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Structure of Polymer and Particle Aggregates in Hydrogel Composites
€ ns Hilborn,1 Adrian R. Rennie3
Ida Berts,1,2 Yuri Gerelli,2 Jo
1
€ m Laboratory, Uppsala University, Box 538, 75121 Uppsala,
Science for Life Laboratory, Department of Chemistry–Ångstro
Sweden
2
Institut Laue-Langevin, 6 rue Jules Horowitz, BP 156, 38042 Grenoble, France
3
Department of Physics and Astronomy: Materials Physics, Uppsala University, Box 516, 75120 Uppsala, Sweden
Correspondence to: I. Berts (E-mail: [email protected])
Received 5 November 2012; accepted 19 November 2012; published online 18 December 2012
DOI: 10.1002/polb.23230
ABSTRACT: Knowledge of the structure of a biomaterial is
usually vital to control its function. This article provides a
structural characterization of a hyaluronan scaffold that has
demonstrated good biocompatibility and is used to induce
bone regeneration. Hyaluronan hydrogels are appealing materials that can function as a matrix to incorporate both organic
and inorganic substances to enhance tissue growth. Because
of the intrinsic properties of this swollen matrix, one needs a
very sensitive technique that can be applied in situ to determine the organization of the polymers in a gel. Small-angle
neutron scattering is used to determine the characteristics of
the inhomogeneous structure of the hydrogel both with and
without added particles. The results are interpreted using
models of structure with two length scales that are beyond
the traditional picture of homogeneous gels. The observed
structure and the dimensions can explain the previously
reported rheological properties of gels containing different
amount of polymers. Hydroxyapatite nanoparticles added to
the gel are frozen in the gel matrix. We are able to determine
the distribution and shape of these particles as they aggregate around the polymer chains. We have also concluded, in
this case, that the particle structure is concentration independent. Information about the nanostructure for an applicable biomaterial guides the formulation, preparation, and use
that should lead to further understanding of its exploitation.
C 2012 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym.
V
Phys. 2013, 51, 421–429
INTRODUCTION Hydrogels are three-dimensional crosslinked
polymer networks that are capable of absorbing large
amounts of water.1 These polymer networks with either
chemical or physical crosslinks provide stable structures
and, for the past 35 years, have been extremely useful due
to their resemblance of natural tissue and their biocompatibility.2 Hydrogel matrices based on hyaluronan are popular
materials for biomedical applications such as drug delivery,3
tissue engineering,4 and aiding wound repair.5 Recent developments within tissue engineering, pharmaceutical, and cell
encapsulation applications involve in vivo injectable systems.
These materials can form a temporary scaffold in situ that
functions as an artificial extracellular matrix which can
accommodate cells and promote their growth in threedimensions and thus form new tissue.6 A recent review has
focused on materials for bone and cartilage regeneration
using polymer gel scaffolds that are able to incorporate components that aid growth, such as cells or bioactive substances, and inorganic particles.7
rounding tissues, retention, and protection of the growth promoting agents and in addition mechanisms for controlled
release. Synthetic modification of natural polymers is a good
strategy to obtain new properties that are significantly different from their precursors.8,9 For example, a two-component
system based on lightly derivatized water-soluble aldehydemodified sodium hyaluronan (HAA) and hydrazide-modified
polyvinyl alcohol (PVAH) that forms a gel upon mixing has
been developed.10–14 Hydrozone formation provides covalent
crosslinks between the two components in aqueous solution:
this may take place in situ to minimize invasive surgical procedures. Incorporating nanoparticles into polymer matrices
has been widely investigated, as nanocomposites have several
advantages in respect of their mechanical properties.15 For
bone regeneration, it is particularly useful to increase the mechanical strength of the scaffold. Calcium phosphate ceramics
are widely considered as bone substitutes.16 Nanosized hydroxyapatite particles (nano-HAP) have been successfully dispersed in polymer gels. Particles with a diameter of roughly
20 nm have been shown to enhance the proliferation of mesenchymal stem cells.17,18 Moreover, the nano-HAP induces
bone growth with a higher density than that formed using
A gel scaffold based on a natural polymer can provide properties such as strong attachment and integration to the sur-
KEYWORDS: biopolymers;
correlation length;
hydrogels; nanocomposites; neutron scattering
hyaluronan;
C 2012 Wiley Periodicals, Inc.
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other calcium phosphate additives. This could be due to the
nanocrystals being directly incorporated in the regenerated
bone.19
The structure of gels and their properties can depend sensitively on the details of preparation and composition. These
factors are not well understood. The modification of the
structure by particles and their state of aggregation are also
important influences on physical properties of materials. Parameters that characterize the hydrogel network structure
include the molecular mass (or the polymer chain length)
between two neighboring crosslinks, the corresponding
mesh size, and the effective network density.20,21 These are
related to elastic properties by the theories of rubber elasticity that, for example, can be used to calculate swelling by
solvent.22,23 According to Peppas et al.,24 the molar ratio of
crosslinking agent to monomer, which is directly related to
the structure of hydrogels, is one of the most important factors that affects the swelling. The average correlation distance between two adjacent crosslinks can be calculated
using the Flory–Rehner model,25 which states that a polymeric gel in equilibrium with surrounding solvent is a result
of the balance of two opposing forces: the pressure arising
from the thermodynamics of mixing and the retractive force
that arises from the changes in entropy of the polymer
chains. For hydrogels, it may also be necessary to take into
consideration the additional contributions from ions. Numerous studies have focused on the determination of the degree
of polymerization between crosslinks.26,27 The outcome of
the swelling studies, however, depends strongly on how the
gels are prepared, and the conditions of swelling such as
temperature, solvent, and the other thermodynamic state
variables of the sample. Studies by Piskounova et al.28 have
shown that different solvents give rise to different swelling
profiles. It was observed that gels in cell culture medium
swelled significantly more than in phosphate buffered saline.
The degree of mixing of the gel components can affect the
crosslinking, which in turn also alters the structural and mechanical properties of the gel. Another problem with the
swelling performance is the stability of the hydrogels. Many
hydrogels are very brittle and rupture or decompose easily
in solvent if they are not constrained. Keeping the gels in
containers on the other hand, limits the diffusion of solvent
into the gel. The two-component hydrogel systems also have
to be assumed to be homogenous for the calculations of the
crosslinked molecular mass.29 An alternative characterization
of crosslinked hydrogels using high-resolution, magic-angle
spinning NMR spectroscopy was presented by van Vlierberghe et al.30 and applied to gelatin-based hydrogels where
the samples were freeze-dried and reswollen in solvent. This
quantifies unreacted crosslinkable groups.
In this study, small-angle neutron scattering (SANS) measurements on hydrogels were used to determine the correlation length inside the gel, how the local structure of the gel
is perturbed by different amounts of particles as well as to
explore the range over which fractal aggregates of nano-HAP
are found in the materials. Previous SANS experiments
performed by Mangiapia et al.31 on freeze/thaw treated PVA
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hydrogels suggested a bicontinuous phase structure with
polymer-rich regions dispersed in water-rich regions. The
work of Jha et al.32 involved spherical hyaluronan hydrogel
particles. They compared the scattering exponent in the low
momentum transfer regimes between the hydrogels particles
with traditional bulk hydrogels, and suggested that the
hydrogel particles increase the network density. Horkay
et al.33 compared the scattering of hyaluronan solutions and
hyaluronan crosslinked with bifunctional low molecular
weight crosslinkers and suggested that the crosslinking only
increased the observed intensity in the intermediate range of
the SANS spectra. The disadvantage in using bifunctional low
molecular weight crosslinkers is that they may cause single
chain looping or self crosslinking which does not contribute
to the mechanical integrity of a hydrogel. Using a two-component system based on different polymers that crosslink on
contact as described in this article can provide a more stable
gel with more predictable structure. SANS is an outstanding
tool for quantitative evaluation of hydrogel networks. Neutrons interact weakly with the nuclei of the sample therefore
do not cause radiation damage. The penetration of the neutrons and the microscopic resolution provides nanometer
scale determination of structures within thick samples.34
Moreover by using contrast matching, one could prepare gels
in mixtures of H2O and D2O such that the scattering arises
mainly from the polymeric matrix or from the composite.
EXPERIMENTAL
Materials
The HAA, modified with a 6% aldehyde functionality of
repeating units and a molecular mass of 180 kDa,11 was provided by Termira AB, Stockholm, Sweden, The PVAH, modified
with a 9% hydrazide functionality of repeating units and a
molecular mass of 14 kDa, was prepared as described
previously.13 The nano-HAP particles were prepared as
described by Hu et al.17 Transmission electron microscopy
studies performed by Hu et al. showed that the particles were
crystalline and grain-like. Dynamic light scattering confirmed
a uniform particle size distribution with an average diameter
of 20 nm and 20% polydispersity.17 For the neutron experiments, deionized water, passed through a purification system
(Milli-Q, resistivity ¼ 18.2 MX cm), was used in all experiments. D2O was supplied by EURISO-TOP, CEA, Saclay.
Hydrogel Preparation
Gel samples are prepared with a total polymer concentration
of 5, 15, and 30 mg mL"1 using D2O as solvent. The
5 mg mL"1 gel sample were prepared from separate solutions with 8.89 mg mL"1 HAA and 1.11 mg mL"1 PVAH. The
15 mg mL"1 sample used 26.70 mg mL"1 HAA and 3.30 mg
mL"1 PVAH. Gels prepared by mixing 53.36 mg mL"1 HAA
with 6.64 mg mL"1 PVAH has a total concentration of 30 mg
mL"1. The concentrations of each component are calculated
to have a 1:1 ratio of the respective crosslinking groups. The
gels were formed during rapid mixing and injecting of the
two polymer components into the sample holder. The nanocomposite samples were all prepared with the 15 mg mL"1
gel as matrix to form samples that contain a total of 5, 10,
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TABLE 1 Properties of Materials Used in the Study
Names
Formula
Molecular
Mass (g mol"1)
Density
(g cm"3)
SLD
(10"6 Å"2)
Hyaluronan monomer
C14O11H21N
401
1
1.39–2.28
HAA monomer
C16O11H24N2
420
–
–
PVA monomer
C2OH4
44
1.26
0.72
PVAH monomer
C5O3H9N3
159
–
–
Hydrogel
–
–
1
2.23a
Nano-HAP
Ca10(PO4)6(OH)2
1004
3.16
4.19–4.51
D2O
D2O
20
1.1
6.35
H2O
H2O
18
0.997
"0.56
a
Calculated based on the fraction of HAA and PVAH in a gel with the respect to the deuterium-hydrogen
exchange.
and 20 wt % nano-HAP. The nano-HAP particles were dispersed in the HAA component before mixing with the PVAH
component for gelation. Different contrasts were used to be
able to apply contrast variation in the evaluation of the scattering intensity for the gel in the nanocomposite samples.
These samples were made in D2O, polymer matrix matched
water (2.23 # 10"6 Å"2), nano-HAP matched water (4.19 #
10"6 Å"2) and additional solvent contrasts of scattering
length density (SLD) of 3 and 5 # 10"6 Å"2. A summary of
the physical properties of the materials used is provided in
Table 1, which includes molecular mass, density, and SLD.
The hydrogel samples were prepared 1 day before the measurement so that they reached equilibration as stable networks. They were mixed and contained in 1-mm path length
fused quartz cells. Figure 1 shows one gel sample and one
nanocomposite sample, each in a sample cell.
SANS Measurement
SANS measurements were performed on the small-angle diffractometers D11 and D22 at the Institut Laue Langevin, Grenoble.35,36 The wavelength was selected as either 6 or 10 Å. A
two-dimensional 1 # 1 m2-position sensitive detector at three
different sample-to-detector distances (1.78, 8, and 39 m for
D11 and 1.4, 5, and 17 m for D22) measured scattering from
the samples. These configurations allowed the collection of
scattering data in an interval of transferred momentum [Q ¼
(4p/k) sin h] between 0.0015 and 0.5 Å"1, where 2h is the
scattering angle and k is the wavelength. The measurement
times ranged between 30 min and 1.5 h per sample. Raw data
were corrected for the electronic background, detector efficiency, and empty cell scattering using the programs RNILS
and SPOLLY37 and software provided by ILL. Incoherent scattering subtraction was performed on the absolute intensity
curves to remove the background contributions from the solvent and from the sample itself. Data modeling were performed using Origin Pro and the SASfit program.38
it is often convenient to describe two different components
of the structure. These components may be taken as giving
rise to two separate contributions to the observed scattering:
I tot ¼ I S þ I L
(1)
in which IS is the scattered intensity that arises from small
structures and IL comes from the large structures. The term
IS dominates the large-Q region, here the Ornstein-Zernike
(Lorentzian) equation is commonly used to describe the solution scattering from overlapping polymer coils39–41 as
found in gels:
I S ¼ A=ð1 þ Q2 n2 Þ
(2)
where n is the thermal correlation length, usually ascribed to
the mesh size of the gel.42 This equation has been used by
MODELS FOR GEL STRUCTURES
Several models have been suggested for the structure of
gels. Many gels are not homogeneous at all length scales and
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FIGURE 1 Gel sample (left) and nanocomposite sample (right)
in 1-mm path length fused quartz sample cells.
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Geissler et al.,41 and was previously described by de Gennes43 and by Higgins and Benoı̂t.34 The scattering depends
on the thermal fluctuations and the factor A is thus given by
the difference in SLD between the polymer and the solvent
(Dq2), Boltzmann’s constant kB, the absolute temperature T,
the volume fraction of the polymer u, and the osmotic modulus Mos.44 Mos governs the scattering properties of the gel,
and is related to the polymer concentration fluctuations.45
The constant in eq 2 is related to the model as:
A ¼ Dq2 # ðk B Tu2 =M os Þ
(3)
For polymer solutions, the expression for the scattering intensity shows the same Q dependence as eq 2. However, the
factor A has a different physical meaning. The observed scattering intensities from gels are usually larger than those of
the corresponding polymer solutions.45 This is because the
compression modulus K for a gel is smaller than that of the
corresponding solution, as there is a significant shear modulus G.45–47 The relation between these quantities and the
elongational or Young’s moduli, E, is given by:
K ¼ E " 4G=3
(4)
For solutions Mos in eq 3 is replaced by Kos. Kos differs from
Mos as it scales with the polymer concentration and is independent of the number of monomers between two neighboring crosslinks in gel.23,25
A modification of eq 2 for IS has been suggested by Horkay
et al. for semiflexible polysaccharides that exhibit rod-like
structures.33,48 The assumption is made that polymer segments randomly distributed at all length scales could be
modeled as stiff chains. The rod-like structure of the polymer
gives rise to intensity given by:
I 0 s ¼ A=½ð1 þ QLÞð1 þ Q2 r 2c Þ(
For neutral gels, the large scale inhomogeneities IL may be
described by the Debye-Bueche structure factor.49 For the
concentration fluctuations of the network44 that are influenced by the physical constraints imposed by the crosslinks.41 These clusters of crosslinks dominate the scattering
at low Q.
(6)
The large structures in a gel are sometimes difficult to define
from scattering data due to the size of the clusters that is
generally larger than the length scale probed, and uncertainties in the small Q region. Horkay et al.33 suggested a power
law decay with a general form:
I 0L ¼ C # Q"d
(8)
where C is a constant and the exponent d is an arbitrary
number, describing the slope in a log–log plot.50 Clearly, eq 8
could approximate the functional form of eq 6 when data
are only available in a restricted Q range. Equations 5 and 8
can be combined to give
I 0tot ¼ I 0S þ I 0L
(9)
Itot is eq 5 in the article of Horkay et al.33 This model for the
total intensity has been adopted to describe sodium polyacrylate gels, DNA gels and hyaluronan solution and hyaluronan gels crosslinked with ethylene glycol diglycidyl ether.
0
RESULTS AND DISCUSSION
where factor B is
B ¼ Dq2 # 8pN3 <Du2 >
(7)
the correlation length N can be related to the average distance between polymer-rich regions in an inhomogeneous
gel but the contribution of IL to the total intensity is negligible in the large-Q region.43 < du2 > is the mean square am424
plitude of the fluctuations of the volume fraction. Equation 1
can be taken as the sum of eqs 2 and 6. This describes a
two-phase system presenting two correlation lengths with
two different length scales in a gel; n as the correlation
length inside the polymer clusters, and N as the distance
between the clusters. A schematic diagram that shows how
the two different correlation lengths can be interpreted is
given in Figure 2.
(5)
in which L is the length of the rod and rc is its radius of
cross section. For polysaccharides like hyaluronan, the rcvalue has been taken as 5 Å.33
I L ¼ B=ð1 þ Q2 N2 Þ2
FIGURE 2 Schematic diagram of the inhomogeneities in a gel
network. The two different correlation lengths, n and N, are
shown.
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Scattering from the HAA Solution
As the main component of the hydrogel, a polymer solution
of 15 mg mL"1 HAA in D2O was measured to compare with
the corresponding gel (Fig. 3). Inspection of the figure shows
that the scattering intensity is approximately the same for
the solution as for the gel, with an increased scattering intensity for the gel in the intermediate Q range. It has been
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at this concentration; however, the low scattering from the
sample in the high-Q region, where the intensity is significantly less than that for pure D2O, is the main source of the
uncertainty in the fitted parameters. A fit using eqs 8 and 2
are very similar to the fit using eq 9, the same parameters
were applied for both fits except for L and rc, which are
replaced by n ¼ 9 6 1. This indicates that the stiffness and
influence of rod-like structures is negligible on the scattering
from the sample.
FIGURE 3 SANS spectra of a 15-mg mL"1 HAA solution (n),
with the fit (solid line through the data points), compared with
the corresponding gel sample (h). The data presented are in
absolute units and show the same intensity.
suggested that this arises from crosslinks.33 Hyaluronan is a
semiflexible, wormlike chain with a persistence length of
about 8 nm.51 Because of its long persistence length and the
stiffness of the chains, the polymers in solutions exhibit an
extended random-coil configuration, and the long chains permeate a large volume.52 Consequently, the chains overlap at
very low concentrations. According to Fraser et al.,52 for hyaluronan of molecular mass 0.1–10 MDa, entanglement starts
at concentrations as low as 0.5–1.0 mg mL"1. The figure also
shows high intensity in the low-Q region that is interpreted
as large-scale inhomogeneities in the HAA solution of 15 mg
mL"1 that arise from association between the large stiff
polymer molecules similar to those that arise from entanglements or crosslinks. This region is dominated by a linear
behavior (in log-log scale) with a slope ranging between "3
and "4 that is comparable with the work of Horkay et al.33
A fit to this part of the data using eq 8, gives d ¼ 3.3 that
lies in the expected range 3 < d < 4 which would occur for
surface scattering from clusters that are very large (>1000
Å). This is characteristic for polyelectrolyte solutions.53
Equation 9 was used to fit the complete range of data from
the solution sample. The parameters used are shown in Table 2. However, the apparent length of the rods (L) is much
lower than the persistence length of the polymer in dilute
solutions with a low concentration of salt. This may be due
to the interactions that occur between hyaluronan molecules
Scattering from the Gel
The scattering data obtained from the gels at different concentrations are shown in Figure 4. The measured intensity
for the 5 and 15 mg mL"1 samples scales almost linearly
with the concentration, whereas the 30 mg mL"1 varies
more strongly with Q below 10"2 Å"1. It is clear that the
low-Q region is dominated by a linear behavior with a slope
ranging from "2.6 for the lowest gel concentration up to
"3.0 for the highest gel concentration. The SANS data for
both the samples of crosslinked gel and the corresponding
uncrosslinked HAA solution could be fitted with the same
model, eq 9, this strategy was also used by Horkay et al.33
The data and a model fit for the gel concentration of 15 mg
mL"1 in D2O are shown in Figure 5 with the total fit and
each of the two components (eqs 5 and 8). The data and
model fit for the 30 and 5 mg mL"1 gel are shown in Figures
6 and 7, respectively. The parameters used are listed in Table 2.
Equation 9 provides the best fit to the data while using eqs
2 and 6, or eqs 2 and 8, does not give a fit that is as satisfactory. This could indicate presence of stiff structures within
short-range regions caused by the crosslinks, best described
by eq 5. In such systems, the length of the rod L describes
the distance between the crosslinks inside the polymer clusters (i.e., the local structure of the gel). One can assume that
the PVAH polymers with a significantly lower molecular
mass compared with the hyaluronan polymers, act as crosslinkers and contributes to the dense crosslinking described
by L. The parameter A increased with the concentration of
the hydrogel polymer components, suggesting a constant osmotic modulus for the 5 and 15 mg mL"1 gel samples. The
derived Mos of about 30 kPa is similar to the value obtained
by Horkay et al. for an hyaluronan gel prepared using small
molecular crosslinkers.33 The correlation length, L, could be
considered as a constant with concentration indicating that
the local structure of these network portions is similar, and
L is close to the persistence length of the hyaluronan polymer. The value of 12 Å for the lowest gel concentration is
TABLE 2 Parameters from the Fits to Solutions and Gels According to eq 8, Including the Derived Values of Kos
Sample
Description
Concentration
(mg mL"1)
A
(#10"3 cm"1)
C
(#10"7 cm"1)
L
(Å)
rc
(Å)
D
Mos
(kPa)
HAA solution
15
661
0.20 6 0.04
661
5
3.32 6 0.03
260 6 45 (Kos)
HAA-PVAH gel
5
561
562
12 6 7
5
2.60 6 0.07
35 6 7
HAA-PVAH gel
15
49 6 16
661
72 6 28
5
2.73 6 0.04
32 6 12
HAA-PVAH gel
30
85 6 20
861
63 6 18
5
2.95 6 0.02
74 6 18
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FIGURE 4 SANS data for the hydrogels of 5 (*), 15 (n), and
30 mg mL"1 (~) in D2O, scaled to 1 mg mL"1.
also not determined precisely due the low scattering from
the sample. A fit using 60 Å < L < 70 Å also lies within the
error bars of the data points; however, we report the parameters that gave the lowest residual in the fit.
The persistence length L for the gel is much higher than for
the corresponding solution. This can be explained by the
effect of local conformation: there are permanent crosslinks
for gels and fluctuating entanglements for the chains that
are mobile in solutions. For gels, the fluctuations depend on
elastic moduli that include a significant shear component as
described in eq 4. This directly relates to the lower scattering intensity for the polymer solution shown in Figure 3.
For the larger structures, eqs 6 or 8 indicate the presence of
aggregates or clusters in the form of polymer-rich regions in
an inhomogeneous gel. The exponent, d, of eq 8 increases
with the sample concentration. This behavior is most significant for the 30 mg mL"1 sample with d)3. This equation
FIGURE 5 SANS data and fitted model for the 15 mg mL"1
hydrogel in D2O. The fit (solid line) consists of two compo0
0
nents: IS is the dashed line and IL is the dotted line, shown
separately.
426
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FIGURE 6 SANS data and model for the 30 mg mL"1 hydrogel
in D2O.
provides only limited information regarding the overall size;
however, eq 6 could provide more information. The models
fitted using eq 6 on the smallest range of Q for all three concentrations of gels indicate that N is in the range of 1000 Å
or larger for the two lowest concentrations, whereas for the
30 mg mL"1 gel, this size is even higher (almost double).
Precise values are difficult to determine because of the
uncertainties and limited data in the low Q region. However,
this result suggests that the average distance between polymer-rich regions was larger for the highest concentration.
This result can be related to the rheological properties of the
different gel samples.
The rheological behavior, or viscoelastic properties, of gels
depends on the connectivity between polymer-rich regions.
An oscillatory shear rheology study of the same gel system
has been described by Berts.54 The samples were cast as cylinders of height 0.4 mm and diameter of 19 mm. Frequency
sweeps were performed using parallel plate geometry. The
strain was maintained at 0.5%, and the frequency range was
FIGURE 7 SANS data and model for the 5 mg mL"1 hydrogel
in D2O.
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TABLE 3 Rheological Properties of the HAA-PVAH Gels at 1 Hz
Concentration
(mg mL"1)
G0 (Pa)
5
20 6 5
0.4 6 0.1
15
160 6 40
2.0 6 0.6
30
2.0 6 0.5
0.2 6 0.1
G00 (Pa)
scanned between 0.1 and 10 Hz. The elastic modulus G0 and
viscosity modulus G00 are shown in Table 3 for a frequency
of 1 Hz where the G0 had reached a plateau. Hydrogel samples were found to have higher elastic moduli with increasing polymer concentration. The elastic modulus G0 changed
from 20 to 160 Pa on increasing the concentration from 5 to
15 mg mL"1, and this is explained by the density of links
between polymer-rich clusters. However, the 30 mg mL"1
hydrogel, appeared highly viscous and the final gel still had
a fluid behavior in the rheological tests with G0 about 2 Pa.
The SANS data for this high concentration sample suggests a
structure with many dense clusters, which might not be well
linked together. The value of Mos for the 30 mg mL"1 gel is
double that of the gels with lower polymer concentration,
which suggests that some phase separation occurred.47 This
result is consistent with the rheology data that could be
attributed to poor polymer integration or mixing.
Inadequate mixing of one polymer component into the other
would create only local crosslinks that could increase the
density of local networks and increase the number of these
networks without having extensive crosslinks between them.
An explanation is that the high concentration of polymer
might have caused insufficient mixing due to the difference
in the viscosity of the two components and thus leads to
less efficient crosslinking. Piskounova et al.28 identified that
factors such as polymer molecular mass, concentration,
crosslinking density, polymer mixing, and curing time, all
affected the structural and mechanical properties of chemically crosslinked hydrogels. In their work, insufficient mixing
gave rise to incompletely crosslinked gels with a lower modulus than that found for the same gel system with complete
mixing.
Scattering from the Gel–Particle Composite
The nano-HAP particles were well dispersed in the HAA
component prior crosslinking due to the high viscosity of the
HAA solution. These particles sediment in pure solvent and
low viscosity solutions such as that of the PVAH due to gravity. The motion through HAA solution is much slower due to
the high viscosity of the entangled, long polymer chains. This
mobility of the particles in the gel is hindered by the rapid
crosslinking reaction between the HAA and PVAH that form
a stable 3D gel network. Experiments were made to determine the structures formed by the particles in the gels but
the form factor for single particles could not be determined
by SANS, as it is not possible to disperse the bare particles
in pure water.
The introduction of nano-HAP particles, which have a SLD in
the range 4.19 # 10"6 to 4.51 # 10"6 Å"2 depending on the
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hydrogen–deuterium exchange, changed completely the scattering from the gel. The nano-HAP scatters much more than
the pure gel; a comparison between the integral of the intensity after a background correction in absolute units indicated
that the HAA-PVAH gel contributed about 1000 times less to
the total intensity than the nano-HAP particles. The scattering from the polymer matrix is therefore no longer significant. As it is difficult to match exactly the scattering contrast
of the particles, it was not possible to determine how the
local structure of the gel is perturbed by the particles. Gel
samples that contained 5–20 wt % nano-HAP were measured in solvents that matched the polymer matrix (SLD of
2.23 # 10"6 Å"2), i.e., far from the nano-HAP matching
point. These data are shown in Figure 8, and the various
concentrations differ only in the intensity while the shapes
of the curves are similar.
The shape of these curves suggests that there are aggregates
of small particles. At large Q, the shape and size of the individual particles is dominant while at low Q, the aggregate
structure determines the scattering. The particles have been
characterized by Hu et al.,17 and they have identified dense
crystals with mean radii of about 100 Å that when observed
in an electron microscope were anisotropic. The scattering
can be modeled as aggregates of disk-like particles, i.e., cylinders with a length that is shorter than the diameter. These
models, made with the SASFit38 program, include instrumental resolution are shown in Figures 8 and 9. The radius of
the cylinder was 100 6 10 Å, and the height was 30 6 4 Å
with about 20% polydispersity for all measured contrasts
and concentrations.
The overall size of the aggregates is not determined precisely
from the data that extend only to a lowest Q of about 10"3
Å"1. A lower limit for the cluster size is about 1000 Å. The
fit has used a model with a fractal aggregate structure55
although only a limited range of the data falls within a fractal region. The slope of the plot and hence the fractal
FIGURE 8 SANS data for 15 mg mL"1 hydrogel samples incorporating 5 (n), 10 (*), and 20 wt % nano-HAP (~). The fit with
a model of aggregated particles is shown. Samples are measured in solvent matched to the SLD of the gel matrix.
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427
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CONCLUSIONS
In this article, we measured hydrogels and hydrogel composites using SANS. The information drawn from the scattering
data is related to the physical properties and possible applications of these gels. Several models have been compared and
discussed to provide an understanding of a previously poorly
characterized, inhomogeneous in situ crosslinked hydrogel.
The SANS technique was applied to determine the organization of the polymers in gels. Gels with three different concentrations could all be pictured as densely linked clusters that
are separated by less dense regions with two clearly defined
length scales. The correlation lengths in the gels change with
concentrations. These changes are correlated with the rheological behavior of the gels. In particular, the requirements for
adequate mixing during preparation in order to achieve full
strength are understood from these new results.
FIGURE 9 SANS data for the 15 mg mL"1 hydrogel with 5%
nano-HAP in different solvent contrasts of SLD: 2.23 (n), 3 (~),
4.19 (D), 5 (*), and 6.35 (^) #10"6 Å"2. The contrasts are
scaled with a factor of 1, 10, 100, 1000 and 10000 respectively,
for better visibility.
dimension is approximate. The fitted value lies in the range
from 2.3–2.8. To obtain better estimates of the size of the
aggregates would need measurements in the very low Q
region. Static light scattering is commonly applied to overcome this problem but is not possible for these samples due
to their opacity that gives rise to strong multiple scattering.
Cryoscanning/transmission electron microscopy techniques
on the gel composite system is also not suitable as the particles and dispersion are fragile and the freezing can alter
the structure in the samples.
Although the possible measurements and interpretation of
scattering data are limited, interesting observations can be
made: the dimensions of the single particles are larger than
the smaller correlation length in the pure gel. The lower
limit of the size of the aggregates is in the same range as the
larger correlation lengths for the pure gel. This suggests that
the particles, either singly or as aggregates, are distributed
around the crosslinks and not only in the voids in between
crosslinks. The apparent differences in shape of the curves
measured in D2O/H2O-mixture with scattering length density
close to the nano-HAP arise from the different contributions
of background (Fig. 9).
As shown in Figure 8, gel samples that contained 5–20 wt %
nano-HAP can be superimposed by a scaling according to the
concentration of particles. This suggests that the overall
aggregation of particles in the matrix is not concentration
dependent. The results are interesting when related to applications. For example, studies have shown that higher nanoHAP content in a composites were more favorable for ectopic
bone formation.56 Our results suggest that for this application it is not a changed structure of the nano-HAP aggregate
but an increased amount of nano-HAP that alters the bone
formation.
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JOURNAL OF POLYMER SCIENCE, PART B: POLYMER PHYSICS 2013, 51, 421–429
The distribution of nanoparticles in crosslinked hydrogels
was also studied and the structure was interpreted as populations of fractal aggregates of cylindrical particles. The particle distribution and arrangement are independent of their
concentration. Although an increased concentration is favorable for ectopic bone formation, this is not a consequence of
a structural change in the arrangement of the particles but
rather depends on the amount of material introduced to the
wounded site.
ACKNOWLEDGMENTS
The authors thank the Institut Laue Langevin for providing
beam time and the PSCM facilities. A special thanks to Lionel
Porcar for the SANS experiment on D22. Ralf Schweins and
David Bowyer provided help with D11. The authors are grateful
to Kristoffer Bergman for the aldehyde derivative of hyaluronan
and for discussion of the results. The authors also thank
Qinghong Hu for the information about the nano-HAP.
REFERENCES AND NOTES
1 D. Y. Teng, Z. M. Wu, X. G. Zhang, Y. X. Wang, C. Zheng, Z.
Wang, C. X. Li, Polymer 2010, 51, 639–646.
2 N. A. Peppas, Curr. Opin. Colloid Interface Sci. 1997, 2,
531–537.
3 T. R. Hoare, D. S. Kohane, Polymer 2008, 49, 1993–2007.
4 K. Y. Lee, D. J. Mooney, Chem. Rev. 2001, 101, 1869–1879.
5 J. K. Hansen, S. L. Thibeault, J. F. Walsh, X. Z. Shu, G. D.
Prestwich, Ann. Oto. Rhinol. Laryn. 2005, 114, 662–670.
6 S. Yang, K.-F. Leong, Z. Du, C.-K. Chua, Tissue Eng. 2001, 7,
679–689.
7 I. C. Bonzani, J. H. George, M. M. Stevens, Curr. Opin. Chem.
Biol. 2006, 10, 568–575.
8 M. Zhang, S. P. James, Polymer 2005, 46, 3639–3648.
9 M. Monier, Y. Wei, A. A. Sarhan, D. M. Ayad, Polymer 2010,
51, 1002–1009.
10 K. Bergman, C. Elvingson, J. Hilborn, G. Svensk, T. Bowden,
Biomacromolecules 2007, 8, 2190–2195.
11 K. Bergman, T. Engstrand, J. Hilborn, D. Ossipov, S. Piskounova, T. Bowden, J. Biomed. Mater. Res. Part A 2009, 91,
1111–1118.
JOURNAL OF
POLYMER SCIENCE
WWW.POLYMERPHYSICS.ORG
39,
35 K. Lieutenant, P. Lindner, R. Gahler, J. Appl. Crystallogr.
2007, 40, 1056–1063.
€nnvall, K. Forsberg-Nilsson, J. Hilborn, J.
13 D. Ossipov, K. Bra
Appl. Polym. Sci. 2007, 106, 60–70.
36 D22. Available at: http://www.ill.eu/instruments-support/
instruments-groups/instruments/d22/. Access date: 26 July
2012.
12 D. Ossipov,
1709–1718.
J.
Hilborn,
Macromolecules
FULL PAPER
2006,
14 D. Ossipov, S. Piskounova, J. Hilborn, Macromolecules
2008, 41, 3971–3982.
15 E. Theunissen, N. Overbergh, H. Reynaers, S. Antoun, R.
"rôme, K. Mortensen, Polymer 2004, 45, 1857–1865.
Je
16 D. Baksh, J. E. Davies, S. Kim, J. Mater. Sci.: Mater. Med.
1998, 9, 743–748.
17 Q. Hu, Z. Tan, Y. Liu, J. Tao, Y. Cai, M. Zhang, H. Pan, X.
Xu, R. Tang, J. Mater. Chem. 2007, 17, 4690–4698.
18 Y. Cai, Y. Liu, W. Yan, Q. Hu, J. Tao, M. Zhang, Z. Shi, R.
Tang, J. Mater. Chem. 2007, 17, 3780–3787.
€ m, Q. Hu, K. Bergman, K. B. Jonsson, J.
19 G. Hulsart-Billstro
Åberg, R. Tang, S. Larsson, J. Hilborn, J. Acta Biomater. 2011,
7, 3042–3049.
20 S. Van Vlierberghe, P. Dubruel, E. Schacht, Biomacromolecules 2011, 12, 1387–1408.
37 R. E. Ghosh, S. U. Egelhaaf, A. R. Rennie, ‘A Computing
Guide for Small-Angle Scattering Experiments’; Institut LaueLangevin Internal Publication ILL06GH05T: Grenoble, 2006.
38 J. Kohlbrecher, In SASfit: A Program for Fitting Simple
Structural Models to Small Angle Scattering Data; Paul Scherrer Institut, Laboratory for Neutron Scattering: CH-5232, Villigen, Switzerland, 2008.
39 F. Horkay, A. M. Hecht, S. Mallam, E. Geissler, A. R. Rennie,
Macromolecules 1991, 24, 2896–2902.
40 F. Horkay, W. Burchard, E. Geissler, A. M. Hecht, Macromolecules 1993, 26, 1296–1303.
41 E. Geissler, F. Horkay, A.-M. Hecht, Phys. Rev. Lett. 1993, 71,
645–648.
21 N. A. Peppas, J. Z. Hilt, A. Khademhosseini, R. Langer, Adv.
Mater. 2006, 18, 1345–1360.
42 G. D’Errico, M. De Lellis, G. Mangiapia, A. Tedeschi, O.
Ortona, S. Fusco, A. Borzacchiello, L. Ambrosio, Biomacromolecules 2007, 9, 231–240.
22 M. C. Boyce, E. M. Arruda, Rubber Chem. Technol. 2000, 73,
504–523.
43 P. G. de Gennes, Scaling Concepts in Polymer Physics; Cornell University Press: New York, 1979.
23 L. R. G. Treloar, The Physics of Rubber Elasticity; Oxford
University Press: Oxford, 1975.
44 F. Horkay, P. J. Basser, A.-M. Hecht, E. Geissler, Polymer
2005, 46, 4242–4247.
24 N. A. Peppas, P. Bures, W. Leobandung, H. Ichikawa, Eur. J.
Pharm. Biopharm. 2000, 50, 27–46.
45 M. Shibayama, Soft Matter Characterization; R. Borsali, R.
Pecora, Eds.; Springer: New York, 2008., Chapter 14, pp 787–
790.
25 P. J. Flory, J. Rehner, J. Chem. Phys. 1943, 11, 521–526.
26 M. Sen, A. Yakar, O. Güven, Polymer 1999, 40, 2969–2974.
27 J. A. Deiber, M. L. Ottone, M. V. Piaggio, M. B. Peirotti,
Polymer 2009, 50, 6065–6075.
28 S. Piskounova, R. Rojas, K. Bergman, J. Hilborn, Macromol.
Mater. Eng. 2011, 296, 944–951.
29 K. Nam, J. Watanabe, K. Ishihara, Polymer 2005, 46,
4704–4713.
30 S. Van Vlierberghe, B. Fritzinger, J. C. Martins, P. Dubruel,
Appl. Spectrosc. 2010, 64, 1176–1180.
31 G. Mangiapia, R. Ricciardi, F. Auriemma, C. De Rosa, F. Lo
Celso, R. Triolo, R. K. Heenan, A. Radulescu, A. M. Tedeschi, G.
D’Errico, L. Paduano, J. Phys. Chem. B 2007, 111, 2166–2173.
32 A. K. Jha, R. A. Hule, T. Jiao, S. S. Teller, R. J. Clifton, R. L.
Duncan, D. J. Pochan, X. Jia, Macromolecules 2009, 42,
537–546.
33 F. Horkay, A. M. Hecht, E. Geissler, J. Polym. Sci. Part B:
Polym. Phys. 2006, 44, 3679–3686.
34 J. S. Higgins, H. Benoit, Polymers and Neutron Scattering;
Clarendon Press: Oxford, 1994.
WWW.MATERIALSVIEWS.COM
46 M. Zrinyi, F. Horkay, Polym. Bull. 1992, 29, 445–452.
47 F. Horkay, D. C. Lin, Langmuir 2009, 25, 8735–8741.
48 F. Horkay, A.-M. Hecht, I. Grillo, P. J. Basser, E. Geissler, J.
Chem. Phys. 2002, 117, 9103–9106.
49 P. Debye, A. M. Bueche, J. Appl. Phys. 1949, 20, 518–525.
50 J. Teixeira, J. Appl. Crystallogr. 1988, 21, 781–785.
51 E. Buhler, F. Boue, Macromolecules 2004, 37, 1600–1610.
52 J. R. E. Fraser, T. C. Laurent, U. B. G. Laurent, J. Intern.
Med. 1997, 242, 27–33.
53 Y. Zhang, J. F. Douglas, B. D. Ermi, E. J. Amis, J. Chem.
Phys. 2001, 114, 3299–3313.
54 I. Berts, Properties of Ideal Polymeric Vitreous Substitutes.
M.Sc. Thesis, Uppsala University, Uppsala, January 2009.
55 A. Harrison, Fractals in Chemistry; Oxford University Press:
Oxford, 1995.
56 S. G. Hulsart, Influence of Calcium Phosphate on Osteogenes is in Hydrogel with Bone Morphogenetic Protein-2.
M.Sc. Thesis, Uppsala University, Uppsala, January 2010.
JOURNAL OF POLYMER SCIENCE, PART B: POLYMER PHYSICS 2013, 51, 421–429
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