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Poly(N-isopropylacrylamide)-based
semi-interpenetrating polymer
networks for tissue engineering
applications. Effects of linear
poly(acrylic acid) chains on rheology
Ranee A. Stile , Eugene Chung , Wesley R. Burghardt & Kevin E.
Healy

To cite this article: Ranee A. Stile , Eugene Chung , Wesley R. Burghardt & Kevin E. Healy
(2004): Poly(N-isopropylacrylamide)-based semi-interpenetrating polymer networks for tissue
engineering applications. Effects of linear poly(acrylic acid) chains on rheology, Journal of
Biomaterials Science, Polymer Edition, 15:7, 865-878

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Poly(N-isopropylacrylamide)-based semi-interpenetrating
polymer networks for tissue engineering applications.
Effects of linear poly(acrylic acid) chains on rheology
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RANEE A. STILE 1 , EUGENE CHUNG 2 , WESLEY R. BURGHARDT 3

and KEVIN E. HEALY 2,4,∗

1 Department of Biomedical Engineering, Robert R. McCormick School of Engineering and Applied

Sciences, Northwestern University, Evanston, IL 60208, USA
2 Department of Bioengineering, University of California at Berkeley, 370 Hearst Memorial Mining
Building, No. 1760, Berkeley, CA 94720-1762, USA
3 Department of Chemical Engineering, Robert R. McCormick School of Engineering and Applied
Sciences, Northwestern University, Evanston, IL 60208, USA
4 Department of Materials Science and Engineering, University of California at Berkeley,
370 Hearst Memorial Mining Building, No. 1760, Berkeley, CA 94720-1760, USA

Received 9 October 2004; accepted 30 January 2004

Abstract—Semi-interpenetrating polymer networks (semi-IPNs), comprised of poly(N-isopropyl-

acrylamide-co-acrylic acid) (p(NIPAAm-co-AAc)) hydrogels and linear p(AAc) chains, were synthe-
sized, and the effects of the p(AAc) chains on semi-IPN rheology were examined. Oscillatory shear
rheometry studies were performed and the rheological data were analyzed as a function of tempera-
ture, frequency, and p(AAc) chain amount (weight average molecular weight (Mw ) 4.5 × 105 g/mol).
At 22◦ C, the semi-IPNs, as well as control p(NIPAAm-co-AAc) hydrogels, demonstrated rheologi-
cal data that were representative of soft, loosely cross-linked solids. Furthermore, only the highest
p(AAc) chain amount tested affected the rigidity of the p(NIPAAm-co-AAc)-based semi-IPNs, as
compared to the p(NIPAAm-co-AAc) hydrogels. At 37◦ C, the complex shear moduli (G∗ ) demon-
strated by the p(NIPAAm-co-AAc)-based semi-IPNs were significantly greater than G∗ exhibited by
the p(NIPAAm-co-AAc) hydrogels, and the semi-IPN G∗ values significantly increased with increas-
ing p(AAc) chain amount. These results can be used to develop p(NIPAAm)-based semi-IPNs with
tailored mechanical properties that may function as scaffolds in tissue engineering initiatives.

Key words: Semi-IPN; N-isopropylacrylamide; rheology; oscillatory shear rheometry; acrylic acid.

∗ Towhom correspondence should be addressed at the Department of Materials Science and Engi-
neering. Tel.: (1-510) 643-3559. Fax: (1-510) 642-5792. E-mail: kehealy@socrates.berkeley.edu
 866 R. A. Stile et al.

INTRODUCTION
Poly(N-isopropylacrylamide) (p(NIPAAm)) chains and cross-linked p(NIPAAm)
hydrogels exhibit unique phase properties in aqueous media when heated above
the lower critical solution temperature (LCST) [1– 5], which is approx. 32◦ C [4].
At temperatures below the LCST, p(NIPAAm) chains are soluble in water and
cross-linked p(NIPAAm) hydrogels swell [6– 8], while at the LCST the chains
precipitate out of solution and the hydrogels demonstrate a volume-phase transition,
during which they collapse considerably, expel a large amount of pore water
and become stiff and opaque [6, 7]. This behavior is reversible [8] and can
be modified by polymerizing the NIPAAm monomer with more hydrophobic or
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more hydrophilic comonomers [3, 5]. p(NIPAAm)-based systems (e.g. copolymer

chains, cross-linked hydrogels, interpenetrating polymer networks and grafted
surfaces) have been developed for use in a number of diverse applications, including
tissue engineering, cell encapsulation, cell culture, solute delivery and molecular
separation [6, 8– 22].
Previously, we developed NIPAAm and acrylic acid (AAc) copolymer hydrogels
for use in tissue engineering applications [15, 23]. These p(NIPAAm-co-AAc)
hydrogels were loosely cross-linked, which allowed the matrices to be injected
through a small-diameter aperture at 22◦ C. When heated to body temperature (i.e.
37◦ C), the hydrogels demonstrated a significant increase (e.g. 10×) in rigidity
(i.e. complex shear modulus, G∗ ), without exhibiting a change in the volume
or water content of the matrix [15]. In addition, the injectable p(NIPAAm-co-
AAc) hydrogels supported bovine articular chondrocyte viability and promoted
articular cartilage-like tissue formation in vitro [15]. Furthermore, peptide-modified
p(NIPAAm-co-AAc) hydrogels supported rat calvarial osteoblast spreading and
proliferation in vitro [23]. While these results were encouraging, the peptide
functionalization process adversely affected the phase behavior of the p(NIPAAm-
co-AAc) hydrogels (i.e. the matrices collapsed considerably when heated to 37◦ C),
limiting their clinical use in tissue-engineering initiatives.
To circumvent this functionalization issue, we developed injectable semi-inter-
penetrating polymer networks (semi-IPNs), comprised of p(NIPAAm-co-AAc)
hydrogels with linear peptide-modified p(AAc) chains physically entangled within
the network [24]. These semi-IPNs were synthesized by first grafting peptides
to the COO− groups in the linear p(AAc) chains, and then simultaneously
polymerizing and cross-linking NIPAAm and AAc in the presence of the peptide-
functionalized p(AAc) chains. This approach offers important clinical and practical
advantages. First, since the p(AAc) chains are modified prior to the semi-IPN
synthesis, the p(NIPAAm-co-AAc) hydrogel is unaffected by the functionalization
process, decreasing the potential that the matrix will collapse once injected into
the body. Additionally, in practice, it is harder to modify three-dimensional (3D)
polymer matrices than linear polymer chains (e.g. degree of substitution, removal
of reactants, etc.). Furthermore, if different types of peptides or macromolecules
are required in a single hydrogel, it is possible that different functionalization
 Semi-IPNs for tissue engineering application 867

schemes would be needed, thus requiring that the reactions be performed in series.
It is also possible that subsequent reactions could adversely affect the peptides
or macromolecules that were previously grafted. In the case of a semi-IPN, the
peptides or macromolecules may be attached to the linear p(AAc) chains in parallel
prior to the semi-IPN synthesis, creating user-defined admixtures of functionalized
p(AAc) chains, and one functionalization scheme would not affect any other. Using
this methodology, we showed that p(NIPAAm-co-AAc)-based semi-IPNs modified
with peptides that bind to hyaluronic acid (HA) adhered to HA-grafted substrates in
an aqueous environment [24].
Recently, we analyzed the effects of linear non-functionalized p(AAc) chains on
the injectability and phase behavior (i.e. transparency, volume change, LCST and
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phase transition) of p(NIPAAm-co-AAc)-based semi-IPNs [25] and we found that

the p(AAc) chains did not affect the LCST or volume change of the semi-IPNs,
as compared to control hydrogels. As a continuation of this work, we performed
the current study, in which we used rheological methods to examine the effects of
linear non-functionalized p(AAc) chains on the rigidity of p(NIPAAm-co-AAc)-
based semi-IPNs. These rheological experiments are significant from a tissue-
engineering perspective. If the p(NIPAAm-co-AAc)-based matrices are to be used
as injectable scaffolds in a clinical setting, they must be injectable at temperatures
below body temperature, which requires that the materials demonstrate low rigidity.
Once being injected into the body, the matrices must provide adequate mechanical
integrity to support tissue formation, which necessitates that the materials exhibit
increased rigidity. While it is widely known that cross-linked p(NIPAAm) networks
demonstrate a change in rigidity at the LCST, relatively few reports have been
published detailing the use of rheological techniques to measure p(NIPAAm)
hydrogel rigidity [15, 26– 29]. More commonly, p(NIPAAm)-based rheological
studies have focused on p(NIPAAm) microgels (i.e. cross-linked p(NIPAAm)
hydrogel particles dispersed in a liquid medium) [30– 33] and aqueous p(NIPAAm)
solutions [34– 38].
One rheological technique that has been used to examine the rigidity of
p(NIPAAm)-based hydrogels is oscillatory shear rheometry [15, 28], in which an
oscillatory shear stress or shear strain is imposed on a material at frequency ω
and the resulting oscillatory shear strain or shear stress, respectively, is determined
[39, 40]. The functions that are generally examined in oscillatory shear rheometry
studies are the shear storage modulus (G , a measure of the energy stored and re-
covered per cycle), the shear loss modulus (G , a measure of the energy dissipated
or lost as heat per cycle), the complex shear modulus (G∗ , defined mathematically
as G∗ = G + iG , |G∗ | = (G2 + G2 )1/2 ) and the phase angle (δ, the phase
difference between the input variable and the output variable; tan δ = (G /G ))
[39– 43]. Importantly, these functions can be used to characterize materials as vis-
coelastic solids or liquids. For example, viscoelastic solids exhibit G values that
are both independent of ω and considerably larger than G at low ω [42, 44, 45].
In contrast, viscoelastic liquids demonstrate G and G values that approach 0 with
 868 R. A. Stile et al.

decreasing ω, scaling as G ≈ ω2 and G ≈ ω; hence G  G as ω approaches 0

[40, 42]. This means that δ → 90◦ as ω → 0 for liquids, which is in sharp contrast
to the corresponding result, δ → 0◦ as ω → 0, for ideal solids.
In this work, oscillatory shear rheometry studies were performed on p(NIPAAm-
co-AAc) hydrogels and p(NIPAAm-co-AAc)-based semi-IPNs. In the p(NIPAAm-
co-AAc) hydrogel studies, the molar ratio of NIPAAm : AAc in the feed was varied,
and in the p(NIPAAm-co-AAc)-based semi-IPN studies, the amount of the p(AAc)
chains (weight average molecular weight (Mw ) 4.5 × 105 g/mol) was varied. The
rheological data were examined as a function of both temperature (i.e. 22◦ C and
37◦ C) and ω (i.e. 0.001 Hz to 10 Hz).
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MATERIALS AND METHODS

Materials
NIPAAm, AAc, N,N  -methylenebisacrylamide (BIS) Chemzymes™ Ultrapure
grade, ammonium peroxydisulfate (AP) Chemzymes™ Ultrapure grade, N,N,N  ,N  -
tetramethylethylenediamine (TEMED) Chemzymes™ Ultrapure grade and linear
p(AAc) chains (Mw 4.5 × 105 g/mol, acid form) were obtained from Polysciences
(Warrington, PA, USA). Dulbecco’s phosphate-buffered saline (PBS; 1.51 mM
KH2 PO4 , 155 mM NaCl and 2.7 mM Na2 HPO4; without CaCl2 , without MgCl2 ;
pH 7.2 ± 0.1; ionic strength 0.165 M) was obtained from Gibco-BRL (Grand Is-
land, NY, USA). All materials were used as received.

p(NIPAAm-co-AAc) hydrogel synthesis

The p(NIPAAm-co-AAc) hydrogels were synthesized according to methods pub-
lished previously [15, 23] as shown in Scheme 1. NIPAAm, AAc, 0.005 g
(0.0325 mmol) of BIS and 50 ml of PBS were bubbled with dry nitrogen gas in
a two-neck flask for 15 min to remove dissolved oxygen. Two NIPAAm : AAc mo-
lar ratios were used: 96.25 : 3.75 (2.4395 g (21.59 mmol) of NIPAAm and 0.0605 g
(0.84 mmol) of AAc) and 96.5 : 3.5 (2.443 g (21.62 mmol) of NIPAAm and 0.057 g
(0.79 mmol) of AAc). Following the nitrogen gas purge, 0.020 g (0.0876 mmol) of
AP and 200 µl (1.3 mmol) of TEMED were added as the initiator and accelerator,
respectively. The mixture was stirred vigorously for 15 s and allowed to polymerize
at 22◦ C for 19 h under regular fluorescent lighting in a 250-ml glass beaker covered
with a glass plate. Following the polymerization, the p(NIPAAm-co-AAc) hydro-
gels were washed three times, 15–20 min each, in excess ultrapure water (UPW;
18 M-cm; pH between 5.5 and 7) to remove unreacted compounds.

p(NIPAAm-co-AAc)-based semi-IPN synthesis

The p(NIPAAm-co-AAc)-based semi-IPNs were synthesized as reported previously
(Scheme 2) [25]. All samples contained 2.4395 g (21.59 mmol) of NIPAAm and
 Semi-IPNs for tissue engineering application 869
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Scheme 1. Synthesis of the p(NIPAm-co-AAc) hydrogel.

Scheme 2. Synthesis of the p(NIPAm-co-AAc)-based semi-IPN.

 870 R. A. Stile et al.

0.0605 g (0.84 mmol) of AAc (i.e. a NIPAAm : AAc molar ratio of 96.25 : 3.75).
The synthesis followed exactly that described for the p(NIPAAm-co-AAc) hydro-
gels, with the addition of linear p(AAc) chains (Mw 4.5 × 105 g/mol) in amounts of
0.001 g (2.22 nmol), 0.0055 g (12.2 nmol) and 0.013 g (28.9 nmol).

Rheology studies

The rheology studies were performed based on methods used previously [15].

p(NIPAAm-co-AAc) hydrogels. Small samples (approx. 0.5 ml) of the

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p(NIPAAm-co-AAc) hydrogels were collected from the top of the matrices using
a 3-ml syringe with a 2 mm diameter aperture. The ‘top’ is defined as the side of
the matrix that did not polymerize in contact with the glass beaker. The rheology
of the hydrogels was then examined on a Bohlin VOR rheometer (Bohlin Rheologi,
Cranbury, NJ, USA) using a parallel plate (30 mm diameter) configuration in os-
cillatory mode. Sample thickness ranged from 0.782 to 1.128 mm, depending on
the sample volume. The samples were injected onto the lower plate using the 3 ml
syringe, the upper fixture was lowered, and a humidity chamber was placed around
the sample to prevent dehydration during data collection. Each sample was tested
at 22◦ C and then at 37◦ C. The temperature of the lower plate was maintained with a
recirculating water bath. Linear viscoelastic data were collected using a 3.61 g cm
torsion bar over a ω range of 0.001–10 Hz. Depending on the sample thickness, the
shear strain ranged from 18.1 to 28.6%. Preliminary experiments demonstrated that
the rheological properties were independent of the applied strain in this range.

p(NIPAAm-co-AAc)-based semi-IPNs. 3-ml samples of the p(NIPAAm-co-

AAc)-based semi-IPNs were collected from the top of the matrices using a 10-ml
syringe with a 2 mm diameter aperture. The rheology of the semi-IPNs was then ex-
amined on a Paar Physica MCR 300 rheometer (Paar Physica, Ashland, VA, USA)
using a parallel plate (50 mm diameter) configuration in oscillatory mode. Two dif-
ferent rheometers were used due to Dr. Healy’s move to Berkeley. Data collected
using the same p(NIPAAm-co-AAc)-based semi-IPNs on both instruments were
nearly identical and fell almost exactly on the same lines, indicating that the results
were independent of the instrument they were collected on, as they should be. The
p(NIPAAm-co-AAc)-based semi-IPN samples were 1.00 mm thick. The samples
were injected onto the lower plate using the 10-ml syringe, the upper plate was low-
ered, excess sample was trimmed and a humidity chamber was placed around the
sample to prevent sample dehydration. Each sample was tested at 22◦ C and then
at 37◦ C. The temperature of the lower plate was maintained with a Peltier heating
element connected to a recirculating water bath. Linear viscoelastic data were col-
lected over a frequency range of 0.001–10 Hz. The shear strain ranged from 18 to
25%.
 Semi-IPNs for tissue engineering application 871

Statistical analysis
Analysis of variance (ANOVA) tests and Newman-Keuls post-hoc analyses were
performed using StatSoft Statistica 5.0 (StatSoft, Tulsa, OK, USA) and SAS JMP
5.0 (SAS, Cary, NC, USA). Statistical significance was determined at a value of
P < 0.05.

RESULTS AND DISCUSSION

At 22◦ C, the p(NIPAAm-co-AAc) hydrogels and p(NIPAAm-co-AAc)-based semi-

IPNs demonstrated G∗ and δ curves that were representative of extremely soft,
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loosely cross-linked solids (Figs 1 and 2, solid lines), consistent with p(NIPAAm-
co-AAc) hydrogel data published previously [15]. With decreasing ω, G∗ ap-
proached a constant value in the range of 3–10 Pa (Figs 1a and 2a, solid lines),
and δ decreased (Figs 1b and 2b, solid lines), indicating that G was considerably
greater than G at low ω (i.e. at least 2× greater; Fig. 3, solid lines). When heated
from 22◦ C to 37◦ C, the hydrogels and semi-IPNs became more rigid and solid-like,
as signified by the significant increase in G∗ (P < 0.05 versus the 22◦ C data; Figs 1a
and 2a, dashed lines) and the decrease in δ (i.e. δ closer to 0◦ ; Figs 1b and 2b, dashed
lines). At 37◦ C, G was roughly an order of magnitude greater than G (Fig. 3,
dashed lines). This change in rigidity is commonly demonstrated by p(NIPAAm)-
based hydrogels when the temperature is increased above the LCST [6, 7]. From
a tissue-engineering perspective, the rigidity of the p(NIPAAm-co-AAc) hydrogels
and p(NIPAAm-co-AAc)-based semi-IPNs at 37◦ C was comparable to that of type-I
collagen gels (Table 1).
Interestingly, the shapes of the G∗ and δ curves at 37◦ C were more representative
of those obtained from amorphous glassy and crystalline linear polymers, rather
than those obtained from cross-linked viscoelastic solids [42]. G∗ was not constant
over a wide range of ω but demonstrated a weak power law dependence on ω
(Figs 1a and 2a, dashed lines), such that δ was relatively constant (Figs 1b and 2b,
Table 1.
G values for p(NIPAAm)-based matrices and collagen gels at 37◦ C

Formulation G Reference
p(NIPAAm-co-AAc) hydrogelsa 68–128 Pa at 0.1 Hz This paper and Ref. [15]
p(NIPAAm-co-AAc)-based semi-IPNsb 99–169 Pa at 0.1 Hz This paper
Type-I collagen gel (1.6 mg/ml) 78 Pa at 0.3 Hz [54]
Type-I collagen gel (2.3 mg/ml) 30 Pa at 0.3 Hz [55]
Type-I collagen gel (3 mg/ml) 20 Pa at 0.02 Hz [56]
a The p(NIPAAm-co-AAc) hydrogels contained NIPAAm : AAc ratios of 96 : 4, 96.25 : 3.75 and

96.5 : 3.5.
b The p(NIPAAm-co-AAc)-based semi-IPNs contained a NIPAAm : AAc ratio of 96.25 : 3.75 and

p(AAc) amounts of 0.001 g, 0.0055 g and 0.013 g.

 872 R. A. Stile et al.

(a)
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(b)
Figure 1. (a) G∗ and (b) δ as a function of frequency and temperature for p(NIPAAm-co-
AAc) hydrogels with a NIPAAm : AAc molar ratio of 96.5 : 3.5 (22◦ C, —Q—; 37◦ C, - -Q- -) and
96.25 : 3.75 (22◦ C, —"—; 37◦ C, - -"- -). Previously published rheology data [15] are also included
for p(NIPAAm-co-AAc) hydrogels with a NIPAAm : AAc molar ratio of 96 : 4 (22◦ C, —4—; 37◦ C,
- -4- -). The 22◦ C G∗ data sets overlap each other. Frequency is on a log scale. Each data set is
the average of at least 3 experiments. The lines were added for illustration purposes only and do not
indicate modeling of the data. If error bars cannot be seen, they are smaller than the size of the symbol.
∗ indicates that the data sets are significantly different from all other 37◦ C G∗ data sets at P < 0.05.

dashed lines). These results were likely influenced by the complex interactions
that occur during the phase transition when p(NIPAAm)-based materials are heated
above the LCST [5, 7, 13, 46– 48].
The p(NIPAAm-co-AAc) hydrogel G∗ data were not affected by the NIPAAm :
AAc molar ratio at 22◦ C (P > 0.05; Fig. 1a, solid lines); however, the G∗
data were affected by the NIPAAm : AAc molar ratio at 37◦ C (Fig. 1a, dashed
lines). As the NIPAAm : AAc molar ratio increased from 96 : 4 to 96.25 : 3.75
to 96.5 : 3.5, G∗ significantly increased (P < 0.05), consistent with the addition
of NIPAAm monomer to the p(NIPAAm-co-AAc) hydrogel. The more NIPAAm
(and consequently, the less AAc) present in the hydrogel, the more hydrophobic
aggregates present at 37◦ C (i.e. above the LCST) and the more rigid the matrix.
Only the highest p(AAc) chain amount tested (i.e. 0.013 g) significantly affected
the rigidity of the p(NIPAAm-co-AAc)-based semi-IPNs at 22◦ C (P < 0.05;
 Semi-IPNs for tissue engineering application 873
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(a)

(b)
Figure 2. (a) G∗ and (b) δ as a function of frequency and temperature for p(NIPAAm-co-AAc)
hydrogels (22◦ C, —"—; 37◦ C, - -"- -), and p(NIPAAm-co-AAc)-based semi-IPNs containing 0.001
g (22◦ C, —F—; 37◦ C, - -F- -), 0.0055 g (22◦ C, —2—; 37◦ C, - -2- -) and 0.013 g (22◦ C, —×—;
37◦ C, - -×- -) of p(AAc) chains (Mw 4.5 × 105 g/mol). The 22◦ C G∗ data sets overlap each other.
Frequency is on a log scale. All hydrogels and semi-IPNs contained a NIPAAm : AAc molar ratio of
96.25 : 3.75. Each data set is the average of at least 3 experiments. The lines were added for illustration
purposes only and do not indicate modeling of the data. If error bars cannot be seen, they are smaller
than the size of the symbol. † indicates that the data set is significantly different from all other 22◦ C
G∗ data sets at P < 0.05. ∗ indicates that the data sets are significantly different from all other 37◦ C
G∗ data sets at P < 0.05.

Fig. 2a, solid lines). At lower p(AAc) chain amounts (i.e. 0.001 g and 0.0055 g),
the p(NIPAAm-co-AAc)-based semi-IPN G∗ data were not significantly different
from the p(NIPAAm-co-AAc) hydrogel G∗ data (P > 0.05; Fig. 2a, solid lines).
We hypothesized that the p(AAc) chains would increase the rigidity of the semi-
IPNs at 22◦ C, due to COO− repulsion (e.g. repulsion between the p(AAc) chains
and the AAc groups in the p(NIPAAm-co-AAc) hydrogel and/or between adjacent
 874 R. A. Stile et al.
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(a)

(b)
Figure 3. (a) G (22◦ C, —"—; 37◦ C, - -"- -) and G (22◦ C, — —; 37◦ C, - - - -) for p(NIPAAm-
co-AAc) hydrogels and (b) G (22 C, —×—; 37◦ C, - -×- -) and G (22◦ C, — —; 37◦ C, - - - -) for
 ◦

p(NIPAAm-co-AAc)-based semi-IPNs containing 0.013 g of p(AAc) chains (Mw 4.5 × 105 g/mol).
For clarity, panels (a) and (b) include insets with a different scale to show the 22◦ C data. The hydrogels
and semi-IPNs contained a NIPAAm : AAc molar ratio of 96.25 : 3.75. Frequency is on a log scale.
Each data set is the average of at least 3 experiments. The lines were added for illustration purposes
only and do not indicate modeling of the data. If error bars cannot be seen, they are smaller than the
size of the symbol.
 Semi-IPNs for tissue engineering application 875

p(AAc) chains) and processes analogous to second-phase strengthening [49, 50].

As discussed in our previous report [25], two-polymer systems, such as semi-
IPNs, generally consist of two immiscible phases [51]. Thermodynamically, the
immiscibility is driven by the low entropy of mixing that ensues when two polymers
are blended. The resulting phase separation leads to the formation of phases that
vary in amount, size, shape, interfacial sharpness and degree of continuity. These
phases constitute the microscopic and macroscopic morphology of the material, and
the morphology then directly influences the material properties of the system. In
our previous work [25], we observed phase separation in the p(NIPAAm-co-AAc)-
based semi-IPNs at 22◦ C, and the opacity of the matrices increased with increasing
p(AAc) chain amount (i.e. semi-IPNs containing 0.0055 g and 0.013 g of p(AAc)
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chains were opaque at 22◦ C). Changing the p(AAc) chain amount most likely
altered the p(AAc)-rich phase within the semi-IPNs (e.g. in amount, size, continuity,
etc.), leading to changes in the microscopic and macroscopic morphology of the
system (i.e. the opacity changed). The morphology changes then altered the material
properties of the semi-IPNs, as the injectability of the matrices at 22◦ C decreased
with increasing p(AAc) chain amount (i.e. the semi-IPNs containing 0.013 g of
p(AAc) chains were not injectable at 22◦ C) [25]. This decrease in injectability may
have been due to the increase in rigidity demonstrated by the p(NIPAAm-co-AAc)-
based semi-IPNs containing higher amounts of p(AAc) chains (i.e. 0.013 g). The
data in both the previous and present studies suggest that the effects of the p(AAc)
chains on the p(NIPAAm-co-AAc)-based semi-IPNs at 22◦ C, whether related to
morphology (e.g. opacity) or material properties (e.g. injectability and rigidity),
are not discernable until a critical amount of p(AAc) chains is present within the
matrices.
In contrast to the 22◦ C data, the 37◦ C G∗ data were affected by the presence of
the p(AAc) chains at all amounts tested (Fig. 2a, dashed lines). The p(NIPAAm-co-
AAc)-based semi-IPNs were significantly more rigid than the p(NIPAAm-co-AAc)
hydrogels at 37◦ C (P < 0.05), independent of p(AAc) chain amount. The 37◦ C data
suggest that the increase in G∗ resulted from interactions between the p(AAc)-rich
phase and phase-separated segments of the p(NIPAAm-co-AAc) hydrogel. Given
that the p(NIPAAm) phase transition in aqueous media has not been completely
elucidated [5, 13], the ternary system that comprises the p(NIPAAm-co-AAc)-based
semi-IPNs (i.e. p(NIPAAm-co-AAc) hydrogels, PBS solvent and p(AAc) chains)
undoubtedly increased the complexity of the environment within the matrices at
temperatures above the LCST, making it difficult to interpret the nature of the
interactions at 37◦ C.
As well as being affected by the presence of the p(AAc) chains, G∗ was also
a function of p(AAc) chain amount at 37◦ C, since the p(NIPAAm-co-AAc)-based
semi-IPN G∗ values significantly increased with increasing p(AAc) chain amount
(P < 0.05; Fig. 2a, dashed lines). In addition to the aforementioned interactions
between the p(AAc)-rich phase and phase-separated components of the p(NIPAAm-
co-AAc) hydrogel at 37◦ C, these data indicate that as the p(AAc) chain amount
 876 R. A. Stile et al.
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Figure 4. G∗ as a function of the relative molar amount of p(AAc) chains (Mw 4.5 × 105 g/mol)
in the p(NIPAAm-co-AAc)-based semi-IPNs at 37◦ C and 0.1 Hz. The relative molar amount of
p(AAc) chains was calculated as follows: (mol p(AAc) chains/(mol NIPAAm + mol AAc in the
hydrogel))×100%. Each data point is the average of at least 3 experiments. The line was added for
illustration purposes only and does not indicate modeling of the data. The semi-IPNs contained a
NIPAAm : AAc molar ratio of 96.25 : 3.75.

increased, alterations in the p(AAc)-rich phase led to changes in the material

properties of the semi-IPNs (i.e. G∗ increased); however, the mechanism by which
the p(AAc)-rich phase varies with p(AAc) chain amount at 37◦ C is unknown. Figure
4 shows a plot of G∗ as a function of the relative molar amount of p(AAc) chains in
the p(NIPAAm-co-AAc)-based semi-IPNs at 37◦ C and 0.1 Hz. The relative amount
of p(AAc) chains was calculated as follows: (mol p(AAc) chains/(mol NIPAAm
+ mol AAc in the hydrogel))×100%. The effect of the p(AAc)-rich phase on
the rheology of the p(NIPAAm-co-AAc)-based semi-IPNs is comparable to that
observed in other two-phase systems. For example, changing the content of styrene-
butadiene block copolymers changes the properties of the glassy and elastomeric
phases, altering the mechanical properties of the system [52]; additionally, adjusting
the epoxy content in acrylic-epoxy IPNs affects the phase continuity within the
system, leading to changes in the swelling behavior of the matrix [53].

CONCLUSIONS
Poly(NIPAAm-co-AAc) hydrogels and p(NIPAAm-co-AAc)-based semi-IPNs were
synthesized, and the effects of numerous reaction conditions on the rheology of
the matrices were analyzed. In the p(NIPAAm-co-AAc) hydrogel studies, the
NIPAAm : AAc molar ratio in the feed was varied, and in the p(NIPAAm-co-AAc)-
based semi-IPN studies, the amount of the linear p(AAc) chains (Mw 4.5 × 105
g/mol) was modified. Oscillatory shear rheometry studies were performed on the
hydrogels and semi-IPNs as a function of temperature (i.e. 22◦ C and 37◦ C) and ω
(i.e. 0.001–10 Hz). At 22◦ C, the rigidity of the p(NIPAAm-co-AAc) hydrogels was
not affected by the NIPAAm : AAc molar ratio, and only the highest p(AAc) chain
 Semi-IPNs for tissue engineering application 877

amount tested significantly affected the rigidity of the p(NIPAAm-co-AAc)-based

semi-IPNs. At 37◦ C, the p(NIPAAm-co-AAc) hydrogel rigidity significantly in-
creased with increasing NIPAAm : AAc molar ratio, the p(NIPAAm-co-AAc)-based
semi-IPNs were significantly more rigid than the p(NIPAAm-co-AAc) hydrogels,
and the rigidity of the p(NIPAAm-co-AAc)-based semi-IPNs significantly increased
with increasing p(AAc) chain amount.

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