Structural Factors That Mediate Scleral Stiffness
David S. Schultz,1,2Jeffrey C. Lotz,2Shira M. Lee,1Monique L. Trinidad,3and
Jay M. Stewart3
PURPOSE. The intent of this study was to correlate measures of
structurally relevant biochemical constituents with tensile me-
chanical behavior in porcine and human posterior sclera.
METHODS. Posterior scleral strips 6 ? 25 mm were harvested
from 13 young porcine and 10 aged human eyes and stored
frozen at ?20°C. Mechanical hysteresis from 10 consecutive
load cycles to a peak stress of 1.0 MPa was recorded via a
custom-built soft tissue tester. In a parallel study, tissue adja-
cent to the mechanical test specimens was apportioned for
each of five assays measuring: total collagen content, nonen-
zymatic cross-link density, elastin content, glycosaminoglycan
content, and water content.
RESULTS. The average porcine scleral modulus at 1% strain was
75% less than that measured for human tissue (0.65 ? 0.53 MPa
versus 2.60 ? 2.13 MPa, respectively; P ? 0.05). However, the
average strain energy absorbed per loading cycle was similar
(6.09 ? 2.54 kJ/m3vs. 5.96 ? 2.69 kJ/m3for porcine and
human sclera respectively; P ? 0.05). Aged human sclera had
relatively high fluorescence due to nonenzymatic cross-link
density (2200 ? 368 vs. 842 ? 342; P ? 0.05) and low
hydroxyproline content (0.79 ? 0.17 ?L/mL/g versus 1.21 ?
0.09 ?L/mL/g; P ? 0.05) while other measured biochemical
factors were statistically similar (P ? 0.05).
CONCLUSIONS. Aged human tissue had superior mechanical stiff-
ness despite reduced collagen content, partially because of the
accumulation of nonenzymatic cross-links. Differences in col-
lagen content and cross-link density either had no effect or
offsetting effects on the ability of the tissues to absorb strain
energy. (Invest Ophthalmol Vis Sci. 2008;49:4232–4236) DOI:
spur the growth of defects in the choroid and retina. Since eye
shape is defined by scleral mechanical properties, their
changes have been linked to the pathogeny of several disease
states, the most prevalent of which is progressive myopia.
During myopization, a retinal–scleral photomechanotransduc-
pproximately one third of the world’s population has a
suboptimal eye shape that impairs visual acuity and can
tion pathway mediates matrix remodeling that, in turn, leads to
reduced stiffness associated with excessive creep deforma-
tion.1–4Although the precise mechanism of this process re-
mains unknown, biochemical alterations of the sclera and sub-
sequent creep behavior underlie progressive myopia and may
result in severe dysfunction.5
The determinants of scleral mechanical properties are the
content and architecture of structural proteins. Glycosamino-
glycans (GAGs) and collagen type I are proteins known to be
most influential in determining how the sclera responds to
load.6,7Increased levels of GAGs have been observed during
scleral remodeling and may be a precursor to a decreased
modulus,8whereas under normative conditions collagen con-
tent and architecture have been implicated as primary deter-
minants of stiffness.9Less common structural proteins such as
elastin may also influence viscoelastic behavior, altering mod-
ulus and the energy absorbed on a load cycle. In addition,
cross-links, or covalent bonds between structural proteins, can
bolster stiffness. Recent studies into exogenous cross-linking
strategies have shown that cross-links can increase scleral stiff-
ness.10Despite this progress, we have yet to understand how
naturally occurring nonenzymatic cross-links that accumulate
with age may affect scleral stiffness. The objective of this study
was to test the hypothesis that collagen content and age-related
nonenzymatic cross-links are predominant biochemical factors
that affect posterior scleral stiffness. In addition, data from
human sclera were compared with data from porcine sclera, to
gain a better understanding of how human data may relate to
more prevalent animal models.
Thirteen porcine eyes (Sierra for Medical Science; Whittier, CA) and 10
human eyes (San Diego and Alabama Eye Banks) were harvested from
fresh cadaveric tissue within 24 hours of death and immediately stored
in a humidified chamber at 4°C for shipping. The eyes were dissected
after storage at 4°C for an average of 4 days for porcine eyes and 30
days for human eyes. The human tissue ranged in age at death from 66
to 89 years with a mean age of 83. The human tissue was assumed to
be representative of the aforementioned age group, and lacking spe-
cific ophthalmic disease, as a review of all available medical informa-
tion that the eye bank provided about each donor revealed no oph-
thalmic diseases or relevant systemic conditions. Human tissue was
used in accordance with the Declaration of Helsinki regarding medical
research. The porcine tissue ranged in age from 2 to 3 years. In all eyes,
a circumferential incision around the cornea allowed for removal of
the intraocular contents. A tissue pen was then used to ink the tips of
medical calipers used to place marks spaced 6 mm apart along the
length of a posterior strip containing the posterior pole. Once the
strips were marked, a retinal surgeon equipped with a surgical micro-
scope and scissors cut along the guidelines. The width dimension
along of each scleral strip was verified after excision by using the
calipers. From each eye a posterior scleral strip measuring 6 ? 25 mm
was removed (Fig. 1). Adjacent globe tissue was apportioned and
From the1Department of Mechanical Engineering, University of
California, Berkeley, Berkeley, California; and the Departments of2Or-
thopaedic Surgery and3Ophthalmology, University of California, San
Francisco, San Francisco, California.
Supported by That Man May See, Inc. and Research to Prevent
Submitted for publication March 3, 2008; revised May 6, 2008;
accepted August 11, 2008.
Disclosure: D.S. Schultz, None; J.C. Lotz, None; S.M. Lee, None;
M.L. Trinidad, None; J.M. Stewart, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Jay M. Stewart, University of California, San
Francisco, Department of Ophthalmology, 10 Koret Way, K301, San
Francisco, CA 94143-0730; email@example.com.
Investigative Ophthalmology & Visual Science, October 2008, Vol. 49, No. 10
Copyright © Association for Research in Vision and Ophthalmology
weighed for each of five assays to be performed later, measuring water
content, elastin content, glycosaminoglycan content, hydroxyproline
content, and cross-link density. Strips for mechanical testing were
stored at ?20°C, along with adjacent globe tissue for biochemical
A quasistatic (d?/dt ? 0.001/s) uniaxial tensile test was used to mea-
sure mechanical response to loading. Before the test, tissue stored at
?20°C was removed from the freezer, kept sealed in a plastic bag and
allowed 1 hour to defrost. Evidence suggests that a moderate storage
time of 3 days does not significantly affect results from stress relaxation
experiments conducted on rabbit sclera.11To further bolster our
confidence that a moderate frozen storage time in a sealed bag would
not significantly affect results, we conducted a pilot study in which
three porcine sclera specimens and one human specimen were me-
chanically tested within 2 days of death and then tested again after a
week of frozen storage (at ?20°C). We found that differences in
mechanical results for each tissue specimen were statistically nonsig-
nificant. From this evidence we made the assumption that our storage
regimen that allowed up to 1 week of frozen storage would have a
minimal effect on relative mechanical properties.
Average specimen thickness in the central portion of the specimen
to be mechanically tested was determined by taking the mean of five
readings in different locations near the center of the specimen with an
ultrasonic pachymeter (Carl Zeiss Meditec, Inc., Dublin, CA). This
number was then used to approximate the specimen’s cross-sectional
area. A 3 ? 3 mm grid of nine uniformly spaced strain targets was made
to adhere to the tissue by blotting it dry and using a minimal amount
of cyanoacrylate. We determined average tissue strain in the direction
of loading by the method described by Bass et al.12Specimens kept
moist at room temperature (23°C ? 1°C) with physiologic saline were
cyclically loaded to a peak engineering stress of 1.0 MPa for 10 cycles.
Although this peak stress is well above physiologic stress levels, pilot
testing showed that results at this stress level were repeatable and
therefore assumed to be nondamaging. Choosing a relatively high level
of peak stress allowed observation of an expanded range of the stress–
The custom soft tissue tester and experimental protocol were
adapted from previous tensile experiments conducted on the annulus
fibrosus.13Briefly, the custom testing apparatus consisted of the fol-
lowing: acrylic and aluminum grips designed to hold strips of sclera
without significant out-of-plane compression, a precision force trans-
ducer (25 N Load Cell Model LCCA; Omega Engineering, Stamford, CT)
to record forces, and a computer-controlled imaging system to calcu-
late the strains in real time. Algorithms were adapted from software
(Labview and IMAQ Vision; National Instruments, Austin, TX) that
captured images of the specimen and strain targets; generated a con-
trast-thresholded image and located the targets within the view frame;
calculated the average strain in the direction of the applied force; and
calculated engineering stress as the load reading normalized by the
estimated cross-sectional area (average thickness from pachymeter
readings ? initial width before test).
The potentially confounding effect of residual bending stresses due
to differences in thickness and curvature of each eye was mitigated by
establishing a reference state at the beginning of each test and by
placing strain targets only on the external outermost surface (as op-
posed to inserting strain marking pins through the full thickness of the
sclera). The reference state was established for each specimen by
loading the tissue in the grips under slight compression initially and
then adjusting the grip location until a slight tensile load of 0.01 MPa
was measured. Once this reference state was established, the initial
distances between strain targets were recorded, and the precondition-
ing routine was allowed to proceed.
The first nine load cycles preconditioned the tissue such that a stable
response to loading could be achieved. Samples were considered
properly preconditioned if the peak strain of the final preconditioning
cycle was within 2% of the previous cycle. According to the method
described by Fung,14a continuous exponential curve of the form
?(?) ? A/B(eB?? 1) was fit to the discrete stress (?) versus strain (?)
data (Fig. 2).14For each sample, distinct loading, and unloading coef-
ficients were optimized using a Levenberg-Marquardt algorithm such
that a minimum of 90% of the variation in the data could be explained
by the curve fit. In addition, a simplistic parameterization of modulus
(E) was reported as the derivative of the fitted loading curve at 1%
strain, the upper end of what was identified by Downs et al.15to be a
physiologic range of strains. Finally, the strain energy absorbed on a
load cycle was reported. The area contained within the hysteresis loop
delimited by the loading and unloading curve fits gives a measure of
strain energy absorbed (W) on a loading cycle in SI units of Joules per
posterior sclera tensile sample rela-
tive to the optic nerve (left) and a
photograph of a test sample with
strain targets attached under tensile
Anatomic location of the
curve fit coefficients from each loading and unloading half-cycle (Sam-
ples P63 and H16 shown). The curve fits are of the form ?(?) ?
Representative hysteresis data presented with exponential
IOVS, October 2008, Vol. 49, No. 10
Structural Factors That Mediate Scleral Stiffness4233
cubic meter. Practically, this parameter was determined by calculating
the difference between integrals of the loading curve fit, ?l(?), and the
unloading curve fit, ?u(?), up to the peak strain, ?max
Five biochemical assays were run to determine water content, elastin
content, sulfonated glycosaminoglycan content, hydroxyproline con-
tent, and nonenzymatic cross-link density.
Water Content. Scleral samples adjacent to the mechanical test
specimen were removed for assays. Each biochemical sample was
weighed after extraction to establish a wet weight. The sample was
then minced to increase exposed surface area. The set of samples from
each tissue was deposited into a test tube. The tubes were laid hori-
zontally and uncapped in a 60°C oven. After 48 hours, the tissue
samples were weighed, establishing a dry weight. Before-heating and
after-heating weight measurements were used to determine the wet-
Elastin Content. An elastin assay and procedure (Fastin; Bio-
color Ltd., Newtownabbey, Northern Ireland, UK) were used to mea-
sure elastin content via a dye-binding method. Insoluble elastin was
extracted from sclera in a soluble elastin form using a 0.25-M oxalic
acid digest. Bound dye was quantified using absorbance readings from
a plate reader (Molecular Devices Corp., Sunnyvale, CA). The mea-
sured amount of elastin in tissue samples was determined as a percent-
age of tissue dry weight according to the known tissue wet weight and
Glycosaminoglycan Content. Glycosaminoglycan content,
hydroxyproline content, and cross-link density were assessed for each
tissue sample using a papain digest and distinct assay protocols. Tissue
samples were cut from the posterior globe to obtain a wet weight of 10
to 25 mg. The wet weight was recorded, and each tissue was minced
and placed in a microcentrifuge tube for papain digestion. To each
tissue sample was added 1 mL of a 10-mM solution of DTT in a
digestion buffer (0.1 M Na acetate/2.4 mM disodium EDTA). To each
tissue sample was then added 20 ?L of a 1-mg papain/1-mL digestion
buffer solution. The tissue was incubated at 60°C for 12 hours, and
then 20 ?L of the papain solution was added again. After 12 more
hours of incubation, the tubes were centrifuged at 10,000 rpm for 12
minutes. The supernatant was removed and used to perform glycos-
aminoglycan, hydroxyproline, and cross-link assays.
Similar to the elastin experiment, sulfonated glycosaminoglycan
(s-GAG) content was measured with a dye-binding method (Blyscan
assay; Biocolor Ltd.). Sulfonated glycosaminoglycan content of each
tissue sample was determined as the percentage of tissue dry weight
according to the known tissue wet weight and water content.
Hydroxyproline Content. After the papain digest, the total
hydroxyproline (HPr) content of each tissue sample was determined
with the Woessner assay.16HPr content was normalized by sample dry
Nonenzymatic Cross-link Density. Fluorescence of the pa-
pain digest supernatant is an indicator of nonenzymatic cross-link
content.17–19Nonenzymatic, glycation-type cross-links form as the re-
sult of the attachment of the carbonyl group of glucose, followed by a
ketoamine rearrangement. The cross-links formed due to the Maillard-
type reactions that follow then yield fluorophores characteristic of a
reaction of a sugar with a protein. To measure fluorescence, a 100-?L
aliquot of each papain supernatant was removed to a black ELISA plate,
and fluorescence was measured in the aforementioned plate reader at
an excitation wavelength of 370 nm with emission sensitivity set at 440
The porcine and human samples were not different in water
content, s-GAGs, and elastin (Table 1; P ? 0.13). The mean
hydroxyproline content in aged human tissue was 53% less
than that of porcine tissue (0.79 ? 0.17 vs. 1.21 ? 0.09; Table
1; P ? 0.05). Mean fluorescence due to nonenzymatic cross-
links for human sclera was 1.6 times that of porcine sclera
(2200 ? 368 vs. 842 ? 342; Table 1; P ? 0.05).
Human sclera had a modulus (E) roughly three times that of
porcine tissue (2.60 ? 2.13 vs. 0.65 ? 0.53; Table 2; ? ? 1.0%,
P ? 0.03). Despite this difference in the stress-versus-strain
profiles, the average strain energy absorbed on a load cycle (W)
was very similar for each group; 5.96 ? 2.69 kJ/m3for human
sclera and 6.09 ? 2.54 kJ/m3for porcine sclera. Human tissue
tended to have a higher modulus while retaining its ability to
absorb strain energy (Fig. 3).
We hypothesized that collagen architecture and associated
cross-links define sclera biomechanical behavior. Our data in-
dicate that the principal determinant of scleral stiffness is
collagen cross-linking: strikingly, while aged human sclera con-
tained significantly less collagen than the porcine sclera, it
achieved superior stiffness via increased collagen cross-link
density. It has been established that, in mammals, nonenzy-
TABLE 1. Biochemical Results Summary
s-GAGElastin HPr* NE x-Links*
Porcine (n ? 13)
Human (n ? 10)
H2O given as (wet wt./dry wt.); s-GAG & HPr in ?L/mL digest/dry wt.; Elastin in % dry wt.; N.E. x-links
* Statistically significant difference between groups (P ? 0.05).
TABLE 2. Mechanical Results Summary
Porcine (n ? 13)
Human (n ? 10)
E given in MPa; W in kJ/m3; A and B are unitless mean coefficients from ? ?
B(eB?? 1) curve fit.
* Statistically significant difference between groups (P ? 0.05).
4234Schultz et al.
IOVS, October 2008, Vol. 49, No. 10
matic cross-links accumulate with age and cause stiffening, and
this may be the predominant factor mediating scleral stiffness
in aged humans.20The human sclera tended to have a higher
modulus than the porcine sclera while having a comparable
ability to absorb strain energy at a low strain rate. This suggests
that sclera can have a relatively high cross-link density and
retain its ability to absorb energy.
This study bolsters a shortage of published information on
human scleral mechanics. Curtin reported on the importance
of a wide-angle collagen weave and swelling as factors that
rendered the human posterior sclera more extensible than
anterior sclera.21The histologic analysis of microstructure in
this study, although interesting, provided no quantifiable bio-
chemical data. Avetisov et al.6advanced the field of sclera
research by reporting fundamental trends in soluble collagen
content and tensile strength with age.6They noted a strongly
pronounced inverse correlation between the soluble collagen
fraction and the modulus of elasticity. Our data support trends
identified by Avetisov et al. and provides a more rigorous
quantification of how biochemical constituents may influence
In addition to expanding data available from human sclera,
our study showed how human data may compare with data
from an animal model. Many animal models have been used in
recent scleral research, including chick, monkey, rabbit, pig,
and tree shrew.9,10,15,22Clinically, these models are valuable
because of the assumption that similar constituents and similar
biological activity in an animal model may have results that are
translatable to humans. To date, this assumption has not been
adequately evaluated, as there are no previous parallel studies
that offer data from both animal and human sclera. Our study
indicates that juvenile animal models likely have lower nonen-
zymatic cross-link density that ought to be taken into account
for appropriate comparisons with elderly human eyes. The
human sclera data presented herein may help contextualize
data from other animal models.
Future research calls for a more rigorous biochemical char-
acterization of both human and animal sclera. In this study, we
assumed that potential postmortem enzymatic activity and the
aforementioned storage regimen would not have a significant
effect on the relative outcome of assays, although we saw no
trends in our data indicating that differences in storage times
had a pronounced effect on results. Nevertheless, our human
tissue biochemical and mechanical data are limited by the fact
that the human eyes were stored at 4°C for an average of 30
days before dissection. Further biochemistry determining the
effect of postmortem tissue processing on structurally relevant
biochemical constituents should be performed to strengthen
our initial assumption.
Further work is also needed to quantify all types of cross-
links present in various models. In this study, we quantified
relative levels of nonenzymatic cross-links via fluorescence
reading. This accounts only for the cross-links due to collagen’s
inherent glycosylated lysine and hydroxylysine residues. As in
most connective tissues, collagen in the mature sclera also
undergoes cross-linking due to nonreducible hydroxypyri-
dinium residues. Our conclusions rely on the assumption that
both enzymatic and nonenzymatic cross-links accumulate with
age in a similar fashion and that neither plays a disproportion-
ate role in the mechanics. This assumption should be verified,
such that all the covalent bonds that serve to stabilize the
collagen network can be evaluated. Further study focusing on
the measurement of cross-links should be conducted with a
pentosidine assay or high-pressure liquid chromatography
techniques to quantify exhaustively the cross-links present in
A question prompted by the data in this study is whether
age-related cross-links accumulate in a manner that might serve
to stabilize the myopic eye. Given that cross-linking seems to
be a principal factor that determines scleral modulus, exoge-
nous cross-linking strategies may be promising treatments for
stabilizing the sclera. Indeed, several studies on this topic have
been published, and others are under way.10,24Conversely, it
has been suggested in a vascular model of age-related macular
degeneration that the stiffening of the sclera with age may spur
macular degeneration.25These studies highlight the growing
need to understand how structural constituents, particularly
collagen cross-links, influence mechanical properties.
In summary, accumulated collagen cross-links probably ac-
count for a significant increase in modulus in human tissue and
suggest an effective means of modulating sclera properties.
Animal model research viewed in the context of this and other
human data may yield a more valuable clinical surrogate. Fi-
nally, further biomechanics research accounting for both en-
zymatic and nonenzymatic cross-links is needed.
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