Lens
Elastic Properties of Human Lens Zonules as a Function of
Age in Presbyopes
Ralph Michael, Marek Mikielewicz, Carlos Gordillo, Gustavo A. Montenegro, Laura Pinilla
Cortés, and Rafael I. Barraquer
PURPOSE. To investigate the elastic properties of human lens
zonules as a function of age in presbyopes.
METHODS. We studied 16 presbyopic human donor eyes (ages
47–97). Anterior eye sections with crystalline lens, zonules,
ciliary body, and sclera were stretched radially. The stretching
device consisted of a chamber filled with balanced salt solution
and eight radial hooks to hold the anterior eye section. Radial
stretching was created with a stepper motor connected to a
digital outside micrometer for linear displacement and digital
balance for force measurement. Three eye globes were used to
test our methodology. For 13 eye globes, the spring constant,
elastic modulus of the zonular system, and Young’s modulus of
the zonules were calculated.
RESULTS. We found linear dependence for force-elongation and
force-strain relationships at all ages. In young presbyopic eyes
(ages 47–60), the Young’s modulus of the zonules was 340 mN/
mm2, whereas in older eyes (ages 83–97) it was significantly
lower at 270 mN/mm2. However, the correlation coefficient
between Young’s modulus and age (47–97 years) was not
significant with P ¼ 0.063.
CONCLUSIONS. The zonular system in presbyopic eyes was linear
elastic, and the Young’s modulus of the zonules decreased 20%
from presbyopic age to late presbyopic age. However, there
was no significant correlation between Young’s modulus and
age in presbyopes. (Invest Ophthalmol Vis Sci. 2012;53:6109–
6114) DOI:10.1167/iovs.11-8702
he human accommodative system consists of four struc-
T tures: crystalline lens, zonules of Zinn, ciliary muscle, and
choroid. To understand how this system functions, it is
necessary to know the mechanical properties of each part.
Various mechanical characteristics must be estimated. The
primary characteristic is force-elongation, with its slope
representing the spring constant (mN/mm). The force-strain
characteristic, which is the force as a function of relative
elongation, has a slope that describes an elastic modulus of the
tissue as a whole (mN). We call this the elastic modulus of the
zonular system. The force per cross-sectional area as a function
of relative elongation is the stress-strain characteristic, whose
slope is the elastic modulus of the material, the Young’s
modulus (mN/mm2).
From the Institut Universitari Barraquer, Universitat Autònoma
de Barcelona, Barcelona, Spain.
Submitted for publication September 30, 2011; revised April 12
and June 28, 2012; accepted July 23, 2012.
Disclosure: R. Michael, None; M. Mikielewicz, None; C.
Gordillo, None; G.A. Montenegro, None; L. Pinilla Cortés, None;
R.I. Barraquer, None
Corresponding author: Ralph Michael, Institut Universitari Barra-
quer, Laforja 88 - E-08021 Barcelona, Spain; ralphm@barraquer.com.
Investigative Ophthalmology & Visual Science, September 2012, Vol. 53, No. 10
Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc.
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Two earlier studies estimated the Young’s modulus of
zonular fibers. Fisher estimated indirectly the force applied to
the lens and zonules by comparing lens thickness changes
during stretching of anterior eye sections to the thickness
changes during lens spinning experiments.1,2 He determined
Young’s modulus for the zonules to be 350 mN/mm2, which
was constant for eyes from donors between ages 15 and 45.3
Van Alphen and Graebel used a two-arm stretching device with
one force sensor, and determined Young’s modulus for the
zonules to be 1000 mN/mm2 for 20-year-old donor eyes and
1500 mN/mm2 for 60-year-old donor eyes.4
The objective of our study was to measure the mechanical
properties of the zonules using our own stretching device and
to determine possible changes with age in presbyopes. Such
changes with age would impact several strategies to restore
accommodation in presbyopes.
METHODS
We studied 16 presbyopic human donor eyes (ages 47–97). The donor
eyes were provided by the Banco de Ojos para Tratamientos de la
Ceguera (Barcelona, Spain) and had a mean postmortem time of 2.5
days (range 1–6 days). The donor eyes were kept constantly at 88C.
Typical cause of death was multiorgan or cardiorespiratory failure. We
excluded donors with cancer, HIV positivity, and hepatitis, as well as
those with anterior eye segment abnormalities found during tissue
preparation or with previous ocular surgery. Research using this
material was in conformance with the Declaration of Helsinki, and
complied with Spanish regulations on the use of human postmortem
and donor organs.
The stretching device (Fig. 1A) consisted of a chamber filled with
balanced salt solution (at 258C) and eight radial hooks to hold the
anterior eye section (Fig. 1B). The hooks were connected to a force
sensor below the chamber by means of 6-0 Prolene, blue monofilament
suture (Figs. 1C, 1D). We used the force sensor from a commercial
digital precision balance (Precisa BJ 210C; Precisa Gravimetrics AG,
Dietikon, Switzerland) that allows force readings at 0.1 mN precision. It
was calibrated with eight microextension springs (spring constant 10.0
mN/mm each). The sensor was mounted on top of a digital outside
micrometer. The outside micrometer could move the force sensor up
and down by means of a high-torque stepper motor. The up and down
movement of the force sensor with the connected eight sutures
produced the stretching and relaxing of the anterior eye section in the
experimental chamber while measuring the applied force (Fig. 1C).
One step movement by the motor produced a radial displacement of
approximately 1.25 lm in the experimental chamber. Stepper motor,
force sensor, and digital micrometer were controlled by Labview
software (Labview 6; National Instruments, Austin, Texas).
The human donor eye globes were prepared as follows: We used
the inked border of a test tube to mark a concentric circle on the sclera
around the cornea, approximately 2 mm from the limbus. We then
marked eight radial lines on the center of the cornea with an inked
keratectomy marker (Fig. 2A). Subsequently, we used 7-0 Prolene
6109
 6110 Michael et al. IOVS, September 2012, Vol. 53, No. 10

FIGURE 1. Top view of stretching device for anterior eye sections (A), experimental chamber with eight hooks (B), schematic of main components
(C), and photograph of main components (D).

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 IOVS, September 2012, Vol. 53, No. 10
FIGURE 2. Main surgical steps of the preparation of the anterior eye
section, with the glass beads sutured to the sclera (A), trephination of
the cornea (B), separation of the specimen from the rest of the eye
globe using Westcott scissors (C), and anterior eye section fitted into
the stretching device submerged in balanced salt solution (D).
suture to attach eight glass beads to the sclera around the marked
circumference aligned with the eight radial marks. The cornea then
was trephined with a 10 mm trephine (Fig. 2B), and complete removal
was performed with scissors. The iris was removed entirely with
scissors, and an anterior eye section was separated from the eye globe
by cutting the sclera 3608 with scissors just below the beads (Fig. 2C).
This anterior eye section was placed inside the experimental chamber,
which was filled with balanced salt solution, and the glass beads were
connected to the metal hooks of the stretching device (Fig. 2D). Once
the anterior eye section was in position, the sclera and ciliary body
were divided into eight separate segments by making eight radial
incisions between the beads.
Before performing the measurement, the resting state of the
anterior eye section had to be determined by stretching and relaxing
the hooks until no force variation was detected by the force sensor.
Each measurement was obtained from three cycles consisting of 10
stretching pulses followed by 10 relaxing pulses. Each pulse included
a move of 200 stepper motor steps. This gave a total overall stretch
distance per cycle of 2500 lm. The time between each stretching
pulse and the force measurement was 10 seconds; the time between
the force measurement and the next stretching pulse was 5 seconds.
Three stretching cycles were necessary because the first cycle was
needed to ‘‘condition’’ the eye section and release some internal
friction. The third cycle was useful to check the repeatability of the
measurement. The data from the second cycle were used for
assessment.
We placed a webcam with Carl Zeiss Tesar optics (Carl Zeiss, Jena,
Germany) and a native 2-megapixel sensor above the experimental
chamber to monitor the coronal diameter, and radial changes of the
crystalline lens and the ciliary body during stretching (Fig. 3). Radial
extension of the anterior eye section stretches the crystalline lens,
zonules, and ciliary body. Since the ciliary body and the sclera were
divided into 8 separate segments, each of the segments could be
represented as three springs in series (Fig. 3, upper part). For such a
system in series, the force acting on all components is the same,
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Elasticity of Human Lens Zonules in Presbyopia 6111
FIGURE 3. Measured distances, radius of the ciliary body (RCB) and
radius of the crystalline lens (RL) next to a schematic of crystalline lens,
zonules, and ciliary body as springs in series in our main stretching
experiments (upper part). The effect of keeping the ciliary body intact
is drawn in the lower part of the figure.
making it possible to estimate the mechanical properties of each
component independently. For the zonules, this results in the following
equation:
F ¼ kz 3 D ðRCB  RL Þ
where F is the applied force, kZ the spring constant of the zonular
apparatus, RL the radius of the crystalline lens, and RCB the ciliary body
radius; D stands for the difference in radius before and after stretching.
The radii RL and RCB were measured in our coronal diameter images
taken by the webcam and the mean of the eight measurements from
each hook were used in our calculations. Since the total length of the
zonules (RZ) cannot be seen in our coronal photographs, we estimated
it as the sum of the space between the lens equator and the ciliary
body, the circumlental space (RCB – RL), and the distance from the
insertion of the zonules into the lens equator, the zonular insertion
distance (RZID). The zonular insertion distance RZID was calculated
according to the literature.5,6
RZ ¼ ðRCB  RL Þ þ RZID
RZID ðmmÞ ¼ 0:0311 þ 0:0124 3 age ðyearsÞ
If the ciliary body is not cut radially, this would result in an additional
circumferential tension (lower part, Fig. 3). In this case we would
expect an increased force during the stretching of the anterior eye
segment compared to the situation when the ciliary body is cut radially
(upper part, Fig. 3). To test this hypothesis, we performed an
experiment with 3 donor eyes (75, 81, and 81 years; postmortem
time 2–3 days). The specimen was prepared as described above, but
the ciliary body was kept intact and a first measurement set with three
stretching cycles was done. Then, we cut the ciliary body radially
 6112 Michael et al. IOVS, September 2012, Vol. 53, No. 10

FIGURE 4. Force as a function of radial zonular elongation. The slope represents the spring constant of the zonular system.

inside the experimental chamber and kept all other settings
unchanged, and performed a second measurement set.
The spring constant and the elastic modulus of the zonular system
were calculated from the force-elongation and force-strain data (Figs. 4,
5). To calculate the Young’s modulus of the zonules, we needed the
number and diameter of the zonular fibers. These data were obtained
from Weeber and van der Heijde,7 who based their calculations on the
observations by Kaczurowski8: 100 anterior fibers with 50 lm diameter
and 50 equatorial and 135 posterior fibers, both with 40 lm diameters.
This resulted in a total cross-sectional area for all zonular fibers of 0.427
mm2.
Student’s t-test was applied to compare the following groups: (1)
the two conditions of the ciliary body in three lenses of the pilot
experiment (Table 1), (2) the five youngest eyes (ages 47–60) with the
five oldest eyes (ages 83–97) from the main experiment (Table 2); we
refer to these groups as ‘‘young presbyopic eyes’’ and ‘‘older eyes’’ in
this text, and (3) we compared eyes with short postmortem times (1–
2.5 days, n ¼ 9) to eyes with longer postmortem times (3.5–6 days, n ¼
4) from the main experiment (Table 3). The Pearson correlation
coefficient was calculated for the relationship between Young’s
modulus and age. The significance level for Student’s t-test and Pearson
correlation was set to 0.05.
RESULTS
Our test with respect to the radial cutting of the ciliary body
revealed that the mean force at 10% strain of the zonules was
25 mN with the ciliary body kept intact, whereas with ciliary
body cut radially the force was 10 mN (Table 1). Also all other
parameters calculated were significantly different for both
conditions. For the main experiment we always cut the ciliary
body radially.
Before each measurement, the resting state of the anterior
eye section was set by stretching and relaxing the system until
no force variation was detected by the force sensor. This
resulted in a mean circumlental space of 1046 lm (SD 270
lm), which was constant in both age groups.
We found a linear dependence for the force-elongation
relationship of the zonular system at all ages (Fig. 4). Young
presbyopic eyes had a higher spring constant (slope in Fig. 4)
than older eyes (Table 2). Mean zonular elongation with 30 mN
force was 380 lm in young presbyopic eyes and 575 lm in
older eyes.
Converting the data into relative elongation or strain
changed the linear relationship only slightly, bringing the lines

FIGURE 5. Force as a function of radial zonular strain. The slope represents the elastic modulus of the zonular system.

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 IOVS, September 2012, Vol. 53, No. 10 Elasticity of Human Lens Zonules in Presbyopia 6113

TABLE 1. Zonular Elastic Properties for Different Specimen Prepara- TABLE 3. Zonular Elastic Properties for Different Postmortem Times
tion (Tested in Three Extra Donor Eyes)
Postmortem t-Test;
Ciliary Body Mean t-Test; P Value Time Mean P Value

Spring constant Intact 119 0.007 Spring constant Short 70.7 0.805
(mN/mm) Cut 47.6 (mN/mm) Long 68.1
Elongation at 30 mN Intact 257 0.009 Elongation at 30 mN Short 445 0.688
(lm) Cut 651 (lm) Long 475
System modulus Intact 249 0.010 System modulus Short 132 0.797
(mN) Cut 104 (mN) Long 136
Force at 10% strain Intact 24.9 0.010 Force at 10% strain Short 13.3 0.797
(mN) Cut 10.4 (mN) Long 13.6
Young’s modulus Intact 582 0.010 Young’s modulus Short 308 0.797
(mN/mm2) Cut 243 (mN/mm2) Long 317
Short postmortem times 1 to 2.5 days (n ¼ 9) and long
postmortem times 3.5 to 6 days (n ¼ 4).
for different ages somewhat closer together (Fig. 5). The slope
of this force-strain relationship gives the elastic modulus of the
zonular system, which remained significantly different in both estimated it to be approximately 1000 lm in young eyes (mean
age groups (Table 2). The mean force at 10% strain was 14.6 22 years) and approximately 650 lm in older eyes (mean 63
mN in younger presbyopic eyes and 11.4 mN in older eyes. years).11 Therefore, we should have covered the physiologic
The Young’s modulus for the zonules shows weak range with our stretching experiments, with a mean circum-
dependence on age (Fig. 6). In young presbyopic eyes (ages lental space of 1046 lm and experimental zonular elongation
47–60), the Young’s modulus of the zonules was 340 mN/mm2, between 0 and approximately 700 lm (Fig. 4).
whereas in older eyes (ages 83–97), it was significantly lower at The difference between our measurement temperature of
270 mN/mm2 (Table 2). However, the Pearson correlation 258C and normal body temperature of 368C may have
coefficient was not significant (r ¼ 0.529 and P ¼ 0.63). The influenced our results. Because of the somewhat colder
Pearson correlation test was selected because the Shapiro-Wilk environment, our tissue probably was somewhat stiffer or
test for small samples confirmed that the data were distributed rigid as it may be in the living human. With respect to
normally (P ¼ 0.743). postmortem time, our retrospective comparison of the elastic
There was no significant difference in the elastic properties properties estimated showed no difference between eyes with
of the eyes with respect to postmortem time (1–2.5 days vs. short postmortem times (1–2.5 days) and long postmortem
3.5–6 days, Table 3). times (3.5–6 days, Table 3).
Another concern was the integrity of the ciliary body.
Keeping the ciliary body radially intact results in a more
DISCUSSION homogeneous stretch of the anterior eye section and none of
the zonular fibers is lost by the cutting procedure. On the other
Our objective was to measure the elastic properties of the hand, the intact ciliary body must increase its diameter during
human lens zonules within a physiologic range. In ex vivo stretching, which induces a circumferential tension (Fig. 3).
experiments, Fisher reported a change of 220 lm in the human We have shown this with an extra experiment (Table 1). The
lens radius during accommodation in 20-year-olds, which main problem with the described circumferential tension is
declines linearly to 0 lm in 50-year-olds.3 He found a change that it cannot be predicted with a trivial spring model. When
of 790 lm in the ciliary body radius during accommodation, we cut the ciliary body, we had the lens, zonules, and ciliary
which was independent of age. More recent studies by Stachs body/sclera as springs in series. The intact ciliary body would
et al.9 and Strenk et al.10 using ultrasound revealed that the be an extra spring component circumferentially between our 8
anterior contour point of the ciliary muscle moves approxi- scleral segments (Fig. 3). Therefore, we decided to cut the
mately 360 lm during pharmacologically-induced accommo- ciliary body radially accepting the risk to cut some single
dation in young subjects (34 years) and 180 lm in older zonular fibers and having a less homogeneous stretching shape
subjects (71 years). The circumlental space in young eyes of our anterior eye sections.
(mean 26 years) has been estimated to be approximately 2000
lm using magnetic resonance imaging.10 A more recent study

TABLE 2. Zonular Elastic Properties of the Five Youngest Eyes (Ages

47–60) and Five Oldest Eyes (Ages 83–97)

Age Mean t-Test; P Value

Spring constant Young 80.3 0.003

(mN/mm) Old 53.2
Elongation at 30 mN Young 379 0.003
(lm) Old 574
System modulus Young 146 0.013
(mN) Old 114
Force at 10% strain Young 14.6 0.013
(mN) Old 11.4
Young’s modulus Young 342 0.013
(mN/mm2) Old 268 FIGURE 6. Young’s modulus of the zonules calculated as a function of
age.

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 6114 Michael et al.
It is interesting that we found a clear linear characteristic for
the force-elongation relationship. This linear elastic behavior
means that elongation is proportional to the loading force, and
the elongation returns to zero after unloading. Such behavior
would be expected in a structure that has to mediate a force
between the crystalline lens and ciliary body with the choroid.
Because of this linear relationship it makes sense to give the
spring constant for our experiments (Tables 1–3). The value of
elongation that is induced by a force of 30 mN is given for a
better intuitive understanding.
We also calculated the elastic modulus of the zonular
system, which is interesting for mechanical modeling of the
accommodation system. It excludes the errors introduced into
the calculation of Young’s modulus. This error originates from
the uncertain number, diameter, and length of the zonules.
One part of the length of the zonules, the zonular insertion
distance, could only be estimated with data from the literature
as explained in the methods. The length of the zonules within
the ciliary body remains unknown as the length of the zonule
portion entering the capsule. Therefore, we consider our
absolute values for Young’s modulus of the zonules only a
rough estimate. Still, it allows valid relative comparison of
different age groups. Our results are very similar to those of
Fisher, who found the Young’s modulus of the zonules of 350
mN/mm2 to be constant between ages 15 and 45.3 Our results
are lower than the value reported by van Alphen and Graebel,
who found 1500 mN/mm2 for 60-year-old eyes. 4 This
difference might be explained by the considerable deformation
in a two-arm stretching device, with quite uneven stretching of
the anterior eye section in different quadrants.
We found that the Young’s modulus of the zonules
decreases from presbyopic age to late presbyopic age by
20%. However, there was no significant correlation between
Young’s modulus and age in presbyopes. A follow-up study
with younger human donor eyes (ages 20–40) is under
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IOVS, September 2012, Vol. 53, No. 10
preparation to establish the relation of the Young’s modulus
with age, including the period with accommodation.
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