats rather than the ty pical 4 rats for all other groups. Figure 6 6 Sciatic nerve transection and graft implantation. (A) longitudinal skin incision from the knee to the hip exposing underlying muscles, (B) location of connectiv e tissue separating two muscles, (C) blunt dissection through connective tissue, (D) isolation of sciatic nerve, (E) sutured autograft, (F) sutured PSHU RGD/PCL graft. Although autotomy is one of the most frequent postoperative complications that arise wit h the rat SNI model, self mutilation was limited to slight nibbling of toenails. However, after obtaining footprints for the walking track analysis, severe autonomy was observed for three rats. It is presumed that the finger paint used to record footprints on paper triggered this behavior. Although the hind feet were cleaned and disinfected with chlorhexidine and alcohol after the analysis any remaining residue may have caused the rats to exces sively groom the hind feet leading to the loss of toes However, severe cases

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53 of autotomy were only observed after the walking track analysis and did not affect the footprint recordings. 6.5 Walking track analysis A w alking track analysis was used to assess th e functional recovery using a quantified SFI value to measure sciatic nerve function ( Figure 6 7 ) As nerve function improves, PL decreases, TS increa ses, and ITS increases with increased muscle function. This improvement in sciatic nerve function is reflected by an increase in SFI (Equation 2 1 ) Figure 6 7 Calculated SFI values. Error bars represent standard error of the mean indicates p < 0.05. No significant difference betwee n grafts was observed, but a significant improvement was observed in sciatic nerve function between 4 and 8 w for the PSHU RGD/PCL conduit At 4 w, the SFI val u es for each graft were similar but at 8 w more of a difference was observed. Although not stat istically significantly, the PSHU RGD/PCL c onduit seemed to outperform the autograft This encouraging level of functional regeneration can be attributed to RGD and intraluminal microchannels with aligned nanofibers. However, the PCL conduit also seemed to attribute to equal, if not superior,

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54 functional recovery compared to the autograft. Although the PCL conduit was expected to see a very limited amount of functional recovery the positive effect of intraluminal micro channels with aligned nanofibers may have been underestimated. Axons in the PNS are known to be able to regenerate on their own, but the topical cues associated with the microchannels and aligned nanofibers may have accelerated this regeneration process. 6.6 A nkle motion analysis Ankle motion analysis was performed by recording the movement of the ankle during various phases of walking. The difference in the ankle angle between the grafted and healthy contralateral sides during initial con tact, mid swing, and t oe off were calculated ( Figure 6 8 Figure 6 9 and Figure 6 10 ) Figure 6 8 The difference in the ankle angle between the grafted and healthy contralateral side s during the initial contact phase of walking. Error bars represent standard error of the mean indicates p < 0.05.

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55 Figure 6 9 The difference in the ankle angle between the grafted and h ealthy contralateral side during the m id swing phase of walking. Error bars represent standard error of the mean indicates p < 0.05. Figure 6 10 The difference in the ankle angle between the grafted and healthy contralateral side during the toe off phase of walking. Error bars represent standard error of the mean indicates p < 0.05. No statistical difference was observed between the three grafts for initial contact and mid swing. However, significant r ecovery was seen with initial contact for the autograft and with mid swing for the PCL conduit between 4 and 8 w. When considering only toe

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56 off angle, the PSHU RGD/PCL conduit showed discernible improvement from 4 to 8 w and was statistically different fro m the PCL conduit. When considering all three phases of walking that were analyzed, no solid conclusion can be made on functio nal recovery between each graft. No graft seemed to consistently show improved function recovery with all phases However, it appears that substantial recovery is occurring between 4 and 8 w after nerve transection 6.7 CAP recordings CAP recordings of the nerve grafts were used to assess the recovery of electrophysiological activity across the transection gap. CAP is dependent on axon diamet er and myelin sheath thickness, and therefore, r ecovering electrophysiological activity after nerve transection is an important step towards achieving functional recovery. After stimulating the nerve samples at the proximal end the CAP was r ecorded at the distal end ( Figure 6 11 ) The CAP recordings consisted of two peaks. The first peak is a stimulus artifact that is produced from electr ical stimulation The second peak is the CAP of which the amplitude and AUC were calculated The record ed CAPs for all the grafts had low latency, characteristic of the short delay of the peak after the stimulus artifact. This suggests that the majority of the axons that are contributing to the CAP have high degrees of myelination. Within 4 w after implantation, a CAP was observed for all grafts. This implies that a sufficient number of myelinated axons have regenerated across the entir e length of the nerve gap.

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57 Figure 6 11 Representative CAP recordings 8 w after nerve transection and graft implantation. Immediately a fter sciatic nerve transection, CAPs are expected to be unmeasurable. D ue to the disconnection of axons, the nerve is in capable of propagating electrical impulses across the nerve gap As axons regenerate across the transection site during the regeneration process, the CAP amplitude and AUC are expected to increase. Amplitude will increase as the number of axons increases. However, the degree of myelin thickness varies greatly for axons during regeneration and the amplitude may not always account for more thinly myelinated axons This is characteristic of a broader CAP. There f ore, AUC was also considered for analysis Due to the variability of CAPs of the healthy contralateral sciatic nerves between each rat, a ratio of CAP amplitude and AUC of the grafted nerve to the healthy contralateral nerve was calculated ( Figure 6 12 Figure 6 13 ) The amplitude was determined as the maximum peak value. Calculating the AUC required several steps as the stimulus artifact and the CAP overlapped with each other. AUC was calculated by initially measuring the AUC of both the stimulus artifact and CAP. The s timulus artifact was then model ed as a

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58 Gaussian by using the parts of the peak that did not overlap with the CAP. The AUC of the Gaussian was calculated and subtracted from the area of both the stimulus artifact and CAP. The resulting value was considered the AUC of the CAP. Figure 6 12 CAP amplitude ratio of the grafted to healthy contralateral nerves. Amplitude was determined as the maximum peak value of the CAP. Error bars represent standard error of the mean. indicates p < 0.05. Figure 6 13 CAP AUC ratio of the grafted to healthy contralateral nerves. AUC was calculated by subtracting the area under the stimulus artifact modeled as a Gaussian from the total area under both the stimulus artifact and CAP. Error bars repres ent standard error of the mean.

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59 The CAP amplitude was significantly higher for the PSHU RGD/PCL conduit compared to the autograft and P CL conduit after 4 w. However, n o statistical difference was observed at 8 w, presumably due to the high variability asso ciated with taking recordings and limited sample size Even so, it appears that CAP amplitude increased with the use of PSHU RGD/PCL over the other grafts. No significant difference was observed for AUC. Although it appears that the PSHU RGD/PCL conduit ha s higher AUC attributed by more axonal regeneration and myelination, high variability and limited sample size, prevented a statistically significant result. Although no statistical significant electrophysiological result was observed, the CAP recordings s eem to imply that PSHU RGD/PCL may provide a comparable, if not improved environment for promoting axonal extension and myelination. 6.8 Gastrocnemius muscle mass As a target mus cle of the sciatic nerve the gastrocnemius muscle is affected by the transectio n of the nerve. Muscle mass and the degree of innervation are dependent on the motor neurons located in the sciatic nerve After nerve transection, the gastrocnemius muscle undergoes a process of denervation causing muscle atrophy and the overall loss of muscle mass. Through the use of nerve grafts, it was expected that the gastrocnemius muscle was gradually reinnervated during the regeneration process causing muscle mass to increase Due to variability of muscle mass between animals that can arise (e.g. size) the grafted to healthy contralateral side mass ratio was used for comparison between the different grafts ( Figure 6 14 ) However, n o significant difference in muscle mass was observed between any of the grafts.

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60 Figure 6 14 Gastrocnemius muscle mass ratios of the grafted to healthy contralateral sides. Error bars repres ent standard err or of the mean. 6.9 Histology 6.9.1 IHC of nerve grafts IHC was used to directly observe regenerated axons. Axons were stained with NF M a nd Alexa Fluor 594, while Schwann cells were stained with S100b and Alexa Fluor 488 ( Figure 6 15 ). As measurable CAPs were recorded for all the grafts at both 4 and 8 w time points it was expected to see axonal extension from the proximal end of the graft across the entire length toward s the distal end.

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61

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62 Figure 6 15 Representative images of d ouble immun ostaining for axonal regeneration and Schwann cell activity after 8 w (A B) autograft, (C D) PCL conduit, and (E F) PSHU RGD/PCL conduit. Longitudinal sections from left to right represent proximal to distal ends. Axons were stained with NF M a nd Alexa Fluor 594 and appear in red Schwann cells were stained with S100b a nd Al exa Fluor 488 and appear in green Scale bar represents 500 m. In all grafts, axon s and Schwann cells were observed to span the entire length of the graft. Axonal extension was not always continuous through each section, but the presence of axons at the d istal end was used to infer that axons have spanned the entire length of the transection gap. In the case of the PCL conduit, axonal extension was observed mostly along the outer surface of the conduit with minimal axonal extension through the intraluminal microchannels For the PSHU RGD/PCL conduit, extension of axons was observed along the entire length of an intraluminal microchannel in a continuous linear path Schwann cell activity was observed much more prevalently through out the grafts as Schwann cel ls are involved in the removal of myelin and axona l debris following nerve injury, as well as in the formation of B ngner bands. The myelination of axons is also attributed to Schwann cell s Myelin sheaths are expected to be present in area s where axonal a ctivity and Schwann cells activity overlap. It appears that axons and Schwann cells are much more prevalent in the autograft compared to the two conduits. However, a direct comparison of axon and Schwann cell

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63 density between the conduits cannot be made ba sed on these images. The autograft initially contained axons and Schwann cells with implantation and both of the stains are not specific for only regenerating axons and Schwann cells involved in regeneration Also, the physical characteristics of the condu its prevent direct comparison As the wall thickness of the intraluminal channels is larger than the section thickness of 18 m, axons and Schwann cells with in a specific microchannel may leave the field of view if sections were not cut perfectly parallel to the microchannels Therefore, it can only be inferred that the PSHU RGD/PCL conduit was more effective in supporting axonal growth compared to the PCL conduit due to higher axonal density. 6.9.2 stain muscle. Muscle atrophy occurs with the denervation of the gastrocnemius muscle following sciatic nerve transection and fibrotic tissue is formed With the regeneration o f motor neurons and reinnervation of the muscles the degree of muscle atrophy and fibrotic tissue will gradually decrease. The quantity of collagen present in muscle was used to assess the disease state of the muscle as collagen fibers indicate the format ion of fibrotic tissue ( Figure 6 16 Figure 6 17 )

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64 Figure 6 16 Representative images of gastrocnemius muscle stained with s trichrome stain after 8 w (A) autograft, (B) PCL conduit, (C) PSHU RGD/PCL conduit. Muscle fibers are stained in red while collagen is stained in blue Scale bars represent 100 m. Figure 6 17 The ratio of collagen to muscle fiber by area. The presence of collagen implies the formation of fibrotic tissue. Error bars rep resent standard error of the mean indicates p < 0.05.

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65 The area of collagen to muscle fibers was used to compare the amount of fibrotic tissue formation between the different grafts. At 4 w after implantation, no statistical difference was observed betwe en any of the graft s but after 8 w, the autograft and PSHU RGD/PCL conduit had a significantly lower presence of collagen. These results suggest t hat the PSHU RGD/PCL conduit had comparable motor neuron regeneration and muscle reinnervation characteristics to the autograft.

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66 7 Conclusion The study started with the synthesis and characterization of the functional polymer used for the NGC PSHU RGD was successfully synthe sized and the molecular structure was verified along with the confirmation of RGD conjugation through 1 H NMR and FT IR. The NGC was then evaluated for nerve regeneration in a rat SNI model. After sciatic nerve transection and graft implantation several a ssessments were performed The PSHU RGD/PCL conduit was compared to the gold standard autograft and a PCL conduit associated with poor neuronal activity [16] Functional recovery was investigated using a walking track analysis and ankle motion analysis. Through the walking track analysis, SFI was calculated to quantitatively evaluate functional recovery. A lthough no statistical d ifference was observed, the PSHU RGD/PCL conduit seemed to outperform the autograft. The ankle motion analysis consisted of three measured ankle angles during initial contact, mid swing, and toe off phases of walking. When considering all three phases of w alking the PSHU RGD/PCL conduit showed similar functional recovery compared to the other grafts. Electrophysiological activity was measured by recording the CAP of the grafted nerves and calculating CAP amplitude and AUC. A n increase in CAP amplitude and AUC was observed for the PSHU RGD/PCL graft compared to other grafts but a statistically significant improvement was only observed for CAP amplitude. Gastrocnemius muscle mass was used to assess muscle reinnervation. N o significant difference in musc le mass was observed between any of the grafts.

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67 Histological assessment was used to di rectly observe regeneration and the presence of fibrotic tissue formation. Axons and Schwann cells were observed using IHC with stains specific for neurofilament and calcium binding protein s, respectively Axonal extension from the proximal end of the graft across the entire length towards the distal end was observed for the PSHU RGD/PCL conduit. The morphology associated with the reinnervation of the gastrocnemius mus The quantity of collagen present in muscle was used to assess the disease state of the muscle The PSHU RGD/PCL conduit had comparable motor neuron regeneration and muscle reinnervation c haracteristics to th e autograft and statistically reduced fibrotic tissue formation compared to the PCL conduit. Although nerve regeneration using the PSHU RGD/PCL conduit did not have statistical ly significant improvement s for all assessments considered for evaluation, the N GC consistently showed similar or improved nerve regeneration characteristics. These results are encouraging as autografts are associated with many drawbacks and it was determined that the PSHU RGD/PCL conduit is a functionally comparable a lternative for s urgical treatment of PNI.

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68 8 Future Work 8.1 Increase sample size For several of the grafts, s tatistically significant differences in several of the assessments after sciatic nerve transection and graft implantation were not observed even though some o f the trends implied that there may be significant difference This is due to the high variability in the functional, electrophysiological, and histological assessments, as well as the low sample size. It is expected with the increase in sample size, statistical significant differences will be observed between the various grafts. 8.2 Additional negative control The PCL conduit was used as a negative control as it was associated with poor neuronal activity [16] However, the results from several of the assessments show that the PCL conduit has substantial nerve regeneration characteristics. T he positive effect of intraluminal microchannels with aligned nanofibe rs may have been underestimated as the topical cues associated with the microchannels and fiber alignment may have accelerated the natural regeneration process. T herefore, another negative control should be used to assess conduits with out the intraluminal microchannels with aligned nanofibe rs. This control could be a hollow tube or simply leaving the nerve untreated after transection 8.3 Additional time points Although s tatistical significant differences between several of the assessments were not observed, the trends associated with the nerve regeneration process showed that a possible difference may be observed at time points longer than 8 weeks after implantation. With additional time points, a difference between the grafts may be more apparent.

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69 8.4 E lectrophysiological assessment using muscle action potentials The electrophysiological assessment in this study was performed by taking CAP recordings of just the nerve. This a ssessment only accounts for regenerated axons across the transection site. However, a more comprehensive analysis will account for axons that have reinnervated target muscle. Therefore, c ompound muscle action potential ( CMAP ) recordings may be of more sign ificance as stimulation occurs at the g rafted nerve and the CMAP is recorded at the target muscle. 8.5 Larger animal model Current FDA approved hollow conduits have been shown to effectively regenerate axons across gap s up to 3 cm in length [27] In order for the PSHU RGD/PCL to become a clinically applicable treatment option to PNI repair a study of the conduit must be performed in a model with a longer tr ansection nerve gap. Due to the smaller size of rats the operable nerve length is limited Options for a larger animal model include rabbits and dogs [83] 8.6 Growth factor and Schwann cell integration Although ample nerve regeneration without the need to use growth factors or support cells is ideal for clinical applications, rapid axonal regeneration is often desired. When considering the integration of growth factors and Schwann c ells, there are many options that could potentially maximize the rate of nerve regeneration (Section 2.2.8 Section 2.2.9 )

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