Biochemical Engineering Journal 40 (2008) 211–217

Viscoelasticity and wearability of hyaluronate solutions

Syang-Peng Rwei a,∗ , Saint-Wei Chen a , Ching-Feng Mao b , Hsu-Wei Fang c
a Institute of Organic and Polymeric Materials, National Taipei University of Technology, #1, Sector 3, Chung-Hsiao E. Road, Taipei, Taiwan, ROC
b Department of Chemical and Material Engineering, Southern Taiwan University, No. 1, Nan-Tai Street, YungKung City, Tainan 710, Taiwan, ROC
c Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan, ROC

Received 25 February 2007; received in revised form 10 December 2007; accepted 18 December 2007

Abstract
This work systematically studied the viscoelastic properties of hyaluronic acid (HA) solution, a major component of synovial fluid, under various
testing conditions. The optimum relaxation time of HA solution is around 4.5 s at pH 6.8, in the absence of salt at room temperature, indicating that
synovial fluid is viscous when the shear rate is less than 1/4.5 s−1 but elastic when the shear rate exceeds this critical value. HA viscosity declines
markedly as the following factors are increased in their order, salt concentration > pH level > temperature, demonstrating that these factors weaken
the intermolecular attraction among HA molecules. The non-thixotropic behavior of the HA solution suggests that the breakdown and recovery
of the HA structure proceed through the same intermediate states, reconfirming the strong performance of HA as a main component of synovial
fluid. The wear results reveal that when the shear rate exceeds a critical value of around 20 s−1 , the drop in viscosity leveled out independent of any
further increase in shear rate. However, in a broad range of wear rates (20–300 s−1 ), the HA viscosity sustains for wearing time less than 20 min
but declines without leveling off as the wear duration increases thereafter. Finally, experimental results verify that bovine albumin (BA), in HA
solution, acts as both hydrodynamic and boundary lubricant, substantially improving the wearability of HA.
© 2008 Published by Elsevier B.V.

Keywords: Hyaluronic acid (HA); Relaxation time; Viscoelasticity; Wear result; Bovine albumin (BA); Lubrication

1. Introduction ronment is insufficient. Accordingly, from a rheological point

As a biopolymer, hyaluronic acid (HA) (Scheme 1) is a
glycosaminoglycan that consists of unbranched repeating disac-
charide units. HA was discovered in the vitreous humor of cattle
eyes [1–3]. Later, HA was found to be present in various living
substrates [4], including extracellular matrices and synovial fluid
[5]. It is one of the primary components of synovial fluid, acting
as a lubricant and shock absorber. Although much information
is available on the structure of HA in aqueous solution [6–8],
little is known about the viscoelastic behavior of HA in syn-
ovial fluid. Al-Assaf et al. investigated the extensional viscosity
of HA and concluded that the extension-thickening occurred
at relatively high deformation rates of 500 s−1 and above. Mo
confirmed the above finding and, furthermore, pointed out that
the addition of sodium chloride would enhance the thickening
behavior [9,10]. However, a deep understanding of HA solution
under shearing in various pH, temperatures, or additive envi-
of view, the protection mechanisms for HA acting as a synovial
fluid in lubricating and shock-absorbing applications are still
unidentified.
Synovial fluid has been hypothesized to be able to behave
as a viscous liquid that responds to a slowly moving joint, and
as an elastic liquid to respond to a rapidly moving joint. The
critical factor that governs the dual roles of synovial fluid is
the relaxation time of the HA solution, which must be less than
the duration of a slow motion but much longer than the period
of rapid motion. This work therefore systematically studied the
viscoelastic characteristics of HA solution under various condi-
tions of concentration, pH and salt concentration in the presence
of albumin protein, over a wide range of frequencies at low shear
amplitude. Finally, a study of wear was performed to investigate
the decay of viscoelastic properties in HA solution.
2. Experimental

Rheological characteristics were measured using a Vilastic

∗ Corresponding author. rheometer (Vilastic Scientific Inc.) [11], with which an oscilla-
E-mail address: f10714@ntut.edu.tw (S.-P. Rwei). tory flow of the desired frequency and amplitude was generated,

1369-703X/$ – see front matter © 2008 Published by Elsevier B.V.

doi:10.1016/j.bej.2007.12.021
212 S.-P. Rwei et al. / Biochemical Engineering Journal 40 (2008) 211–217

Scheme 1. Chemical structure of hyaluronic acid (HA).

and the instantaneous pressure and the flow rate were detected.
Fig. 1 reveals that the sample cell was made of aluminum in
a cylindrical tubular configuration. The rheological behavior
of the HA solution was examined under dynamic shear of a
fixed amplitude. Frequency sweep measurements over the range Fig. 2. Typically experimental and simulated results of storage (G ) and loss
10−1 Hz < ω < 102 Hz were made, and the real part G and imag- (G ) moduli for HA solution (0.3 wt%) at pH 6.5, 25 ◦ C, and without any added
inary part G of the shear modulus were recorded under low salt.

deformation conditions. Experimental curves of the viscosities

All of the ingredients were used as received without fur-
as a function of frequency were plotted for various shear rates
ther purification. The wear experiment was conducted using a
by applying Cox-Merz rule [12].
homemade apparatus, which is of a parallel plate device with
In general, the pH values for an arthritic knee range from 6.5
an accurate shear rate control. Circular dichroism (CD) [16]
to 8.1 [13]. The concentrations of NaCl in a regular ox synovial
was employed to study changes in the structure of bovine albu-
fluid were reported from 0 to 0.3658 m [14,15]. The experi-
min due to wearing. The gel permeation chromatography (GPC)
mental conditions employed in this study, therefore, focused on
was performed on a JASCO PU-1580 GPC with water column
the range mentioned above to simulate the synovial fluid under
calibrated by standard PEO & PEG.
dynamic shearing.
HA and bovine albumin (BA) were commercially obtained
3. Results and discussion
from Acros: HA from rooster comb had a MW over 2 × 106 ,
and BA was a fatty acid and globulin-free with a MW of around
Fig. 2 presents the typical result of a dynamic rheological test.
6 × 104 . The pH of the HA solution was adjusted using acetic
The storage (G ) and loss (G ) moduli increase with frequency.
acid (Acros) and ammonia solution (Acros) and monitored using
The storage modulus usually represents the elastic character and
a pH meter (25 ◦ C). Notably, the concentration of HA solution
the loss modulus describes the viscous behavior. This result sug-
prepared in this study are fixed in 0.3% by weight to simulate
gests that the enhancement in structural entanglement increases
the real composition in a synovial fluid [1,10].
the storage modulus, while structural breakdown increases the
loss modulus. At lower frequencies, both structural buildup and
breakdown are enhanced as the frequency increases. However, as
the frequency increases further, the loss modulus decays beyond
the cross-over point, while the storage modulus increases toward

Fig. 1. Setup of Vilastic rheometer to measure the dynamic viscoelastic prop- Fig. 3. Viscosity as a function of shear rate for HA solutions (0.3 wt%, pH 6.5,
erties of HA solutions. saltfree) at various temperatures.
 S.-P. Rwei et al. / Biochemical Engineering Journal 40 (2008) 211–217 213

Table 1 Table 2
The rheological properties of HA solution under various experimental conditions The rheological properties of HA solution added with BA under various exper-
imental conditions
Experimental condition Parameter
Experimental conditions Parameters
λc (s) G∞ η (P) (shear rate = 1 s−1 )
λc (s) G∞ ␩ (P) (shear rate = 1 s−1 )
Temperature (pH = 6.5, saltfree)
15 ◦ C 4.45 3.5 3.09 BA concentration (25 ◦ C, pH = 6.5, saltfree)
25 ◦ C 3.41 3.5 2.37 None 4.01 2.7 2.44
40 ◦ C 2.77 3.5 1.89 6 mg/ml 2.91 3.0 1.76
50 ◦ C 2.17 3.5 1.49 12 mg/ml 2.10 3.0 1.30
60 ◦ C 1.94 3.5 1.20 24 mg/ml 1.93 3.0 1.12
pH value (25 ◦ C, saltfree) pH value (25 ◦ C, saltfree)
2.5 1.01 2.1 0.35 6.5 1.70 3.2 1.36
3.5 1.46 3.0 0.82 6.7 2.29 3.0 1.45
5.4 2.22 3.5 2.07 6.9 2.74 2.7 1.40
6.8 4.47 3.5 2.32 7.9 2.31 2.7 1.13
9.3 4.42 2.8 2.05 9.9 1.92 2.5 0.93
11.4 3.07 2.8 1.39
Salt concentration (25 ◦ C, pH = 6.5)
Salt concentration (25 ◦ C, pH = 6.5) None 2.10 3.0 1.30
None 4.01 2.7 2.44 0.01 M 0.83 4.5 0.82
0.01 M 1.73 2.7 0.94 0.10 M 0.59 3.1 0.33
0.10 M 0.81 2.5 0.37 1.0 M 0.38 3.1 0.20
1.0 M 0.25 3.0 0.13

trations, respectively. The variation in temperature from 288 to

a saturation point. Notably, the inverse of the cross-over point
of G and G yield a single relaxation time of the HA polymer
that is dissolved in aqueous solvent. The single relaxation time
is physically defined as the longest time required for the elastic
structures in the fluid to relax [17]. Moreover, a dimensionless
group Deborah number, De, is defined as the relaxation time
divided by the process time (or observation time). An elastic
solid has a De number higher than one but a viscous liquid, pos-
sesses a De number lower than one. The entanglement-structure
of the HA polymer is believed to be enhanced more at higher
frequencies, but the transition cycle is too short for structural
breakdown or relaxation. Consequently, the Deobrah number
of the system markedly exceeds one, causing the suspension to
become more solid-like [18–20]. G thus tends to increase while
G decreases. Interestingly, the Maxwell Model (Eq. (1)) [21],
comprising an elastic component (spring) in series with a vis-
cous component (dashpot), was found to well match the results
presented herein (Fig. 2).
333 K in Fig. 3, had a weak effect on the viscosity of HA solu-
tion. The transition of concentrated polymer solutions from the
shear-independent region to the shear-thinning region generally
reflects the extent of intermolecular interaction. The subsequent
decline in viscosity as shear increases is attributable to the pro-
gressive breakdown of the intermolecular network. When the
shear rate falls below the intrinsic rate at which molecules regain
their random coil structure, the hydrodynamic radius of the
sphere Rh typically does not vary with the flow rate; a Newtonian
fluid is therefore observed. In higher shear rates, polymer relax-
ation becomes less competitive with shear, and shear-thinning
occurs because a shear that exceeds the critical value reduces
the extent to which neighboring molecules become entangled,
and the flow resistance decreases.
The variation in pH values of HA (Fig. 4) affects viscosity
more than it affects temperature, as discussed in Fig. 3. The vis-

G0 ω 2 τ 2 G0 ωτ
G = ; G = (1)
1 + ω2 τ 2 1 + ω2 τ 2
where ω is the applied frequency, G0 is the fitting parameter, and
τ is the relaxation time determined by the inverse of the cross-
over point of G and G . The simulated results (solid line) in
Fig. 2 agree closely with the experimental data (points) at applied
frequencies from 0.01 to 30 Hz. The experimental results are
consistent with the well known fact that synovial fluid not only
acts as a viscous liquid in low-frequency regions, responding
to slowly moving joints, but also acts as an elastic behavior in
high-frequency regions, responding to rapidly moving joints.
Tables 1 and 2 present all of the fitted results herein, and will be
discussed in detail later.
Figs. 3–5 plot the viscosity as a function of shear rate for HA Fig. 4. Viscosity as a function of shear rate for HA solutions (0.3 wt%, 25 ◦ C,
solutions at various temperatures, pH values and salt concen- saltfree) at various pH values.
214 S.-P. Rwei et al. / Biochemical Engineering Journal 40 (2008) 211–217

formation of helices. Such conformational changes significantly

reduces the shear modulus and shear viscosity. Briefly, the results
presented herein reveal that HA viscosity can be reduced as the
following factors are increased in the order of importance, salt
concentration > pH level > temperature.
Figs. 2 and 6(a) and (b) present the frequency-dependence
of G and G for 0.3 wt% HA solutions with and without NaCl.
Fig. 2 reveals that the experimental results are strongly consis-
tent with the Maxwell Model (Eq. (1)) without any added salt.
However, Fig. 6(a) and (b) demonstrates that G from Maxwell’s
prediction when NaCl salt is added to a concentration of 0.01 M
and 0.1 M, respectively. The deviation is greater at lower fre-
quency. The Maxwell model can be generally interpreted by
a Rouse chain model [23] with a mono-dispersed molecular
weight. The Rouse model considers that the polymer chain has n
Fig. 5. Viscosity as a function of shear rate for HA solutions (0.3 wt%, pH 6.5, completely flexible repeating units with an end-to-end distance
25 ◦ C, saltfree) at various concentrations of NaCl. that obeys Gaussian statistical motion in viscous surroundings.
However, adding salt induces the formation of shrunken coil,
cosity reaches a maximum value of 2.3 P when the pH value even forming a locally helical conformation sometimes, from
reaches 6.8. Both acidic and alkaline environments reduce the an expanded random coil. This conformation change may sub-
viscosity by a factor of two to six. Most random coil polysac- stantially alter the Gaussian statistics to another asymmetric
charides undergo a large drop and a slight increase in solution distribution of the end-to-end distances of HA. Additionally,
viscosity at low and high pH, respectively, because deioniza- cations that shield the electrostatic repulsion weaken the inter-
tion and ionization of hydroxyl groups reduce and increase the molecular attraction [24–26] and cause the breakdown of the
dimensions of the coil by intramolecular electrostatic interac-
tion at low and high pH, respectively. HA, in contrast, shows
a reduction in viscosity under alkaline conditions at high pH.
Table 1 reveals that the viscosity reaches a maximum at a pH of
6.8, indicating that the coil dimensions are maximal and the opti-
mum viscoelastic behavior enhances the cushion performance
of the synovial fluid. Notably, the relaxation time λ, given by the
inverse of the G and G cross-over frequency, is also maximal
at a pH of 6.8. The consistency between the points at which λ
and viscosity are optimal reconfirms the aforementioned chain
conformation hypothesis. Interestingly, the optimal relaxation
time of the HA solution, about 4.5 s (Table 1), is roughly char-
acteristic of a synovial fluid, which acts as a viscous liquid at a
slow shear rate of under 1/4.5 s−1 but exhibits elastic behavior
at a high shear rate that exceeds this critical value.
Fig. 5 reveals that the salt concentration substantially affects
the drop in HA viscosity. The decrease in viscosity with
increasing NaCl concentration is attributable to the shielding of
electrostatic repulsion between anionic groups in HA molecules,
which produces the contracted forms of random coils. As is
well known, adding cations promotes the aggregation of dou-
ble helices for ␬-carrageenan gels [22]. In such a case, the
cations shield the electrostatic repulsion of sulfate groups in HA
molecules, promoting the formation of the helices. HA is a lin-
ear polyelectrolyte that consists of glucosamine and glucuronic
acid. HA is believed to exist in a complex with proteins that
extend the spatial domain of the molecule and confer rigidity
upon it. However, due to the intermolecular attractions, includ-
ing hydrogen bonding or ionic attraction, among HA molecules
are considerable, HA solutions exhibit the behavior of an exten-
sively hydrogen-bonded network. Adding salt appears to weaken Fig. 6. (a) and (b) The experimental and simulated results of storage (G ) and loss
the intermolecular attraction, increasing the freedom of the chain (G ) moduli for HA solution (0.3 wt%) at pH 6.5, 25 ◦ C, NaCl concentrations
or its extension under shearing, and moreover, leading to the of 0.01 and 0.1 M, respectively.
 S.-P. Rwei et al. / Biochemical Engineering Journal 40 (2008) 211–217 215

Fig. 9. Viscosity as a function of shear rate for HA solutions (0.3 wt%, 25 ◦ C,

saltfree) with added BA (12 mg/ml) at various pH values.
Fig. 7. Hysteresis cycle of shear stress against shear rate for HA solution
(0.3 wt%, pH 6.5, 25 ◦ C, saltfree).
trations, pH values and salt concentrations, respectively. The
concentration of albumin in a normal and an artificial synovial
hydrogen-bonded network [27] into numerous locally attracted
fluid ranges from 7 to 18 and from 20 to 39 mg/ml, respectively
HA blocks, which behave more like poly-dispersed moving
[13]. BA concentration of 12 mg/ml is therefore employed as the
chains. Both phenomena are stronger at a lower frequency and
testing condition herein. As demonstrated by Fig. 8, the viscosity
can be employed to explain completely the deviation from the
decreases as the concentration of BA increases, but levels out at
Maxwell model with a single relaxation time.
12 mg/ml. The BA is thought to act as a hydrodynamic lubricant
The suppression of the thixotropy phenomenon is important
that reduces the viscosity of the HA solution. Adding albumin
in evaluating the quality of synovial fluid. Thixotropy is observed
may shield the repulsive force produced by the net charges on
when the shear viscosity measured at a progressively increasing
the HA molecules. The tribological properties of HA are there-
shear rate differs from that measured at a progressively decreas-
fore altered. Notably, BA is known to function not only as a
ing shear rate. Fig. 7 plots the hysteresis cycle of the HA solution.
hydrodynamic lubricant but also as a boundary lubricant [30].
The curve of the shear stress against the shear rate of the HA
The adherence of albumin to the artificial joint surface reduces
solution, even when exhibiting a nonlinear behavior, falls on the
the coefficient of friction, promoting boundary lubrication at
same trace and illustrates the time-independent characteristics.
low speed. A wear study is performed and described below to
The non-thixotropic behavior of the HA solution indicates that
elucidate this point.
the recovery of change in the HA conformation that is induced
Like Figs. 4 and 5, Figs. 9 and 10 plot the dependence of
by the flow is too short to leave any thixotropic track. Restated,
viscosity on shear rate for HA when BA is added at various
the breakdown and recovery of the HA structure proceed through
pH values and salt concentrations. Unsurprisingly, the results
the same intermediate states, indicating that HA acts effectively
demonstrate that an increase in pH or salt concentration reduces
as a synovial fluid.
HA viscosity. However, a comparison between Figs. 5 and 10
Figs. 8–10 plot viscosity versus shear rate curves for HA
shows that adding BA reduces the sensitivity of the viscos-
solutions with the addition of BA [28,29] at various BA concen-

Fig. 8. Viscosity as a function of shear rate for HA solutions (0.3 wt%) with Fig. 10. Viscosity as a function of shear rate for HA solutions (0.3 wt%, pH 6.5,
added BA in various BA concentrations. 25 ◦ C) with added BA (12 mg/ml) at various concentrations of NaCl.
216 S.-P. Rwei et al. / Biochemical Engineering Journal 40 (2008) 211–217

Table 3
GPC results for HA solutions experienced various wearing times

Wearing time Mn × 10−6 Mw × 10−6 Polydispersity

Fresh 1.84 2.77 1.50

20 min 1.77 2.65 1.50
40 min 1.36 2.28 1.67
5h 1.30 2.23 1.71
24 h 1.09 1.90 1.74
50 h 1.00 1.78 1.76
100 h 0.76 1.38 1.81

perhaps because a long rotation period offers more opportuni-

ties for the shearing stress to act on the weakest point of the
chain to trigger chain-breaking. This finding can be extended to
the important conclusion that continuously mild exercise over
Fig. 11. Viscosity decrease as a function of wear rate for HA solutions (0.3 wt%,
pH 6.5, 25 ◦ C, saltfree) experienced various wear time.
a long period damages synovial fluid more severely than harsh
exercise of short duration. Another interesting result in Fig. 11
is that a 20 min exercise would not cause any drop in HA viscos-
ity of the HA to the changes in the concentration of the salt.
ity in a broad range of wear rates (0–300 s−1 ). Table 3 presents
The formation of helices induced by cations is hypothesized to
the GPC results, number and weight average of HA molecular
be interrupted by the BA protein, reducing the drop in shear
weight, against the wearing time at a given wear rate (250 s−1 ).
modulus and shear viscosity of HA.
The results reconfirm above conclusion that the chain-breakings
Fig. 11 presents perhaps the most important result of this
occur after wear over 20 min and increase with wearing time
work, regarding the wear of HA. Fig. 11 shows that shear rate
thereafter.
and the wear time are the main factors that govern wear perfor-
Fig. 12, which presents the CD signal for BA before and after
mance. The wear time applied herein is defined as the duration
shearing, demonstrates that the curve shape does not change but
of the wearing test at a given shear rate. When the shear rate
the peak height varies somewhat. This result suggests that after
exceeds a critical value, about 20 s−1 , the decrease in viscosity
a long period (100 h) of shearing at a high shear rate (250 s−1 ),
stops independent of any further increase in the shear rate. How-
some BA molecules may deteriorate, but most survive. Fig. 13
ever, the viscosity keeps decreasing without leveling off when
plots viscosity against shear rate for samples that had and had
the wear time increases. This result indicates that wear time is
not been worn for 100 h. Adding BA only slightly reduces the
more important than shear rate. Shear stress typically gener-
viscosity of HA solution, demonstrating remarkable resistance
ates a torque on a polymer molecular and causes the polymer
to wearing. However, the viscosity of normal HA differs sig-
chain to rotate in a molecular scale. Once the shear rate is suf-
nificantly between samples that had and had not undergone a
ficiently large and the duration of rotation is sufficiently long,
wear test. This result verifies the preceding claim that BA acts as
the polymer chain cannot resist the rotating environment, and
both hydrodynamic and boundary lubricant, with a very positive
chain-breakings therefore occur. The experimental data herein
effect on the wear resistance associated with HA solution.
demonstrate that the shearing duration influences the deteriora-
tion of HA viscosity more severely than does the shearing stress,

Fig. 13. Viscosity as a function of shear rate for HA solutions (0.3 wt%, 25 ◦ C,
Fig. 12. CD spectra for fresh and post-wear BA solutions (12 mg/ml). saltfree) with and without added BA (12 mg/ml) after 100 h, 250 s−1 wear.
 S.-P. Rwei et al. / Biochemical Engineering Journal 40 (2008) 211–217
4. Conclusions
This work systematically studied the viscoelastic properties
of HA solution under various conditions. The optimum relax-
ation time of HA solution is around 4.5 s at pH 6.8, in the
absence of salt at room temperature, indicating that synovial
fluid is viscous when the shear rate is less than 1/4.5 s−1 but
elastic when the shear rate exceeds this critical value. HA vis-
cosity declines markedly as the following factors are increased
in their order, salt concentration > pH level > temperature, sug-
gesting that these factors weaken the intermolecular attraction
among HA molecules. They therefore increase chain freedom
and extension, and promote the formation of helices under
shearing, reducing the shear modulus and shear viscosity. The
non-thixotropic behavior of the HA solution suggests that the
breakdown and recovery of the HA structure proceed through
the same intermediate states, reconfirming the strong perfor-
mance of HA as a main component of synovial fluid. The wear
results reveal that when the shear rate exceeds a critical value of
around 20 s−1 , the drop in viscosity leveled out independent of
any further increase in shear rate. However, the viscosity keeps
decreasing without leveling off as the wear duration increases.
Finally, the results herein verify that BA, in HA solution, acts
as both hydrodynamic and boundary lubricant, substantially
improving the wearability of HA.
Acknowledgement
The authors would like to thank the National Science Council
of the Republic of China (Taiwan) for financially supporting this
research under Contract No. NSC-95-2221-E-027-062-MY3.
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