Injectable Bioadhesive Hydrogels for Nucleus Pulposus Replacement

and Repair of the Damaged Intervertebral Disc

A Thesis
Submitted to the Faculty
of
Drexel University
By
Jennifer Vernengo
in Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
January 2007






















Copyright 2007
Jennifer Vernengo. All Rights Reserved.














Dedications









To my parents,
MariaInez and Guillermo Vernengo










Acknowledgements

First and foremost, I would like to thank my parents, to whom I feel gratitude beyond
what I can articulate. They have provided me with unwavering support, reassurance,
generosity, and advice. Without them, I could not have completed this thesis. I could
spend a lifetime trying to give back what my parents gave to me.

I am also very grateful to my advisor, Dr. Tony Lowman. Without having met him
during my senior year at Drexel University, I would not be here today. He was an
exceptional advisor and has given me constant encouragement, strong support, and
invaluable guidance. I will always look back on my experience in graduate with
fondness, and this is due, in large part, to having him as my advisor. Tony, thank you for
giving me the opportunity to work in your laboratory.

I would also like to thank my committee members who have been advising me since my
first proposal in 2004: Garland Fussell, Michele Marcolongo, Guiseppe Palmese, and
Yossef Elabd. In particular, I would like to thank Garland for the great deal of guidance
and humor he has provided over the years.

Speaking of humor, I would also like to acknowledge the current and former members of
the Lowman Lab who have become my extended family. I hope these friendships will
ii
extend beyond the time we shared together in the Lowman lab. Specifically, I would like
to thank Jonathan Thomas, Tony Tuesca, Kara Spiller, Noelle Comolli, Vanessa Vardon,
Sam Laurencin, Karri Momyer, Jason Colman, Jenna Tulskie, Erik Brewer, Kris Kita,
Eric Perakslis, and Jamie Ostroha. Meredith Hans deserves a special thanks not only for
her friendship, but for her technical contributions to my project as well. I would be
remiss without mentioning Larry Matthews. Having him around to help during my last
year has been priceless. I would also like to thank Dr. Hung Lee and Dr. Dunja Radisic
for their assistance with NMR, as well as Amy Arthur for her guidance with cadaver
testing and mechanics.
iii
Table of Contents



List of Figuresvi
List of Tables......ix
Abstract...x
Chapter 1 - INTRODUCTION.......................................................................................... 1
Chapter 2 - BACKGROUND............................................................................................. 4
2.1 Anatomy of the Spine .......................................................................................................... 4
2.2 Intervertebral Disc Biomechanics.................................................................................... 12
2.3 Intervertebral Disc Degeneration..................................................................................... 14
2.4 Treatment for Lower Back Pain ...................................................................................... 18
2.4.1 Conservative Treatments ............................................................................................................18
2.4.2 Current Surgical Interventions....................................................................................................21
2.4.3 Nucleus Pulposus Replacement ..................................................................................................39
2.5 Poly(N-isopropylacrylamide)............................................................................................ 47
2.5.1 Thermodynamics of the Phase Transition...................................................................................47
2.5.2 LCST Determination...................................................................................................................50
2.5.3 LCST Modulation.......................................................................................................................53
2.5.4 PNIPAAm-based Hydrogels.......................................................................................................56
2.5.5 PNIPAAm-based Systems for Drug Delivery.............................................................................60
2.6 Poly(ethylene glycol).......................................................................................................... 68
2.7 Poly(ethylene imine) .......................................................................................................... 74
2.8 Bioadhesive Polymers for Soft Tissue Repair ................................................................. 79
Chapter 3 - RESEARCH GOALS ................................................................................. 105
Chapter 4 - SYNTHESIS OF PNIPAAM-PEG BRANCHED COPOLYMERS......... 107
4.1 Introduction ..................................................................................................................... 107
4.2 Experimental Section ...................................................................................................... 109
4.2.1 Materials ...................................................................................................................................109
4.2.2 Methods ....................................................................................................................................109
iv
4.2.2.1 Methacrylation of PEG .....................................................................................................109
4.2.2.2 Copolymer Synthesis ........................................................................................................110
4.3 Results and Discussion .................................................................................................... 111
4.3.1 Methacrylation of PEG.............................................................................................................111
4.3.2 Synthesis of PNIPAAm-PEG Branched Copolymers...............................................................111
4.4 Conclusions ...................................................................................................................... 113
Chapter 5 - CHARACTERIZATION OF PNIPAAM-PEG BRANCHED
COPOLYMERS.............................................................................................................. 119
5.1 Introduction ..................................................................................................................... 119
5.2 Experimental Section ...................................................................................................... 120
5.2.1 Methods ....................................................................................................................................120
5.2.1.1 LCST Characterization......................................................................................................120
5.2.1.2 Gel swelling and Dissolution ............................................................................................121
5.2.1.3 Mechanical Properties.......................................................................................................122
5.2.1.3.1 Compressive Studies .................................................................................................122
5.2.1.3.2 Stress Relaxation Studies ..........................................................................................122
5.2.2. Statistical Analysis...................................................................................................................123
5.3 Results............................................................................................................................... 123
5.3.1 LCST Characterization .............................................................................................................123
5.3.2 Swelling and dissolution experiments.......................................................................................124
5.3.3 Compressive mechanical studies ..............................................................................................125
5.3.3.1 Network Analysis with Rubber Elasticity Theory.............................................................126
5.3.4 Stress Relaxation Studies..........................................................................................................128
5.4 Discussion........................................................................................................................ 128
5.5. Conclusions ..................................................................................................................... 132
Chapter 6 - OPTIMIZATION STUDIES TO PREVENT WATER LOSS IN VIVO.. 142
6.1 Introduction ..................................................................................................................... 142
6.2 Experimental Section ...................................................................................................... 143
6.2.1 Materials ...................................................................................................................................143
6.2.2 Methods ....................................................................................................................................143
6.2.3 Statistical Analysis....................................................................................................................144
6.3 Results and Discussion .................................................................................................... 145
v
6.3.1 Osmotic Swelling Studies .........................................................................................................145
6.3.2 Thermal Cycling Studies...........................................................................................................146
6.4 Conclusions ...................................................................................................................... 147
Chapter 7 - SYNTHESIS AND CHARACTERIZATION OF BIOADHESIVE
PNIPAAM-PEG/PEI BLENDED COPOLYMERS..................................................... 152
7.1 Introduction ..................................................................................................................... 152
7.2 Experimental Section ...................................................................................................... 155
7.2.1 Materials ...................................................................................................................................155
7.2.2 Methods ....................................................................................................................................156
7.2.2.1 PNIPAAm-PEG/PEI Blend Preparation and Characterization .........................................156
7.2.2.2 PNIPAAm-PEG/PEI Crosslinking with Glutaraldehyde ..................................................157
7.2.2.3 Free Glutaraldehyde Release.............................................................................................157
7.2.2.4 PNIPAAm-PEG/PEI Gel Swelling ...................................................................................158
7.2.2.5 PNIPAAm-PEG/PEI Compressive Mechanical Properties...............................................159
7.2.2.6 Bioadhesive Force Studies ................................................................................................159
7.2.2.7 Statistical Analysis............................................................................................................160
7.3 Results............................................................................................................................... 161
7.3.1 PNIPAAm-PEG/PEI Blend Characterization ...........................................................................161
7.3.2 Gel Crosslinking with Glutaraldehyde......................................................................................161
7.3.3 Free Glutaraldehyde Release ....................................................................................................162
7.3.4 PNIPAAm-PEG/PEI Gel Swelling...........................................................................................163
7.3.5 PNIPAAm-PEG/PEI Compressive Mechanical Properties.......................................................164
7.3.6 Bioadhesive Force Studies........................................................................................................164
7.3 Discussion......................................................................................................................... 166
7.4 Conclusions ...................................................................................................................... 170
Chapter 8 - CONCLUSIONS AND FUTURE RECOMMENDATIONS.................... 181
8.1 Conclusions ...................................................................................................................... 181
8.2 Recommendations for Future Work.............................................................................. 184
References ..188
Vita..218

vi
List of Figures



Figure 2.1 Anterior view of the vertebral column showing the cervical, thoracic, lumbar,
and sacral regions...................................................................................................... 90

Figure 2.2 The division of the vertebrae into three regions. ............................................. 91

Figure 2.3 (A) Lateral view, (B) superior view, (C) inferior view of the vertebra........... 92

Figure 2.4 The structure of the intervertebral disc. It consists of a nucleus pulposus (NP)
surrounded by an annulus fibrosis (AF). Both are sandwiched between two
vertebral endplates (VE). .......................................................................................... 93

Figure 2.5 A cylindrical interbody fusion cage. A, anterior; P, posterior......................... 94

Figure 2.6 Photograph of the Type III SB Charit............................................................ 95

Figure 2.7 The Charit artificial disc consists of a free-floating biconvex sliding core
encased in concave endplates.................................................................................... 96

Figure 2.8 Photograph of the Prodisc-L............................................................................ 97

Figure 2.9 Photograph showing the Maverick disc. ......................................................... 98

Figure 2.10 An implanted Prosthetic Disc Nucleus (PDN) device................................... 99

Figure 2.11 The Newcleus spiral implant. ...................................................................... 100

Figure 2.12 The chemical structure of poly(N-isopropylacrylamide) (PNIPAAm) ....... 101

Figure 2.13 The chemical structure of poly(ethylene glycol) (PEG).............................. 102

Figure 2.14 The chemical structure of poly(ethyelene imine) (PEI) .............................. 103

Figure 2.15 The chemical structure of glutaraldehyde. .................................................. 104

Figure 4.1 Schematic for the synthesis of PEGDM. ....................................................... 114

Figure 4.2 Schematic for the synthesis of PNIPAAm-PEG branched copolymers ........ 115

Figure 4.3
1
H NMR spectrum for PEGDM in deuterium oxide at 25C ........................ 116

vii
Figure 4.4 The changes in spectra over reaction time for PNIPAAm-PEG branched
copolymers. ............................................................................................................. 117

Figure 4.5. NMR Spectra for purified PNIPAAm-PEG branched copolymer s in
deuterium oxide at at 25C...................................................................................... 118

Figure 5.1 LCST and enthalpy of transition as a function of weight percent PEG in
PNIPAAm-PEG branched copolymers................................................................... 135

Figure 5.2 Effect of PEG block molecular weight on gel water content ........................ 136

Figure 5.3 Effect of PEG block size on the equilibrium compressive modulus of
PNIPAAm-PEG branched copolymers................................................................... 137

Figure 5.4 Stress-strain behavior of PNIPAAm-PEG branched copolymers at 90 days
immersion in PBS, normalized to account for the swelling of the gel ................... 138

Figure 5.5 Modulus, G, for the high PEG content PNIPAAm-PEG branched copolymers
at 90 days immersion in PBS, normalized to account for differences in the swelling
behavior of the gels................................................................................................. 139

Figure 5.6 The effective molecular weight between crosslinks, M
e
, for the high PEG
content PNIPAAm-PEG branched copolymers at 90 days immersion in PBS ...... 140

Figure 5.7 Effect of immersion time in vitro on the compressive modulus at 15% strain of
high PEG content (a) and low PEG content (b) PNIPAAm-PEG branched
copolymers.............................................................................................................. 141

Figure 6.1 Effect of immersion media osmotic pressure on the water content of
PNIPAAm-PEG (4600 g/mol) ................................................................................ 149

Figure 6.2 Effect of solution concentration at room temperature on equilibrium water
content of PNIPAAm-PEG (4600 g/mol) gels........................................................ 150

Figure 6.3 Effect of thermally cycling the polymer solution by equilibrating in a 37 C
osmotic environment, followed by cooling to room temperature........................... 151

Figure 7.1 The
1
H NMR spectrum for PNIPAAm-PEG/PEI (M
n
=60,000 g/mol) blend in
deuterium oxide at 25C.......................................................................................... 173

Figure 7.2 The chemical stability of PNIPAAm-PEG/PEI (M
n
=10,000) and PNIPAAm-
PEG/PEI (60,000 g/mol) blends ............................................................................. 174

Figure 7.3 The FTIR spectra for a dry (A) PNIPAAm-PEG/PEI gel, (B)
glutaraldehyde/PEI mixture, (C) PNIPAAm-PEG/PEI gel injected with
glutaraldehyde......................................................................................................... 175
viii

Figure 7.4 FTIR difference spectroscopy for the analysis of PNIPAAm-PEG/PEI
crosslinked gels....................................................................................................... 176

Figure 7.5 Release profile of free glutaraldehyde from the PNIPAAm-PEG/PEI network
................................................................................................................................. 177

Figure 7.6 The water content over a 30 day immersion in PBS for PNIPAAm-PEG/PEI
blends with and without glutaraldehyde injection .................................................. 178

Figure 7.7 The compressive modulus of PNIPAAm-PEG/PEI gels at 15% strain with and
without glutaraldehyde crosslinking at 7 days immersion in PBS ......................... 179

Figure 7.8 Typical stress strain curves for the in vitro bioadhesive force studies .......... 180

ix

List of Tables




Table 5.1 Equilibrium water contents and compressive moduli for PNIPAAm-PEG after
90 days immersion in vitro ..................................................................................... 134

Table 7.1 The mean maximum force and work of adhesion required to detach the
PNIPAAm-PEG and PNIPAAm-PEG/PEI gels from the biological substrates. .... 172


x
Abstract
Injectable Hydrogels for Nucleus Pulposus Replacement
and Repair of the Damaged Intervertebral Disc

Jennifer Vernengo
Anthony Lowman, Ph.D.




Researchers have begun to realize the potential benefits of treating intervertebral disc
degeneration by replacing the nucleus pulposus. One of the material classes being
studied for nucleus replacement is the hydrogel, a three-dimensional, hydrated polymer
network. The development of an injectable hydrogel nucleus replacement would have
important clinical consequences because it could be injected non-invasively using a
needle. Aqueous solutions of poly(N-isopropylacrylamide), or PNIPAAm, have a lower
critical solution temperature (LCST) between room and body temperature, making it
suitable for an injectable implant material. Aqueous polymer solutions could be injected
as a free flowing liquid at 25C and solidify to a gel within the body at 37C.

At physiological temperatures, PNIPAAm homopolymer gels hold little water and show
poor elastic recovery. In the work, the swelling and mechanical properties of PNIPAAm
gels are tailored by polymerizing NIPAAm in the presence of poly(ethylene) glycol
dimethacrylate (PEGDM), thereby creating PNIPAAm-PEG branched copolymers. The
effect of PEG molecular weight and NIPAAm/PEG molar ratio on the water content,
stiffness, and elasticity of the hydrogels was determined. In addition, a suitable material
candidate for nucleus pulposus replacement was chosen from this class of hydrogels. The
copolymer formulation was optimized to minimize implant water loss following
xi
implantation into the intradiscal environment, allowing the implant to remain space
filling in the nuclear cavity. Bioadhesive properties were then imparted to the hydrogel
system by blending it with the amine containing polymer poly(ethylene imine) (PEI).
After gelation in the disc, the implant will be crosslinked with itself and with the
surrounding tissues by the injection of a dialdehyde. This approach will help secure the
implant in place, reducing the risk of implant migration or extrusion. The bioadhesive
material also has the potential to function as tissue adhesive for the repair of the damaged
annulus fibrosis.






































1
Chapter 1 - Introduction

Lower back pain, one of the most common pain conditions in the western world, is
responsible for significant economic and social costs. In the United States alone, it
accounts for more than sixty billion dollars annually in health care costs
1
. One of the
major causes of lower back pain is intervertebral disc degeneration
2
. Disc degeneration
is the result of damage to or dehydration of the nucleus pulposus, which reduces its
hydrostatic pressure on the internal surface of the annulus fibrosis. This reduced
hydrostatic pressure results in abnormal compressive stresses on the annulus, potentially
causing tears, cracks and fissures after repeated loads. Back pain can develop as a result
of nucleus tissue migrating through the annulus and impinging on nerve roots
2,3
.

Currently, the most common surgical treatment for the degenerative disc is spinal fusion
4
, but this causes loss of spinal mobility and increases stress on adjacent discs,
accelerating degeneration of these joints
3,4
. Total disc arthroplasty is emerging as a
viable alternative to fusion because more spinal motion is retained
4
. However, because
these treatments involve highly invasive surgeries, a promising alternative is the
replacement of the nucleus pulposus alone. There are several pre-formed polymeric
implants currently under investigation for nucleus pulposus replacement. While some of
these materials are promising, using an in situ forming nucleus replacement could have
important clinical consequences, because it can be injected non-invasively using a small
gauge needle.

2
In situ gel formation can be achieved using the thermo-responsive polymer poly(N-
isopropylacrylamide) (PNIPAAm). Aqueous solutions of PNIPAAm undergo a phase
transformation at its lower critical solution temperature (LCST), typically around 32C,
allowing it to form a free flowing solution in water at ambient temperatures and a gel at
body temperature
5
. This property makes it ideal for an injectable nucleus pulposus
replacement.

This work focuses on designing an in situ forming PNIPAAm-based polymeric nucleus
pulposus replacement. Because the PNIPAAm homopolymer holds little water and
shows poor elastic recovery, the swelling and mechanical properties of PNIPAAm gels
are tailored by polymerizing NIPAAm in the presence of a small amount of
poly(ethylene) glycol macromers, thereby creating PNIPAAm-PEG branched
copolymers. It is hypothesized that a suitable candidate from this class of materials can
be developed, which upon implantation, could prevent or postpone the annular
degeneration process by restoring the healthy biomechanics of the intervertebral disc and
alleviate the pain associated with degenerative disc disease.

The work will begin with the synthesis of a class of hydrogels composed of PNIPAAm
and PEG. The molecular weight of the PEG chains and the relative proportions of
NIPAAm and PEG units will be varied. Then, the range of pertinent material behavior
for the PNIPAAm-PEG family of copolymers, and how it varies with polymeric structure
will be examined. Based on these characterization studies, a material candidate which
exhibits suitable, stable material properties will move on for further evaluation as a
3
material candidate for nucleus pulposus replacement. The copolymer formulation will
then be optimized to minimize water loss and subsequent volume loss, following
injection into the intradiscal environment due to the de-swelling of the polymer network.
Finally, the copolymer chemistry will be modified to impart bioadhesive properties to the
hydrogel system. This will help secure the implant in place, reducing the risk of implant
migration or extrusion. With these bioadhesive characteristics, the material will also
have the potential to perform as a closure material for the repair of annular defects.
4

Chapter 2 - BACKGROUND

2.1 Anatomy of the Spine

The back, or dorsum, is the posterior part of the body and includes the skin, muscles,
vertebral column, spinal cord, nerves, and blood vessels
6
. The vertebral column consists
of 24 moveable vertebrae (Figure 2.1), 7 cervical, 12 thoracic, and 5 lumbar. Below the
lumbar vertebrae are 5 sacral vertebrae, which in the adult body, fuse to form the sacrum.
The three to five lowermost vertebrae fuse in late adult life to form the coccyx. These
bones are joined together by a series of joints to form the vertebral column
7
.

The main functions of the human spine are to support the body, protect the spinal cord
and spinal nerve roots, and allow for movement of the trunk. These functions are carried
out not only by the vertebrae, but the soft tissues surrounding them. The adult vertebral
column has several visible curves. The cervical and lumbar regions are anteriorly
convex, and the thoracic and sacral areas are posteriorly convex. The spinal curves are
able to dissipate loads more efficiently than if the spine were a straight column
7
.

A typical vertebra may be divided into three main regions: the vertebral body, the
pedicles, and the posterior elements (Figure 2.2)
8
. The anterior part of each vertebra is a
large block of bone called the vertebral body. The pedicles connect the back of the
vertebral body to the posterior elements
8
. The posterior elements consist of the lamina,
5
superior articular arch, inferior articular, transverse, and spinous processes (Figure 2.3)
7
.
The lamina are formed from projections of bone extending from the pedicles. Two
lamina meet one another to form the neural arch, which houses the neural elements of the
vertebral column. The right and left inferolateral corners of each lamina extend
downwards to form a large mass of bone called the inferior articular process. Similarly,
the superior right and left portions of the lamina extends upwards to form the superior
articular process. Each of these four processes possess a smooth region on the surface
covered by articular cartilage, known as the articular facet of each process. Projecting
posteriorly from the junction of the two lamina is a blade of bone called the spinous
process. These bones form visual projections under the skin. Extending laterally from
the junction of the pedicle and the lamina is a flat bar of bone called the transverse
process
8
.

The vertebral body is designed to impart load-bearing characteristics to the vertebrae.
The flat superior and inferior surfaces of the vertebral bodies contribute to the stability of
the vertebrae under longitudinal, or compressive, loads. Its internal architecture also
contributes to its ability to support weight. The bone in this region is composed of an
outer layer of dense cortical bone and a core of spongy cancellous bone. While the
cortical bone allows the bone to sustain static loads, the cancellous bone makes the
vertebral body resilient, or able to withstand sudden and dynamic loading. The
cancellous bone is composed of thin rods of bone called trabeculae that extend in the
vertical and transverse directions. Applied compressive loads are first supported by the
vertical trabeculae and are then transferred to the horizontal trabeculae in transverse
6
tension
8
. Both the vertical and transverse trabeculae increase in numbers after prolonged
or increased compressive loading. Also, the transverse diameter of the vertebral bodies
increases from the cervical to the lumbar regions of the spine, resulting from the fact that
the latter carries a greater load. The width then diminishes from the first sacral segment
to the tip of the coccyx
7
.

The spaces between the trabeculae serve as channels for blood supply
8
. Small veins are
present in the superior and inferior parts of the vertebral body. These smaller veins enter
into larger tributaries in the center of the vertebral body, forming a dense network of
veins. The cortical bone also contains small veins and nutrient arties, as well as some
nerve endings
7
.

Because there are no mechanisms on the surfaces of the vertebral bodies that prevent
sliding and twisting motion, the vertebral bodies are dependent on the posterior elements
for stabilization in the horizontal direction. The inferior articular processes form hooks
that lock into the superior articular process of the vertebra below, forming a locking
mechanism that prevents sliding. The posterior elements are designed to receive the
dynamic forces placed on the spine. Loads on the processes are eventually transmitted to
the pedicles and, eventually, the vertebral bodies. In addition, the posterior elements
provide areas for muscle attachments, so muscle forces are transmitted to the posterior
elements and then to the vertebral bodies through the pedicles
8
.
7

Because the pedicles play such an important role in transmitting all forces to the vertebral
bodies, they must possess a structure which enables them to resist bending, tension, and
compression
8
. Interestingly, the amount of cortical bone on the pedicles varies from one
region of the spine to the other, being higher in regions which are subjected to more
motion. The middle cervical and upper lumbar pedicle regions contain more cortical
bone than the thoracic regions. The pedicles in the thoracic region are composed mostly
of cancellous bone
9
. These trends may be explained by the fact that there is less spinal
motion in the thoracic region.

Adjacent vertebral bodies are separated by a layer of strong yet flexible soft tissue, called
the intervertebral disc. This tissue is strong enough to sustain weight yet can deform to
accommodate rocking motions
8
. It also helps to carry compressive loads placed on the
spine
7
and transfer loads to the vertebral bodies
8
. It has been suggested that its
mechanical efficiency improves with use
10
. Applied stresses affect cellular activity, so
the disc can remodel itself to minimize that stress
11
. However, the extent to which
remodeling is possible may be limited, and high stresses are also thought to lead to disc
degeneration
12
. The shape of the disc is determined by the shape of the adjacent
vertebral bodies. Also, the thickness of the discs varies with spinal level. The discs are
thickest in the lumbar region and thinnest in the thoracic region. The discs of the cervical
and lumbar regions are thicker anteriorly than posteriorly, while thoracic discs have a
constant thickness throughout
13
.

8
The main components of the disc are water, proteoglycans, and collagen
7
. Proteoglycans
are molecules consists of several glycosaminoglycan (GAG) chains covalently linked a
core protein. GAGs are a class of chemicals present in most forms of connective tissue,
such as skin, bone, cartilage, and tendons. They are long chains of polysaccharides,
which consist of a repeating sequence of two molecules, a sugar and a sugar with an
amine attached. The GAGs found in the intervertebral disc are principally chondroitin-6-
sulphate, chondroitin-4-sulphate, keratin sulphate, and hyaluronic acid. As many as 60
GAGs can be attached to the core protein. Proteoglycans can aggregate when several of
them link to a chain of hyaluronic acid. However, proteoglycans that do not aggregate
with hyaluronic acid are the major proteoglycans that occur in the disc. These
proteoglycans play a key role in allowing the disc tissues to attract and retain water
8
.

The fundamental unit of collagen is the tropocollagen molecule
8
. A tropocollagen
molecule consists of three polypeptide chains that are twisted around one another in a
helical fashion and held together by hydrogen bonding
8
. When these molecules align
side by side, a collagen fibril is formed. Aggregates of collagen fibrils form a collage
fibre
7
. There are 19 different types of collagen, differing in chemical composition and
structural arrangement. The types of collagen found in the disc are predominantly type I
and II, which have the helical structure described above
8
. These rope-like molecules
give the disc its ability to withstand forces and loads
7
.

The disc is composed of three basic structures: a central nucleus pulposus, a peripheral
annulus fibrosis, and two layers of cartilage covering the top and bottom called vertebral
9
endplates (Figure 2.4)
8
. These components differ in the arrangement of proteoglycans
and collagen in the tissue, as well as the relative concentrations of each. In healthy discs,
the nucleus is a fluid like mass of mucoid material
8
. The nucleus is thickest in the
lumbar region and thinnest in the thoracic region. It is most centrally placed in the
cervical region and is more posteriorly placed in the lumbar region
10
. The nucleus
pulposus is approximately 80% water in youth and gradually dehydrates beginning
between ages 20 and 30
2
. Proteoglycans comprise about 65% of the dry weight of the
nucleus
14
. Only about 25% of these proteoglycans exist in the aggregate form. About
20% of the dry weight of the nucleus is composed of collagen. Bundles of type II
collagen help to hold the proteoglycan matrix together
15
.

The main component of the annulus fibrosis is water, accounting for 70% of its weight.
About 60% of the dry weight of the annulus is composed of type I and type II collagen
fibres, arranged in concentric sheets called lamellae. The lamellae are thickest toward the
center of the disc
16
. Posteriorly, they are finer and more tightly packed, so the posterior
of the annulus is thinner that the anterior
17
. In the lumbar region, the fibres run parallel
to one other at a 65 angle with the vertical plane, although the direction of the lamella
can vary from person to person
10
. The spaces between lamellae are filled with
proteoglycan gel which binds the collagen fibres together
8
. These proteoglycans make
up about 20% of the dry weight of the annulus.

The vertebral endplates are attached to both the disc and the adjacent vertebral body.
They are approximately 1mm thick peripherally and 3mm thick centrally
7
. The chemical
10
structure of these plates resembles that of the nucleus pulposus and the annulus fibrosis
because they are composed of proteoglycans and collagen fibers. Directly above the
nucleus, the endplate contains more proteoglycans than collagen. However, at the
periphery of the endplates, the opposite is true
8
. The inner lamellae of the annulus attach
to the vertebral endplate and the outer lamellae anchors itself to the cortical bone that
exists on the rim of the vertebral body
7
. Even though the disc tissues are attached to the
endplates, the endplates are only weakly attached to the vertebral bodies. For this reason,
the endplates are considered part of the disc, instead of part of the vertebral body
18
.

The mature human intervertebral disc has a very low density cell population compared to
other tissues in the body. Cells occupy approximately 0.25-0.5% of the tissue volume in
the disc. The cells in the nucleus and inner annulus are chondrocyte-like and round
19
.
On the other hand, the cells in the outer annulus are thin and extend along the collagen
fibrils
20
. In the endplates, chrondocytes are aligned along the collagen fibrils, too
8
. The
chondrocytic cells are responsible for the synthesis of proteoglycans and collagen in the
disc, as well as proteases, which degrade old proteoglycans and collagen
8
. The health of
these cells is vital to the disc because degeneration will ultimately occur if the rate of
tissue breakdown increases over the matrix synthesis rates
21
.

Cell activity requires oxygen, glucose, and other substrates necessary for tissue synthesis.
However, the disc is the largest avascular tissue in the body, since cells are located 6-8
mm from the nearest blood supply. Nutrients are supplied to the outer annulus via blood
vessels at its periphery
22
. The inner annulus relies on nutrients supplied from capillary
11
beds in the vertebral body, which terminate at the vertebral endplate
23
. Small solutes
such as oxygen, glucose, and lactate diffuse through the endplate in order to enter the
center of the disc
22
.

One key feature of the disc is its ability to maintain a high water content, even under
compressive loads. This ability is attributed to the high osmotic pressure exerted by the
proteoglycans in the tissue
24
. The osmotic pressure of the proteoglycans is mainly
attributed to the ionic carboxyl and sulfate radicals on the GAG chains, which impart a
high negative fixed charge to the tissue matrix. Positively charged molecules are
attracted into the tissue to balance the negative charge of the GAGs. The osmotic
pressure generated by this ion flux is responsible for the ability of disc tissue to attract
and retain water. The size of the GAG chains has little effect on the osmotic pressure of
the disc tissues. In fact, the water binding capacity of the proteoglycans has been shown
to be proportional to their concentration, which increases from the outer to inner annulus
8
, and is highest in the nucleus pulposus
21
.

The water content of the disc not only depends on proteoglycan concentration, but the
pressure applied on it under loading
21
. In human lumbar discs, the pressure inside the
nucleus is lowest when laying prone, at 0.1-0.2 MPa, and increases by 5-8 times when
standing or sitting
25
. The disc fluid is expressed as pressure increases and under
sustained loads, however this fluid loss occurs very slowly. The proteoglycans account
for the fine pore size of the disc tissues, which restricts fluid flow under compression
26
.
Thus, the disc water content rarely achieves equilibrium
21
. Approximately 20-25% of
12
the disc water is expressed during daily loading, but this water is regained during a
nights rest
27
.

2.2 Intervertebral Disc Biomechanics

It has been shown experimentally that the annulus can support briefly applied
compressive loads and transmit them to adjacent vertebrae without the nucleus pulposus
10,28
. Its densely packed collagen lamellae make the annulus a relatively stiff body, and
the collagen fibres are prevented from buckling due the presence of the proteoglycan gel.
However, if the annulus is subjected to sustained loads, the proteoglycan gel will not be
strong enough to prevent deformation. The fibres will start to buckle and annulus will be
slowly squashed
8
.

The primary function of the nucleus pulposus is to provide a bracing mechanism for the
annulus under sustained loads. Because of its low hydraulic permeability, the nucleus
volume initially will not be compressed under vertical loading. Instead, it deforms by
expanding radially, exerting a hydrostatic pressure on the annulus, and pushing its
collagen lamellae outwards. The annulus fibres resist this stretch, and exert an opposing
force on the nucleus. Vertical loads are then transferred to the annulus by the nucleus in
circumferential tension, preventing the fibers from buckling under sustained loads.
2,8
. In
addition, because the annulus is stiff enough to resist bulging, nucleus deformation in the
radial direction is limited. This results in increased vertical pressure against the
endplates. This pressure serves to transmit part of the applied load to the adjacent
13
vertebral bodies, lessening the weight on the annulus. Ultimately, however, the load is
fully transmitted to the adjacent vertebrae
8
.

Another characteristic of the healthy disc is its elasticity. It has the capacity to store
energy. When the expanding nucleus pushes the fibres in the annulus outwards, they
stretch like springs due to the elasticity of the collagen. Once the load is released, the
elastic recoil of the collagen fibers allows the energy stored in them to be transferred the
nucleus, aiding it to restore any deformations caused by the loading
8
.

Together, the nucleus and annulus also act as a shock absorbing system for the vertebrae.
When a compressive force is rapidly applied to the disc, it is first transferred to the
annulus in the tension, before being transmitted to the vertebral bodies. While this does
not lessen the magnitude of the force, slowing the rate at which it is transferred to the
vertebrae will help protect the bones
8
. The shock absorbing capacity of the disc lessens
when the same disc is loaded repetitively. Movements such as jumping or subtle
bouncing, which may be experience during driving, are associated with disc injury
7
.

The fluid property of the nucleus pulposus is essential for maintaining synergy between
itself and the annulus fibrosis. Its high swelling pressure is the basis for the healthy
biomechanical function of the disc. The normal mechanical function of the disc will
depend on the ability of the nucleus to attract and maintain water. Any changes in the
proteoglycan concentration or water content of the disc tissues will alter the mechanical
properties of the disc
8
.
14

In traction, sliding, and twisting movements the annulus acts as a ligament, helping to
stabilize the spine by restraining movement. During traction, the collagen fibres in the
annulus are all pulled an equal vertical distance from the vertebral body. The annulus
resists this strain. The capacity of the disc to resist this strain is illustrated by how well
the spine can sustain the load of the trunk and legs when one is hanging by the hands
8
.
Even under traction, however, the spine is still under some compressive load due to
muscle action
7
. In slide movements, the fibres that line parallel to the direction of
movement will be strained and thus provide resistance to movement. Twisting is also
only resisted by the collagen fibres inclined in the direction of movement. Movements
such as these, which cause half of the annulus fibers to be stretched while the other half
stays relaxed, are most likely to cause injury to the disc
8
.

2.3 Intervertebral Disc Degeneration

Decreases in nutritional supply to the disc due to structural changes in the vertebral
endplates are thought to be a major cause of disc degeneration
29
. As a result of aging,
the blood vessels in the vertebral bodies become gradually occluded
30
, resulting in a
decrease in nutrients delivery to the disc. Nutrient transport may also be inhibited by
endplate calcification, which also naturally occurs in aging discs
29
, as well smoking and
vibration
31
. This impaired nutrition to the cells in the disc may be the underlying cause
for biochemical changes seen as the result of degeneration
8


15
With aging, the rate of proteoglycan synthesis decreases. In early adulthood,
proteoglycans comprise approximately 65% of the dry weight of the nucleus
14
, but this
drops to 30% by age 60
32
. The proteoglycans that are synthesized are generally lower in
molecular weight
33
. In addition to their molecular weight, the chemical nature of the
proteoglycans changes with age. The concentration of chondroitin sulphate GAGs falls,
while keratin sulphate content stays the same. Because chondroitin sulfate possesses
both sulphate and carboxyl radicals, it has twice the water binding capacity of keratin
sulfate, which only carries one sulphate radical. The water binding capacity of the tissue
therefore decreases with the drop in chondroitin sulphate content
34
. In addition, the
frequency of collagen-proteoglycan binding increases
35
, leaving fewer ionic groups on
the proteoglycans available to attract water
36
. Because of these biochemical changes, the
disc tissues dehydrate with age. In fact, by age 75, the water content of the nucleus can
drop to as low as 65%
14
. Added to this fluid loss, the disc tissues exhibit increasing
hydraulic permeability with age, so water loss will occur more quickly under loading
37
.

The overall collagen content of the disc also increases with age
38
. Type X collagen is
found in aged discs, and may be connected to calcification of the disc tissues
39
. The
fibre diameter of the type II collagen in the nucleus pulposus increases, making it
resemble the type I collagen in the annulus
38
. The collagen fibre diameter in the annulus
decreases, making it resemble the type II collagen in the nucleus
40
. This may be one
reason that the boundary between the nucleus and the annulus become indistinguishable
with age. Also, the annular rings thicken and become more disorganized
18
. The tensile
16
strength of the collagen decreases as well
41
, making the disc tissues more susceptible to
injuries associated with daily movements.

These fundamental biochemical changes alter the biomechanical behavior of the disc and
can initiate a degenerative cascade, ultimately leading to back pain. The dehydration of
the nucleus results in an overall volume loss, making it less able to exert a fluid pressure
on the annulus when it is compressed
42
. In other words, the nucleus loses its ability to
transmit weight directly to the annulus
8
, altering the uniform load distribution
characteristic of healthy discs
43
. The annulus then bears a greater portion of any load
placed on the disc
8,43
. Repeated stresses cause the lamellae to buckle
8
and annular
fissures to form
2,8,21
. These defects reduce the weight bearing capacity of the annulus,
leading to further tearing and loss of tissue integrity
44
. Some finite element models have
shown that loss of nucleus volume can lead to inward bulging of the annulus during
compression
45
. This increases the shear stresses between the lamellae
46
and tears the
layers apart
47
.

The nucleus may then migrate from the center of the disc, and perhaps eventually through
all layers of the annulus, resulting in disc herniation. Consequently, the nucleus is no
longer sheltered from the immune system, causing an inflammatory response from the
body. Studies on human patients have shown that herniations attract macrophages,
fibroblasts, and lymphocytes
48
. A variety of inflammatory chemicals are also produced,
such as phospholipase A
2

49
and interleukin 12
50
. The presence of chemical pain
17
mediators activates nociceptors, which send the signals to the spinal cord and brain to
cause back pain
51
.

In the healthy human disc, nerves extend into the outer third of the annulus. However,
degenerated discs often exhibit innervation that penetrates much deeper into the annulus,
and in some extreme cases into the nucleus pulposus
52,53
. The migrating nucleus also
may impinge on nerve roots, leading to additional pain
3
. Therefore, pain can occur even
in discs where there is no protrusion of nuclear material into or beyond the periphery of
the annulus fibrosis. Typically, fissures reaching into the inner third of the annulus are
not painful, but 70% of fissures extending into the outer third of the annulus cause pain
for the patients
54
.

With age, there is also an overall decrease in the bone density of the vertebral bodies and
a corresponding decrease in bone strength
7,55-57
. With ageing, the horizontal trabeculae
are absorbed and not replaced
57
. This phenomenon occurs most dramatically in the
central region of the vertebral body, directly above or below the nucleus pulposus
8
. The
vertebral endplates then must share a greater portion of every load because it lacks
support from the weakened underlying bone
58
. Repeated loading causes microfractures
to occur in the endplate and a gradual bow into the vertebral body
57
. If the fracture in the
endplate is large enough, nuclear material may migrate into the vertebral body, forming a
Schmorls node. The incidence of these is highest in the thoracic region of the spine
59
.
The fracture of an endplate can also lead to the introduction of inflammatory cytokines
into the nucleus. The presence of these chemical mediators actually slow metabolic and
18
reparative processes in the disc and lead to further degradation
60
. Cytokines can also be
a direct source of pain
61
.

2.4 Treatment for Lower Back Pain
2.4.1 Conservative Treatments

Current treatment for lower back pain begins with conservative interventions, the goal
being to relieve pain and restore function quickly
62
. Conservative treatment consists of
rest, medication, and physical therapy
63
. In a study on 71 athletes with disc herniation, it
was found that conservative treatment was satisfactory in controlling symptoms.
Approximately 79% of the athletes were able to resume their sporting activities an
average of 4.7 months after the start of treatment. Resumption of activity occurred when
the subjective symptoms were reduced by at least 80%
64
. Overall, however, the relative
effectiveness of common conservative treatment strategies is not well understood
65
.

The most common initial treatment for lower back pain is rest. Two days of total bed rest
is often prescribed after a back injury. This can help to reduce inflammation, relax
muscles, and allow healing to occur. Once healing has begun, a patient may resume light
activity. Although activity can increase the risk of relapse, periods of prolonged rest can
lead to weakness and depression
63
. Four days or longer of bed rest has been reported to
be detrimental to recovery
66
. Therefore, proper modifications to normal activities should
be made to avoid recurrence of the injury
62
.
19

The benefit a patient gains from bed rest may depend on the type of low back pain being
treated. Hagen et al.
67
performed a randomized trial comparing the effect of two days of
bed rest on the symptoms of acute lower back pain (without neurological deficits) and
sciatica (nerve root compression)
8
. For patients with acute lower back pain, staying
active was slightly more effective than staying in bed. For patients with sciatic back
pain, there were no differences between staying active and bed resting
67
.

There is substantial evidence that exercise helps in the management of lower back pain
62,65,67-70
. Exercises can help regain range of motion
62
, strengthen abdominal muscles to
help compensate for injured spinal tissues, and increase endurance of spinal
muscles
62,70,71
. Exercise may also help prevent future episodes of chronic pain and
modify pain perception
72
.

There are several different modes of exercise and physical therapy. Cold therapy can
reduce inflammation and pain, whereas hot therapy can aid in muscle relaxation and
improve circulation
62
. Transcutaneous electrical stimulation can block nerve impulses
from being sent to the cerebral cortex that are interpreted as pain
73
. Traction, where
systems of weights, cables, and pulleys are used to reduce pressure on the vertebrae, is
used by 41% of 1239 physical therapists interviewed in the United Kingdom
74
. Clarke et
al.
75
reported results of a randomized trial of 2177 patients, approximately half of which
received traction. There were no significant differences in the short or long-term
outcomes of continuous or intermittent traction and placebo, with or without sciatica.
20

Many questions still remain regarding the exact application of exercise. Mannion
maintains that an exercise program must target the special needs of the patient and be
balanced with the healthcare budget of the provider
76
. Hayden et al.
70
demonstrated that
individually designed exercise programs improved outcomes for non-specific chronic
lower back pain patients over a 12 week period. Hurwitz et al.
65
followed 681 patients
with lower back pain for 18 months and found that focusing on recreational physical
activity rather than specific lower back exercises was more effective at reducing pain and
increasing psychological health. Linton et al.
77
studied the effects of combining physical
therapy with cognitive behavioral therapy on back pain related sick leave. They reported
no significant differences in the recovery of patients who received cognitive behavioral
therapy in addition to physical therapy compared to patients who received physical
therapy alone.

If symptoms do not improve over 4 to 6 weeks, pharmacologic pain control can be
considered based on symptoms and history
66
. Muscle relaxants and oral nonsteriodal
anti-inflammatory drugs are often prescribed. The nonsteriodal anti-inflammatory drugs
are prescribed at a much higher frequency, however
78
. They block prostaglandin
production and reduce inflammation, effectively relieving pain. Epidural anesthetic
injections are also used to minimize nerve irritation. A series of injections several weeks
apart may be needed
79
. The relief may last up to a few months. However, a risk
associated with this procedure is accidental puncture of the dural sac, leading to the
leakage of cerebral spinal fluid
62
.
21

Only after exhaustive conservative treatments should surgical interventions be employed.
In addition, surgery can only provide relief when the correct source of the pain is
identified. For this reason, unresolved persistent back pain requires repeated, in depth
evaluation. There are several diagnostic tests which can supplement a physical
examination. Radiographs can be used to identify breaks in the vertebral body or
posterior elements. They can also be used to detect spondylolisthesis, which is the
slippage of one vertebral body over the other. Computed topography (CT) scans and
magnetic resonance imaging (MRI) can be used to identify fractures, herniated discs,
tumors, and infections. Discography can allow for the identification of a painful disc by
following the injection of a contrast medium into the nucleus pulposus. Radiographic
imaging will show leakage during a herniation. In addition, low injection pressures will
invoke intense pain in degenerated discs
61
. Despite these tools, in 80% of patients, no
obvious cause of back pain can be found
80
.

2.4.2 Current Surgical Interventions

Many current surgical interventions for back pain fall under one of two categories:
decompression or stabilization procedures. Laminotomy, laminectomy, and discectomy
are decompression procedures
62
. They involve relieving pressure on the nerve elements
by excision of disc, bone, or ligament material
2
. In a laminotomy, the surgeon will
attempt to relieve nerve compression by making a small hole in the disc material to free
the nerve root. However, if this is not sufficient to free the nerve, a laminectomy can be
22
performed. This is the removal of a small part of the disc tissue, facet joints, or any
portions of the disc impinging on nerves. This procedure will provide relief as soon as
the inflammation subsides
79
.

A discectomy is used in cases where complete herniation has occurred. The portion of
the nucleus pulposus which is causing pressure on the nerve root is removed.
Discectomy is sometimes performed in conjunction with laminectomy. Discectomy can
be done via open, micro, or percutaneous routes or using a laser. In open discectomy, an
incision over the posterior midline is made, and the ligaments and muscles are moved in
order to access the affected disc. In microdiscectomy, this is done with a small incision.
Percutaneous discectomy involves the use of a cannula to suction out the offending
tissue, and the surgery is monitored with a fluoroscope
62
. Laser discectomy is similar to
percutaneous discectomy, however the tissue is removed with a laser, which burns the
tissue for its removal
81
.

To date, however, microdiscectomy has been used as the gold standard because no other
discectomy technique has been able to match or exceed its outcomes
66
. One popular
microdiscectomy system is the METRx (Medtronic Sofamor Danek, Memphis, TN).
The necessary incision with this system is less than one inch long and damage to muscles
is minimal compared to open discectomy. The surgery is made minimally invasive by
accessing the surgical area with a series of tubes, called dilators, which increase in
diameter. The tubes are sequentially inserted though the muscle to split the fibers,
creating an opening large enough through which the disc tissue can be removed
66
.
23

A review was performed on 873 consecutive patients who were treated for lumbar disc
herniation with the METRx system. The Oswestry disability index was used to quantify
pain. At 28 months post surgery, significant improvements were seen in the mean
preoperative and postoperative score. The average hospital stay was 4.8 days and it took
an average of 15 days to return to work. The average blood loss during the surgery was
44 mL
82
.

Wu et al.
83
performed one of the only studies which compares the outcome of
percutaneous discectomy to that of conservative treatment. Surgery was performed on
patients with herniations which were symptomatic for 6-12 weeks. At a 2 year follow up,
there were no clinically significant differences in pain intensity and quality of life
between the two groups. However, the discectomy group was associated with a more
rapid initial recovery.

Another study involved a 25 year or longer follow-up on patients who received
discectomy for lumber herniation. Patients who underwent surgery reported a higher
quality of life and less pain than non-surgically treated patients
84
. Despite these results,
there has been little other research into the long term effects of discectomy
85
, making
long term outcomes of the treatment questionable
86
. While the procedure is successful in
relieving pain after surgery due to nerve root decompression, it does not restore the
healthy biomechanics to the disc. The degenerative cascade can therefore continue, and
has been reported to be accelerated due to alterations made to the disc
87-89
.
24

Vertebroplasty, kyphoplasty, and spinal fusion are stabilization procedures.
Vertebroplasty was developed in France in the 1980s, but it was not used in the United
States until 1994
90
. It involves the injection of bone cement into an area of vertebral
compression facture. By filling in a bone defect in the vertebral body, the vertebral body
is stabilized because mechanical support is rapidly regained. A CT scan is used to verify
that the void is completely filled
62
.

Generally, curing poly(methylmethacrylate) is used as the augmentation material
62,91
.
The bone will not bond with the polymer and it will remain a foreign body in the
vertebral body
91
. Consequently, the patient may experience fever and increased pain
from the inflammatory response
62
. It is also possible to sustain tissue damage due to the
exothermic polymerization reaction
91
. Calcium phosphates have also been investigated
as a bone replacement material because it is biocompatible, osteoconductive,
nonexothermic and injectable
92
. However, a bone substitute in the vertebral body must
be able to withstand cyclic loading, so there are concerns over the brittleness of calcium
phosphate cements
93
. Wilke et al.
91
investigated the fatigue behavior of calcium
phosphate cement and poly(methylmethacrylate) in intact human osteoporotic specimens
over 100,000 loading cycles. Both of the cements exhibited decreases in height with
increasing number of load cycles. The poly(methylmethacrylate) lost 2.8 mm, while the
calcium phosphate ceramic lost 4.2 mm. Cryosections revealed the presence of small
cracks in the calcium phosphate, but there were no signs of fatigue for the
poly(methylmethacrylate).
25

After vertebroplasty, patients have reported a decrease in pain and increase in function
90,94
. However, for patients with malignancies, vertebroplasty has been shown to offer
adequate vertebral stabilization, but inadequate pain relief
90
. In addition, Berlemann and
Polikeit et al. both found that vertebroplasty increases stress in adjacent vertebrae,
leading to fracture
95,96
. Another drawback to vertebroplasty is that vertebral height is not
completely restored
62
. Kyphoplasty involves the percutaneous insertion of a balloon into
a vertebral defect. The balloon is inflated to compress the cancellous bone in order to
create a cavity into which bone cement can be injected, thereby restoring height to the
vertebral body
97,98
. Kyphoplasty and vertebroplasty have been shown to provide similar
pain relief.
99
. A correlation between vertebral body height restoration and pain relief
was not found by Kasperk et al.
99
However, a higher rate of cement leakage was
reported for vertebroplasty. The rate of other adverse events, such as pulmonary
embolism or neurological injury, was low for both procedures
99
.

Spinal fusion, another stabilization procedure, is one of the most rapidly increasing forms
of inpatient surgery in the United States. Currently, it is recognized as a treatment for
degenerated discs and segmental instability
100
. Between 1979 and 1990, the rate of
lumbar spinal fusion doubled
101
. Then, in the 1990s, the rate tripled
102
. Possible
reasons for this are changes in population, technological advances in the surgical
methods, uncertainty about the indications, and financial incentives for doctors and
hospitals
103
.

26
The basic idea behind fusion is to use arthrodesis to prevent motion across the pain
generating disc. The disc material of the affected segment is removed, and the surface of
the two opposing vertebral bodies are roughened and packed with bone material. This
fills the gap between bones, allowing them to grow into a single segment.
3,103,104
.

Today, metal implants are often used to stabilize the vertebrae until the fusion solidifies
62
. However, uninstrumented fusions have been performed, and are indicated for spine
conditions where there is no instability
104
. In these fusions, bone grafts are place on the
laminae, spinous processes, or transverse processes in order to eliminate motion. The
patient would be required to wear a brace to provide additional stabilization to the
segment
104
.

Reported rates of internal fixation with instrumentation doubled from the 1980s to the
1990s
105
. The most popular implants for internal fixation are metal pedicle screws
103
.
Using pedicle screws to stabilize the vertebral body provides significant immobilization,
allowing for complete arthrodesis to occur
106-108
. Wood et al. concluded that patients
with degenerative discs who undergo uninstrumented fusion are 24 times more likely to
have pseudoarthrosis
109
, or incomplete fusions. The screws also provide continued
support after the maturation of the fusion
110
. One disadvantage of pedicle screws is that
less space is made available for the bone graft material
104
. An alternative method is to
use facet screws for fixation. Here a screw is used to transverse the facet joints. While
the procedure for using facet screws is more technically challenging than pedicle screws,
27
there is less need for soft tissue dissection. High fusion rates have also been reported for
facet screws
111
.

There has been some debate about the clinical value of internal fixation with screws.
There have been several studies indicating that, while it may help accelerate fusion rates,
there has been little impact on the clinical results of fusion
112-115
. For instance, Thomsen
et al conducted a randomized clinical trial which showed no significant differences in the
functional outcomes of patients who received fusions with or without pedicle screw
supplements
114
Moller et al. showed that the level of pain between the two groups 2
years after surgery was very similar
112
. On the other hand, there have been other studies
which showed a close relation between fusion rate and clinical outcome. One group
reported an 80% clinical success rate and 80% successful fusion
116
. Another group
reported a 73% successful fusion rate and a 74% clinical success rate
117
.

Fusion rates in the United States rose 77% between 1996 and 2001. This is because in
1996 the Food and Drug Administration (FDA) approved intervertebral fusion cages
(Figure 2.5)
102
. Fusion cages are another alternative to providing stabilization to the
fused segment. The advent of fusion cages reduced the incidence of graft collapse
because they provide mechanical support while the bone matures
100
. The bone graft is
placed inside the cage
3,118
. Cages are also associated with improved fusion rates over
screw fixation and uninstrumented fusions
119,120
. The high stiffness of some metal cages
can cause endplate subsidence
3
, therefore titanium or carbon cages have been
28
recommended
100,118
. Carbon cages have an advantage over metal cages because they do
not interfere with radiographs, tomography scans, or MRI
100
.


Spinal segments can be fused anteriorly, posteriorly, or circumferentially (360 fusion)..
The method employed will depend on the area of the defect. In anterior lumbar fusions,
the surgeon retracts the abdominal contents and pulls the aorta and vena cava to the right
side of the spine in order to access the anterior spinal area. The anterior approach is
advantageous because the back muscles and nerves remain undisturbed, resulting in
lower recovery time. In addition, Brady et al. maintain that fusing the anterior spine
produces better results when the anterior portion of the disc is compressed
118
. Still, the
posterior approach has also gained wide acceptance as a method for lumbar fusion. It
allows for the decompression of neural elements via a posterior midline incision
118
.
While both of these approaches have had clinical success
122-125
, there is still debate
regarding the effectiveness of an isolated arthrodesis because it may not totally eliminate
motion through the segment
126
. Posterior lumbar fusion can be supplemented by the
addition of a posterolateral fusion, creating a 360 circumferential fusion
100
. All of these
techniques may or may not be assisted with internal fixation implants
100
.

The bone graft used in spinal fusions can either be an autograft or allograft. Autografts
are bone that is usually removed from the patients iliac crest, either requiring an
additional incision or tunneling under the skin to reach the patients hip. Allograft is
obtained from a donor cadaver. The bone is ground and applied over the area to be fused.
29
Autografts are generally considered the golden standard because of the availability and
minimal risk of rejection. However, many patients elect to use bone from a donor
because of the pain associated with an autograft
127
. Patients have reported donor site
pain to be greater than that of the spinal incision
62
and it can persist for years after the
surgery
127
. It takes between three months and two years for the fusion to become solid
127
.

The usage of osteoinductive agents can enhance clinical outcomes for patients. Bone
morphogenic protein (BMP) can be mixed with the bone in order to induce bone growth
127
. Usage of these materials can also eliminate the need for autogenous bone harvesting
all together
104
. Recombinant BMP (rh-BMP) has been reported to give superior results
to autogenous bone grafts and more rapid clinical improvement
121,128,129
. Berkus et al.
followed 46 patients who received spinal fusions with rh-BMP-2 threaded with cortical
allograft. Clinical results were compared to patients who received autograft from the
iliac crest. At 12 and 24 months, patients in the investigation group had higher rates of
fusion and there were no unanticipated adverse events related to the rh-BMP-2.

While spinal fusion undoubtedly provides pain relief for some patients, its efficacy for
treating degenerative disc disease remains unclear
103
. Mofidi et al.
100
surveyed 65
patients who received posterior lumbar fusion with carbon cages between 1993 and 2000.
Clinical outcomes were assessed by measuring the postoperative Oswestry Disability
Index, return to work, and overall satisfaction with the outcome. Approximately four
years after the surgery, there was an 84% satisfaction rate and 61% of the patients were
30
able to return to pre-disease activity level, and there was a significant improvement in the
Oswestry Disability Index. In another prospective assessment, 35 patients who received
360 fusion with screw fixation were asked about their outcome approximately 31 months
after surgery. There was 71% overall satisfaction rate, but only 28.6% rated their
outcome good or excellent. There was also a 75% reduction in medication usage
130
.

The UK Medical Research Council followed 349 patients with a 12 month history of
chronic low back pain. Approximately half of the patients received a fusion, though the
surgical approach and instrumentation varied among patients. The other half received an
intensive rehabilitation program for three weeks in addition to cognitive behavioral
therapy. At 24 months after the surgery, the patients who received fusion had slightly
better Oswestry scores than patients who participated in the rehabilitation program, but
the groups did not differ in any other outcomes (anxiety, depression, or any adverse
effects)
131
. In Norway, a similar study was done where 64 patients were studied after
either receiving 360 fusion with pedicle screws or 3 weeks of physical exercises and
cognitive behavioral treatment for lumbar degeneration. No differences were found
between the Oswestry Disability Index for both groups one year after the surgery
132
.

The Swedish lumbar Spine Study Group did a similar study where they followed 249
patients with chronic lower back pain and radiological evidence of lumbar degeneration.
A 2 year study was done on a non surgical group, who received a variety of conservative
treatments such as physical therapy, acupuncture, electrical nerve stimulation, and
cognitive behavioral therapy, and a surgical group, who received three different fusion
31
techniques (posterior, anterior, and circumferential). Initially, the surgical group reported
more pain relief and a quicker return to work. Pain and functioning improved by 33% for
the surgical group, versus 7% for the non-surgical group. At the 2 year follow-up,
however, the improvement gradually deteriorated for the surgical group and only 63% of
the patients who received fusions considered themselves better or much better
133
.

Spinal fusion is also associated with several surgical complications such as infection
100
,
nerve injury
114
, and high blood loss
124
. Complications at the donor site and vascular
complications have also been reported
134
. In addition, a Cochrane review of randomized
controlled trials on the surgical treatment of lumbar disc degeneration concluded that
there is no evidence for the efficacy of fusion for the treatment of spinal instability
135
.

Like with discectomy, the long term results of fusion are questionable. Several
biomechanical studies have shown that fusion causes increased stress to be experienced
by spinal segments adjacent to the fusion site
126,136-139
. This promotes degeneration of
these segments and the cycle leading to back pain begins again. Miyakoshi et al.
139

found that all intervertebral disc heights adjacent to the site of fusion decreased. Chen et
al.
140
used finite element modeling to confirm that the largest stress after fusion is placed
on the disc adjacent to the surgical site. Then, Chosa et al.
141
showed specifically that
the stress increase in these discs is concentrated on the vertebral endplate and annulus
during flexion and extension loadings. They also showed that firm fusions increased the
stress imposed on adjacent vertebral segments. This has also been seen experimentally
by other researchers
142
.
32

Theoretically, replacing the diseased disc with a prosthesis would have an advantage over
fusion because it could more closely mimic the loading and motion characteristics of a
healthy spine
143
. It is hypothesized that motion preservation will decrease stress on
adjacent segments, leading to favorable long term results
142,144
. Total disc replacement is
therefore emerging as a viable alternative to fusion since it can be used to treat the same
symptomatic pathology as fusion. In addition to allowing for the removal of the painful
disc, restoring disc height, and improving stability, proponents of this method say a
prosthetic disc may help restore a healthy pattern of load bearing to the spine
144
.

The artificial disc with the longest clinical history is the Charit artificial disc. The
prosthesis was developed in the early 1980s and it consisted of a polyethylene core
sandwiched between to metal endplates. Since then, there have been three revisions to
the implant, in order to minimize complications such as subsidence and fatigue failure.
The current generation of the product is called the Type III SB Charit or the LINK SB
Charit (Depuy Spine, Johnson & Johnson, Raynham, MA) (Figure 2.6)
145
. This version
of the Charit was the first total disc implant for the lumbar spine to gain approval from
the FDA
144
. It is indicated for patients between the ages of 18-60 with symptomatic
single-level degeneration and failure to achieve pain relief for at least six months.
Patients who have spinal fracture, bone disease, or spinal tumors should not receive the
implant
143
.

33
The implant consists of two concave metal endplates made from a cobalt chromium
molybdenum alloy. The surfaces of the endplates facing the vertebral endplate are
covered in porous titanium and coated with calcium phosphate to encourage bonding with
the bone. In addition, the endplate surfaces also contain six teeth which are used to
physically anchor the implant into the vertebral body. Encased between the two
endplates, there is a free-floating biconvex sliding core made of ultra-high-molecular-
weight polyethylene (Figure 2.7). The mechanical behavior of the core mimics the major
movements of the intervertebral segment: flexion, extension, and translation
146
.

The technique for the surgical implantation of all artificial discs is very similar to anterior
lumbar fusion
144
. An anterior midline incision, approximately 5 cm in length, is made in
order to access the spine and remove the diseased disc tissue. A spreader instrument is
used to distract the disc space. The surfaces of the vertebral endplates are smoothed in
order to maximize contact area with the prosthesis. The artificial endplates are then
positioned so that the inner surfaces of the plates are parallel and then they are implanted
with the aid of a mallet to secure the teeth in the bone. The polyethylene core is then
inserted in place between the endplates
143
.

An FDA regulated prospective, randomized study was performed for the purpose of
gaining FDA approval for the Charit. The objective of the study was to compare the
safety and effectiveness of the Charit artificial disc to anterior lumbar fusion for the
treatment of single level lumbar degeneration and determine if there exists a correlation
between implant placement and clinical outcome. A total of 304 patients were followed
34
over 24 months, 205 being in the investigational group, and the remainder receiving
fusion with a BAK cage and an iliac crest bone graft
147,148
. Overall, patients in both
groups improved significantly after surgery. However, patients who received the Charit
recovered faster, had lower disability levels, and statistically lower pain scores. The
hospital stay was also significantly shorter for the disc replacement group
147
. Patients in
the investigational group had a 13.6% mean increase in mean flexion/extension ROM,
while the fusion group had an 82.5% decrease. The investigational group also had
significantly better restoration of disc height and less subsidence. The flexion/extension
ROM and prosthesis function improved with surgical technical accuracy of placement.
At the 24 month follow up, no implant wear and creep was found
148
.

The study has received major criticism because the reference procedure, fusion with the
BAK cage, has a very poor clinical history and is rarely used by surgeons due to its high
failure rate
149
. This control was chosen because it was the only FDA approved anterior
cage at the time of the study. Currently there are other cages available that could have
produced better scores for the fusion group
150
. It has also been suggested that the claim
that the Charit disc produces less subsidence is not a valid argument for the superiority
of disc replacement. The BAK cage rests on the vertebral endplate, and because the area
of the cage is small, this produces a stress point that leads to subsidence. The Charit
endplates are much larger, so it is only logical that less subsidence would occur
150
. In
addition, despite the improvements in pain scores for both groups, 64% of the people in
the investigational group and 80% of the people in the fusion group remained on
narcotics 2 years after the surgery
147
.
35

While the purported advantage of disc replacement technology is the preservation of
adjacent vertebral segments, there was no conclusive evidence presented in the FDA
regulated study to support this hypothesis. Van Ooij et al.
142
reported on 27 patients who
received the Charit disc replacement and were suffering from complications. At the 53
month follow-up, 7 patients exhibited degeneration at another spinal level that was not
present prior to surgery, despite accurate placement of the prosthesis. The authors
conjecture that it is unlikely that this degeneration was simply caused by the degenerative
disease itself. They maintain that it is highly unlikely that the Charit or other disc
replacements can completely mimic the normal movement of the disc, making adjacent
segment degeneration inevitable. Physiologically, the center of rotation in normally
functioning segments is positioned posteriorly in the disc, and in the Charit this is
centrally or anteriorly located
142
. In addition, the authors claim that the metal and
polyethylene do not possess the same shock absorbing capacity as the healthy disc.

Van Ooij et al.
142
also reported 16 cases of subsidence into the vertebral bone. Because
the central endplate is weaker than the outer rim, the metal plates of the implant must be
large enough to rest on the periphery of the endplate. Several of the patients who
experienced subsidence had too small of a prosthesis inserted. However, a disadvantage
to larger plates is that there is a greater chance of nerve impingement. Because a great
many blood vessels must be mobilized for correct insertion of the implant, this poses
bleeding and thromboembolic risks
151
, which increase with implant size
142
. Van Ooij et
al. also reported anterior migration of the implant, causing iliac vein compression
142
.
36

The ProDisc-L (Synthes Spine, West Chester, PA) (Figure 2.8) was developed in the late
1980s and the second generation, the Prodisc, has been available since 1999
144
. The
implant consists of two metal endplates made from cobalt chromium molybdenum alloy,
the surface of which is coated with porous titanium. Implant stability is achieved by a
spike located on each endplate, called a keel
144
. During surgery, keel cuts in the bone are
made with a chisel. The ultra-high-molecular-weight polyethylene core is snapped into
the lower endplate during the operation after endplate distraction
152
. Unlike the Charit,
which has a free floating biconvex sliding core, the Prodisc inlay does not move, thus the
center of rotation is fixed
153
. In 2006, the FDA approved the ProDisc-L for lumbar disc
replacement in skeletally mature patients with discogenic back pain and degeneration.

Chung et al.
154
reported positive clinical outcomes for 36 patients who received a lumbar
total disc replacement with the ProDisc. The mean score for lower back pain improved
significantly and 7 out of 10 preoperatively unemployed patients were able to return to
work. The Oswestry disability index scores also improved. Mean range of motion and
disc height also significantly increased. There were no significant complications in this
study. Similar results were reported by Bertagnoli et al.
152
. At 3 months
postoperatively, statistical improvements were reported in Oswestry scores and patient
satisfaction, which were maintained at 24 months. There were increases in disc height
and range of motion. There were no significant complications or the need for any
surgical revisions. Mayer et. al.
155
followed 37 patients postoperatively after receiving
the ProDisc prosthesis. After an average of 5.8 months, 76% of the patients had no low
37
back pain. In addition, 61% of the patients were completely satisfied and 21.7% were
satisfied. Radiographs showed no changes in function of the implant over time and
there were no incidences of subsidence.

Zigler et al.
156
report the early results of an ongoing FDA trial at 19 US centers. One
report concerns 39 patients at a center in Plano, TX who received either the Prodisc or a
360 lumbar fusion. The fusion was a more substantial operation involving anterior
fusion with femoral allograft followed by posterior fixation with pedicle screws and an
iliac bone graft. It is not surprising, then, that lower blood loss, shorter operating times,
and briefer hospital stays were reported for the disc replacement group. Zigler et al. also
reports that, at three months postoperatively, the Oswestry disability scores were
significantly lower for the disc replacement group and they exhibited significantly
improved range of motion.

Delamarter et al. reported 30 month follow-up data on 78 patients in Santa Monica, CA,
who were participating in the same FDA trial. Early results were similar to those
described above; the disc replacement group had an early advantage in pain and recovery.
However, at 12, 18, and 24 months, there were no significant differences in the pain,
disability or mobility for the two groups
157
.

The complications associated with surgical implantation of the ProDisc appear similar to
those associated with the Charit. Those include nerve root impingement
155
, endplate
subsidence
158
, malpositioning
158
, dislodgement of the polyethylene core
155,156
, and iliac
38
vein laceration
156
. Shim et al.
159
reported a split fracture of the vertebral body, which
was attributed to the keel design of the ProDisc. In the authors opinion, the need for
chiseling increases the risk of vertebral fracture, and the height of the keel is
unnecessarily high, especially for Asian people who have smaller vertebral bodies than
Caucasians.

There are other total disc replacements besides the Charit and ProDisc under
investigation, though the literature data concerning them is sparser. The Maverick disc
(Medtronic, Minneapolis, MN) (Figure 2.9) has similar design to the ProDisc, but is a
metal-on-metal implant. It consists of two flat metal endplates with a ball and socket-
joint sandwiched between them. The center of rotation is fixed in the posterior of the
disc space. Stabilization in the vertebral bones is achieved by two large keels, similar to
those on the Prodisc. Due to the height of the keels, usage of the disc may be limited to
larger vertebral bodies
144
. Barzilay et al.
160
reported 1 year follow-up results on the first
30 consecutive patients to receive single level Maverick discs. At 6 months and 1 year,
patients experienced an improvement in back pain and achieved a normal range of
motion. There were no major device related complications. The Flexicore disc (Stryker
Corp, Kalamazoo, MI), is also a metal-on-metal implant with a ball-and-socket joint.
The endplates are dome-shaped to adapt to the concavity of the endplates. Because of
this shape, the implant can be implanted either anteriorly or anterolaterally. Also, the
device is implanted as one single unit
144
.
39

2.4.3 Nucleus Pulposus Replacement

Based on the clinical outcomes of spinal fusion and total disc replacement, it appears that
re-establishing the healthy biomechanics of the degenerated spine is still a challenge that
needs to be realized by spine medicine. Other than total disc replacement, another non-
fusion alternative being investigated is the replacement of the nucleus pulposus alone. A
synthetic nucleus replacement could be designed to recreate the biomechanical function
of the healthy nucleus pulposus by applying tension to the annulus under compressive
loads
161,162
. In doing so, a nucleus replacement would restore stability to the spinal
segment
162
. In addition, motion could be preserved and disc height could also be
restored
161-163
, the latter helping to lessen compressive forces on the facet joints
163,164
.
Further tearing and nerve irritation could be prevented or postponed by the nucleus
replacement relieving or lessening the shear forces on the annulus
165
. In addition, the
implantation procedure for a nucleus replacement has the potential to be less invasive
than a total disc replacement or spinal fusion
166
, so the morbidity associated with those
surgeries can be avoided.

There are no clear standards establishing the degree of degeneration for which nucleus
replacement is acceptable
167
. Some propose that nucleus replacement be intended for
people in the earlier stages of degeneration, when the annulus is still intact
168
and there
have been no previous operations
167
. Proponents of this philosophy view nucleus
replacement as an early therapy, rather than a replacement for spinal fusion or a
40
prosthetic disc
161
. An intact annulus would be better able to contain the implant,
reducing the risk of migration
163
. This is especially important for in situ forming nucleus
replacement materials, which are injected as liquid and form a solid in the body. A
competent annulus is necessary to prevent the spread of liquid beyond its periphery
169
.

The problem with this approach is that by the time discogenic symptoms occur, the
annulus may already be in the later stages of degeneration
167
. Nucleus replacement has
also been proposed as an adjunct to discectomy or nucleotomy. Removing offending disc
material and filling the space with a synthetic material could help to restore function to
the disrupted disc
168
. The nucleus replacement would lessen the load on the remaining
disc tissues and transfer compressive forces to the annulus in tension again, preventing or
postponing future degeneration. However, the disc height must still be sufficient to allow
insertion of the implant
163
. One major consideration to be taken into account when using
nucleus replacements for this indication is that, by the time the patient becomes
symptomatic, nerve impingement may not be the only cause of back pain. Pain can be
generated by inflammatory mediators which can access and stimulate the nociceptors in
the tears of the outer annulus. Mayer et al. maintains that replacing the nucleus in these
situations would not bring relief to the patient, because the annulus fibrosis will remain
insufficient
167
. However, if a soft tissue adhesive were used to close the annular fissures,
and no vertebral fractures are present, the patient theoretically should not feel this pain
anymore, because inflammatory chemical mediators would not be able to reach the
nociceptors in the outer annulus.

41
There are several material requirements for a synthetic nucleus replacement. First, it
must be able to endure cyclic fatigue without failure or formation of significant particle
debris
163,166,169
. An implant is expected to withstand approximately 100 million cycles
over 40 years
170
. It must also possess elastic characteristics which would allow for shape
memory under repetitive loading. The stiffness of the material must be such that the load
distribution on the endplates and vertebral bodies will not cause subsidence or stress
shielding, leading to bone resorption
47,169
. The material should completely fill the disc
space in order to avoid excessive movement of the implant
169
. In addition, intimate
contact between a nucleus replacement and the inner annulus is necessary for the full
restoration of biomechanical function
171
. Furthermore, the implant should be delivered
to disc non-invasively, with minimal tissue trauma, which would reduce the risk of
implant migration or expulsion
166,169
.

Currently, the most widely studied material candidates for nucleus replacement are
hydrogels. Hydrogel-based materials have been investigated for this application for over
15 years
168
. In the literature, these three dimensional hydrated polymer networks have
been favored for nucleus replacement because they have the ability to exude water under
loads and re-imbibe it when the load the released
161,172
. Authors often note that this
behavior is similar to the healthy nucleus pulposus
161,166,169
. Under sustained loading,
however, the nucleus only undergoes a gradual fluid loss
173
. To more closely mimic the
mechanical behavior of the disc, it may be favorable to design a material with a low
hydraulic permeability, which would allow it to maintain a hydrostatic pressure on the
annulus under sustained loading.
42

Currently, hydrogel materials for nucleus pulposus replacement can be categorized as
either preformed or in situ curing. Preformed hydrogels are of a predetermined size and
shape. In order to minimize the invasiveness of the implantation procedure, preformed
hydrogels are often implanted in a dehydrated or partially dehydrated state. They then
expand in the nuclear cavity due to the presence of physiological fluids. The main
challenge associated with using preformed materials is matching the final size and shape
of the implant to the cavity in the disc
169
.

The largest clinical experience with a preformed hydrogel nucleus replacement comes
from the Prosthetic Disc Nucleus (PDN) (Raymedica, Inc., Bloomington, MN). This
copolymer hydrogel is composed of polyacrylamide and polyacrylonitrile, encased in a
polyethylene jacket which constrains the swelling of the hydrogel pellet, reducing risk of
endplate fracture
174
. Earlier versions of the device were implanted as two separate units;
a tapered anterior unit and a rectangular posterior unit. The two-unit system allowed the
device to be implanted less invasively than a larger single unit. The two units are
connected with a tethering suture in the disc
162
(Figure 2.10). Currently, the
invasiveness of the implantation is thought to fall between a discectomy and fusion or
total disc replacement
162
. The product is indicated for patients with degenerative disc
disease and at least 5mm residual disc height. Patients with annular defects are excluded
as candidates for receiving the PDN
162
.

43
Biocompatibility testing of the device revealed no systemic toxicity, carcinogenicity, or
genotoxicity
175
. Scanning and transmission electron microscope examinations
demonstrate no evidence of a giant-cell reaction or evidence of wear debris
176
. Fatigue
testing revealed that the device is able to continue functioning as designed after 50
million cycles
175
. In addition, Eysel et al.
177
implanted the PDN in nucleotomized
cadaveric lumbar spinal segments and found that the device restored disc height and
stabilized the motion segment to the intact pre-nucleotomized condition.

In 1996, the first feasibility study for the PDN device was conducted. Twenty four
patients received the implant. With the hydrogel pillow positioned perpendicular to the
anteroposterior dimension of the disc, a success rate of 83% was seen. Then, a second
series of studies were performed on 17 additional patients in 1997 and 1998. They tested
an angle shaped hydrogel which was meant to conform better to the concavity of the
endplates. They also used a softer polymer to reduce stress on the endplates.
Unfortunately, this version only produced a 62% success rate. There was a high rate of
device expulsion and several revision surgeries to remove the implant and perform a
fusion. In 1998-1999, a new trapezoid shaped model, designed to reduce the migration
rate, was implanted in 26 patients. The success rate improved to 79%
162
.

In 1999, surgical instrumentation was modified to facilitate implantation of the device. A
series of dilators were developed to help stretch the annulus fibers, rather than cut them.
Fluoroscopic imaging was used to ensure proper placement of the device. A more
44
restrictive post-operative procedure was developed which required patients to wear a
brace for 6 weeks. With these changes in place, the success rate increased to 91%
162
.

The clinical results have been encouraging. After four years, patients report significant
improvements in Oswestry scores. Disc heights increased and implanted segments were
stabilized
162
. However, there was still problems with device migration, the average
migration rate being 12 percent
178
. For this reason, a new surgical technique was
introduced. While the device was traditionally implanted via a posterior approach,
similar to discectomy, Bertagnoli et al.
179
developed an anterolateral transpsoatic
approach. An incision is made in the lateral region overlaying the disc, and the
lateroabdominal muscles are dissected. The disc can be accessed without disrupting the
posterior structures of the spine. A small study group of 8 patients were implanted with
this approach. While the Oswestry scores improved and disc heights increased, there was
an increase in the migration rate. The magnitude of the increase was not disclosed in the
report, but the authors note that it may have been an artifact of the small patient
population.

The Newcleus Spiral Implant (Centerpulse Orthopedics, Winterthur, Switzerland) has
also been implanted in humans as a treatment for the degenerative disc
163
. The implant
is a polycarbonate urethane having the shape of a memory coiling spiral (Figure 2.11).
The device is intended to be implanted into the disc after a discectomy, performed via a
microsurgical posterior approach. During the discectomy, the nucleus material is
removed to create space for the implant. The spiral can then be inserted into the disc
45
space through the same opening in the annulus created by the discectomy. The spiral is
loaded into a special insertion instrument and pressure is applied to advance it into the
disc. Gradually, a spiral forms in the disc space. The volume of the implant is not fixed.
When the cavity is filled, the surgeon can cut the spiral and close the wound
163
.

Fatigue tests were run up to 50 million cycles with no significant wear observed. The
device was also implanted into human cadaveric specimens. Flexion and extension were
measured in the intact condition, after the nucleotomy, and after insertion of the spiral.
After the nucleotomy, an increase in rotation and facet joint translation was seen
(decrease in spinal stability). After implantation, a return to the intact condition was seen
163
.

Five patients have been implanted with the Newcleus thus far. Low back pain decreased
postoperatively in all the patients. Four out the five people were able to return to
employment and there were no intraoperative or postoperative complications that
occurred. MRI pictures show no abnormal displacements. Five years post-implantation,
disc height and rotational mobility were maintained. Long term clinical studies are now
under way
163
.

The Aquarelle (Stryker Spine, Allendale, NJ) is another preformed nucleus pulposus
replacement. It is primarily composed of poly(vinyl alcohol). The implant is inserted
laterally or posteriorly through an annulotomy using a 4 to 5mm tapered cannula. The
46
implanted hydrogel contains approximately 80% water. It has shown good
biocompatibility in animal models and mechanical durability up to 40 million cycles
166
.

In situ forming hydrogels are injectable materials which can be delivered into the disc
space as a liquid through a cannula or needle and harden in the body
166
. This minimally
invasive approach reduces the risk of implant migration or expulsion
166,169
. These
materials can conform to the shape of the cavity, completely filling the disc space,
allowing for better stability of the vertebral segment
166
. The most common injectable
nucleus replacements are self-curing elastomers. The drawback to these systems is the
potential for leakage of unreacted monomer into the physiological environment. It is
critical that the polymerization reaches completion, eliminating the presence of toxic
leachables
180
. In addition, the concentration of unreacted monomer necessary to achieve
the mechanical strength necessary for a nucleus replacement is quite high
181
.

The DASCOR Disc Arthroplasty Device (Disc Dynamics, Inc., Eden Prairie, MN) is a
nucleus replacement consisting of self curing polyurethane. An 18C monomer mixture
is injected into the disc via a catheter and cures in 12-15 minutes to form an elastomer
168
.
The reaction mixture is contained in a polyurethane balloon
166
. The balloon also serves
to create a space in the disc to accommodate the injectable material
168
. The device has
been implanted in 16 patients in Europe, but the clinical results have not been published
yet
166
.

47
Also being developed is the Biodisc (Cryolife, Kennesaw, GA). It is an injectable protein
hydrogel based on Cryolifes surgical adhesive BioGlue. The Biodisc material, the
detailed components of which have not been reported, is injected into the disc space and
cures within 2 minutes. The polymerization process occurs with negligible exothermic
heat production. This produces a solid with properties similar to the natural nucleus
pulposus. Cadaver tests on calf lumbar segments showed that the posterior injection of
the hydrogels restored denucleated disc height and segment stability. Minimal reduction
in height was seen after 10 million loading cycles
182
.

Another injectable nucleus replacement device is the IDN (Injectable Disc Nucleus)
(Spine Wave, Shelton, CT). The hydrogel is composed of a synthetic silk-elastin
copolymer created through DNA bacterial synthesis fermentation. It is injected through a
needle and forms a gel within a few minutes with no measurable exotherm. Preliminary
testing indicates that the IDN is able to restore disc height under loads, and cadaveric
tests have shown no evidence of extrusion
168
.

2.5 Poly(N-isopropylacrylamide)
2.5.1 Thermodynamics of the Phase Transition

It is common for the miscibility of a polymer-solvent system to increase with increasing
temperature. Complete miscibility is reached at an upper critical solution temperature
(UCST). This phase behavior can be used to describe several binary mixtures, often in
48
systems composed of a polar liquid and paraffin hydrocarbon
183
. The miscibility of such
a system can be explained in terms of fundamental thermodynamics. A system tends to
minimize its free energy, which can be described by Equation 2.1. At low temperatures,
a system minimizes its free energy by minimizing its enthalpy. However, at high
temperatures, increasing the entropy by a small amount can result in a large decrease of
free energy of the system. At high temperatures, binary systems with a UCST tend to
maximize their compositional entropy by distributing the molecules randomly, therefore
forming a miscible mixture
184
.

G H T S = Equation 2.1

There are some polymer-solvent mixtures, however, which exhibit increased solubility
with decreasing temperature. This phase equilibrium results in a lower critical solution
temperature (LCST), below which complete miscibility occurs. LCST behavior occurs in
systems where both components have polar groups which interact through hydrogen
bonding at lower temperatures. This hydrogen bonding lowers the enthalpy of the
system, since molecules that are bound together are at a lower energy state than free
molecules. However, hydrogen bonds have very low orientational entropy because the
bonds between the polar groups on the polymer and the solvent can only be formed when
the molecules are in specific orientations. Hydrogen bonds have an angular spread of 10
degrees, so if the orientation of the molecules relative to one another changes by more
than 10 degrees, the bond will break. At increased temperatures, the hydrogen bonds are
not thermodynamically favored because of the high losses in orientational entropy. The
increase in molecular rotations caused by the temperature increase will break the
49
hydrogen bonds between the water and polymer. It will be replaced by van der Waals
attractions between like polymer chains. The solute then segregates itself from the
solvent, forming an immiscible phase. The enthalpy of the system is minimized by the
aggregation of like molecules, and this loss in enthalpy outweighs associated gains in
orientational entropy associated with this conformation. Some systems exhibit both
LCST and UCST behavior, meaning that the miscible phase may once again reappear at
higher temperatures. This is because the high orientational entropy associated with the
randomly distributed molecules minimizes the free energy of the system, causing
miscibility to be thermodynamically favored
184
.

Aqueous solutions of poly(N-isopropylacrylamide) (PNIPAAm) exhibit an LCST,
typically occurring around 32-34C
185,186
. The chemical structure of PNIPAAm is shown
in Figure 2.12. Below the LCST, water hydrates PNIPAAm chains by forming hydrogen
bonds with its polar amide and carbonyl groups. Above the transition temperature, the
polymer becomes hydrophobic, so its behavior is dominated by the isopropyl group and
backbone, resulting in a dramatic phase separation
187-189
. In linear PNIPAAm systems
which are soluble in water below the LCST, the phase transition will result in a sol to gel
transition. In crosslinked systems, hydrated PNIPAAm networks exhibit a swelling-
shrinking transition at the LCST
190
. It has also been observed that the phase transition of
PNIPAAm is reversible
191-193
.


50
2.5.2 LCST Determination

The first reported value of the LCST of PNIPAAm was in 1967 by Scarpa et al
194
. They
reported a dramatic precipitation of the polymer when a 2% solution was heated to 31C.
This temperature was determined by simple visual observation of the phase separation
upon heating. Because this method is simple and convenient, other researchers have
employed this method of LCST determination
195-197
, and it has become known as the
cloud point method
198
. Since then, efforts have been directed at developing more
quantitative techniques for cloud point determination, such as UV-Vis
spectrophotometry. Fundueanu et al.
199
measured the temperature dependence of the
absorbance at 450nm using a UV-Vis coupled to a temperature controller. The group
used 1 w/v% aqueous solutions of poly(N-isopropylacrylamide-co-methyl methacrylate-
co-methacrylic acid) and heated the solutions at 0.2C/min and 0.02C/min in the vicinity
of the LCST. The cloud point, found to be 36C, was taken as the temperature at the
inflection point of the absorbance versus temperature curve. This method of analysis is
consistent with that of Feil et al
200
.

Differential scanning calorimetry (DSC) is another very common method of LCST
determination. Heskins and Guillet
195
first reported that the transition temperature can
be detected on a DSC thermogram by an endothermic peak. The heat of phase
separation, AH, can also be determined from the thermogram. This is the energy required
to break the hydrogen bonds formed between water and the amide and carbonyl groups
on PNIPAAm
198,201
. Schild and Tirrell
187
reported a value of 6.3 kJ/mol for the
51
enthalpy, which is consistent with those found from other researchers
200,202,203
and in the
typical range for hydrogen bonded interactions
198
. The entropy changes associated with
the transition can also be calculated using Equation 2.1, where AG is taken as zero at the
transition
204
.

Schild and Tirrell
187
determined that LCST of aqueous PNIPAAm solutions determined
with DSC is consistent with results from cloud point measurements. Transition
temperatures were found to be independent of heating rate in the range below 30C/hr.
While the shape of the endotherm varied with molecular weight of the PNIPAAm, it was
independent of polymer concentration in water in range 0.4-4.0mg/mL. These results are
in contrast to those of Boutris et al.
201
, who varied the polymer concentration in water
between 0.5 and 22 wt%. There was a significant dependence of transition temperature
on polymer weight fraction in the range below 5 wt%; however the phase diagram tended
to plateau in the range of 15-20%. This group also compared the phase transition
temperatures obtained from DSC and cloud point measurements with UV. They reported
that the techniques yielded similar values, but those obtained from DSC were slightly
lower. This was attributed to the fact that DSC detects the energy change associated with
the destabilization of hydrogen bonds, which is the initial step in the phase separation
process. Cloud point techniques only detect the macroscopic phase separation which
follows.

In the literature, when DSC is used to detect the LCST of PNIPAAm, the temperature has
been defined as either the onset of the endotherm
200,205
or the temperature at the peak
52
minimum
187,200
. Boutris et al. reported that the peak minimum can shift to higher
temperatures with increasing sample mass, while the peak onset is not affected by this
variable. However, the peak onset can depend on the polydispersity of the polymer. For
these reasons, the group defined the transition temperature as the temperature
corresponding to the maximum of the first derivative of the thermogram
201
.

The LCST of PNIPAAm has also been studied through light scattering techniques. This
route may be considered advantageous over DSC and cloud point techniques because it
reveals information about the size of the polymer chains
206
. Light scattering will detect
the change in chain conformation from coil to globule as the polymer is heated above its
LCST
206,207
. Dilute solutions must be used in order to accurately measure the radius of
gyration of a single coil. Because the phase transition begins with the collapse of
individual chains, followed by chain aggregation, decreasing the concentration in solution
will decrease interchain aggregation, making the measured size changes for a single
polymer chain more precise
207
. Kubota et al. measured changes in the hydrodynamic
radius and radius of gyration of linear PNIPAAm chains of varying molecular weights
between 1.63E6 and 2.52E7 g/mol
206
. They found a sharp decrease in the dimensions of
the polymer at the LCST. They were also one of the first groups to report a
polydispersity for the polymers, 1.3-1.4, making the measured changes in chain
dimensions more reliable. Longer chains will precipitate first, perturbing experimental
results
207
.

53
There are a variety of other LCST characterization techniques that have been used in the
literature. The changes in solution viscosity associated with PNIPAAm chain
aggregation have been studied. Heskins and Guillet have reported a decrease in viscosity
with increasing temperature for dilute solutions
195
. IR spectroscopy has also been used
to detect changes in inter- and intramolecular bonding that occur at the LCST. A study
on aqueous PNIPAAm solutions using Fourier Transform Infrared Spectroscopy (FTIR)
revealed that the peak associated with the polymer hydrogen bond with water (C=OHO-
H) at 1625cm
-1
decreases as temperature increases. In addition, a peak at 1650cm
-1
,
representing intersegment hydrogen bonding (C=OH-N) appears at temperatures above
the LCST. This group also reported that the intersegmental hydrogen bonds act as
crosslinking points, allowing the chain aggregates to swell like a gel
208
.

2.5.3 LCST Modulation

Many studies have focused on modulating the LCST by modifying PNIPAAm with
hydrophilic, hydrophobic, or ionic comonomers. Acrylic acid is one of the most
commonly used comonomers for increasing the LCST of PNIPAAm. The LCST of
random block copolymers of PNIPAAm and acrylic acid were characterized by DSC and
cloud point techniques. The copolymers composed of 2 mol% acrylic acid exhibited a
3.6C increase in transition temperature compared to the PNIPAAm homopolymer.
Furthermore, the LCST of the copolymer composed of 7 mol% acrylic acid was found to
be approximately 60C
209
. The group asserts that this increase in transition temperature
is tied to increase in hydrophilicity of the copolymers due to the presence of acrylic acid.
54
As the temperature is raised, the hydrogen bonds between the water and polymer are
weakened, and eventually broken, when the polymer precipitates. When the hydrophilic
acrylic acid chains are present, these losses in hydrogen bonding are offset by the
hydrogen bonds between the acrylic acid units and the water. This results in an increase
in the LCST.

The above hypothesis was also put forth by Feil et al, who synthesized poly(NIPAAm-
co-butyl methacrylate) copolymers with butyl methacrylate (hydrophobic), acrylamide
(hydrophilic), and acrylic acid (anionic)
200
. It was found that the changes in LCST were
proportional to comonomer content. The increase in LCST was largest for the anionic
comonomer, followed by the cationic and hydrophilic comonomers. The group also
varied the charge density on the anionic and cationic copolymers by changing the pH of
the swelling medium. They reported an increase in LCST with increasing charge density,
and attributed this to the same mechanism by which hydrophilic components increase the
transition temperature.

Hoffman et al. reported similar increases in the LCST of PNIPAAm as a result of
copolymerization with acrylic acid
210
. In addition, Brazel et al. reported increases in the
LCST of PNIPAAm as a result of copolymerization with methacrylic acid
211
. David et.
al. created block copolymers of PNIPAAm and poly(n-acetylimino)ethylene. They
showed that increasing the chain length and frequency of the hydrophilic blocks
increased the LCST
212
.

55
By the same token, incorporating hydrophobic comonomers into PNIPAAm networks
decreases the LCST. Ma et al. showed that creating PNIPAAm copolymers with the
hydrophobic isopropyl methacrylate lowered the transition temperature. The
experimental results showed a linear correlation between the LCST and the amount (mol
%) of isopropyl methacrylate
213
. Xue et al. prepared hydrogels composed of PNIPAAm
with hydrophobic di-n-propylacrylamide, di-n-octylacrylamide, or di-dodecylacrylamide.
The group hypothesized that use of comonomers containing two alkyl chains would
impart stronger hydrophobic interactions to the polymer network. It was found that the
incorporation of the comonomers lowered the LCST of the PNIPAAm. However, the
temperature was independent of hydrophobic comonomer. Also characterized was the
transition temperature of gels formed from aqueous solutions containing the anionic
surfactant sodium dodecyl sulfate. The surfactant causes an appreciable increase in the
LCST of the polymer, this being attributed to electrostatic repulsions between polymer
chains due to the binding of the sodium dodecyl sulfate to the PNIPAAm chains
192
.

Shin et al. observed anomalous swelling behavior for an interpenetrating network of
PNIPAAm and hydrophilic poly(acrylic acid). They observed a decrease in the LCST of
PNIPAAm as a result of the formation of the interpenetrating network and hypothesized
that the free carbonyl groups on the PNIPAAm hydrogen bonded with the acid group on
the poly(acrylic acid), leaving less polar groups on the PNIPAAm available for hydrogen
bonding with water. This increased the overall hydrophobicity of the PNIPAAm,
lowering its LCST
214
.

56
Han and Bae
215
performed an extensive study on the precipitation of a high molecular
weight PNIPAAm block copolymer with 2 mol% acrylic acid The LCST of the
copolymer, determined by cloud point measurements, was 32C. Solutions composed of
5 wt% polymer were dissolved in phosphate-buffered saline (pH 7.4). The solutions,
which were initially clear, turned cloudy when heated to 27C. When the temperature was
increased to 35C, an immobile, bulk phase appeared. Upon heating to 43C, the solid
began to shrink due to the expulsion of water from the network. The authors noted that
this gel deformed without shape memory. In other words, it exhibited very poor elastic
properties. Based on these visual observations, it was speculated that polymer chains
with lower acrylic acid contents precipitated first, forming globules, but those with higher
acrylic acid contents remain in a partially collapsed conformation. The globules become
entangled with the partially collapsed chains, forming hydrophobic regions with physical
junctions
215
.

2.5.4 PNIPAAm-based Hydrogels

In the past several years, temperature sensitive and in situ-forming hydrogel systems have
gained extensive interest for biomedical and pharmaceutical applications. PNIPAAm-
based systems have been one of the most commonly proposed thermosensitive materials,
not only because its phase transition conveniently occurs between ambient and body
temperature, but because copolymers of NIPAAm with different types of monomers can
result in materials with a range of different properties. For instance, copolymers of
thermosensitive N-isopropylacrylamide and gelatin were prepared which form an elastic
57
hydrogel above 34C or below 10C
216
. In another study, functionalized PNIPAAm
chains were grafted onto alginate to form a comb-like polymeric structure which, when
heated above the LCST, formed extremely porous hydrogels, allowing for rapid
deswelling kinetics. These materials were investigated as a rapid stimuli-responsive drug
delivery system
217
.

Some researchers have investigated copolymeric hydrogels of NIPAAm with a
hydrophobic comonomer by characterizing the effect that the hydrophobic component
has on the swelling and mechanical properties of the hydrogels. Hydrogels composed of
PNIPAAm and hydrophobic comonomers 2,2,3,3,4,4,5,5-octafluoropentyl methacrylate
and n-butyl methacrylate were evaluated by Lee et al. It was hypothesized that the
presence of the hydrophobic comonomer would increase the deswelling kinetics of
hydrogel system, decrease the swelling capacity of the gel network, and increase the
mechanical properties. The group found that the gel swelling ratio decreased and the
stiffness increased with increasing content of the hydrophobic comonomer. This effect
was more pronounced in the hydrogels composed of NIPAAm and 2,2,3,3,4,4,5,5-
octafluoropentyl methacrylate, attributing this to the fact that the atomic size of the
fluorine is much larger than hydrogen, causing the molecular chain packing in these gels
to be much denser than that of the n-butyl methacrylate gels
218
.

Conversely, the incorporation of hydrophilic comonomers tends to increase the swelling
capacity of PNIPAAm networks. For example, PNIPAAm copolymers with dextran were
synthesized by free radical polymerization of NIPAAm and a dextran macromer
58
containing multiple hydrolytically degradable oligolactate-2-hydroxyethyl methacrylate
units. It was found that the swelling capacity of the gels was strongly dependent on
temperature and copolymer composition. While all of the copolymers exhibited
decreases in water content as the temperature was raised above the LCST, the swelling
ratios increased with increasing dextran content. This was attributed to overall increase
in hydrophilicity of the network due to the presence of the dextran. After six days
immersion in PBS, the gels began de-swelling at a higher rate, due to the degradation of
the dextran within the gel work, resulting in an overall increase in the hydrophobicity of
the polymer
219
.

In addition, Lin et al. investigated the structure-property relationship of block or star
copolymers of PNIPAAm and poly(ethylene glycol) (PEG) hydrogels. The PEG lengths
used in this study were approximately 2000 Da. These copolymers formed injectable
solutions at 5C and at physiological temperatures formed strongly associated networks.
The group reported that these networks were stabilized by either crosslinking, in the case
of the networks formed from multiple armed PEG, or micellar packing and
entanglements in the case of the linear block copolymers. The gelation kinetics of the
hydrogel system were rapid, occurring in less than one minute, and there was no
significant syneresis after 2 months. The networks formed from the four-armed PEG
exhibited the highest strength and deformability. These materials are being investigated
for use as a drug delivery or cell encapsulation system
193
.

59
The incorporation of ionizable groups into PNIPAAm hydrogels can improve the
swelling of the network more effectively than non-electrolyte hydrophilic components
190
. For instance, Shin et al. synthesized interpenetrating networks of PNIPAAm and
poly(acrylic acid). They found the swelling behavior to be highest above pH 5.3,
conditions under which poly(acrylic acid) is charged. They also found that the
compressive stiffness of the networks was directly related to the swelling of the network,
and therefore pH of the immersion media. Gels swelled at lower pH, such as 3, were
stiffer than those swelled at pH 4.6 and 5.3
214
.

In another study, block copolymers of PNIPAAm and acrylic acid were loosely
crosslinked with N-N-methylenebis(acrylamide), forming a transparent, pliable solution
at ambient temperatures. These gels possessed improved volume retention capabilities
compared to gel networks composed of the PNIPAAm homopolymer. This was
attributed to the fact that the acrylic acid containing networks exhibited 75% water
retention after 6 days immersion in phosphate buffered saline, whereas the homopolymer
network lost half of its water during that time period. The authors postulated that the
improved swelling properties of the acrylic acid containing networks were due to
repulsions of the COO
-
groups, whereas the homopolymer chains can collapse
substantially and expel pore water above the transition temperature
220
.

PNIPAAm-based hydrogels have also been investigated as three dimensional matrices for
cell encapsulation. The acrylic acid copolymers described above
220
were shown to
support chondrocyte viability, resulting in the formation of cartilage-like tissue inside the
60
matrix. Consequently, the hydrogels are a viable material for the repair of cartilage
defects. Cho et al. encapsulated human mesenchymal stem cells in a PNIPAAm matrix
grafted with chitosan. The cell-thermosensitive gel complex was injected into the
submucosal layer of the bladder of a rabbit, resulting in cartilage growth. This system
could be used as a treatment for vesicoureteral reflux or reflux esophagitis
221
.

Solutions of PNIPAAm were also proposed as an extracellular matrix for an artificial
pancreas. It has been shown that islets of Langerhans, groups of pancreatic cells that
produce and secrete hormones, need to be repeatedly replaced after implantation to avoid
malfunction. Solutions of PNIPAAm loaded with islets can be placed in a pouch and
implanted into diabetic patients. The PNIPAAm gel, formed at body temperature, has
been shown to immobilize islets in rats without impairing insulin secretion. Periodically,
the pouches can be cooled, allowing the solutions to be withdrawn, and refilled with a
fresh polymer/cell suspension
222-224
.

2.5.5 PNIPAAm-based Systems for Drug Delivery

PNIPAAm-based hydrogels can also be used to immobilize drugs, enzymes, antibodies,
and other biomolecules. The LCST phase transition can be used as trigger for drug
release. In many of these cases, therapeutics are loaded inside a swollen matrix at T<
LCST and is released when placed in a medium with T>LCST
225
. An initial burst
release of surface drug, or other biologically active agent, can occur. This is followed by
the formation of a dense skin on the outside of the hydrogel as a result of the temperature
61
gradient across the gel. Drug from within the matrix is squeezed out due to a buildup of
hydrostatic pressure inside the gel. Lastly, as PNIPAAm chains throughout the entire
network collapse, drug release can occur via a diffusion process through the newly
formed gel
225
.

Interestingly, the LCST phase transition can be used as an on or off switch for drug
release. Hoffman et al. have studied the release rate of methylene blue from copolymers
of NIPAAm and methacrylic acid, as well as from PNIPAAm homopolymer gels. In
both cases, above the LCST, there were two distinct regions of release where the amount
of methylene blue liberated was proportional to the square root of time. The first region
was the initial rapid release and it was followed by a slow Fickian diffusion through the
network at long times
225
. Gutowska et al.
226
synthesized PNIPAAm copolymers with
acrylic acid which were crosslinked with ethylene glycol dimethacrylate. They reported
a zero order release profile for acetaminophen at pH of 7.4, which was dictated by the
des-welling kinetics of the gel network. This release occurred as a response to heating
the polymer above its LCST, in contrast to the NIPAAm copolymer system with butyl
methacrylate studied by Bae et al
227
. They reported that the release of indomethacin
occurred at temperatures lower than the gel LCST. Above the LCST, the release was
inhibited by the formation of a dense outer layer on the polymer surface. The formation
of this layer was found to be controlled by the concentration of hydrophobic comonomer.
The presence of the hydrophilic component in the PNIPAAm copolymers with acrylic
acid prevented the dense skin formation and allowed for release above the phase
transition of the gel
226
.
62

Drug loading into, and release from, a hydrogel network will be controlled by pore
volume fraction, pore size and their interconnections, the size of the drug, interactions
between the polymer and the drug, among other factors
228
. Some research efforts have
focused controlling the release rate of certain drugs from PNIPAAM systems.
Specifically, efforts have been directed into understand how to reduce a burst release of
drug, which could lead to toxic levels of drug in the blood plasma
229
, as well as
modifying the system to prolong dosing of the drug.

For instance, Zhang et al.
230
prepared hydroxyl-functionalized glycerol poly(c-
caprolactone) based microspheres loaded with ovalbumin and physically entrapped them
within a crosslinked PNIPAAm hydrogel. At physiological temperature, the PNIPAAm
hydrogel alone release 90% of the ovalbumin within 24 hours, but only 40% of the model
drug was released after 60 days from the combined microsphere/hydrogel delivery
system. The release curve for the latter had a typical biphasic pattern, with an initial
burst release followed by a slow sustained release. Release from the combined system
was also examined at 22C, and the release characteristics were similar to those at 37C,
but the burst release was smaller in magnitude. This was attributed to the collapse of the
PNIPAAm chains extruding ovalbumin from the network. A more sustained release was
achieved at 37C because the collapsed PNIPAAm matrix slowed the release of the drug.

In another study, interpenetrating networks of temperature sensitive PNIPAAm and
hydrophobic poly(ethylene acrylate) were investigated for the release of hydrophobic
63
drug daidzein. The release profiles were compared to those from interpenetrating
networks composed of the PNIPAAm homopolymer. The poly(ethylene acrylate) created
hydrophobic reservoirs, present even below the LCST when the gel is in the hydrated
state. This increased the diffusion resistance of the drug through the network and slowed
its release rate, thereby avoiding the burst release seen with the homopolymer network
231
.

Wu et al.
228
evaluated the release of bovine serum albumin (BSA) and insulin from a
crosslinked PNIPAAm network. Drug loading was achieved by soaking the crosslinked
gels in aqueous solutions of the proteins for 4 days, and it was found that loading
decreased with increased network density, which limited the adsorption of the protein. It
was found that the insulin was released in its entirety within 48 hours, while the release
of the encapsulated bovine serum albumin was incomplete at the end of the study. The
group hypothesized that the release of the BSA was slowed by a strong protein-gel
interaction due to hydrogen bonding or hydrophobic interactions. Other researchers have
also reported an association between PNIPAAm and BSA whichcan be described as an
intramolecular complex of a PNIPAAm chain with several bound albumin molecules
232
.
Zhang et al. demonstrated that release bovine serum albumin from interpenetrating
polymer networks of PNIPAAm lowered the burst release compared to non-
interpenetrating PNIPAAm networks. This is due to the higher polymer density in the
interpenetrating network, inhibiting protein diffusion and increasing its interaction with
the BSA. In addition, they also noted an incomplete release of the albumin
233
.

64
Other studies on drug release from PNIPAAm networks have focused on the use of micro
or nanosized PNIPAAm-based gel carriers. Microgels composed of PNIPAAm
copolymers with acrylic acid were prepared. BSA was loaded into the microgels by
incubating them in a solution of BSA and phosphate buffered saline for 12 hours,
allowing the albumin to adsorb onto the particles. It was found that without the acrylic
acid component in the copolymer, albumin adsorption did occur, though in small
quantities. The authors maintain that the driving force for the adsorption was the
hydrogen bonding interaction occurring between the carboxyl groups on the albumin and
the amide groups on the PNIPAAm. However, adsorption was enhanced considerably by
the presence of acrylic acid, due to the affinity between the carboxyl groups on the
acrylic acid and the CONH- groups on the protein. BSA loading was maximum at pH
between 4 and 5, since the acrylic acid dissociates, initiating an electrostatic attraction
with the positive charges on the protein molecule
234
.

Another group studied the potential for using PNIPAAm-based nanogels to prevent
proteins against denaturation agents. Their studies demonstrated that nanospheres of
various sizes between 765 and 1146 nm composed of PNIPAAm crosslinked with PEG
dimethacrylate 400 and 1000 had the ability to protect insulin against high temperature.
After 12 hours at 80C, 25% of the insulin loaded into the nanoparticles could still be
detected by reverse phase high pressure liquid chromatography, compared to 0% for the
control, which was an insulin solution in phosphate buffered saline
235
.

65
Nanoparticles consisting of interpenetrating polymer networks of poly(acrylic acid) and
PNIPAAm were also investigated as a potential drug delivery system
236
. The
nanoparticles, loaded with dextran markers of various molecular weights, can be
dispersed in an aqueous medium at ambient temperatures and injected into the body,
where the dispersion becomes a gel as a result of the phase transition of PNIPAAm. The
resulting gel was described by the authors as a physically crosslinked system of
nanoparticles. The bonding between nanoparticles can be turned on or off using the
gelation characteristics of PNIPAAm. The release studies, conducted at 37C, indicated
that lower molecular weight dextrans were released from the network much faster than
the higher molecular weight markers. Decreasing the particle radius did not have a
significant impact on release.. Additionally, decreasing the weight percent of copolymer
in the particles increased the release rate due to the formation of pores through which the
model drug molecules could diffuse.

Hsieu et al.
237
studied two PNIPAAM-based systems designed for the controlled release
of ophthalmic agents for drug release. The first system was composed of linear
PNIPAAm chains, which formed an entangled network above 32C through which the
drug could diffuse. The second was a mixture of linear PNIPAAm chains combined with
physically entrapped crosslinked PNIPAAm nanoparticles. The intraocular pressure-
lowering effect was measured in order to evaluate the release characteristics of the
systems. It was found that the composite system produced a longer, sustained release.
The intraocular pressure drop lasted 32 hours for the composite system, compared to 24
66
for the linear PNIPAAm network. Both delivery systems produced an improved effect
over traditional eye drops, the pressure drop duration of which only lasts 6 hours.

One of the most interesting applications for nano and microscale PNIPAAm delivery
systems is targeted cancer treatment based on magnetic particles. Magnetite
nanoparticles were conjugated to functionalized PNIPAAm chains
238
. Because the
magnetite particles generate heat in response to an alternating magnetic field, the system
exhibited a temperature-induced aggregation in response to the magnetic field. In
addition, the nanoparticles also possessed free carboxyl groups, which could be used as a
conjugation point for drugs.

Muller-Schulte et al.
239
studied the release characteristics of a similar system. Magnetic
colloids and model drugs rhodamine B or methylene blue were encapsulated together
inside spherical micro and nanoparticles composed of crosslinked PNIPAAm. Those
model drugs were chosen due to similarities in molecular mass and structure to the
anticancer drug doxorubicin. When an alternating magnetic field was applied, the
particles were heated above the transition temperature of the PNIPAAm, due to heat
transfer between the magnetic colloid and the polymer matrix, resulting in the complete
release of the model drugs within 16 minutes, in concentrations that would be adequate
for cancer therapy. Methylene blue had a slightly slower release, attributed to an
interaction between the positive charge on the methylene blue and the negative charge on
the magnetite.

67
Nanoscale delivery systems composed on polymeric micelles have gained significant
importance in biomaterials over the last two decades
240
. In an aqueous environment,
amphiphilic block copolymers can form micelles, with the hydrophobic block becoming
the hydrophobic corona of the micelle, and they hydrophilic forming the outer shell, or
corona.. The highly hydrated corona of the micelle reduces recognition of the particle by
phagocytic cells, increasing circulation time in the bloodstream
241
. In addition, the
hydrophobic core can serve an environment for the incorporation hydrophobic drugs
241
,
which protected against external stresses by the outer shell. These hydrophobic drugs
would otherwise have a very low bioavailability in the body
240
.

Temperature-sensitive PNIPAAm copolymer-based micelles all function by a similar
mechanism. At ambient temperatures, hydrated PNIPAAm chains for the micelle corona,
while a hydrophobic comonomer, conjugated the PNIPAAm, stabilizes a hydrophobic
drug in the core. Drug release occurs as a result of increasing temperature, which causes
the PNIPAAm to become hydrophobic, causing structural changes in the polymeric
micelles. For instance, amphiphilic graft polyphosphazene, constructed with hydrophilic
PNIPAAm oligomers and hydrophobic ethyl 4-aminobenzoate side groups can assemble
into micelles at 25C. Above the LCST of PNIPAAm, occurring at 32.6 C, the micelles
aggregate due to interactions with neighboring PNPAAm chains, resulting in an abrupt
increase in the measured particle size
242
.

Temperature and pH responsive micelles were constructed from a diblock copolymer of
poly(t-butyl acrylate-co-acrylic acid) and PNIPAAm. At room temperature and pH 5.8,
68
the copolymers form micelles with the t-butyl acrylate as the core, and the hydrophilic
acrylic acid and PNIPAAm chains stretched out to form a two component shell. When
the temperature is raised from 25C to 32.4C, the LCST of the PNIPAAm, water
progressively becomes a poorer solvent for the PNIPAAm, so these chains collapse into
the core and the acrylic acid composes the corona. At this temperature, a significant
decrease in the hydrodynamic size distribution of the particles could be measured. As the
temperature is raised further to 50C, the size of the particles reaches a constant value
because the collapse of the PNIPAAm chains stop. In addition, with the temperature held
constant at 25C, and the pH lowered, the 3.0, the acrylic acid chains collapse to the core
and PNIPAAm becomes the corona. The solubility of the acrylic acid depends on the
degree of ionization and is enhanced at increasing pH values
243
.

2.6 Poly(ethylene glycol)

Poly(ethylene glycol) (PEG) is a hydrophilic, non-ionic, crystalline polymer. Its
chemical structure is shown in Figure 2.13. It is soluble in water via hydrogen bonding
interactions
244
. PEG is known for its biocompatibility. It has been approved by the FDA
for internal consumption
245
.

Because of its biocompatibility, PEG has received ample attention in the biomaterials
community of recent years. PEG is a component in several injectable biodegradable
polymer systems intended for drug delivery, such as ABA or BAB block copolymers,
where A is PEG and B is poly(DL-lactide-co-glycolide). These systems are soluble in
69
water just below room temperature due to the dominating behavior of hydrogen bonding
that exists between the PEG and water. At slightly higher temperatures, typically above
30C, the behavior of the hydrophobic poly(DL-lactide-co-glycolide) dominates,
allowing it to form associative physical crosslinks, causing a sol to gel transition. These
systems have been shown to be effective carriers for both hydrophilic and hydrophobic
drugs, such as 5-fluorouracil and indomethacin
246
. Other PEG based sol to gel systems
have also been investigated for biomedical applications, such as copolymers with
polyglycolide, c-caprolactone, sulfamethazine
247
, poly(butylene oxide)
248
, and
poly(propylene oxide)
249
.

In many cases, research has been directed at using PEG-based materials for scaffolds that
support cell growth and function. For instance, Sims et al.
250
developed a technique for
delivering a mixture of bovine chondrocytes and PEG subcutaneously into rats. The cells
were suspended in a 20 wt/vol% solution of PEG 100,000 g/mol. It was hypothesized
that the PEG could provide a biocompatible scaffold in which the chondrocytes could
proliferate and secrete extracellular components. The researchers saw an increase in the
glycosaminoglycan content of the rat cartilage. However, the PEG scaffold exhibited
rapid dissolution and poor mechanical properties
251
.

Subsequent work showed that the properties of these networks can be enhanced by the
preparation of crosslinked or interpenetrating PEG networks. For example, PEG
dimethacrylate-based hydrogels were prepared by UV photopolymerization. By varying
the crosslinking density of the network, hydrogels were prepared with shear moduli
ranging from 10 kPa to 1 MPa. In addition, supplementing the reaction mixture with a
70
small amount of glycidyl methacrylate comonomer created reactive sites within the
network through which biomolecules could covalently bond with the PEG network.
Textured surface topographies, in the form of parallel grooves, were created on the
hydrogels by casting them on microstructured silicon wafers. The cellular response to the
surface topography and varying mechanical properties will be investigated
252
.

Crosslinked PEG scaffolds can also be rendered biodegradable by the incorporation of
hydrolytically degradable components into the hydrogel matrix. Crosslinked hydrogels
were prepared from photopolymerization of PEG dimethacrylate. Three degradable
lactide units were grafted to each PEG molecule. Additionally, neural cells were
photoencapsulated into the network, which were shown to differentiate, proliferate, and
generate neural tissue which penetrated through the open space in the network. The
distance between crosslinks in the network, or the mesh size, was shown to be a
controlling factor in the rate of tissue ingrowth. If the average mesh size was too narrow,
neural growth was inhibited. Because the network mesh size increased as the lactide
units degraded, one way to control the rate of tissue ingrowth is to vary the lactide
content of the network. This work also demonstrated that the PEG-based material was
well tolerated by the neural cells, making it a viable carrier for cell transplantation into
the central nervous system
253
.

Conventional tissue engineering scaffolds which only provide a temporary, 3-D structural
support are being replaced with scaffolds which are composed of more interactive
biomaterials, such as biomolecules. which not only support cell migration and
71
proliferation, but initiate it
254
. In this new direction in tissue engineering, PEG still
remains a major component in the hybrid biomaterials. For instance, PEG-protein
hydrogels have been studied for applications in tissue engineering because the protein
sequences naturally possess cell signaling domains for adhesion and protease degradation
254
. Meanwhile, the PEG domains impart structural integrity to the scaffold.

Benoit et al. created PEG hydrogels functionalized with heparin to use as a culture
system for human mesenchymal stem cells
255
. Heparin has been shown to promote cell
adhesion
256
and interact with proteins associated with osteoblast and osteoblast
progenitor cell adhesion
257
. In this study, 94% of the stem cells survived over the five
week study, compared to only 47% in previous studies with unfunctionalized PEG
hydrogels
258
. This improvement was attributed to the ability of heparin to bind with
fibronectin and bone morphogenic protein.
255
. Hwang et al.
259
incorporated
glucosamine into embryonic stem cell-containing photocrosslinked PEG hydrogels.
Glucosamine has been shown to play a role in enhancing the synthesis of cartilage,
tendons, ligaments, bone, and other connective tissues
260
. The researchers found that the
presence of glucosamine enhanced extracellular matrix production and, in doing so,
helped improve the structural integrity of the scaffold.

PEG has also been shown to be a beneficial component in mucoadhesive drug delivery
systems. Mucoadhesive devices can be applied to, and deliver drugs locally to, mucosal
tissues, including the gastrointestinal, nasal, and buccal areas
261
. Mucoadhesion is the
attachment of a natural or synthetic polymer to the mucosa
262
. This occurs via the
72
interpenetration, and subsequent hydrogen bonding, between the polymer chains in the
chemical carrier with the glycoproteins present in the mucus
263
. Acrylicbased polymers
have been extensively used in mucoadhesive applications due to their high adhesive bond
strength with the tissue
264,265
. However, PEG can be used as a mucoadhesion promoter,
though the polymer is not mucoadhesive itself. PEG chains that are grafted onto the
chemical carrier will enhance interpenetration of the material into the mucosa, increasing
mucoadhesive strength
262,263,266-268
. For instance, Serra et al.
263
showed a 480% increase
in the work of adhesion value for a poly(acrylic acid) copolymer with PEG 1000 g/mol
tethers compared to pure poly(acrylic acid).

Arguably, the most important and extensive use of PEG is in colloidal polymer systems
for biological and pharmaceutical applications. In fact, PEG is used universally as the
hydrophilic block in polymeric micelles
241,269
. There are several reasons for its
widespread use. Firstly, PEG provides steric stabilization to colloidal particles such as
liposomes or micelles
241
. The hydrated PEG chains create steric forces which compete
with interparticle van der Waals attractive forces. Particle coagulation is prevented
because the repulsive forces overwhelm the attractive forces between particles
270
. The
larger particle sizes caused by coagulation would increase the chances of the drug
delivery vehicle being cleared by the immune system, reducing circulation time in the
body
241
.

Secondly, PEG inhibits the surface adsorption of biological molecules
241
, such as
proteins, which typically occurs within the first few minutes of exposure to physiological
73
fluids. As proteins approach a PEG surface, the PEG chains become compressed,
reducing their conformational entropy and increasing their enthalpy, making protein
adsorption thermodynamically unfavorable. Also, the increase in particle concentration
at the PEG surface due to the presence of proteins causes an influx of water caused by an
osmotic pressure gradient, forcing the protein to separate from the surface
271
. Protein
adsorption onto drug delivery vehicles could damage the particle or interfere in
biochemical pathways
241
. Moreover, PEG chains prevent surface adsorption of proteins
which belong to the reticuloendothelial system. The adsorption of these proteins would
attract phagocytic macrophages to ingest the drug delivery vehicle, clearing it from the
body. Grafting PEG onto particle surfaces will therefore impart stealth characteristics
to the drug delivery system, increasing circulation time in the body
272
.

The work of Tobio et al.
273
is a prime example of how PEG can improve the stability and
bioavailability of nanoparticle delivery systems. The work investigated the influence of a
PEG coating around poly(lactic acid) nanoparticles on their stability in GI fluids and their
ability to transport proteins though the intestinal mucosa. The authors hypothesized the
PEG coating would help to overcome the challenges involved in oral application of drug
carriers, specifically the instability of the particles in the GI tract and low intestinal
uptake of the drugs. PLA nanoparticles have been shown to degrade significantly in
intestinal fluids
274,275
, but the protein repellent properties of PEG could help overcome
this. In addition, the ability of PEG to improve mucoadhesion could increase transport
through the intestinal mucosa. Their studies revealed that, after four hours of incubation
in digestive fluids the PEG coated particles were only slightly degraded (3% of the
74
poly(lactic acid) was converted to lactate, compared to 9% for the uncoated particles).
Additionally, the levels of radioactive antigen in the blood stream, which was
encapsulated in the nanoparticles, were higher for the coated system
273
.

Similarly, in another study, using a hydrophilic PEG corona in polymeric micelles (with
cyanoacrylate-co-hexadecyl cyanoacrylate as the hydrophobic block) for the delivery of
active therapeutic molecules increased the half life of the particles in the blood. Results
also showed a higher uptake of the drug by the target organs, the spleen and brain,
compared to non-PEGylated micelles, the composition of which was not expressly stated
276
.

2.7 Poly(ethylene imine)

Poly(ethylene imine) or PEI is a cationic polymer consisting of primary, secondary and
tertiary amines. Its chemical structure is shown in Figure 2.14. It is available in a wide
variety of molecular weights and degrees of branching. It exhibits a very high charge
density over a large range of pH values. In fact, PEI exhibits one of the highest charge
densities among organic molecules
277
. The pKa value of the primary amine is around 9,
the secondary amine around 8 and the tertiary amine around 67
278
. At pH of 10, the
amine groups are uncharged, and the swelling of PEI under these conditions can be
mainly attributed to the hydrophilicity of the amine groups. Lowering the pH results in
protonation of the amine groups. In salt solutions, swelling under these conditions is
determined by a balance between the polymer network osmotic pressure and elasticity.
75
The osmotic pressure resulting from the high concentration of counterions in the interior
of the PEI network causes an influx of water which swells the polymer. If the PEI chains
are crosslinked, they will oppose this expansion
279
.

At the present time, the biological properties of PEI are not completely understood
280
, as
there have been few investigations into the biocompatibility of PEI toward human cell
lines. Larkard et al.
281
coated semiconducting electrodes with polymer films and
examined the adhesion, proliferation, and morphology of rat neuronal cell lines on the
polymer surfaces. After 8 hours in a cell culture medium, a high percentage of the cells
were found to be attached to the PEI. After 72 hours, the cells were found to proliferate
faster on PEI than on the other polymers (polypropyleneimine and polypyrrole). It was
therefore determined by this group that PEI is a satisfactory coating for the cultivation of
neuronal cells. In another study, the adhesion and proliferation of human Schwann cells
was compared on substrates composed of fibronectin, laminin, crosslinked gelatin, or
PEI. It was found that adhesion and proliferation was satisfactory on all coatings, but the
relative performance of PEI was poor. Cell proliferation was moderate compared to the
other polyelectrolytes, and while choice of substrate was deemed not to be an important
consideration in the culture of Schwann cells, PEI was not recommended.

Based upon these studies, it may be inferred that the biocompatibility of PEI may depend
on cell type. However, the molecular weight of PEI used in the aforementioned research
articles was not reported by the authors. Morimoto et al.
282
reported the cytotoxicity of
76
PEI to be molecular weight dependent. A comparison among the cytotoxicity of PEI
1800, 10,000, and 70,000 Da showed increasing cytotoxicity with molecular weight.

In another study, PEI 70,000 Da was deposited onto a titanium alloy surface, and it was
found that osteoblasts and fibroblast proliferation and adhesion decreased over a 7 day
period
280
. A group who studied PEI 750,000 Da as a potential coating for bone implants
evaluated the response of human osteoblasts-like cells and periodontal ligament cells.
While maximum adhesion occurred within twenty minutes for the coatings composed of
poly(sodium 4-styrenesulfonate), poly(allylamine hydrochloride), poly(L-glutamic acid),
or poly(L-lysine), cell adhesion to PEI was similar to that of glass or plastic. In addition,
no proliferation was seen for either cell type on the PEI substrate. The cell nuclei became
condensed or fragmented, and after 24 hours, cell apoptosis was observed. These results
are consistent with those of Moghimi et al.
283
, who showed that branched PEI of
molecular weight 25,000 and 750,000 Da induced necrotic changes within 30 minutes in
human umbilical vein endothelial cells and hepatocyte-like cells. Within 24 hours, cell
apoptosis was initiated. While the authors note that the molecular basis for the
cytotoxicity is not understood, this property of PEI may have beneficial aspects when
applied to tumor gene therapy.

Despite its potential cytotoxicity, the presence of the ionizable groups on PEI has
motivated researchers to investigate its potential in drug delivery. Vinegradovs group
developed nanosized gel networks composed of crosslinked PEG and PEI. While the
PEG aids in the stabilization of the aqueous nanogel dispersions, the cationic PEI chains
77
allow for the immobilization of negatively charged biologically active molecules, such as
indomethacin. Prior to drug loading, the nanogels form expanded networks due to the
osmotic pressure generated by the fixed positive charges on the PEI. After drug loading
occurs, due to the formation of an electrostatic complex with the charged drug molecule,
the nanogels dehydrate, resulting in network collapse and decrease in particle size.
Release studies showed that 17.5% of the indomethacin was released during the first
hour, followed by an 82% release by 24 hours. Interestingly, the release of the drug
causes the nanogel to swell again, due to the increase in net ion concentration inside the
network
279
.

Some researchers have diverged from using PEG as the hydrophilic corona for micellar
structures in favor of using PEI. The cationic nature of PEI allows the micelles to
interact with negative charges on cell surfaces
284,285
, helping to increase cell uptake.
Tian et al.
284
synthesized novel amphiphilic biodegradable block copolymers based on
PEG, hyperbranched PEI, and poly(-benzyl L-glutamate). In aqueous solutions, these
copolymers self-assembled to micellar structures. Characterization studies on the
micelles revealed that particle size was dependent on the ionic state of PEI. As its charge
density increased, the particle size decreased. The authors attribute this change to an
increase in macromolecular aggregation, which impeded hydrophobic block interactions.
In addition, micellar solutions were added to plasmid DNA, which readily became
immobilized on the micelles.

78
Another group prepared micelles composed of PEI and poly(D,L-lactide-co-glycolide).
They also found that the micelle size depended on the charge density of the PEI, the size
decreasing as charge density increased. This was once again attributed to the inhibition
of hydrophobic block interactions. This group incubated micellar solutions with human
keratinocyte cells and found that the particles not only adsorbed onto the cell membranes,
but translocated into the nucleus. PLGA nanoparticles were also studied as a control, and
minimal adsorption and diffusion into the cell surface was exhibited
285
.

Based on the above findings, it is obvious why the most prominent application of PEI is
gene delivery
286
. DNA is condensed with PEI due to its electrostatic interaction with the
positive charges on PEI. In the condensed form, DNA is protected against digestion of
enzymes and can be delivered toward a targeted cell, and internalized via endocytosis
287
.
Polycations destabilize cell membranes, causing their rupture, releasing the PEI/DNA
complex into the nucleus, allowing it to be expressed
288,289
. It has been reported that the
size of the PEI has a strongly affects the transfection efficiency. Low molecular weight
PEI (less than 2000 Da), though proven to be non-toxic, produces low transfection
activity. High molecular weight PEI (greater than 25 kDa) has shown high transfection
activity combined with significant cytotoxicity
290,291
.

While the reason for its low transfection activity remains unclear
292
, there have been
some efforts to chemically modify low molecular PEI in order to increase its transfection
efficiency while retaining its low cytotoxicity. Peterson et al. created PEI block
copolymers with star-shaped PEG molecules
292
. They hypothesized that chemically
79
modifying low molecular weight PEI to increase its size would increase the transfection
efficiency, and that the charges on PEI do not critically contribute to condensation.
Based on the obtained results, the authors maintain that low molecular weight PEI
exhibits poor transfection efficiency because it only moderately condenses the DNA,
insufficiently protecting it. However, its condensation potential was enhanced by
coupling it to star PEG molecules with branch sizes of 10 or 15 kDa. These conjugates
had similar size to high molecular PEI, and formed compact, stable complexes upon
mixing with plasmid DNA. The transfection activity and cytotoxicity of these complexes
are still under investigation.

Similar copolymers, based on linear copolymers of PEG 5 kDa and PEI 22 kDa, have
been complexed with DNA to deliver genes to tumors which encode for the tumor
necrosis factor
293
. Tumor growth inhibition was shown to occur in three murine tumor
models. It has also been demonstrated that the PEG molecules prevent non-specific
interactions with the biological environment and prolong the circulation time of the DNA
complex
294
.

2.8 Bioadhesive Polymers for Soft Tissue Repair

Bioadhesive polymers are natural or synthetic materials that can be used for soft tissue
repair, including wound closure, achieving hemostasis after a surgical procedure, or
80
fistula repair. Bioadhesive materials can supplement the use of sutures or replace them
altogether
295
.

Cyanoacrylates are a widely used class of bioadhesive compounds. The use of these
adhesives in medicine began in the 1960s
296
. Cyanoacrylates are synthetic glues that
polymerize in the presence of water or blood. N-butyl-2-cyanoacrylate (Histoacryl, B
Braun, Melsungen, Germany) and N-butyl-2-cyanoacrylate (Glubran, GEM S.r.l.,
Viareggio, Italy) were recently approved for endoscopic use in Europe. Although
Histoacryl and Glubran are not commercially available in the United States, 2-octyl-
cyanoacrylate (Dermabond, Ethicon, Inc., Somerville, NJ) was approved by the FDA for
superficial wound closure, but not for endoscopic applications
297
.

Cyanoacrylate sealants have had wide ranging clinical applications. For example,
injection therapy with cyanoacrylate adhesives has shown to be an effective treatment for
gastric varices
298
. In one clinical study, this treatment was shown to be superior to band
ligation, a widely accepted treatment for gastric variceal bleeding
299
. The use of
cyanoacrylates for the treatment of gastric varices has been widely adopted outside of the
United States
297
. In addition, N-butyl-2-cyanoacrylate was used to repair esophageal
variceal rupture, and this method was found to be superior to sclerotherapy with
ethanolamine oleate solution
300
. The same adhesive was used to repair a peptic ulcer,
and it was shown to be superior to using hypertonic saline. While the two treatments
showed similar initial bleeding rates, the re-bleeding rate was lower for the cyanoacrylate
adhesive
301
. In another study, 8 out 12 pancreatic fistulas were successfully closed with
81
N-butyl-2-cyanoacrylate
302
. 2-octyl-cyanoacrylate was used to close a corneal
perforation, and the patient went on to full visual recovery
303
.

Mixing cyanoacrylates with lipid soluble contrast agent lipidol increases the radiopacity
of the adhesive, enhancing imaging. Lipidol also retards the polymerization process,
allowing the adhesive to be applied endoscopically via needle, with less risk of premature
solidification. When closing superficial wounds, Chigira et al. recommends combining
2-octyl-cyanoacrylate with reinforcing skin closure tape, which significantly increases its
tensile strength. Another group proposes preparing composites of 2-octyl-cyanoacrylate
and poly(L-lactic-co-glycolic acid) to increase tensile strength.

Cyanoacrylates form solid impermeable plastics in situ after polymerization is complete
304
. Therefore, possible complications include inflammatory response to a foreign body
and local tissue necrosis
305
. Wippermann et al.
296
created distal coronary anastomoses in
porcine hearts and subsequently sealed with butyl cyanoacrylate. Immediate hemostasis
was obtained with the application of the cyanoacrylate adhesive. Histological
examinations were performed to determine the immunologic response to the adhesive.
The inflammatory response to the adhesive was found to be moderate. Microscopic
evaluation revealed the presence of foreign body giant cells. Three months after the
surgery, evidence of an inflammatory response still existed and glue fragments were still
present, though there was no scar tissue. Another group used cyanoacrylate to close
anastomotic blood vessels in adult dogs. They reported necrosis rapidly occurring in the
region surrounding the glue, which was later replaced by fibrous tissue. They propose
82
that this was caused by thermal injury as a consequence of the heat generated from the
exothermic polymerization
306
.

Fibrin adhesives have been investigated experimentally since the 1970s
307
. They act as a
hemostatic plug by mimicking the last stage of blood clotting. Fibrin sealants contain
components such as fibrinogen, factor XIII, and thrombin. Thrombin catalyzes the
conversion of fibrinogen to fibrin, and factor XIII catalyzes the formation of crosslinks
within fibrin to form an insoluble fibrin clot
308
. The clot is resorbed within days or
weeks by macrophages and fibroblasts
309
, allowing healing to occur at the site of
adhesion. Because they are natural materials, fibrin sealants are completely
biocompatible
310
.

They are available commercially from Tisseel (Baxter, Westlake Village, CA) and
Hemaseel (Hemacure, Sarasota, FL)
311
. They are approved for topical application and
the closure of anastomoses in cardiovascular and colorectal surgery
297
. Tisseel is
composed of four main components. Fibrinogen, which is obtained from human donor
plasma, is dissolved in bovine aprotinin. Dry human thrombin is reconstituted in calcium
chloride, and the two solutions are mixed and applied simultaneously
312
.

On the whole, studies comparing this fibrin adhesive to other hemostatic agents show
fibrin to be equivalent or superior
297
. One such trial compared the use of the fibrin
sealant to the injection of polidocanol for the repair of peptic ulcers. Patients who
received daily applications of the fibrin sealant had less re-bleeding and fewer acute
83
treatment failures. However, patients who received only a single fibrin treatment did not
experience significantly different outcomes than those in the control group
313
. In a
randomized trial involving patients with esophageal variceal bleeding, the outcome of
using sclerotherapy with ethanolamine in addition to human thrombin was compared to
using sclerotherapy alone. All clinically important outcomes were deemed to be
equivalent
314
. Fibrin adhesives have also been concluded to be effective sealants for
corneal incisions, LASIK flaps, and conjunctival and skin grafts
304
.

Although fibrin sealants are not associated with inflammatory or foreign body reactions
310
, some components in fibrin adhesives may present complications to patients. Because
the fibrinogen is obtained from human plasma, theoretically there is the risk of viral
transmission. Because aprotinin is extracted from bovines, there is risk or immunologic
or allergic response when administered to humans
312
.

Glutaraldehyde (Figure 2.15) has achieved widespread acceptance as a successful
chemical agent for tissue crosslinking
315
. Glutaraldehyde is inexpensive, can form
miscible solutions with water, and reacts with tissue in a short period of time.
Glutaraldehyde is an aliphatic organic molecule with aldehyde groups at each end. The
di-aldehydes are able to react readily with the amines on proteins of the tissue
extracellular matrix, via a Schiffs base reaction
316
, resulting in covalent crosslinks.
Lower concentrations of glutaraldehyde, rather than high concentrations, have been
shown to more effective for bulk tissue crosslinking. High concentrations produce rapid
84
crosslinking of the tissue surface and inhibit diffusion of glutaraldehyde into the tissue
317
.

Because of its ability to rapidly react with tissues, a major group of tissue adhesives are
based on glutaraldehyde. BioGlue (CryoLife Europa Ltd, Hampshire, United Kingdom)
is composed of BSA and glutaraldehyde. The material does not become active until the
two components are mixed at the point of application. A rigid mechanical seal forms
within seconds
318
. The glutaraldehyde forms covalent crosslinks between the BSA
molecules and also reacts with the tissue at the location of application, forming an
adhesive bond at the repair site
319
. Currently, BioGlue is approved in the United States
to repair aortic dissection
320
. However, in Australia, the Therapeutic Goods
Administration has approved BioGlue for all surgical specialties, including cardiac care
321
. The glue exhibits very slow biodegradation, as remnants have been shown to exist in
the body as long as two years after surgery
321,322
. The cohesive strength of BioGlue is
higher than the fibrin glue Tisseel
321
. As a consequence of this strength, Bioglue is not
recommended for use in children because it could inhibit growth when applied
circumferentially
321
.

Passage et al. produced a large scale evaluation of 115 consecutive patients who
underwent a range of cardiac procedures such as aortic root, aortic wall, or valve
replacement, and coronary artery bypass grafting. BioGlue was used for the achievement
of hemostasis, tissue adherence, or tissue strengthening. All of the surgeons believed that
85
BioGlue facilitated the operation. However, they recommended more studies to assess
the long term patient outcomes
321
.

BioGlue was also investigated for use after a thoracotomy to reduce alveolar air leaks.
These air leaks could not be repaired by conventional tissue closure techniques. It was
found that BioGlue reduced air leak duration and resultant chest drainage, as well as
hospital stay. The surgeons recommended that the volume of BioGlue applied be kept to
a minimum, while, of course, ensuring tight closure of the air leaks
323
. BioGlue was also
applied for the achievement of hemostasis after a partial nephrectomy. Adequate
hemostasis was achieved, in addition to decreased blood loss and transfusion rate.

The authors recommended investigation into using BioGlue in renal surgery
324
. The
adhesive was also used to repair a dural tear which occurred during lumbar
decompressive surgery. Two years after application, during re-operation for an onset of
sciatica, fragments of the glue were found adjacent to the dura. They were not encased in
fibrous tissue and there was no evidence of an inflammatory response. Complete dural
healing occurred
322
.

Despite its high adhesive strength, the potential toxicity of glutaraldehyde based
adhesives needs to be considered
318
. Inflammatory responses have been associated with
glutaraldehyde application and have been ascribed to its cytotoxicity
325
. Application of
BioGlue to the bovine pericardium resulted in the presence of inflammatory cells
surrounding the fixed tissue for as long as 26 weeks
326
. In another study, BioGlue was
86
used to repair bronchial anastomesis in sheep. While tight closures were obtained with
the adhesive, microscopic evaluation revealed the presence of polymorphonuclear
neutrophils, macrophages, granulation tissue, and foreign body giant cells after two
weeks. This response did not occur in the sutured group
327
.

A valuable study was performed by Frst et al
318
. The group cured portions of BioGlue
and overlaid it with saline solution. The supernatant was analyzed for glutaraldehyde
content and applied to human embryo fibroblasts or mouse myoblasts, so the in vitro
cytotoxic effects of the glue could be evaluated. In addition, the in vivo toxicity of
BioGlue was evaluated in the lung, liver, and aortic tissues of rabbits. The application of
the supernatant to both cell lines caused changes in the morphology and density of the
cells. Even diluted supernatants, which contained 10% of the initial concentration of
glutaraldehyde, resulted in a complete loss of cell viability. The in vivo studies revealed
severe inflammatory responses in the lung and liver tissues, characterized by necrosis,
edema, granulation tissue, and detected giant cells. In contrast, the aortic model showed
no significant differences between the tissue reactions to BioGlue and the biocompatible
fibrin sealant
318
.

While several authors have demonstrated that inflammatory effects are a potential
consequence of glutaraldehyde-based adhesives, this is the only study which
systematically compares the physiological response of different tissues. The results in
this study indicate that the consequences of glutaraldehyde toxicity are strongly
dependent on the location of application. The authors postulate that the inflammatory
87
response was less severe in the aortic tissues than in the lung or liver because the aorta is
contains larger amounts of extracellular matrix and fewer cellular components, rendering
them less sensitive to the cytotoxicity
318
. In addition, Matsuda et al.
328
suggested that a
lesser inflammatory response would be elicited by the adhesive if the aldehyde groups
were immobilized on the surface of the implanted material. This would prevent diffusion
deep into the natural tissues, a scenario which could cause a large scale, persistent
inflammatory response due to large scale damage and impairment.

It is important to note that it is not clear whether the inflammatory responses to BioGlue
have had any long term negative effects on the patient
329
. Herget et al.
327
, who repaired
bronchial anastomesis in sheep with BioGlue, noted that healing was not complicated by
the foreign body reactions, and that the adhesive layer was eventually replaced by
connective tissue. However, another group reported tissue healing in glutaraldehyde
treated tissues was retarded due to the microenvironment created by the inflammatory
responses
325
.

A number of adhesive systems have been developed specifically for the repair of the
damaged intervertebral disc tissues. Repair of a defective annulus can be achieved by
applying a curable material, which forms a solid that closes the defect. Slivka et al.
330

proposes the delivery of a crosslinking agent into a damaged intervertebral disc in order
to stabilize the disc structure, help prevent prolapse of the disc material, and restore or
retain biomechanical function. If delivery of the crosslinking agent is targeted at the
center of the disc, it would result in crosslinking of the native nucleus pulposus. This
88
would shift its material properties from a viscous gel to a viscoelastic solid, reducing the
risk of herniation. The crosslinking agent can also be used to adhere the nucleus tissues
to the surrounding annulus, further reducing its mobility in a degenerated disc.
Furthermore, this technology could be used in conjugation with current surgical
treatments for lower back pain, such as discectomy, to close the herniated portion of the
annulus
330
.

Slivka et al. proposes using a range of crosslinkers, such as diepoxy compounds,
diisocyanates, bis(sulfosuccinimidyl suberate), or glutaraldehyde. The patent holders
note that the crosslinker molecular weight should be over 100 Da, otherwise the agent
can too easily diffuse into the tissues surrounding the disc. They also propose modifying
the polysaccharides or collagen in the native tissue with aldehyde groups using an
oxidizing agent, then allowing spontaneous crosslinking to occur
330
. A similar method
has also been proposed by Liu et al., who development a method for the oxidation of
exogenous polysaccharides using periodic acid or periodate
331
. The aldehyde
functionalized polysaccharides will crosslink the collagen molecules in the tissue matrix.
Growth factors can be incorporated into the system before or after crosslinking, providing
a matrix for the therapeutic repair of the tissue.

Tardy et al.
332
put forth a treatment method for collagen that should improve its stability
and mechanical characteristics. The tissues are heated via a laser, electrode, magnetic
field, or heated aqueous solutions. This shrinks the collagen in the tissue matrix, and then
crosslinking is achieved with a solution of periodic acid or periodate. This invention was
89
intended for the treatment of joint stability problems or the manipulation of skin structure
and properties, but it could also be applied to repair of disc tissues. However, heating
could produce tissue damage or a non-uniform skin morphology
330
.
90



Figure 2.1 Anterior view of the vertebral column showing the cervical, thoracic, lumbar, and sacral
regions
7
.

91

Figure 2.2 The division of the vertebrae into three regions
8
.





92


Figure 2.3 (A) Lateral view, (B) superior view, (C) inferior view of the vertebra
7
.

93

Figure 2.4 The structure of the intervertebral disc. It consists of a nucleus pulposus (NP) surrounded
by an annulus fibrosis (AF). Both are sandwiched between two vertebral endplates (VE)
8
.

94

Figure 2.5 A cylindrical interbody fusion cage. A, anterior; P, posterior
121
.

95

Figure 2.6 Photograph of the Type III SB Charit
144
.



96

Figure 2.7 The Charit artificial disc consists of a free-floating biconvex sliding core encased in
concave endplates
146
.
97

Figure 2.8 Photograph of the Prodisc-L
144
.
98

Figure 2.9 Photograph showing the Maverick disc
144
.
99

Figure 2.10 An implanted Prosthetic Disc Nucleus (PDN) device
162
.

100

Figure 2.11 The Newcleus spiral implant
163
.
101
H
2
C CH
O
NH
CH
C H
3
CH
3

Figure 2.12 The chemical structure of poly(N-isopropylacrylamide) (PNIPAAm).
102
H
O
OH

Figure 2.13 The chemical structure of poly(ethylene glycol) (PEG).
103
N H
2
NH
NH
NH
N
NH
2
NH
2

Figure 2.14 The chemical structure of poly(ethyelene imine) (PEI).
104
O
H
H
O

Figure 2.15 The chemical structure of glutaraldehyde.
105

Chapter 3 - RESEARCH GOALS

This work focuses on investigating the properties of a family of in situ forming
PNIPAAm-based nucleus pulposus replacements. The first objective in this work was to
synthesize a class of hydrogels composed of PNIPAAm and PEG. Because the
PNIPAAm homopolymer holds little water and shows poor elastic recovery, the swelling
and mechanical properties of PNIPAAm gels were tailored by the addition of the PEG.
The molecular weight of the PEG chains and the relative proportions of NIPAAm and
PEG units were varied.

Then, the range of pertinent material behavior for the PNIPAAm-PEG family of
copolymers, and how it varies with polymeric composition was examined. These
properties include lower critical solution temperatures, gel swelling, dissolution, stiffness,
and elasticity. Evaluation of the swelling and mechanical properties was carried out over
long-term in vitro studies to determine the stability of the materials. A material candidate
from this class of materials was chosen for further evaluation as a synthetic nucleus
pulposus replacement. The copolymer formulation was then optimized to minimize
water loss following implantation into the intradiscal environment, allowing the implant
to remain space filling in the nuclear cavity.

The last objective in this project was to impart bioadhesive properties to the injectable
hydrogel system. The PNIPAAm-PEG copolymer was blended with the amine
containing polymer poly(ethylene imine) (PEI). After gelation in the disc, the implant is
106
crosslinked with itself and with the surrounding tissues by the injection of a dialdehyde.
This approach can help secure the implant in place, reducing the risk of implant
migration or extrusion. The material also has the potential to function as tissue adhesive
for the repair of the degenerated intervertebral disc. The specific aims in the project are
as follows:

Specific Aim 1: Synthesize a class of polymers composed of PNIPAAm and PEG.
Specific Aim 2: Characterize the gelation, swelling and mechanical properties and long
term stability of this family of hydrogels.
Specific Aim 3: Optimize the copolymer formulation to minimize water loss, and
subsequent volume loss, due to the de-swelling of the polymer network following
injection into the intradiscal environment.
Specific Aim 4: Modify the copolymer chemistry to impart bioadhesive properties to the
hydrogel system and evaluate its capacity for tissue adhesion in vitro.

107

Chapter 4 - SYNTHESIS OF PNIPAAM-PEG BRANCHED
COPOLYMERS

4.1 Introduction

The preparation of PNIPAAm-PEG branched copolymers begins with the synthesis of
PEG dimethacrylate (PEGDM) from PEG diol and methacryloyl chloride (Figure 4.1).
Synthesis was achieved using a modified procedure based on that of Bryant et al.
333
.

PNIPAAm-PEG branched copolymers are prepared by polymerizing the monomer
NIPAAm in the presence of PEGDM, thereby creating a lightly crosslinked polymer
network (Figure 4.2). The PEGDM will be used in low enough quantities for the product
to form a flowing solution in water below the LCST of PNIPAAm. Most examples in the
literature of crosslinked PNIPAAm systems form hydrated, insoluble polymer networks
below the LCST of PNIPAAm
226,227,334
.

While PNIPAAm copolymerizations with methacrylate functionalized PEG chains have
been reported before, most of these studies used short PEG chains. Bae et al.
215
and
Gutowska et al.
226
both prepared PNIPAAm networks that were crosslinked with
ethylene glycol dimethacrylate (EGMDA). Another group synthesized microspheres
composed of PNIPAAm networks that were crosslinked with a combination of PEG (400
and 1000 g/mol) dimethacrylate. Still, there are some examples where researchers have
108
explored the use of higher molecular weight PEG chains. PNIPAAm microgels,
crosslinked with PEG (8000 g/mol) diacrylate
334
and PNIPAAm copolymers with star-
shaped PEGs with arms approximately 2000 g/mol
193
were both studied for drug
delivery.

Alava et al. studied the reactivity ratios of NIPAAm and PEG monomethacrylate. The
values were determined to be r
NIPAAm
=1.2 and r
PEGM
=0.13
335
. This indicates that
NIPAAm is more reactive than PEG monomethacrylate to both propagating species.
Copolymers of these two species would contain larger amounts of the more reactive
monomer, NIPAAm, in random placement
336
.

In this work, a family of copolymers was synthesized by varying the molecular weight of
the PEG blocks from 1000 to 10,000 g/mol and the relative amounts of NIPAAm and
PEG. Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance
(NMR) spectroscopy were used to characterize the chemical nature of the polymers.
Specifically, these analytical tools were used to verify the successful polymerization of
NIPAAm, complete removal of residual monomer, and the covalent incorporation of
PEG along the polymer backbone.


109
4.2 Experimental Section
4.2.1 Materials

N-isopropylacrylamide (NIPAAm) monomer (Sigma Aldrich) was recrystallized in n-
hexane before use. Polyethylene glycol (PEG) diol (M
n
= 2000, 4600, 8000, and 10000
g/mol) (Sigma Aldrich), initiator 2,2-Azobisisobutyronitrile (AIBN), 98% (Sigma
Aldrich) poly(ethylene glycol) dimethacrylate (PEGDM) (n=1000) (Polysciences), and
deuterium oxide (Sigma Aldrich) were all used as received. All solvents and other
chemicals were of analytical grade.

4.2.2 Methods
4.2.2.1 Methacrylation of PEG

PEGDM, with molecular weight between 2000 and 10,000 g/mol, was synthesized from
PEG diol
333
. The diol was dissolved in methylene chloride and reacted with an excess of
methacryloyl chloride and triethylamine under nitrogen atmosphere for 12 h at 4C and
an additional 24 h at room temperature. The product was then precipitated in ether
multiple times to ensure purification and the excess ether was removed in the vacuum
oven. The average methacrylation efficiency of the reaction was determined by
1
H NMR
(Varian 300, Palo Alto, CA) in deuterium oxide at 25C.

110
4.2.2.2 Copolymer Synthesis

NIPAAm was polymerized in the presence of PEGDM to create PNIPAAm-PEG
branched copolymers. Copolymers composed of five different block sizes were
synthesized by using PEG 1000, 2000, 4600, 8000, or 10,000 g/mol dimethacrylate. For
each PEG molecular weight, a high and low PEG content copolymer was produced by
using a molar ratio of NIPAAm monomer units to PEG branches of 700:1 and 1600:1,
respectively. The free radical polymerization, initiated by AIBN, took place in methanol
at 65C for 48 hours. The methanol was evaporated from the reaction mixtures after
polymerization was complete. The reaction product was purified by treatment with n-
hexane at 45C. The purified polymer was isolated by vacuum filtration and dried under
vacuum overnight. It was subsequently ground into powder and analyzed with
1
H NMR
to verify the molar ratio of NIPAAm monomer units to PEG branches.

Solutions containing 15 wt% polymer were prepared from the ground polymer and
deionized water. The polymer was dissolved overnight at 4C then centrifuged (Kendro
Sorvall Legend Mach 1.6/R, Asheville, NC) at 15C and 10,000 rpm for 45 minutes to
remove entrapped air from the viscous solutions.




111
4.3 Results and Discussion
4.3.1 Methacrylation of PEG

The
1
H NMR spectrum for the PEGDM can be seen in Figure 4.3. The signals
corresponding to the functional groups are shown in the figure. The average
methacrylation efficiency of the reaction was tracked over a 12 month period and was
found to be 90.8 9.2 %. This was calculated by comparing the area under the integral
for the vinyl protons (o=5.7 ppm, o=6.1 ppm) to that of the PEG backbone (o=3.5 ppm).

4.3.2 Synthesis of PNIPAAm-PEG Branched Copolymers

The extent of polymerization of the NIPAAm can be evaluated with FTIR
337
. Shown in
Figure 4.4 is the FTIR spectra of the reaction mixture after 24 and 48 hours reaction time
at 65C. The time point t=0 refers to the FTIR spectrum of the reaction mixture prior to
heating. The peaks at 1650cm
-1
, 1543cm
-1
, and 1458cm
-1
correspond to the Amide I
338-
340
, Amide II
338-340
, and C-H bending
341
on the NIPAAm and PNIPAAm. In addition,
the peaks at 1368cm
-1
, and 1387cm
-1
correspond to the two isopropyl groups on the N-
isopropylacrylamide
338
. Therefore, these peaks are present at all time points. At t=0, the
peak at 1620cm
-1
has been attributed to the C=C stretch on the NIPAAm monomer
341
. In
addition, the peaks at 984 cm
-1
and 912 cm
-1
correspond to out of plane C-H bending of
an alkene with an RCH=CH
2
structure, like the NIPAAm monomer. The disappearance
of these peaks over the course of the reaction confirms polymerization of the NIPAAm.
112

It is important to note that peaks for PEG or PEGDM are not seen in the FTIR spectra,
likely because the instrument was not sensitive enough to detect the presence of PEG.
The ether C-O stretch in PEG in typically seen around 1106 cm
-1
and the C-H bending on
an alkene with a methacrylate group structure (R
2
C=CH
2
) occurs between 880-900 cm
-1
341
. Therefore, this FTIR analysis cannot be used to verify the covalent incorporation of
PEG along the polymer backbone.

A typical
1
H NMR spectrum for PNIPAAm-PEG branched copolymers is shown in
Figure 4.5. The peak assignments have been previously described by Ma et al.
342
,
however the group neglected to assign a peak to the methyl protons on the methacrylate
group of the PEG (A), which were incorporated along the polymer backbone during
polymerization. The covalent incorporation of PEG along the polymer backbone can be
verified by the presence of the characteristic peaks for PEG and PNIPAAm in the spectra,
which are designated on the figure, and the absence of the sharp singlets at 5.7 and 6.1
ppm, which represent the vinyl protons on the double bond of the NIPAAm monomer or
PEG dimethacrylate. To quantify the relative amounts of NIPAAm and PEG in the
copolymer, the area of the peak at o=3.5ppm, characteristic of the protons of the
(CH
2
CH
2
O-)
n
- segment on the PEG, was compared to the area of the peak at o=3.6 ppm
for the CH protons adjacent to the isopropyl group of the NIPAAm. Based on these
calculations, all copolymers had molar ratios satisfactorily close to the targets of 700:1
and 1600:1.

113
4.4 Conclusions

The successful polymerization of NIPAAm was shown with FTIR. This result was
verified with
1
H NMR. In addition,
1
H NMR was used to verify the covalent
incorporation of PEG along the polymer backbone and quantify the molar ratio of
NIPAAm monomer units to PEG blocks in each of the copolymers. A family of
copolymers was created by varying the PEG block molecular weight between 1000 and
10,000 g/mol. For each PEG block molecular weight, a high and low PEG content
copolymer was synthesized by keeping the molar ratio of NIPAAm monomer units to
PEG blocks at either 700:1 or 1600:1. The material properties of this family of
copolymers will be examined in subsequent chapters.
114

CH
2
CH
3
Cl
O
O H CH
2
CH
2
OH C H
3
CH
2
O
O CH
2
CH
2
O
O
CH
2
CH
3
n
n
+
Methacryloyl Chloride
PEG diol
polyethylene glycol dimethacrylate

Figure 4.1 Schematic for the synthesis of PEGDM.
115
C H
2
O
NH
C H
3
CH
3
C H
3
CH
2
O
O CH
2
CH
2
O
O
CH
2
CH
3
NIPAAm
poly(ethylene glycol) dimethacrylate
Branched copolymer
H
2
C CH
O
NH
CH
(CH
3
)
2
CH
2
C
CH
3
O
O
CH
2
CH
2
O
O
C CH
2
CH
3
n
+

Figure 4.2 Schematic for the synthesis of PNIPAAm-PEG branched copolymers.

116


Figure 4.3
1
H NMR spectrum for PEGDM in deuterium oxide at 25C.
117

Figure 4.4 The changes in spectra over reaction time for PNIPAAm-PEG branched copolymers.

118

Figure 4.5. NMR Spectra for purified PNIPAAm-PEG branched copolymers in deuterium oxide at
at 25C.
119

Chapter 5 - CHARACTERIZATION OF PNIPAAM-PEG
BRANCHED COPOLYMERS

5.1 Introduction

There have been very few studies investigating PNIPAAm-based materials for orthopedic
applications. This may be because the polymer exhibits poor elastic properties and holds
little water at physiological temperature due to its hydrophobic nature. Ho et al.
343

investigated PNIPAAm-PEG (1000 g/mol) copolymers for soft tissue fixation or repair.
The authors found that by engineering a third component into the hydrogel system,
trimethacryloxypropyltrimethoxysilane (MPS), the mechanical properties of the network
could be tuned. The MPS aided in network formation and increased the mechanical
properties of the gels. In addition, the authors note that by using different permutations
of PNIPAAm, PEG, and MPS, the modulus of the hydrogels could also be tailored to
match that of trabecular bone.

This work focuses on tailoring the swelling and mechanical properties of PNIPAAm
network by adding PEG blocks to the network. It is proposed that lightly crosslinking
PNIPAAm with a long chain, hydrophilic component will increase its water retention
capability and elasticity. PEG was chosen due to its biocompatibility, hydrophilicity,
ease of functionalization, and availability in a range of molecular weights. PNIPAAm-
PEG branched copolymers were synthesized by polymerizing the monomer NIPAAm in
120
the presence of PEG dimethacrylate (PEGDM). In the previous chapter, it was shown
that a family of copolymers was created by varying the molecular weight of the PEG
chains between 1000 and 10,000 g/mol. For each PEG block molecular weight, a high
and low PEG content copolymer was synthesized by keeping the molar ratio constant at
700:1 or 1600:1, respectively.

In the following experiments, the structure-property relationship in these copolymers was
studied. Specifically, gel water content, stiffness, and elasticity were investigated as a
function of PEG content and PEG block molecular weight. It is hypothesized that a
material candidate from the class of PNIPAAm-PEG branched copolymers can serve as a
synthetic nucleus pulposus replacement exhibiting suitable, stable material properties
over long term immersion in the physiological environment.

5.2 Experimental Section
5.2.1 Methods
5.2.1.1 LCST Characterization

The LCST and transition enthalpy of each copolymer was characterized using a DSC
Model 2010 (TA Instruments, Wilmington, DE) by placing 7-10mg of solution in a
hermetically sealed allodized aluminum pan, and heating from 10 to 60C at a rate of
10Cmin
-1
. The phase transition of the polymer is detected as an endothermic peak on
the DSC thermogram, with the temperature at the peak onset taken as the LCST. The
121
area of the peak, normalized to the total mass of solution
337
, was taken as the transition
enthalpy.

5.2.1.2 Gel swelling and Dissolution

To characterize gel swelling, approximately 2mL of each polymer solution was heated to
37C within a closed vial for 3 hours. The precipitated gels were then immersed in
Dulbeccos phosphate buffered saline (PBS) at 37C. A set of gels were evaluated at 0,
7, 14, 30, 60, and 90 days, weighed, and subsequently dried under vacuum and weighed
again. Time 0 refers to a set of gels that were weighed immediately after precipitation
and never immersed in PBS. The gel water content was calculated according to Equation
5.1, where m
wet,37C
(t) is the wet gel mass at 37C at time t and m
dry
is its dry mass.

,37
( )
(%) 1 100
( )
o
dry
wet C
m t
Water content
m t

=


Equation 5.1

For the dissolution studies, an identical procedure was used. A set of gels were examined
at 30, 60, and 90 days, dried under vacuum, and weighed. The mass retention of the gels
at time t was calculated according to Equation 5.2, with m
wet,25C
(t=0) being the mass of
polymer solution initially placed in the vial at 25C.

,25
( )
(%) 100
0.15 ( 0)
o
dry
wet C
m t
Mass retention
m t

=

=

Equation 5.2
122
5.2.1.3 Mechanical Properties
5.2.1.3.1 Compressive Studies

Unconfined uniaxial compression tests were performed on gel samples after 7, 30, 60,
and 90 days immersion in PBS. The hydrogel samples were loaded in an Instron
mechanical testing system (Instron Model 3362, Park Ridge, IL) and compressed at a
strain rate of 100%min
-1
while submerged in a 37C PBS bath. Load and displacement
data were recorded with Instron Bluehill 1.2 software and converted to stress and strain
values. The compressive moduli at 10, 15, and 20% strain were approximated as the
slope of the chord drawn between 5 and 15% strain, 10 and 20% strain, and 15 and 25%
strain, respectively.

5.2.1.3.2 Stress Relaxation Studies

The gels ability for elastic recovery was evaluated using stress relaxation experiments,
performed after 2, 7, 14, and 30 days immersion in PBS. Hydrogel samples were loaded
in the Instron mechanical testing system and 20% compressive strain was applied and
held for 1 hour while the gel was submerged in a 37C PBS bath. Load versus time data
was recorded with the Instron Series IX software. Based on the Maxwell model, the
stress of a polymer under constant strain will decay exponentially
344
.

( ) exp t
t
=

0
Equation 5.3

123
Where o(t) is the stress at some time t, and t is the relaxation time constant. The
relaxation time constant for each copolymer was computed as the time needed to lower
the stress from its original value,
o
to

o
e
. The hydrogel height and diameter were also
measured 0, 30, and 55 minutes after unloading.

5.2.2. Statistical Analysis

The sample sizes for each of the previously described tests were n=6. Selected results
were compared statistically using one way ANOVA with a 95% confidence level.

5.3 Results
5.3.1 LCST Characterization

The LCSTs in the family of copolymers is shown in Figure 5.1 A. The LCST of the
PNIPAAm homopolymer was found to be 31.0 0.1C. Every copolymer synthesized
had a significant increase in LCST compared to the homopolymer (p<0.05). No
significant changes in LCST were made by varying the PEG molecular weight or the
molar ratio of NIPAAm monomer units to PEG blocks (p>0.05). All of the LCSTs in
this family of copolymers were between 31.3C and 32.4C, which is in the range
necessary for in situ gel formation. Similarly, the homopolymer had a transition enthalpy
of 4.350.24 J/g. Every branched copolymer had a significant increase in transition
124
enthalpy compared to the homopolymer (p<0.05) (Figure 5.1 B). There were no
significant differences in the enthalpy values among the branched copolymers (p>0.05).
The low PEG content PNIPAAm-PEG (8000 g/mol) and both the high and low PEG
content PNIPAAm-PEG (10,000 g/mol) copolymers were not considered for further
study because they only formed semisolid materials above their LCST, disqualifying
them as candidate materials for nucleus replacement.

5.3.2 Swelling and dissolution experiments

All of the copolymers exhibited a dry mass retention of 97% or greater over 90 days
immersion in vitro. None of the copolymers had a significant change in mass retention
between the 30 and 90 day time points except for the low PEG content PNIPAAm-PEG
(2000 g/mol), which showed some statistically significant increase to 101.8% (p<0.05).
This increase in the dry mass of the polymer could be attributed to ion uptake from the
immersion media
345
, but this was not confirmed.

Holding PEG block molecular weight constant and varying the molar ratio of NIPAAm
units to PEG blocks between 700:1 and 1600:1 produced no significant differences in
equilibrium water content of PNIPAAm-PEG (1000, 2000, and 4600 g/mol) (p>0.05).
Equilibrium water contents at 90 days immersion in vitro are shown in Table 5.1.

The data in Figure 5.2 shows the water content for gels composed of block sizes varying
between 1000 and 8000 g/mol. The molar ratio of NIPAAm monomer units to PEG
125
blocks is held constant at approximately 700:1. At the initial time point 0, the
copolymers with PEG block sizes of 2000, 4600, and 8000 g/mol had significantly higher
water contents (p<0.05) than the homopolymer and PNIPAAm-PEG (1000 g/mol).
However, at 90 days immersion, only PNIPAAm-PEG (4600 and 8000 g/mol) had
significantly higher equilibrium water contents (p<0.05) than the homopolymer control.

All of the branched copolymers and the homopolymer had significant decreases in water
content between the 0 and 7 day time points (p<0.05 in all cases). However, none of the
copolymers exhibited significant changes in water content after the 7 day time point
(p>0.05 in all cases).

5.3.3 Compressive mechanical studies

The compressive moduli at 15% for the family of copolymers at 7, 30, 60, and 90 days
immersion in vitro are shown in Figure 5.3. Equilibrium modulus values at 90 days are
shown in Table 5.1. All of the branched copolymers showed increasing trends in
stiffness over 90 days immersion in vitro. All of the copolymers reached an equilibrium
stiffness by 30 days, except for PNIPAAm-PEG (8000 g/mol), which equilibrated by 60
days immersion. Both the high and low PEG content copolymers (Figures 5.3 A and B,
respectively) exhibited decreasing stiffness as the PEG block molecular weight was
increased. However, there were no significant changes in the equilibrium compressive
modulus as the molar ratio of NIPAAm monomer units to PEG blocks was varied
(p>0.05).
126

5.3.3.1 Network Analysis with Rubber Elasticity Theory

Based on the theory of rubber elasticity, the compressive modulus of the hydrogels, G,
can be expressed in terms of the compressive stress, t
c
, the polymer volume fraction in
the swollen gel
2,s
, and the elongation, u. (Equation 5.4)
336,346
. In tensile testing, u is
defined as the length of the sample at any time divided by its initial length. Because
compressive testing was performed in this case, u was taken as the initial height of the
sample divided by the height at any time.

1/ 3
2,
2
1
c
s
G

Equation 5.4

The stress-strain behavior of the high PEG content copolymers, normalized to account for
differences in swelling behavior, is shown in Figure 5.4. The slope of the curves, or the
modulus G, decreases with increasing PEG molecular weight. The values for G at 90
days immersion in PBS are illustrated in Figure 5.5.

Using rubber elasticity theory, the effective molecular weight between crosslinks, M
e
, can
also be calculated. For a network crosslinked in the presence of a solvent, the following
relationship holds
347
:

127
1/ 3
2.
2,
2,
2
1 2
1
s
r
e n r
RT
M M

Equation 5.5

Where
2,r
and
2,r
are the polymer volume fraction and density in the relaxed state,
respectively, and M
n
is the number average molecular weight if no crosslinks were
introduced. The value for
2,r
is taken as 1, since the network is in its relaxed state when
it is completely dry. The density of the polymer in this state, 0.93 g/mL, was measured
with the heptane density determination system. The value of M
n
for the PNIPAAm
homopolymer was estimated based on the viscosity average molecular weight, M
v
,
(75,000 g/mol), determined previously for the same polymerization scheme by Thomas et
al. using Ubbelohode capillary viscometry
337
. For a linear polymer in a theta (O) solvent
which possesses the most probable molecular weight distribution, M
v
can be related to M
n

and the weight average molecular weight, M
w,
through the following expression:

M
n
: M
v
: M
w
= 1: 1.67 : 2 Equation 5.6

It has been shown previously that water is a O solvent for PNIPAAm
348
. Using the ratios
in Equation 5.6, the estimated value of M
n
for the PNIPAAm homopolymer was 44,910
g/mol. The calculated effective molecular weight between crosslinks, M
e
, was
determined for the high PEG content copolymers (Figure 5.6). The values for M
e

increased steadily between PEG block molecular weight 2000 and 8000 g/mol.
128

5.3.4 Stress Relaxation Studies

The relaxation time constants, as determined by Equation 4.3, over 30 days immersion
time in vitro are shown in Figure 5.7. All of the copolymers showed a significant
increase in the value of the time constant compared to the homopolymer control (p<0.05).
For the high PEG content copolymers (Figure 5.7 A), there was a significant increase in
the time constant between PNIPAAm-PEG (2000 g/mol) and PNIPAAm-PEG (4600
g/mol) (p<0.05). However, varying the PEG block molecular weight did not produce any
significant changes in the relaxation time constant for the low PEG content gels ( (Figure
5.7 B) (p>0.05). All materials recovered between 85 and 98% of their original height
within 55 minutes after unloading.

5.4 Discussion

The PNIPAAm-PEG copolymers had higher LCSTs and transition enthalpies than the
PNIPAAm homopolymer due to the presence of the hydrophilic PEG, which hinders the
collapse and subsequent dehydration of the PNIPAAm chains
211
. However, there were
no significant shifts in LCST or transition enthalpy as the copolymer structure was
varied; signifying that the hydrophilic/hydrophobic balance was not impacted
significantly by the range of PEG compositions studied.

129
It was investigated whether the water content of the precipitated phase is related to the
weight percent of hydrophilic component in the gel. If this were the case, the high PEG
content PNIPAAm-PEG (2000 g/mol) would have the same water content as a low PEG
content PNIPAAm-PEG (4600 g/mol) gel, since they contain approximately the same
weight percent of PEG. On the contrary, the PNIPAAm-PEG (4600 g/mol) held
significantly more water at all time points. This result suggests that overall water content
of the precipitated phase is tied to network mesh size. Since the PEG blocks do not
collapse like the thermoresponsive PNIPAAm chains after the LCST transition, large
spaces in the polymer network are created, allowing the gel to retain more water. The
low PEG content PNIPAAm-PEG (8000 g/mol) copolymers, as well as both PNIPAAm-
PEG (10000 g/mol) copolymers may have produced semi-solid gels because the high
mesh sizes allowed the network to be overwhelmed with free water after the LCST
transition. Conversely, PNIPAAm-PEG 1000 g/mol did not hold significantly more
water than the homopolymer, likely because of the tighter network structure.
Furthermore, the increases in equilibrium gel water content seen experimentally between
the high PEG content PNIPAAm-PEG (2000 g/mol) and PNIPAAm-PEG (8000 g/mol)
correspond well with the steady increases in M
e
predicted by rubber elasticity theory.

The gels exhibited an overall decreasing trend in compressive modulus as the PEG block
molecular weight was increased. This is likely due to the fact that the higher molecular
weight PEG blocks caused increases in gel water content. Water in hydrogels acts as a
plasticizer, causing increased flexibility of the polymer chains, decreasing the mechanical
properties as water content increases
349
. Additionally, varying the PEG content of the
130
gels produced no significant changes in equilibrium modulus. This result was also
consistent with the swelling results, which indicated that the molar ratio of NIPAAm
monomer units to PEG blocks had no effect on gel water content. The increases in gel
stiffness seen during the 90 day study is attributed to the gradual de-swelling of the gels,
which increases the polymer content, enhancing the mechanical properties. The longer
equilibration time for gels in the mechanical study compared to the swelling study is due
to the gel geometry. Water in the larger cylindrical samples used in the mechanical study
took longer to diffuse out of the gel network than that in the flat discs used in the swelling
study.

In addition, Figures 5.4 and 5.5 illustrate the compressive mechanical behavior of the gels
normalized to account for differences in swelling behavior. The effect of network
crosslinking density on the polymer modulus, G, can be determined from this data.
Because G decreased with increasing PEG block molecular weight, it can be inferred that
high molecular weight PEG blocks result in a looser network structure.

Joshi et al. determined that a polymeric hydrogel implant with an unconfined
compressive modulus of 50 kPa at 15% strain could successfully restore intervertebral
disc stiffness
350
. The mechanical results presented here can therefore be used as an
indication of whether or not there are suitable candidates in this family of copolymers to
serve as a nucleus pulposus replacement. The compressive mechanical results
demonstrated that, after 7 days immersion in vitro, all of the high PEG content
copolymers, except for PNIPAAm-PEG (8000 g/mol), and all of the low PEG content
131
copolymers, except for PNIPAAm-PEG (4600 g/mol), met this value of stiffness. By 60
days immersion, all of the copolymers met this value of stiffness.

In addition to meeting this minimum stiffness, the material must be designed so that its
elastic recovery is maximized. In this analysis, the relaxation time constant is used as a
measure of material elasticity. More elastic gels will exhibit a slower decrease in stress
over time and hence have higher relaxation times.

The elastic behavior of macromolecules can be linked to how their monomer units
interact with one another and with surrounding solvent molecules. Above the LCST of
PNIPAAm, its polymer chains collapse into densely packed globular conformations due
to the positive attractions between monomer units and between chains. In other words,
the energy of interaction between monomer-monomer molecules is lower than that of
monomer-solvent molecules. Compressing a PNIPAAm gel increases the frequency of
monomer-monomer contacts. Since this is the lowest free energy configuration for the
chains, they do not a have a tendency to return to a more stretched out conformation after
the external force is released. On the contrary, PEG molecules swell in water because the
monomer units prefer to be surrounded by the solvent molecules. When the PEG chains
are uniaxially compressed, the repulsive interactions between monomer units cause the
free energy of confinement for PEG chains to be high
351
. This is manifested in a high
elastic force compared to PNIPAAm chains. For this reason, all PNIPAAm-PEG
branched copolymers in this study had significantly higher relaxation time constants than
the homopolymer.
132

The end-to-end distance, R, of a polymer chain increases with molecular weight. The
free energy of confining a real linear chain in a good solvent into a slit of spacing D or a
cylinder with diameter D is proportional to R
3/5

351
. Consequently, high molecular weight
PEG chains exhibit high free energies of confinement. This property accounts for the
increase in elasticity between the high PEG content PNIPAAm-PEG (2000 g/mol) and
PNIPAAm-PEG (4600 g/mol). It is likely that the low PEG content copolymers showed
no changes in time constant as the PEG block molecular weight was increased because
there were not enough PEG chains in the system to impact the elasticity of the gel.

5.5. Conclusions

All of the copolymers had LCSTs in the range necessary for injectability and exhibited
satisfactory mass retention over 90 days immersion in vitro. PNIPAAm-PEG (4600 and
8000 g/mol) had significantly higher mass swelling ratios than the PNIPAAm
homopolymer, with the latter having the highest water content of all the copolymers.
After 7 days immersion in vitro, the high PEG content PNIPAAm-PEG (1000, 2000, and
4600 g/mol) and the low PEG content PNIPAAm-PEG (1000 and 2000 g/mol) met the
minimum stiffness of 50 kPa for the restoration of intervertebral disc stiffness. After 60
days in vitro, all of the copolymers met the minimum stiffness. The branched
copolymers showed improved elasticity over the PNIPAAm homopolymer, but
PNIPAAm-PEG (4600 and 8000 g/mol) had the highest relaxation time constants,
indicating maximum elasticity. Because of its high water content and suitable
133
mechanical properties, the high PEG content PNIPAAm-PEG (4600 g/mol) will be
further assessed as a material candidate for nucleus pulposus replacement.
134

Table 5.1 Equilibrium water contents and compressive moduli for PNIPAAm-PEG after 90 days
immersion in vitro.
PEG molecular
weight (g/mol)
PEG
content
Water content
Compressive modulus at
15% strain at 90 days
immersion in vitro (kPa)
Low 0.35 0.02 205 54
1000
High 0.35 0.03 127 64
Low 0.47 0.11 115 40
2000
High 0.41 0.01 119 38
Low 0.51 0.01 141 36
4600
High 0.52 0.02 90.3 9.3
8000 High 0.71 0.00 52.3 8.9

135


30.5
31.0
31.5
32.0
32.5
33.0
0 2 4 6 8 10 12
Weight % PEG
L
C
S
T

(
o
C
)

2
3
4
5
6
7
8
9
0 2 4 6 8 10 12
Weight % PEG

H

(
J
/
g
)

Figure 5.1 LCST and enthalpy of transition as a function of weight percent PEG in PNIPAAm-PEG
branched copolymers.

A
B
136
0
20
40
60
80
100
0 20 40 60 80 100
Time (days)
W
a
t
e
r

c
o
n
t
e
n
t

(
%
)
PNIPAAm-PEGDM (8000 g/mol)
PNIPAAm-PEGDM (4600 g/mol)
PNIPAAm-PEGDM (2000 g/mol)
PNIPAAm-PEGDM (1000 g/mol)
homopolymer control

Figure 5.2 Effect of PEG block molecular weight on gel water content.
137

0
50
100
150
200
250
300
PNIPAAm-PEG
(1000 g/mol)
PNIPAAm-PEG
(2000 g/mol)
PNIPAAm-PEG
(4600 g/mol)
PNIPAAm-PEG
(8000 g/mol)
K
P
a
7 day 30 day
60 day 90 day


0
50
100
150
200
250
300
PNIPAAm-PEG
(1000 g/mol)
PNIPAAm-PEG
(2000 g/mol)
PNIPAAm-PEG
(4600 g/mol)
K
P
a

Figure 5.3 Effect of PEG block size on the equilibrium compressive modulus of PNIPAAm-PEG
branched copolymers.
A
B
138
0
5
10
15
20
25
30
35
40
0 0.2 0.4 0.6 0.8 1 1.2
v
2,s
-1/3
(-1/
2
)

(
k
P
a
)
PNIPAAm-PEG (2000 g/mol)
PNIPAAm-PEG (1000 g/mol)
PNIPAAm-PEG (4600 g/mol)
PNIPAAm-PEG (8000 g/mol)

Figure 5.4 Stress-strain behavior of PNIPAAm-PEG branched copolymers at 90 days immersion in
PBS, normalized to account for the swelling of the gel.
139

0
10
20
30
40
0 2 4 6 8 10
PEG block molecular weight (x10
-3
)
M
o
d
u
l
u
s
,

G

(
k
P
a
)

Figure 5.5 Modulus, G, for the high PEG content PNIPAAm-PEG branched copolymers at 90 days
immersion in PBS, normalized to account for differences in the swelling behavior of the gels.
140
16000
16400
16800
17200
17600
18000
18400
0 2 4 6 8 10
PEG block molecular weight (x10
-3
)
E
f
f
e
c
t
i
v
e

M
o
l
e
c
u
l
a
r

W
e
i
g
h
t

B
e
t
w
e
e
n

C
r
o
s
s
l
i
n
k
s
,

M
e

Figure 5.6 The effective molecular weight between crosslinks, M
e
, for the high PEG content
PNIPAAm-PEG branched copolymers at 90 days immersion in PBS.
141

0
50
100
150
200
2 7 14 30
Days
S
e
c
o
n
d
s
control homopolymer
PNIPAAm-PEGDM (1000 g/mol)
PNIPAAm-PEGDM (2000 g/mol)
PNIPAAm-PEGDM (4600 g/mol)
PNIPAAm-PEGDM (8000 g/mol)

0
50
100
150
200
2 7 14 30
Days
S
e
c
o
n
d
s

Figure 5.7 Effect of immersion time in vitro on the compressive modulus at 15% strain of high PEG
content (a) and low PEG content (b) PNIPAAm-PEG branched copolymers.
A
B
S
e
c
o
n
d
s

142

Chapter 6 - OPTIMIZATION STUDIES TO PREVENT WATER
LOSS IN VIVO

6.1 Introduction

This chapter describes a two part study that was conducted to better understand the
dehydration behavior of the PNIPAAm-PEG (4600 g/mol) gels. The in vitro
characterization studies in Chapter 5 showed that the de-swelling behavior of the gels
upon heating to 37C is characterized by an initial gradual water loss, followed by an
equilibrium condition. To predict the implant behavior in vivo, it is necessary to
understand how the de-swelling rate and equilibrium water content change with osmotic
pressure of the surrounding medium. The disc tissues possess a high osmotic pressure
due to the flux of ions into the tissues generated by the fixed negative charges on the
proteoglycans
8
. J.P. Urbans group performed studies on the osmotic pressure of lumbar
disc tissues and reported the osmotic pressure of the healthy inner annulus and nucleus
pulposus to be approximately the same, at 0.2 MPa
26
. The first part of the study will
focus on analyzing the de-swelling behavior of PNIPAAm-PEG (4600 g/mol) gels over a
range of physiologically pertinent osmotic pressures.

PNIPAAm gels also exhibit a decrease in volume as a result of dehydration
352
.
Significant volume loss in vivo will prevent the implant from having intimate contact
with the inner annulus. It has been shown experimentally and through finite element
143
modeling that total nucleus replacements which are oversized, allowing for intimate
contact with the inner annulus, were most effective at restoring the compressive stiffness
of the intervertebral disc
171
. The second part of this dehydration study will focus on
optimizing the copolymer formulation to minimize water loss and subsequent volume
loss upon injection into the intradiscal environment. Ideally, the volume of injected
solution would equal that of the gel formed, allowing the implant to remain space filling
after the thermal transition.

6.2 Experimental Section
6.2.1 Materials

PEG diol (20,000 g/mol) (Serva), dialysis tubing (Spectrum Spectra/Por Biotech cellulose
ester, 500 Dalton MWCO), and 55 mm dialysis clips (Fisher Scientific) were all used as
received.

6.2.2 Methods

Gel swelling under a range osmotic pressures was evaluated using a modified version of
the procedure developed by J.P. Urban
24,26
. Varied concentrations of aqueous solutions
of PEG (20,000 g/mol) were prepared. The osmotic pressure of a macromolecular
solution is given by Equation 6.1, where is osmotic pressure, R is the gas constant, T
is absolute temperature, M
2
is the molecular weight of the macromolecule, B and C are
144
the second and third virial coefficients, and c
2
is the concentration of the macromolecule
in solution
351
.

+ + + = ...
3
2
2
2
2
2
Cc Bc
M
C
RT Equation 6.1


The values used for the second and third viral coefficients were 2.59e10
-3
and 1.35e10
-2
,
respectively
353
. Approximately 2mL of 10, 15, or 25 wt% aqueous PNIPAAm-PEG
(4600g/mol) solution were injected into dialysis tubing and sealed with dialysis clips.
The dialysis bags were then immersed in aqueous PEG solution at 37C. The volume of
PEG solution used was approximately 100 times the volume of PNIPAAm-PEG solution.
After 1, 2, 7, 14, and 30 days, the gels were removed from the dialysis bags, weighed,
dried under vacuum, and weighed again. Time 0 refers to solutions that were not
immersed in PEG solution, but immediately weighed, dried under vacuum, and weighed
again to determine water content prior to swelling. The water content was calculated at
each time point according to Equation 4.1.

6.2.3 Statistical Analysis

The sample sizes for each of the previously described tests were n=3. Selected results
were compared statistically with one way ANOVA and a 95% confidence level.

145
6.3 Results and Discussion
6.3.1 Osmotic Swelling Studies

The water content of PNIPAAm-PEG (4600 g/mol) gels over 30 days immersion in 37C
aqueous PEG (20,000g/mol) solution, with an osmotic pressure of 0.2 MPa, is shown in
Figure 6.1. As a comparison, a set of gels were also swollen in 0.4 MPa and pure water.
Between the 0 and 2 day time points, the gels swollen in 0.4 MPa dehydrated faster than
those swollen in 0.2 MPa osmotic pressure and water. By 7 days immersion, there were
no significant differences between the water contents of the gels swollen in 0.2 and 0.4
MPa (p>0.05). By 30 days, there were no significant differences in the water contents of
all three sets of gels (p>0.05).

These results indicate that the equilibrium gel water content for PNIPAAm-PEG (4600
g/mol) is not significantly affected by varying osmotic pressures in the range studied.
Osmotic pressure measurements are based on the use of a semi-permeable membrane
through which solvent molecules pass freely and polymer molecules cannot pass
354
. It is
possible that higher osmotic pressures did not appreciably affect equilibrium water
contents of the gels because the remaining water in the gel is sufficiently associated with
the polymer through hydrogen bonding such that the osmotic pressures studied did not
generate enough thermodynamic drive to cause further dehydration.

To ensure the implant remains space filling, it is necessary to eliminate the drastic water
loss occurring between the 0 and 2 day time points in Figure 6.1. The first avenue
146
explored was analyzing the relationship between the water content of the polymer
solution at room temperature and that of the gel at equilibrium. Figure 6.2 shows the
water content of gels swelled for 30 days at 37C in aqueous solutions of PEG (20,000
g/mol) with an osmotic pressure of 0.2 MPa. The gels were formed from aqueous 10, 15,
or 25 wt% PNIPAAm-PEG solutions at room temperature. Initially, the gels swelled
under the highest osmotic pressure dehydrated at the quickest rate. However, by 30 days
immersion, the water content of all three sets of gels was very similar, between 0.4 and
0.5.

Logically, it follows, that in order to minimize the magnitude of water loss after gelation,
the solution from which the gels are formed must be more concentrated. However,
solutions above 25 wt% polymer cannot be prepared directly by combining dry polymer
and water because they will not form uniformly.

6.3.2 Thermal Cycling Studies

Water loss upon gelation was minimized by thermal cycling. Solutions composed of 15
wt% PNIPAAm-PEG (4600 g/mol) were injected into dialysis bags at room temperature
and allowed to equilibrate at 37C and 0.4 MPa for 48 hours. When these gels were
subsequently cooled to room temperature they formed concentrated, homogenous
polymer solutions and the water content was measured. The water content of the
solutions before and after this 2 day cycle is indicated in Figure 6.3 as thermal cycles 0
and 1. As expected, there was significant water loss during this period (p<0.05), which
147
was large in magnitude. These concentrated polymer solutions were subsequently
injected into dialysis bags again at room temperature and re-immersed in the same 37C
osmotic environment for an additional 48 hours. The water content of the solutions after
this second thermal cycle is shown in Figure 6.3 as thermal cycle 2. There were no
significant changes in water content (p>0.05) compared to cycle 1. The cycle was
performed an additional 3 times to ensure repeatability. There were no significant
changes in water content between cycles 1 and 3 (p>0.05). However, there were slight
decreases in water content between cycles 1 and 4 and 1 and 5, which were statistically
significant (p<0.05), though small in magnitude. During the initial 48 hours immersion,
the solutions only retained approximately 25% of their initial volume. However, there
were no more significant changes in solution volume thereafter (p>0.05).

While homogenous polymer solutions cannot be prepared from dry powder at these high
concentrations, these results indicate that reversibility of the PNIPAAms phase transition
can be exploited in order to form them. Dilute polymer solutions can be thermally cycled
by equilibration in a 37C osmotic environment. These gels can then be cooled to room
temperature, forming more concentrated solutions that should show minimal water loss
once injected into the intradiscal environment.

6.4 Conclusions

The osmotic swelling studies revealed that while osmotic pressure influenced the rate of
PNIPAAm-PEG (4600 g/mol) gel dehydration, the equilibrium water content was not
148
dependent on osmotic pressure of the immersion media. Furthermore, equilibrium water
content of the gels is not appreciably affected by the concentration of the polymer
solution from which they were formed. Significant implant water loss upon gelation can
be prevented by equilibrating dilute polymer solutions in a 37 C environment. Upon
cooling to room temperature, the polymers will form uniform, concentrated solutions
which should show minimal water loss when gelled again.
149


0
20
40
60
80
100
0 5 10 15 20 25 30 35
Day
W
a
t
e
r

c
o
n
t
e
n
t

(
%
)
0.4 MPa 0.2 MPa water

Figure 6.1 Effect of immersion media osmotic pressure on the water content of PNIPAAm-PEG
(4600 g/mol).

150
0
20
40
60
80
100
0 5 10 15 20 25 30 35
Time (days)
W
a
t
e
r

c
o
n
t
e
n
t

(
%
)
25 wt% polymer solution
10 wt% polymer solution
15 wt% polymer solution

Figure 6.2 Effect of solution concentration at room temperature on equilibrium water content of
PNIPAAm-PEG (4600 g/mol) gels.
151
0
20
40
60
80
100
0 1 2 3 4 5 6
No. thermal cycles
W
a
t
e
r

c
o
n
t
e
n
t

(
%
)
0
20
40
60
80
100
(
V
t
/
V
t
=
0
)

x

1
0
0
Water content
Volume retention

Figure 6.3 Effect of thermally cycling the polymer solution by equilibrating in a 37 C osmotic
environment, followed by cooling to room temperature.

152

Chapter 7 - SYNTHESIS AND CHARACTERIZATION OF
BIOADHESIVE PNIPAAM-PEG/PEI BLENDED
COPOLYMERS

7.1 Introduction

Imparting bioadhesive properties to this hydrogel system will reduce the risk of implant
migration or expulsion out of the annular defect created by injection of the nucleus
replacement. Additionally, bioadhesive properties will increase the potential for using
this material as nucleus replacement in discs which are in the later stages of degeneration,
which possess tears, cracks, and fissures that would normally increase the risk of implant
migration or expulsion.

Because of its injectable nature, this bioadhesive could be used as a closure material for
the repair of a damaged annulus. The technology could be used in conjunction with a
discectomy, to close the herniated disc, or with a total nucleus replacement, to close the
defect created by injection of the material and help anchor the implant in the center of the
disc. The bioadhesive could also repair annular tears associated with degeneration.
Using a hydrogel material to close these annular defects would help stabilize the disc
structure, prevent prolapse of disc material, and restore biomechanical function
330
. This
strategy can also be used to address pain produced by cytokines and inflammatory
mediators that can migrate through annular fissures and produce pain by stimulating the
153
nociceptors in the outer annulus
51
. Sealing annular fissures will prevent the diffusion of
these molecules to the nerve roots in the outer annulus.

The plan for imparting bioadhesive properties to the hydrogel system begins with the
incorporation of a third component in the hydrogel system, poly(ethylene imine) (PEI).
PEI is a highly branched polyamine
277
. The pKa value of the primary amine is around 9,
the secondary amine around 8 and the tertiary amine around 67
278
. At or above a pH of
10, the amine groups are uncharged
279
. In the pH range from 10 to 4, approximately
60% of the total amount of amino groups are protonated
355
.

PEI will be incorporated in the PNIPAAm-PEG (4600 g/mol) network by physical
blending. The blends will be prepared by co-dissolving PNIPAAm-PEG and PEI in
water at room temperature. PEI will comprise approximately 4.25 wt% of the aqueous
solution, while the PNIPAAm-PEG copolymer content will be fixed at 15 wt%. This
weight percent of PEI was chosen because it is approaching the maximum amount of PEI
that can be co-dissolved with 15 wt% PNIPAAm-PEG. Above the LCST of PNIPAAm,
a collapsed network will form, physically entrapping the PEI chains within it.

The three component system will be injected into the disc and allowed to thermally
transition, forming a gel. This will be followed by the injection of aqueous
glutaraldehyde solution into the gel core. The aldehyde groups of the glutaraldehyde will
become covalently bonded with the primary amines of PEI, crosslinking PEI with itself.
This reaction between amine and aldehyde functionality is known as a Schiffs base
154
reaction
356
(Equation 7.1) and has been studied with Fourier Transform Infrared (FTIR)
spectroscopy. For this reason, FTIR will be used to verify the crosslinking chemistry in
the PNIPAAm-PEG/PEI blends.

NH
2
+ O=CH- N=CH + H
2
O Equation 7.1

In the disc, after the glutaraldehyde crosslinks the PEI, unreacted glutaraldehyde will
continue to diffuse through the gel and crosslink it to the tissues in contact with the
implant by reacting with the amine functions present in the proteins of the tissue
extracellular matrix. Because only small amounts of the reacting dial will be injected
directly into the gel, reaction with tissues not in immediate proximity to the gel should
not occur.

It is postulated that this system is feasible for use in the intervertebral disc despite the
potential cytotoxicity of PEI
280,283,290,291,357
and glutaraldehyde
318,325,327,328,358
. Because
the disc tissues contain large amounts of extracellular matrix proteins and fewer cellular
components
19,318
, they are less sensitive to the cytotoxicity, and the inflammatory
response from the body can be minimized with a controlled release profile of
glutaraldehyde.

Preliminary work for this project has indicated that injecting larger volumes of dilute
glutaraldehyde solutions results in a more uniform crosslinking than small volumes of
highly concentrated solutions. Therefore, the swelling, mechanical, and adhesive
155
properties of the hydrogels will be evaluated based on the injection of 0.62 mL of a 5
wt% glutaraldehyde solution in water into the gel core. As a comparison, the properties
of the system were also evaluated using 10 and 20 wt% glutaraldehyde solutions.

The overall objective of these studies is to successfully impart bioadhesive properties to
the injectable PNIPAAm-PEG hydrogel system. The basic synthesis and characterization
techniques will be established, laying the foundation for future optimization of the
adhesion system.

7.2 Experimental Section
7.2.1 Materials

Poly(ethylene imine) (PEI) (M
n
=10,000 g/mol, water-free), Copper(II) sulfate
pentahydrate, Iron(III) chloride, 3-Methyl-2-benzothiazolinone hydrazone hydrochloride
monohydrate (MBTH), and deuterium oxide (D
2
O) were obtained from Sigma Aldrich
and used as received. PEI (M
n
=60,000 g/mol, 50 wt% in water) and glutaraldehyde (70
wt% in water) were also obtained from Sigma Aldrich and diluted before use to the
concentrations specified in the method descriptions. Fresh porcine skin was obtained
from a butcher.

156
7.2.2 Methods
7.2.2.1 PNIPAAm-PEG/PEI Blend Preparation and Characterization

A polymer solution was prepared at ambient temperatures composed of 15 wt%
PNIPAAm-PEG (4600 g/mol), 4.25wt% PEI (M
n
= 60,000 g/mol), and the balance water.
The solutions were heated to 37C for gelation to occur. The precipitated gels were
lyophilized overnight and re-dissolved in D
2
O. They were analyzed with
1
H NMR
(Varian 300, Palo Alto, CA) to verify and quantify the incorporation of PEI into the gel
network.

The chemical stability of the blends was evaluated by comparing the amount of PEI
released from the network upon gelation to the amount dissolved in the room temperature
solution. Approximately 2 mL of the PNIPAAm-PEG/PEI (M
n
=10,000) or (60,000
g/mol) solutions were heated inside a closed vial for 10 minutes. Then, 10 mL of
deionized water, preheated to 37C, was added to each vial. The vials were agitated at 40
rpm for 2 minutes. Then 500 L of each immersion media was added to 500 L of 0.4
wt% aqueous copper(II) sulfate pentahydrate. Upon the addition of copper (II) ions, PEI
forms a blue cuprammonium complex
359
, allowing the PEI to be quantified
spectrophotometrically. The absorbance of these solutions was measured at 550 nm with
a plate reader (BIO-TEK, model ELx800, Winooski, VT). The concentration of PEI in
the gel immersion media was determined using a standard curve of known PEI
concentrations. A linear relationship (r
2
=0.999 for PEI M
n
=60,000 g/mol and r
2
=0.993
for PEI M
n
=10,000 g/mol) was found between the absorbance at 550 nm and the amount
157
of PEI in concentration range tested, which was 0.001 to 6.67 mg/mL. Each optical
density measurement was performed in triplicate and the sample size was n=3.

7.2.2.2 PNIPAAm-PEG/PEI Crosslinking with Glutaraldehyde

For this and all subsequent characterization studies, uniformly sized cylindrical gel
samples were formed by injecting aqueous solutions of 15 wt% PNIPAAm-PEG (4600
g/mol) and 4.25wt% PEI (M
n
= 60,000 g/mol) into molds and heating to 37C for 10
minutes. The precipitated gels were removed from the molds and 0.62 mL of 5 wt%
aqueous glutaraldehyde was injected into the gel core with a 26 gauge needle at rate of 40
cc/hr, using a syringe pump (Autosyringe, model 5A, Hooksett, NH). The gels were then
lyophilized overnight and analyzed with FTIR (Nicolet Magna IR 560, Madison, WI) to
verify the covalent crosslinking of the PEI with the glutaraldehyde.

7.2.2.3 Free Glutaraldehyde Release

Uniformly sized gel samples formed from aqueous solutions composed of 15 wt%
PNIPAAm-PEG and 4.25 wt% PEI (M
n
=60,000 g/mol) were injected with either 5, 10, or
20 wt% glutaraldehyde. Once again, 0.62 mL of glutaraldehyde solution was injected
into the gel core with a 26 gauge needle at a rate of 40 cc/hr. This process was carried
out while the gels were fully immersed in 150 mL of deionized water. The gels were
swelled in this same immersion media throughout the study. The amount of free
158
glutaraldehyde in the immersion media over time was then quantified with the MBTH
method
360
. At t=10 mins, 3, 24, 48, and 96 hours, 300 L of immersion media was
combined with 300 L of 0.4 wt% aqueous MBTH and 0.2 wt% Iron (III) chloride. The
reaction was allowed to stand for approximately 10 minutes, and then 2 mL of acetone
was added. The optical density of the solution at 405 nm was measured with the plate
reader. The concentration of glutaraldehyde in the immersion media was determined
using the standard curve of known glutaraldehyde concentrations. A linear relationship
(r
2
=0.951) was found between the absorbance and the amount of glutaraldehyde in
concentration range 0.0002 to 0.10 mg/mL. The amount of glutaraldehyde released at
each time point was compared to the total amount injected into the gel core. Each optical
density measurement was performed in triplicate and the sample size was n=3.

7.2.2.4 PNIPAAm-PEG/PEI Gel Swelling

To characterize the swelling properties of the blends, uniformly sized cylindrical gel
samples composed of PNIPAAm-PEG/PEI (M
n
=60,000 g/mol) were prepared using
molds and immersed in PBS at 37C. A set of gels were pulled at 0, 1, 7, 14, and 30
days, weighed, and subsequently dried under vacuum and weighed again. Time 0 refers
to solutions that were not heated to 37C or immersed in PBS, but weighed, dried under
vacuum, and weighed again to determine water content prior to gel formation and
swelling. This procedure was repeated 3 additional times with a set of gels which were
injected with 5, 10, or 20 wt% glutaraldehyde solutions, according to the procedure
159
described above prior to immersion in PBS. The gel water content was calculated
according to Equation 4.1.

7.2.2.5 PNIPAAm-PEG/PEI Compressive Mechanical Properties

Unconfined uniaxial compression tests were performed on gel samples injected with 5,
10, or 20 wt% glutaraldehyde after 7 days immersion in PBS. As a comparison, a set of
gel samples which were not injected with glutaraldehyde were also tested. The hydrogels
were loaded in an Instron mechanical testing system (Instron Model 4442, Park Ridge,
IL) and compressed to 50% strain at a rate of 100%min
-1
while submerged in a 37C
PBS bath. Load and displacement data were recorded with the Instron BlueHill 1.2
software and converted to stress and strain values. The compressive moduli at 10, 15,
and 20% strain were approximated as the slope of the chord drawn between 5 and 15%
strain, 10 and 20% strain, and 15 and 20% strain, respectively.

7.2.2.6 Bioadhesive Force Studies

The bioadhesive capacity of PNIPAAm-PEG/PEI (M
n
=60,000 g/mol) blends was
determined using an Instron mechanical testing system (Instron Model 4442, Park Ridge,
IL). Uniformly sized cylindrical gels were fixed to the upper support with cyanoacrylate
adhesive. Sections of fresh porcine skin were cut and cleaned to remove excess fat. The
substrate was fixed to the bottom support with cyanoacrylate adhesive. The gel was
160
lowered slowly until full contact with the skin was achieved. Subsequently, the gels were
injected with 0.62 mL of 5, 10, or 20wt% glutaraldehyde at 40 cc/hr using a 26 gauge
needle. The gels were kept in contact with the substrate for 10 minutes. The upper
fixture was then withdrawn at a rate of 2mmmin
-1
and the load-displacement data was
recorded by the Instron BlueHill 1.2 software until the polymer became completely
detached from the skin. During the test, the skin and gel were submerged in a 37C PBS
bath. The sample size was n=3. A fresh piece of tissue was used for each repeat.

The load versus displacement data was converted to stress strain values and the
maximum force required to detach the gels from the biological samples was read directly
from this the curve. In addition, the work of adhesion was calculated by fitting the stress
strain curve for each sample to a sixth order polynomial, and calculating the area under
the curve between the strain values of 0 and that after which complete detachment
occurred.

7.2.2.7 Statistical Analysis

The sample sizes for each of the previously described tests were n=3. Selected results
were compared statistically with one way ANOVA and a 95% confidence level.

161
7.3 Results
7.3.1 PNIPAAm-PEG/PEI Blend Characterization

The successful physical incorporation of PEI (M
n
=60,000 g/mol) in the PNIPAAm-PEG
(4600 g/mol) network was verified with the
1
H NMR. The NMR spectrum for the blends
is shown in Figure 7.1. The incorporation of PEI was verified by the presence of the
peak for the CH
2
protons on the PEI at o=2.2 ppm. Additionally it was determined that
approximately 10.34.2% of PEI (M
n
=60,000 g/mol) that was dissolved in the solution at
room temperature was released from the network upon gelation (Figure 7.2). As a
comparison, the same measurement was performed on blends composed of PEI
(M
n
=10,000 g/mol) and it was found that 56.74.0% was released.

7.3.2 Gel Crosslinking with Glutaraldehyde

The FTIR spectra for a dry PNIPAAm-PEG/PEI (M
n
=60,000 g/mol) gel,
glutaraldehyde/PEI mixture, and a gel injected with glutaraldehyde are shown in Figures
7.3 A, B, and C, respectively. As can be seen in 7.3 A, the spectrum for the blend is
completely dominated by PNIPAAm, the characteristic peaks being the Amide I and II
bands at 1649 and 1543 cm
-1

338-340
. The peaks for PEI are not seen in this spectrum,
likely because the FTIR is not sensitive enough to detect its presence. The peak for free
NH
2
amines on the PEI should occur at 1596 cm
-1

316,361
, the C-H bending at 1456 cm
-1

361,362
, and C-C vibration and C-N stretch between 1300 and 1000 cm
-1

341,361
.

162
Illustrated in 7.3 B is the spectrum for a dried 1:1 mixture of PEI (M
n
=60,000 g/mol) and
5 wt% glutaraldehyde. The spectrum is shown simply to indicate that the peak at 1661
cm
-1
represents the C=N- covalent bond formed as a crosslink between the NH
2
on PEI
and the HC=O group on the glutaraldehyde
316,363
. In addition, the amide II band (HN=C-
) on the crosslinked PEI occurs at 1560 cm
-1

316
. These peaks could not be detected in the
spectrum of the PNIPAAm-PEG/PEI (M
n
=10,000 g/mol) blend that was injected with
glutaraldehyde (7.3 C) because PNIPAAm once again dominates the spectrum.

When this overlap occurs, the spectrum of one component can be subtracted from the
combined system in order to reveal the peaks of interest
364
. In this case, the spectrum for
the PNIPAAm-PEG/PEI blend was subtracted from that of the same blend injected with
5% glutaraldehyde. The results of the spectral subtraction are shown in Figure 7.4.
Peaks in the characteristic range for the C=N- and HN=C- bonds occur at 1668 cm
-1
and
1540 cm
-1
. The results of the FTIR difference spectroscopy indicated that covalent
crosslinking of the PEI occurring in this system can be described as a Schiffs base
reaction.

7.3.3 Free Glutaraldehyde Release

Shown in Figure 7.5 are the release profiles of free glutaraldehyde from the gels.
Initially, 10, 21, and 16% of the glutaraldehyde injected was released from the gels
injected with 5, 10, and 20% glutaraldehyde, respectively. This corresponds to a total of
3.00.4, 13.37.4, and 20.210.0 mg of glutaraldehyde. After 96 hours, 47, 83, and 59%
163
of the glutaraldehyde was released from the gels injected with 5, 10, and 20%
glutaraldehyde, respectively. This corresponds to a cumulative total of 14.53.9,
51.53.4, 73.214.8 mg of glutaraldehyde, respectively.

7.3.4 PNIPAAm-PEG/PEI Gel Swelling

The water content of PNIPAAm-PEG/PEI (M
n
=60,000 g/mol) blends with and without
glutaraldehyde injection over a 30 day immersion in PBS is shown in Figure 7.6. All of
the PEI-containing gels, including those that were crosslinked with glutaraldehyde, held
significantly more water than the PNIPAAm-PEG copolymer alone (p<0.05). At 30 days
immersion, only the gels crosslinked with 20% glutaraldehyde underwent a significant
decrease in water content compared to the polymer solution from which it was formed
(p<0.05). Furthermore, the PNIPAAm-PEG/PEI blend alone exhibited a significant
increase in water content between 0 and 30 days, from 78.20.0 to 83.71.2 % (p<0.05).
Though varying the concentration of glutaraldehyde solution injected made no significant
changes in gel water content (p>0.05), all of the crosslinked gels exhibited decreasing
trends in water content compared to the PNIPAAm-PEG/PEI blend. At 30 days
immersion, the gels injected with 10 and 20% glutaraldehyde held significantly less water
than the PNIPAAm-PEG/PEI alone(p<0.05).



164
7.3.5 PNIPAAm-PEG/PEI Compressive Mechanical Properties

Shown in Figure 7.7 are changes in the compressive mechanical properties of PNIPAAm-
PEG/PEI as a result of glutaraldehyde injection. The compressive modulus of the
PNIPAAm-PEG/PEI blend at 15% strain was 6.32.6 kPa. All of the blends exhibited
significant increases in compressive modulus (p<0.05) with glutaraldehyde injection.
However, there were no significant changes in the modulus value as the glutaraldehyde
concentration was varied from 5 to 20 wt% (p>0.05).

7.3.6 Bioadhesive Force Studies

Figure 7.8 illustrates the typical stress-strain curve for the in vitro bioadhesive force
studies. The curves shown in shades of blue represent PNIPAAm-PEG and PNIPAAm-
PEG/PEI hydrogels which were never injected with glutaraldehyde, but fully contacted
with the porcine skin and withdrawn at 2mmmin
-1
. The curves for these samples show a
gradual increase in stress followed by a plateau region. The initial increase in load is
likely caused by two factors. First, as the gel loses contact with the skin, the upper
fixture supports an increasingly greater portion of the weight of the gel itself. Secondly,
as the fixture moves up, it encounters resistance as it moves through water, contributing
to the load. The plateau region represents full loss of gel contact with the substrate.

The curves shown in red, orange, and pink represent samples that were injected with
glutaraldehyde while in contact with the porcine skin and subsequently withdrawn.
165
These samples also exhibited an initial increase in load but they continue to a higher
maximum value in the stress. This is indicative of greater adherence to the substrate.
The stress field caused by the withdrawal of the upper fixture will seek the most
vulnerable point in the adhesive bond and failure is initiated there
365
. This initial failure
is represented on the stress-strain curve as the first drop in stress. The subsequent drops
in stress represent the failure propagating. Again, complete separation from the substrate
is represented by the plateau region. It is worth noting that failure can occur at any of the
adhesive interfaces or by the fracture of the gel sample. In each of these cases, the
separation occurred at the interface between the gel and the substrate.

The mean maximum force required to detach PNIPAAm-PEG and PNIPAAm-PEG/PEI
gels from the porcine skin was taken as the maximum in the stress-strain curve before
complete detachment occurred. These values are shown in Table 7.1. The mean
maximum force of detachment for PNIPAAm-PEG and PNIPAAm-PEG/PEI without
glutaraldehyde injection was 0.640.14 kPa and 0.710.24 kPa, respectively. There were
no significant differences between these two values (p>0.05). All of the PNIPAAm-
PEG/PEI gels injected with glutaraldehyde exhibited significant increases in the mean
maximum force of detachment (p<0.05). Among the gels injected with glutaraldehyde,
varying the concentration between 5 and 20% increased the adhesion strength by a factor
of 1.6. There were no significant differences in the mean maximum force of detachment
for the gels injected with 10 and 20% glutaraldehyde (p>0.05), but both values were
significantly higher than that of the gels injected with 5% glutaraldehyde (p<0.05). The
values for work of adhesion are shown also shown in Table 7.1. While there were no
166
significant differences in work of adhesion for the PEI blends injected with
glutaraldehyde (p>0.05), all of them had significantly higher values that the PNIPAAm-
PEG/PEI blend and the PNIPAAm-PEG copolymer alone (p<0.05).

7.3 Discussion

A chemically stable hydrogel blend will be defined in this context as one in which all of
the PEI dissolved in the room temperature solution becomes physically entrapped in the
gel network during the collapse of the PNIPAAm chains. The blends composed of PEI
(M
n
=10,000 g/mol) released 5 times as much PEI as the blends composed of PEI
(M
n
=60,000 g/mol) immediately after gelation. In other words, the blends prepared from
the higher molecular PEI were more chemically stable. It is likely that the higher
molecular weight PEI allows for a greater degree of physical entanglements with the
PNIPAAm-PEG upon network collapse, reducing the free PEI released. A minimal
release of free PEI is considered a positive scenario since PEI with molecular weight
higher than 2000 Da has been shown to be cytotoxic
290,291
.. It is for this reason that the
PNIPAAm-PEG/PEI (M
n
=60,000 g/mol) gels were further characterized in these studies.

It is likely that the chemical stability of these blended networks will be further enhanced
by the injection of the glutaraldehyde, which will crosslink the PEI to itself. The amount
of PEI released from the network post-injection of the glutaraldehyde cannot be
quantified with the current method because the free glutaraldehyde and PEI in the
immersion media will readily react before they can be quantified. If histological studies
167
indicate a necessary decrease in the release of free PEI, a higher molecular weight PEI
can be utilized in the blend. Another option is to incorporate the PEI along the polymer
backbone as grafts. PEI chains can be functionalized with methacrylate groups and then
polymerized in the presence of NIPAAm and PEG dimethacrylate
366
. It should be
considered, however, that an increase in free PEI release could potentially increase the
adhesive strength of the gels. A thick coating of PEI on the gel surface could play an
important role in stabilizing the adhesive bond between the material and the surrounding
tissue.

The incorporation of PEI into the PNIPAAm-PEG network increased the swelling
capacity of the network appreciably. The equilibrium water content of PNIPAAm-PEG
increased significantly with the incorporation of PEI from 63.5.1.2 to 83.71.2 %. In
addition, the PNIPAAm-PEG/PEI blend exhibited a significant increase in water content
(p<0.05) between the 0 and 30 day time points. At the pH of the PBS, approximately 7.4,
several of the primary and secondary amine groups on the PEI are ionized. In salt
solutions, the charges on the PEI attract an influx of counterions into the polymer
network. The osmotic pressure created by this these ions causes the polymer to swell
279
.
The network expansion could also be due to the ionic repulsions between the positively
charged PEI chains
220
. Overall, these results are consistent with the findings of Qui et
al., who showed that incorporation of ionizable groups into PNIPAAm hydrogels can
improve the swelling of the network more effectively than non-electrolyte hydrophilic
components alone
190
.

168
This increase in water content is responsible for decreasing the mechanical properties of
the PNIPAAm-PEG. While the previous characterization studies have shown the
modulus of this copolymer after 7 days immersion in PBS to be over 50 kPa, the modulus
of the PEI blend at this same strain level was 6.32.6 kPa. The glutaraldehyde
crosslinking did, however, cause an 8 to 12-fold increase in the compressive modulus of
the gels, putting the moduli back above 50 kPa, indicating the potential of the material to
function as a total nucleus replacement by restoring the compressive stiffness of
intervertebral disc stiffness
171
.

Varying the concentration of glutaraldehyde injected did not produce significant changes
in gel water content. In addition, while the crosslinked gels exhibited a small increasing
trend in stiffness with increased glutaraldehyde concentration, the differences were not
significant (p>0.05). These results indicate that the crosslinking density of the gels did
not vary greatly with increasing glutaraldehyde concentration. Furthermore, increasing
glutaraldehyde concentration only produced modest gains in adhesive strength. The
adhesion tests were conducted 10 minutes after glutaraldehyde injection, corresponding
to approximately 3.00.4, 13.37.4, and 20.210.0 mg of free glutaraldehyde release for
the 5, 10, and 20% glutaraldehyde solutions, respectively. The adhesive strength only
increased by a factor of 1.6 in this range.

The above results can be better understood by analyzing the release profile of free
glutaraldehyde shown in Figure 7.6. A sustained release of unreacted glutaraldehyde
from the gel network occurred for 48 hours. It is apparent that a large portion of the
169
glutaraldehyde diffused out of the network unreacted, and only a small portion of the
glutaraldehyde is contributing to the crosslinking of the PEI and the adhesion to the
tissue.

Ideally, a much smaller portion of the glutaraldehyde should be released from the
network, though the target amount will be a balance between the desired adhesive
strength and minimization of large scale tissue damage. A higher concentration of PEI
inside the gel is one way to minimize the long term release of glutaraldehyde. This
would increase the likelihood of the amine-aldehyde reaction, so it could also potentially
increase the crosslink density of the network, lower the water content and increase the
stiffness. Employing more dilute solutions of glutaraldehyde and a slower injection rate
could also minimize the long term release of glutaraldehyde.

Slivka et al.
330
conjectures that the crosslinker molecular weight should be at least 100
Da, otherwise diffusion into the environment surrounding the disc would occur too easily.
Using a high molecular weight dialdehyde, such as PEG dialdehyde, would slow the
diffusion of the molecules through the network, also making reaction more likely.
Perhaps the best option in terms of minimizing the diffusion of aldehydes into the tissue
would be to immobilize the aldehyde groups on the surface of the implanted material
328
.
This could be achieved by conjugating an aldehyde group to one end of a PEG chain that
possesses a methacrylate group on the other end. The methacrylate group can be reacted
in the presence of NIPAAm monomer to form a copolymer
366
.
170
7.4 Conclusions

In these studies, an injectable, bioadhesive hydrogel system was developed which has the
potential to serve as a synthetic replacement for the nucleus pulposus of the intervertebral
disc or as an annulus closure material. Gel samples were formed at 37C from aqueous
solutions at room temperature composed of 15 wt% PNIPAAm-PEG (4600 g/mol) and
4.25 wt% PEI (M
n
=60,000 g/mol).
1
H NMR results indicated the successful physical
incorporation of PEI into the PNIPAAm-PEG network by blending, though 10.34.2 %
of the PEI that was dissolved in the room temperature solution was released from the
network immediately upon gelation. This value increases with decreasing PEI molecular
weight. The covalent crosslinking between the amine functionalities on the PEI and the
aldehyde functionalities on the glutaraldehyde was verified using FTIR difference
spectroscopy. Mechanical characterization of these blends showed a significant increase
in compressive modulus following glutaraldehyde injection, also an indication that
crosslinking occurred. Post-injection of the glutaraldehyde, all of the gels attained a
modulus value of at least 50 kPa at 15% strain, indicating their potential for restoring the
compressive intervertebral disc stiffness as a total nucleus replacement
171
. Varying the
amount of glutaraldehyde injected into the gels at 40 cc/hr did not have an appreciable
effect on the water content or mechanical stiffness of the gels.

The bioadhesive force studies show a significant increase in the mean maximum force to
the break the adhesive bond between the PNIPAAm-PEG and PNIPAAm-PEG/PEI gels
and the porcine skin when 5, 10, or 20 wt% glutaraldehyde was injected into the gel core.
The bioadhesive strength of the gels injected with 10 and 20 wt% glutaraldehyde was
171
significantly higher than that of the gels injected with 5 wt% glutaraldehyde (p<0.05).
The results from this study indicate that the reactivity between amines and aldehyde
functionalities can be exploited in order to impart bioadhesive properties to PNIPAAm-
PEG/PEI copolymers.
172

Table 7.1 The mean maximum force and work of adhesion required to detach the PNIPAAm-PEG
and PNIPAAm-PEG/PEI gels from the biological substrates.

Mean Maximum Force of
Detachment (kPa)
Work of Adhesion (kPa)
5% glutaraldehyde 0.70.3 1.50.7
10% glutaraldehyde 0.60.1 1.6.08
20% glutaraldehyde 1.40.0 1.71.1
PNIPAAm-PEG/PEI 2.50.2 0.10.1
PNIPAAm-PEG 2.20.1 0.00.0

173

Figure 7.1 The
1
H NMR spectrum for PNIPAAm-PEG/PEI (M
n
=60,000 g/mol) blend in deuterium
oxide at 25C.
174

0
20
40
60
80
100
PNIPAAm-PEG/PEI (60,000 g/mol) PNIPAAm-PEG/PEI (10,000 g/mol)
P
E
I

r
e
l
e
a
s
e
d

(
%
)

Figure 7.2 The chemical stability of PNIPAAm-PEG/PEI (M
n
=10,000) and PNIPAAm-PEG/PEI
(60,000 g/mol) blends.
175

Figure 7.3 The FTIR spectra for a dry (A) PNIPAAm-PEG/PEI gel, (B) glutaraldehyde/PEI mixture,
(C) PNIPAAm-PEG/PEI gel injected with glutaraldehyde.
176

Figure 7.4 FTIR difference spectroscopy for the analysis of PNIPAAm-PEG/PEI crosslinked gels.
177
0
20
40
60
80
100
0 20 40 60 80 100 120
Time (hrs)
G
l
u
t
a
r
a
l
d
e
h
y
d
e

r
e
l
e
a
s
e

(
%
)
5% glutaraldehyde 10% glutaraldehyde 20% glutaraldehyde

Figure 7.5 Release profile of free glutaraldehyde from the PNIPAAm-PEG/PEI network.
178
50
60
70
80
90
100
0 5 10 15 20 25 30 35
Time (days)
W
a
t
e
r

c
o
n
t
e
n
t

(
%
)
PNIPAAm-PEG
PNIPAAm-PEG/PEI
5% glutaraldehyde
10% glutaraldehyde
20% glutaraldehyde

Figure 7.6 The water content over a 30 day immersion in PBS for PNIPAAm-PEG/PEI blends with
and without glutaraldehyde injection.
179
0
40
80
120
160
PNIPAAm-
PEG/PEI
5% glutaraldehyde 10%
glutaraldehyde
20%
glutaraldehyde
k
P
a

Figure 7.7 The compressive modulus of PNIPAAm-PEG/PEI gels at 15% strain with and without
glutaraldehyde crosslinking at 7 days immersion in PBS.
180
0
0.5
1
1.5
2
2.5
3
0 50 100 150 200 250 300
Strain (%)
k
P
a
5% glutaraldehyde
10% glutaraldehyde
20% glutaraldehyde
PNIPAAm-PEG/PEI blend
PNIPAAm-PEG branched
copolymer

Figure 7.8 Typical stress strain curves for the in vitro bioadhesive force studies.

181


Chapter 8 - CONCLUSIONS AND FUTURE
RECOMMENDATIONS

8.1 Conclusions

The long term objective of this project was to develop an in situ forming nucleus
pulposus replacement, which upon implantation, could prevent or postpone the annular
degeneration process by restoring the healthy biomechanics of the intervertebral disc. A
class of PNIPAAm-PEG branched copolymers was characterized. From this class, a
material candidate which exhibited suitable, stable material properties over long term
immersion in vitro was further optimized to minimize water loss upon gelation, therefore
remaining space-filling in vivo. Bioadhesive properties were then imparted to the
hydrogel system, increasing its potential for use as a total nucleus replacement or tissue
adhesive in discs with annular degeneration.

First, the successful polymerization of NIPAAm was shown with FTIR. This result was
verified with
1
H NMR. In addition,
1
H NMR was used to verify the covalent
incorporation of PEG along the polymer backbone and quantify the molar ratio of
NIPAAm monomer units to PEG blocks in each of the copolymers. A family of
copolymers was created by varying the PEG block molecular weight between 1000 and
10,000 g/mol. For each PEG block molecular weight, a high and low PEG content
copolymer was synthesized by keeping the molar ratio of NIPAAm monomer units to
PEG blocks at either 700:1 or 1600:1.
182

All of the copolymers had LCSTs in the range necessary for injectability and exhibited
satisfactory mass retention over 90 days immersion in vitro. PNIPAAm-PEG (4600
g/mol) and PNIPAAm-PEG (8000 g/mol) had significantly higher water content than the
PNIPAAm homopolymer, with the latter having the highest water content of all the
copolymers. After 7 days immersion in vitro, the high PEG content PNIPAAm-PEG
(1000, 2000, and 4600 g/mol) and the low PEG content PNIPAAm-PEG (1000 and 2000
g/mol) met the stiffness value of 50 kPa, indicating potential for the restoration of
intervertebral disc stiffness. After 60 days in vitro, all of the copolymers met this value of
stiffness. The branched copolymers showed improved elasticity over the PNIPAAm
homopolymer, but PNIPAAm-PEG (4600) and PNIPAAm-PEG (8000 g/mol) had the
highest relaxation time constants, indicating maximum elasticity. Because of its high
water content and suitable mechanical properties, the high PEG content PNIPAAm-PEG
(4600 g/mol) was further assessed as a material candidate for nucleus pulposus
replacement.

The osmotic swelling studies revealed that while osmotic pressure influenced the rate of
PNIPAAm-PEG (4600 g/mol) gel dehydration, the equilibrium water content was not
dependent on osmotic pressure of the immersion media in the range tested. Furthermore,
equilibrium water content of the gels is not appreciably affected by the concentration of
the polymer solution from which they were formed. Significant implant water loss due to
de-swelling can be prevented by equilibrating 15 wt% polymer solutions in a 37 C
183
environment. Upon cooling to room temperature, the polymers will form uniform, more
concentrated solutions which should show minimal water loss when gelled again.

In the last portion of this project, the hydrogel system was made bioadhesive. Gel
samples were formed at 37C from aqueous solutions composed of 15 wt% PNIPAAm-
PEG (4600 g/mol) and 4.25 wt% PEI (M
n
=60,000 g/mol).
1
H NMR results indicated the
successful physical incorporation of PEI into the PNIPAAm-PEG network by blending.
The chemical stability of the blended network decreased with decreasing PEI molecular
weight. The covalent crosslinking between the amine functionalities on the PEI and the
aldehyde functionalities on the glutaraldehyde was verified using FTIR difference
spectroscopy and can be described by Schiffs base chemistry. The swelling
characterization revealed that PNIPAAm-PEG/PEI blends exhibit significantly better
water retention than PNIPAAm-PEG alone (p<0.05) in PBS. Mechanical
characterization of these blends showed significant increases in compressive modulus
following the injection of 5, 10, or 20 wt% glutaraldehyde. Post-injection of the
glutaraldehyde, all of the gels attained a modulus value of at least 50 kPa at 15% strain,
indicating their potential for restoring the compressive intervertebral disc stiffness as a
total nucleus replacement. Varying the concentration of glutaraldehyde injected into the
gels at 40 cc/hr did not have an appreciable effect on the water content or mechanical
stiffness of the gels. The results from the bioadhesive force study indicate that the
reactivity between amines and aldehyde functionalities can be exploited in order to
impart bioadhesive properties to PNIPAAm-PEG/PEI copolymer blends. A significant
increase (p<0.05) was observed in the mean maximum force to break the adhesive bond
184
between the PNIPAAm-PEG and PNIPAAm-PEG/PEI gels and the porcine skin when 5,
10, or 20 wt% glutaraldehyde was injected into the gel core. The bioadhesive strength of
the gels injected with 10 and 20 wt% glutaraldehyde was significantly higher than that of
the gels injected with 5 wt% glutaraldehyde (p<0.05).

8.2 Recommendations for Future Work

More in-depth mechanical and biomechanical studies on the PNIPAAm-PEG (4600
g/mol) base formulation and PNIPAAm-PEG/PEI adhesive formulation are warranted.
Joshi et al.
350
evaluated solid polymer hydrogels formed from blends of poly(vinyl
alcohol) and poly(vinyl pyrrolidone) for nucleus replacement. The hydrogels were
loaded dynamically at 10 million cycles for compression fatigue. Fatigue testing has also
been performed on a number of other material candidates being investigated for nucleus
pulposus replacement
175,163,182
. Joshi et al.
350
also implanted the hydrogels in cadaveric
specimens to evaluate the compressive behavior of the implanted discs. The authors also
recommended testing the implant under complex loading conditions.

With the addition of PEI to the network, the swelling behavior of the copolymers in PBS
improved dramatically. The swelling behavior of PEI containing gels should also be
evaluated within a range of physiologically pertinent osmotic pressures. Furthermore, the
need for thermal cycling to minimize water loss upon gelation needs to be re-evaluated.
It is likely that the presence of PEI will increase the swelling capacity of the network
such that minimal volume loss upon gelation will be achieved with more dilute polymer
185
solutions at room temperature. The chemical stability of the blends post-glutaraldehyde
injection should also be characterized over long term immersion in vitro and under
repetitive loading conditions.

A target adhesive strength needs to be established for the injectable, bioadhesive
hydrogel system. In order to do so, gels of varying adhesive capacities (determined by in
vitro bioadhesive force studies) should be implanted into the discs of cadaveric
specimens, and subjected to static, cyclic, bending, and torsional loading to seek signs of
implant migration or expulsion. An understanding can be gained about the minimum
bioadhesive strength that is suitable for the application. In addition, if the material were
also used as an adhesive for the repair of annular defects, mechanical tests are warranted
to determine if the requisite mechanical properties differ from that of a nucleus
replacement.

The work in the current studies has led to valuable insight into the parameters that can be
varied in order to modify the properties of the material. Slight increases in adhesive
strength were seen with increasing free glutaraldehyde release into the porcine tissue.
The release of free glutaraldehyde from the network can likely be tailored by using varied
injection flow rates, glutaraldehyde solution volumes, or glutaraldehyde solution
concentrations. It is also recommended that other dialdehyde molecules such as PEG
dialdehyde be studied. Larger crosslinkers would likely diffuse through the network
slower, making them more likely to react, and decreasing their release into the
surrounding medium. If the release profile indicates a prolonged dialdehyde release,
186
adhesion tests should be performed at time points following extended contact between the
material and tissue. To eliminate diffusion of aldehydes into the tissues all together,
aldehyde groups can be immobilized on the surface of the implanted material
328
. This
could be achieved by conjugating an aldehyde group to one end of a PEG chain that
possesses a methacrylate group on the other end. The methacrylate group can be reacted
in the presence of NIPAAm monomer to form a copolymer
366
.

It is also proposed that blending PNIPAAm-PEG with lower molecular weight PEI will
increase the adhesive capacity of the material. The loss of chemical stability of the gel
network, probably due to a lower frequency of molecular entanglements, causes a thick
coating of PEI to form on the surface of newly formed gels which can enhance adhesion
to the tissue. Therefore, the adhesive capacity of blends composed of PEI (M
n
=10,000
and 800 g/mol) should be investigated. The blend needs to be chemically stable enough,
however, to retain enough PEI in the network for uniform crosslinking to occur following
glutaraldehyde injection.

It is likely that the adhesive capacity of the gels will have to be balanced with the
cytotoxic effects of PEI
280,283,290,291,357
and glutaraldehyde
318,325,327,328,358
. Therefore,
histological studies will be warranted to determine the in vivo inflammatory response to
the system. The inflammatory response to the optimized system should be minor and
small in duration. It is also recommended that the use of less cytotoxic crosslinkers, such
as genipin
325
, be evaluated. Genipin is a naturally occurring compound, derived from
187
fruits of Gardenia jasminoides Ellis
367
. Genipin crosslinks molecules containing
primary amines
368
.
188
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218
Vita




Jennifer Vernengo was born on February 8, 1980 in Puebla, Mexico. She
graduated from Highland High School in Blackwood, NJ in 1998 and enrolled in Drexel
University in Philadelphia, PA. At Drexel, Jennifer pursued a Bachelors of Science in
chemical engineering. In 1999, she worked as a co-op at Dupont and assisted in the
operation and analytical support of their wastewater treatment plant. In 2000, Jennifer
worked as a co-op at Dupont Fluoroproducts, where she supervised the production of
purified Zyron C-318 gas via an extractive distillation system. In 2001, Jennifer
completed a final co-op cycle at the Dupont Experimental Station, where she assisted in
the development of a lab reactor system for the production of oxetane and gained
valuable experience with the design, testing, and optimization of chemical processes. In
2003, Jennifer completed her undergraduate degree and began to pursue a Ph.D. in
chemical engineering under the guidance of Dr. Anthony Lowman. Her research focused
on the design and development of injectable, bioadhesive hydrogels for nucleus pulposus
replacement and the repair of the damaged intervertebral disc. During her time at Drexel,
Jennifer has coauthored publications, presented her research at national and regional
conferences, and received numerous awards and honors. Jennifer defended her Ph.D.
thesis on January 31, 2007.