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Journal of Biomechanics 41 (2008) 1840–1846

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Rheological properties of the vitreous and the role of hyaluronic acid

Charles S. Nickersona, John Parkb, Julia A. Kornfielda,, Hampar Karageozianb
a
Division of Chemistry and Chemical Engineering, California Institute of Technology, Chemical Engineering 210-41,
1200 E. California Blvd., Pasadena, CA 91125, USA
b
Vitreoretinal Technologies, Inc., Irvine, CA, USA
Accepted 8 April 2008

Abstract

Coordinated rheological and biochemical measurements provide the linear and nonlinear mechanical properties of the vitreous and
demonstrate the structural role of hyaluronic acid. ‘‘Cleated’’ tools are used to overcome wall slip and avoid tissue compression during
measurements of the dynamic moduli of fresh porcine and bovine vitreous. Shear moduli decreased five-fold from initial to steady-state
values in the first hour after dissection. Steady-state values (porcine: G0 ¼ 2.870.9 Pa, n ¼ 9; bovine: G0 ¼ 7.072.0 Pa, n ¼ 17) are
significantly greater than previously reported. The decrease in modulus after removal from the eye correlates with a decrease in mass:
even in the absence of external driving forces, porcine vitreous expels 5% of its mass within 5 min and continues to decay to a steady-
state mass 10% lower than its initial mass. The expelled fluid has a substantial hyaluronan concentration, but very low protein content.
These results indicate that the vitreous network is under tension at its native volume and its high initial modulus results from this state of
tension. We hypothesize that hyaluronan plays a role in sustaining the ‘‘internal tension’’ by Donnan swelling.
r 2008 Elsevier Ltd. All rights reserved.

Keywords: Rheology; Eye; Hyaluronan; Biomechanics; Tension

1. Introduction which has been associated with macular holes, macular

The vitreous humor is a transparent gel comprised of a
delicate, highly swollen double network of protein fibrils
(primarily collagen type II) and charged polysaccharide
chains (primarily hyaluronan) (Bishop, 2000). By weight,
however, vitreous is 99% water and 0.9% salts; the
network components represent only a small fraction of its
mass. Healthy vitreous is also avascular and nearly acellular
and its functions appear to be primarily structural.
Indeed, the unique mechanical properties of the vitreous
are essential for three of its primary functions: develop-
mental—mediating proper growth of the eye; optical—
maintaining a clear path to the retina; and mechanical—
supporting the various ocular tissues during physical
activity (Sebag, 1989). Its mechanical properties also play
a role in several vision-threatening pathologies: localized
destabilization of the vitreous can lead to retinal traction,
Corresponding author. Tel.: +1 626 395 4138; fax: +1 626 568 8743.
E-mail address: jak@cheme.caltech.edu (J.A. Kornfield).
edema, vitreous hemorrhage, retinal tears, and retinal
detachment (Sebag, 1989).
The vitreous derives its bulk properties from the
hydrated double network of collagen fibrils and high
molecular weight, polyanionic hyaluronan macromolecules
(Bishop, 2000; Sebag, 1989; Sebag and Balazs, 1989)
(Fig. 1). The network of collagen fibrils has been presumed
to serve the load-bearing function because the vitreous
does not collapse during enzymatic removal of hyaluronan
but dissolves completely after digestion with collagenase
(Bishop, 2000; Bos et al., 2001; Sebag, 1989). It was
suggested that swollen hyaluronan macromolecules simply
hydrate the network and fill the space between the fibrils to
prevent aggregation.
The relationship of the composition and microstructure
to the mechanical properties essential to vitreous function
remains poorly understood, hampered by a lack of
sufficient experimental methods to quantitatively define
its mechanical properties. The lubricating ability of its
constituent molecules and its fragile network structure have

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doi:10.1016/j.jbiomech.2008.04.015
 ARTICLE IN PRESS
C.S. Nickerson et al. / Journal of Biomechanics 41 (2008) 1840–1846
made reliable measurements extremely difficult. A number
of creative methods have been devised to quantify the gel
character of the vitreous including bulk measurements
(Bettelheim and Wang, 1976; Pfeiffer, 1963; Tokita et al.,
1984), magnetic microrheology (Lee et al., 1992, 1994a, b),
in vivo visual tracking for humans (Zimmerman, 1980), and
more recently, acoustic techniques (Walton et al., 2002).
These techniques allow for comparative analyses in terms
of phenomenological parameters, but not for quantitative
measurements.
We have recently described a rheological tool—the
‘‘cleat geometry’’—designed to address the difficulties
mentioned above (Nickerson and Kornfield, 2005), allow-
Fig. 1. Schematic depiction of the network structure of the vitreous. The
vitreous is composed of a highly swollen double network of heterotypic
collagen fibrils (primarily type II) and hyaluronic acid.
Fig. 2. Typical time sweep of fresh bovine (A) and porcine (B) vitreous samples showing the rapid modulus decay to a steady state after removal from the
eye (g ¼ 3%, o ¼ 10 rad/s). The loss tangent reached steady state more rapidly, often in as little as 20 min in bovine (tan d ¼ 0.3170.03) and 2 min in
porcine (tan d ¼ 0.2470.09) specimens.
1841
ing direct, quantitative, time-resolved rheological charac-
terization of the vitreous using a fluids rheometer
(Nickerson and Kornfield, 2005). The cleat geometry
suppresses wall slip while causing minimal disturbance to
tissue structure and properties during measurement (see
Section 2). We have used this method to track the softening
that occurs after the vitreous is extracted from the eye.
Prior literature makes no mention of time-dependence of
the modulus of the vitreous post-dissection; and our results
indicated that the literature values of the modulus are
systematically low for bovine and porcine vitreous—even
when compared to the relatively low steady-state values
(Nickerson et al., 2005).
We also apply the cleat geometry to examine vitreous
failure characteristics under high strain, pertinent to certain
injuries and surgical procedures. During severe head
trauma, the vitreous gel can be detached and, in turn,
cause retinal tears. During vitrectomy procedures per-
formed to treat diabetic retinopathy, among other diseases,
surgeons destroy the vitreous in order to remove it from the
eye. However, to our knowledge, there have been no
published studies that demonstrate the maximum stress the
vitreous can bear or the behavior of the tissue as it fails.
Rheological results, together with observations of mass
changes and analytical biochemistry, give insight into the
molecular mechanisms responsible for the bulk behavior.
In contrast to prior literature, we find that hyaluronan does
contribute significantly to the elastic character of the
vitreous. In particular, the swelling pressure of hyaluronan
drives the network of rope-like collagen fibrils into a state
of tension (in accord with their appearance in the presence
of hyaluronan), increasing the rigidity of the network.
2. Methods and materials
Fresh bovine and porcine eyes were acquired through Sierra for
Medical Science (Santa Fe Springs, CA, USA). All specimens were tested
between 3 and 48 h postmortem; storage as an intact globe for up to 60 h
postmortem did not affect rheological results as observed previously
(Weber et al., 1982). Eyes were gently dissected to remove the vitreous
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1842 C.S. Nickerson et al. / Journal of Biomechanics 41 (2008) 1840–1846

with minimal disruption. Intact vitreous was either used directly or a disc- reflect rapid initial changes, making the observed moduli
like section (typically 1.5–2.5 g) was cut with the axis of the disc coinciding
sensitive to the precise time from dissection to loading.
with the anterior-to-posterior axis of the eye. Some of the vitreous discs
were loaded into a cleated 25 mm parallel disc geometry for mechanical Smaller standard deviations for porcine samples were
characterization and the remainder were used for network stability studies. achieved by using a consistent loading time of approxi-
Cone-and-plate geometries are inappropriate for crosslinked gels because mately 1 min from removal of the dissected globe to
high compression near the center of the tool induces a non-uniform inception of measurement. The average steady-state moduli
normal force profile and destroys the vitreous network. The initial for bovine vitreous were G0 fin ¼ 7.072.0 Pa and
modulus values are very sensitive to the time that elapses between
dissection and rheological testing. Therefore, dissection time was kept as G00 fin ¼ 2.270.6 Pa and for porcine vitreous
uniform as possible (2–3 min) and tissue was transferred to the instrument G0 fin ¼ 2.870.9 Pa and G00 fin ¼ 0.770.4 Pa.
immediately after dissection was completed.
Mechanical measurements were made on an ARES-RFS fluids
rheometer from TA Instruments, Inc. (New Castle, DE, USA) using the
3.2. Frequency and strain dependence
‘‘cleat geometry’’ to overcome slip (Nickerson and Kornfield, 2005).
Measurement using previously reported methods for overcoming wall slip, The strain amplitude (3%) and frequency (10 rad/s) for
such as roughened plates or sandpaper, failed because the upper boundary vitreous analysis were optimized to achieve gentle defor-
of the vitreous develops a lubricating layer. Cleated tools succeed by mations close to the linear viscoelastic regime. At 10 rad/s,
penetrating the lubricating boundary layer to achieve an effective no-slip
boundary condition. All measurements were made in a closed, humid
the porcine vitreous responds linearly at or below 3%
atmosphere at 20 1C and with zero normal force on the samples. Shear strain (Fig. 3). To optimize the signal-to-noise ratio,
moduli were monitored as samples were subjected to oscillatory strain oscillatory shear experiments were conducted near the
(g ¼ 3%) at a fixed frequency (o ¼ 10 rad/s) for up to 90 min. Longer upper limit of the linear regime at g ¼ 3%. The broad
experimental times were precluded by drying of the sample edges, which plateau modulus region expected for gels (Ferry, 1980)
artificially raised the apparent modulus of the samples. The conditions for
these experiments were chosen based on the observed dynamic moduli as
appears below 5 rad/s with storage modulus rising
functions of frequency (g ¼ 3%, o ¼ 1–50 rad/s) and strain (o ¼ 10 rad/s, significantly as frequency increases (Fig. 4). To generate
g ¼ 0.5–100%). sufficiently large torques for the instrument specifications
In addition to oscillatory tests, steady shear rate tests were conducted to while operating at small strain amplitudes, a frequency of
determine the minimum shear stress and strain required to destroy the 10 rad/s was required. Thus, the moduli we report may be
tissue. These ‘‘failure’’ tests were conducted after the moduli reach steady
state in oscillatory shear (Fig. 2). A steady shear rate (_g ¼ 0:1 s1 ) was
somewhat higher than the gel’s plateau modulus. The
applied for 150 s while continuously monitoring the resulting shear stress. strain and frequency dependencies of bovine vitreous are
Higher and lower shear rates (_g ¼ 0:3 and 0:01 s1 ) were also probed similar to the porcine data shown.
separately, to demonstrate the rate-dependence of the transient shear stress.
The discovery that vitreous loses elasticity and exudes fluid after
dissection motivated us to measure the time-dependent mass loss and the 3.3. Failure tests
composition of the exuded fluid. Post-dissection weight loss was measured
in four different environments denoted A–D: (A) an intact vitreous body Shear stress rises sharply at the onset of steady shear,
was placed in a dry Petri dish and covered; (B–D) central discs of vitreous rapidly reaches a rate-dependent maximum, and then
were placed in covered Petri dishes that were either (B) dry, (C) filled with
isotonic saline or (D) filled with mechanically liquefied vitreous from other
eyes. Liquefied vitreous was obtained by removing the vitreous of three to
six fresh eyes of the same species immediately prior to the experiment and
slicing them into small fragments, which caused much of the sacrificial
vitreous tissue to liquefy. The mass of each vitreous sample was measured
immediately after removal from the eye (Mo) and only once at the end of
the treatment period (Mf)—excess handling damages the vitreous and
artificially accelerates weight loss. The treatment periods ranged from 5 to
120 min. One specimen was subjected to condition A and photographed at
1, 10, 30, and 90 min from the moment it was placed in the Petri dish.
Using condition C, the components exuded into the saline solution were
analyzed for hyaluronic acid (Hyaluronic Acid Test Kit; Corgenix, Inc.,
Denver, CO, USA) and the presence of protein (ninhydrin assay).

3. Results

3.1. Dynamic shear moduli

The dynamic moduli of vitreous sections decay mono-

tonically to a significantly lower, steady-state value that
persists thereafter (Fig. 2). The average initial storage and
loss moduli of bovine vitreous were G0 init ¼ 32712 Pa Fig. 3. Typical variable strain experiment conducted after the shear
modulus of the vitreous had reached steady state (porcine vitreous
(mean7S.D.) and G00 init ¼ 1777.0 Pa (n ¼ 17), and for 20 min after loading, o ¼ 10 rad/s). Three percent strain is just above the
porcine vitreous G0 init ¼ 1071.9 Pa and linear regime but lower strains do not generate sufficient torque for
00
G init ¼ 3.970.8 Pa (n ¼ 9). The large standard deviations accurate determination of moduli.
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C.S. Nickerson et al. / Journal of Biomechanics 41 (2008) 1840–1846
Fig. 4. Typical variable frequency experiment conducted after the shear
modulus of the vitreous had reached steady state (porcine vitreous,
g ¼ 3%). Ten rad/s is slightly above the plateau regime, but lower
frequencies do not generate sufficient signal for accurate modulus
measurements at low strain amplitude.
Fig. 5. Average failure behavior of porcine vitreous strained under steady
shear at three different shear rates (0.01, 0.1, and 0.3 s1; nX3). Y-axis is
the resulting shear stress on the vitreous, plotted on a log scale.
gradually declines (Fig. 5). Failure stress and strain
increase with shear rate. Shearing at lower rates appears
to allow the network to relax, thereby reducing the stress at
any given strain. Based on the frequency dependence of the
storage modulus in oscillatory tests, a shear rate of g_ ¼
0:1 s1 is sufficiently slow to allow the high frequency
relaxation processes evident at o45 rad/s to relax. How-
ever, slower relaxation mechanisms are apparently active
under high strains because a lower shear rate (_g ¼ 0:01 s1 )
decreases the failure stress from smax ¼ 2473 Pa
(_g ¼ 0:1 s1 ) to smax ¼ 5.770.9 Pa (_g ¼ 0:01 s1 ) and fail-
1843
ure strain from gfail ¼ 418751% (_g ¼ 0:1 s1 ) to
gfail ¼ 259735% (_g ¼ 0:01 s1 ).
3.4. Post-dissection mass loss
Upon removal of the test samples from the rheometer
two obvious changes in the tissue suggest that the steady-
state moduli we measure are lower than the moduli of the
vitreous in vivo: the post-rheology vitreous appears to sag
when lifted with forceps and a small puddle of liquid is left
behind on the instrument where aqueous material has
seeped out of the vitreous.
To explore the possible connection between the loss of
aqueous material and rheological changes, we observed
vitreous shape and mass as a function of time (Fig. 6).
Immediately following dissection, the vitreous partially
retains the native shape of the vitreous cavity. After being
placed in a sealed Petri dish for 10 min, the vitreous visibly
elongates upon lifting. This trend continues until 90 min
after dissection, when the specimen reaches its post-
dissection steady state. Morphological changes coincide
with mass loss, which was rapid immediately after removal
from the eye but stabilized within 2 h. Regardless of the
conditions under which the vitreous was placed, the mass
dropped 5–10% within the first 5 min (Fig. 7). Note the
small change in vitreous size to which this corresponds: if
the tissue is approximated as a contracting, isotropic
sphere, this results in a 2% radial contraction. Beyond
this initial contraction period, C and D stabilized but A
and B continued to exude fluid much longer. The exudate
was rich in hyaluronan, but contained only trace amounts
of protein as determined by enzyme-linked hyaluronic acid
binding protein assay and ninhydrin amino acid assay,
respectively. Fluid loss was very similar in bovine and
porcine vitreous.
4. Discussion
4.1. Rheological properties of the vitreous
Time-dependent modulus changes (Fig. 2) have not
previously been reported. The steady-state moduli are
characteristic of a post-dissection equilibrium network
structure that is not as stiff as the in vivo vitreous. Because
the tissue clearly loses fluid during the course of the
rheology experiment, we believe that the initial moduli are
closest to the in vivo moduli. The steady-state moduli we
have measured provide a lower bound that is significantly
higher than any estimates found in the literature (Bettel-
heim and Wang, 1976; Lee et al., 1994a; Tokita et al., 1984;
Zimmerman, 1980), by up to an order of magnitude
(Tokita et al., 1984).
Regarding the failure behavior, the distribution of
relaxation times and graceful failure indicate significant
heterogeneity in the network: short, thin collagen fibrils
break under small strains, while larger and longer filaments
(aggregated fibrils) survive longer, breaking only after large
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1844 C.S. Nickerson et al. / Journal of Biomechanics 41 (2008) 1840–1846
Fig. 6. This sequence of photographs illustrates the state of a whole vitreous gently removed from a fresh porcine eye, 1, 10, 30, and 90 min after
dissection. The vitreous was kept in a dry, covered Petri dish during the course of the experiment.
Fig. 7. Changes in porcine vitreous sample mass monitored over time
under the following conditions: whole vitreous placed in covered Petri dish
(A), central section of vitreous placed in covered Petri dish that is dry (B),
contains isotonic saline (C), or contains mechanically liquefied vitreous
(D) (n ¼ 4).
deformations (beyond the peak stress). This is in agreement
with the accepted view that the vitreous network contains a
distribution of fibril sizes that randomly associate along
their lengths (Bishop, 2000; Sebag, 1989). The rate-
dependence of failure stress and strain show that the
vitreous stiffens considerably against rapid deformations—
possibly a protective mechanism against head trauma. In
the context of vitreous surgery, it may be advantageous to
remove the vitreous slowly due to the observed failure
characteristics: during vitrectomy, the vitreous may con-
tinue to transfer a substantial fraction of its maximum
stress to the retina long after yielding (Fig. 5).
4.2. Mass loss
The qualitative and quantitative differences observed
under conditions A–D provide insight into the mechanisms
that expel fluid from the vitreous (Fig. 7). Two obvious
driving forces in conditions A and B are gravity and
diffusion, with gravity playing a smaller role in B due to its
flatter shape.
Conditions C and D are significantly different from
A and B because gravity and concentration gradient-driven
diffusion have been eliminated. In C, the saline bath
allows the vitreous to float at neutral buoyancy in a
solution that is isotonic with the primary soluble compo-
nent of the vitreous—NaCl. In D, the vitreous is
completely surrounded by other vitreous material—thereby
completely eliminating the concentration gradient for all
components, including hyaluronic acid. Despite these
changes there is still a mass loss on the order of 7% in
the first 5–10 min. The unexpected observation that mass
loss proceeds in the absence of hydrostatic pressure
gradients or composition gradients suggests that hyalur-
onan is not simply diffusing out of the vitreous but, rather,
that it is driven out.
It is well accepted that hyaluronan draws water into the
fibril network to achieve Donnan equilibrium, adds
chemical stability to the collagen, and separates the fibrils
(Bishop, 2000; Bos et al., 2001; Sebag and Balazs, 1989).
Polyelectrolytes, such as HA, are swollen by the hydration
spheres of their associated counter ions (Flory, 1953). The
counter ions also greatly increase the entropic cost of
molecular overlap because a single overlap event between
two polymers results in the doubling of the local
concentration of many counter ions. Based upon its
approximate concentration (which is not completely
homogeneous) (Ansari et al., 2001; Bishop, 2000), mole-
cular weight (Bishop, 2000), and radius of gyration
(Mendichi et al., 2003) in the vitreous, HA is at or beyond
its overlap concentration under physiological conditions.
Thus, HA is well suited to drawing water into the vitreous
and separating fibrils, as proposed by previous authors
(Bishop, 2000; Bos et al., 2001; Sebag and Balazs, 1989).
We suggest that the swelling pressure and resistance to
overlap also play a direct role in stiffening the vitreous in
vivo and driving fluid out after dissection.
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C.S. Nickerson et al. / Journal of Biomechanics 41 (2008) 1840–1846
4.3. Network tension hypothesis
We hypothesize that hyaluronan increases the moduli of
the vitreous by placing the collagen network under internal
tension as it swells to its Donnan equilibrium hydration
state (Fig. 8A vs. B) (Nickerson et al., 2005). Tension on
individual collagen fibrils would reduce their ability to
deform and, thereby, increase the modulus.
We offer two plausible mechanisms by which hyaluronan
could induce network tension. Flexible and semiflexible
macromolecules can be visualized as entropic springs that
exert an entropically driven elastic force when their end-to-
end distance is increased beyond its equilibrium value
(Ferry, 1980). Thus, vitreous collagen fibrils are loosely
analogous to a stretched web of ropes; the semiflexible
fibers resist stretching to their fully extended length due to
the loss of entropy as they are restricted from undulating
(bending). It is known that hyaluronan is essentially
trapped in the vitreous, and so can be regarded as being
enveloped in a semipermeable membrane. The hydrostatic
pressure (due to hyaluronan-induced Donnan swelling),
exerts an outward force on the semipermeable periphery of
the vitreous network, which may stretch it until the collagen
fibrils are pulled taut. A second possible tension mechanism
arises if the hyaluronan is bound to the network strands, as
suggested by Scott (2001). The affinity of hyaluronan for
water would drive the attachment points to separate. The
fibril would extend until the chemical potential of swelling
was balanced by the conformational entropic loss. Just as in
the first case, swelling would induce internal tension that
would be distributed throughout the network. These two
mechanisms are not mutually exclusive, so both mechan-
isms may contribute. If the collagen fibrils of the vitreous in
vivo are viewed as extended ropes, then upon removal from
the eye the entropically favored reduction in fibril end-to-
end length could provide a thermodynamic driving force for
loss of hyaluronan-rich fluid.
Both scenarios are similar and consistent with all of our
observations. In both cases, tension would be distributed
Fig. 8. Schematic depiction of the collagen fibrils (heavy lines) of the vitreous network under tension and after relaxation. (A) Native state of the vitreous
in the eye and (B) after relaxation by release of hyaluronan (thin coils) after removal from the eye.
1845
throughout the network. The validity of this ‘‘homoge-
neous properties’’ prediction can be verified by comparing
the weight-loss behavior of conditions A and B: early
weight loss driven by the network is nearly identical even
though different portions of the vitreous are being
examined. In both mechanisms, fluid loss should also
soften the vitreous (Figs. 2 and 5). Finally, both mechan-
isms are in accord with Bos’s microscopic observation that
the collagen network appears to ‘‘relax’’ after removal of
hyaluronan (Bos et al., 2001).
While this is the first evidence that network tension
stiffens the vitreous, the concept of hydrostatic structures
(‘‘hydrostats’’) is well known in the biomechanics literature
(Vogel, 2003). One notable example of hydrostatic pressure
contributing to mechanical strength is cartilage (Hasler
et al., 1999; Vogel, 2003). Like the vitreous, cartilage consists
of a hydrated network of collagen II containing fibrils filled
with highly charged HA. Cartilage has a much greater
modulus than the vitreous due to its higher protein content,
but the underlying mechanism appears to be the same—
Donnan swelling due to the high fixed charge density stiffens
the tissue by extending the collagen fibrils of the network.
Our internal tension model suggests that the initial value
of the modulus, measured within minutes of dissection may
resemble the mechanical properties of tissue in vivo, where
the collagen fibrils are stretched. While it appears that our
steady-state moduli are systematically lower than in vivo,
they are also useful for several reasons. First, while they
represent minimum values for the native moduli, they are
higher than any previous estimates found in the literature
(Bettelheim and Wang, 1976; Lee et al., 1994a; Tokita
et al., 1984; Zimmerman, 1980). Second, they provide
useful target values for those striving to create synthetic
vitreous replacements (Chirila et al., 1998; Dalton et al.,
1995; Soman and Banerjee, 2003). Third, the steady-state
network properties are quantitative and reproducible,
suitable for quantifying the effects of pharmacological
vitrectomy agents and examining natural and pathologic
vitreous liquefaction.
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1846 C.S. Nickerson et al. / Journal of Biomechanics 41 (2008) 1840–1846
Conflict of interest
Two of us (J.P. and H.K.) are employees and stock-
holders of Vitreoretinal Technologies.
Acknowledgments
The authors wish to thank Prof. Zhen-Gang Wang and
Jennifer Witman for discussions regarding network ten-
sion. Funding was provided by Vitreoretinal Technologies,
Inc. C.S.N. is an ARCS Foundation scholar.
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