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( ) 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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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.