1054

ARTICLE

Comparison of the mechanical properties

of the anterior lens capsule in senile
cataract, senile cataract with trypan blue
application, and pseudoexfoliation syndrome
Cem Simsek, MD, Sibel Oto, MD, Gursel Yilmaz, MD, Dilek Dursun Altinors, MD,
Ahmet Akman, MD, Sirel Gur Gungor, MD

Purpose: To evaluate the elastic modulus, hardness, and
mechanical properties of the anterior lens capsule in different
types of cataract and to assess the correlation with age.
Setting: Baskent University Hospital, Department of Ophthal-
mology, Ankara, Turkey.
Design: Prospective comparative study.
Methods: Patients were divided into 3 groups. Group 1 comprised
patients with senile cataract, Group 2 patients had pseudoexfoliation
(PXF) syndrome, and Group 3 patients had dye-enhanced cataract
surgery. The capsules were analyzed using a nanoindentation
device. Young’s modulus of elasticity was measured by the
Oliver-Pharr method and capsule hardness by the Martens method.
6.01 G 1.25 GPa in Group 2, and 8.12 G 0.98 GPa in
Group 3. The capsules in Group 2 were more elastic than in
Group 1 and Group 3 (P < .001). The capsules in Group 3
had lower elasticity than in Group 1, although the difference
was not significant (P Z .94). The mean capsule stiffness
was 326.41 G 98.40 MPa in Group 1, 210.5 G 52.32 MPa
in Group 2, and 315.54 G 163.15 MPa in Group 3. The lens
capsules in Group 2 were less stiff than those in Group 1 and
Group 3 (P < .001).
Conclusions: Capsule thickness was positively correlated with
increasing age in all groups. The anterior lens capsules of patients
with PXF had more elasticity and less stiffness than the other
groups. Intracameral trypan blue application had no effect on
capsule elasticity and stiffness.

Results: The study comprised 72 patients, 24 per group. The

mean Young’s modulus was 7.53 GPa G 1.07 (SD) in Group 1, J Cataract Refract Surg 2017; 43:1054–1061 Q 2017 ASCRS and ESCRS

T
he lens capsule is an acellular viscoelastic membrane
that completely surrounds the lens and maintains
its structural integrity. It is the thickest basement
membrane in the body and consists primarily of collagen,
fibrillin, laminin, and heparin sulphate.1 The lens capsule
functions as a selective membrane for biochemical inter-
change of metabolic substrates and waste and it is also
considered to be a reservoir for growth factors. Studies
of the physical and biomechanical properties of the lens
capsule aim to develop better surgical interventions for
cataract surgery. Understanding the viscoelastic properties
of the lens capsule has implications for improving cataract
surgical techniques and for developing measures to
prevent surgical complications.2
Pseudoexfoliation (PXF) syndrome is characterized by a
greenish–white, fibrogranular PXF material at the edge of
the anterior lens capsule and/or pupil, which is observed
via anterior segment examination. Pseudoexfoliation
material can be seen in ocular tissues other than the anterior
lens capsule and iris, including the trabecular structure,
zonular fiber region, ciliary body processes, and anterior
surface of the vitreous.3 Capsule fragility is one of the major
intraoperative risk factors associated with PXF, and
surgeons performing capsulorhexis in PXF patients might
experience capsule-splitting phenomena in which pseudo-
layers of a fragile anterior capsule tear abnormally. The
anterior lens capsule does not have a lamellar structure;
however, a gradual deposition of fibrillary residue caused

Submitted: November 20, 2016 | Final revision submitted: May 3, 2017 | Accepted: May 31, 2017
From the Department of Ophthalmology, School of Medicine, Baskent University, Ankara, Turkey.
Presented in part at the ASCRS Symposium Cataract, IOL and the Refractive Surgery, San Diego, California, USA, April 2015.
Supported in part by a research grant from Baskent University, School of Medicine, Ankara, Turkey.
Corresponding author: Cem Simsek, MD, Department of Ophthalmology, Keio University School of Medicine, Shinanomachi 35, Shinjuku-ku, Tokyo 160-8582, Japan.
E-mail: cemsimsek1@hotmail.com.

Q 2017 ASCRS and ESCRS 0886-3350/$ - see frontmatter

Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jcrs.2017.05.038
 ANTERIOR LENS CAPSULE ASSESSMENT WITH NANOINDENTATION
by microfibrillopathy results in a pseudolayer over the sur-
face of the lens capsule. This layer might break up during
capsulorhexis, giving a false impression of anterior lens
capsule split.4
Trypan blue is administered to the anterior chamber to
facilitate visualization of the anterior capsule when the
red retinal reflex is not adequate. It is thought that admin-
istration of trypan blue causes structural changes to the lens
capsule.5 Some studies have shown that trypan blue staining
of the lens capsule modifies the biomechanical structure of
the capsule and decreases its elasticity6; however, this does
not affect the resistance of the capsular edge to tearing.7
The nanoindentation method is used to determine the
mechanical properties (such as elasticity and hardness) of a
tissue or material, according to the reaction against applied
loading. This computer-based technique calculates the load
applied by an indenter during the loading–unloading cycle
and measures the indenter’s penetration depth.8
The present study aimed to determine the mechanical
properties of the anterior lens capsule, including Young’s
modulus and hardness, as measured using a nanoindentation
device. Patients with no ocular pathology except for cataract,
patients with PXF, and patients administered trypan blue to
the anterior chamber were included in the study. An
additional aim was to compare lens capsule thickness, which
was measured by a scanning electron microscope (SEM)
(Quanta 400F, FEI), between the 3 patient groups.
PATIENTS AND METHODS
Participants and Sample Preparation
The morphology, physiology, and mechanics of the anterior lens
capsule were evaluated in 3 groups of patients who had cataract
surgery at the Ophthalmology Department, Baskent University,
Ankara, Turkey, between January 2014 and June 2014. The study
Figure 1. Scanning electron microscope images of an anterior lens
capsule slightly separated from the PXF material, which is deposited
on the surface of the lens capsule. A:
1600 magnification.
B:
6000 magnification. C:
7071 magnification. D:
60 000
magnification. Red arrows indicate the PXF material on the anterior
lens capsule.
1055
was approved by the institutional ethics committee and adhered to
the tenets of the Declaration of Helsinki. Informed consent was
obtained from all patients after informational materials about
the procedure, the risks and complications associated with the
surgery, and a study description were provided to them.
All surgeons (C.S., S.O., G.Y., D.D.A., A.A., and S.G.G.)
performed the capsulorhexis and collected specimen samples
using a standard technique. During creation of the capsulorhexis,
the anterior lens capsule was removed en bloc. Senile cataract
and PXF samples were collected as consecutive cases, whereas
dye-enhanced cataract surgery was selected from the scheduled
operations.
Group 1 included patients with no pathology other than
cataract, Group 2 included patients with PXF (Figure 1), and
Group 3 included patients administered trypan blue 0.06%
(Blue Rhexis, Care Group India) during cataract surgery
(Figure 2). In each group, anterior lens capsules that were removed
during the anterior capsulorhexis stage of the surgery were evalu-
ated. The size of these capsules was approximately 5.0 to 6.0 mm.
No patient had additional pathologies, a history of drug use, or
systemic diseases. All removed anterior capsule samples were
placed on 1 cm
1 cm sterile glass slides by the same surgeon
(C.S.) and stored in standard plastic containers with a fortified
balanced salt solution (BSS Plus, Alcon Laboratories, Inc.), which
acted as an intraocular irrigating solution for hydration. The
fortified balanced salt solution was placed on top of the lens
capsule to maintain hydration during the experiments. The patient
samples were continuously submerged in the balanced salt
solution from excision until testing to ensure that dehydration
and rehydration did not change the mechanical properties.
To maintain hydration during testing, the lens was covered in a
droplet of the fortified balanced salt solution. All experiments
were performed at room temperature. The samples were evaluated
on the same day at the Central Laboratory of Middle East
Technical University, Ankara, Turkey.
Indentation Technique
Young’s modulus, hardness, and the morphologic properties of
the lens capsule surface were evaluated using a nanoindentation
Figure 2. Scanning electron microscope images at different
magnifications of an anterior lens capsule stained with capsule
dye (trypan blue 0.06%). A:
200 magnification. B:
800 magnifica-
tion. C:
6000 magnification. D:
20 000 magnification level.
Volume 43 Issue 8 August 2017
1056 ANTERIOR LENS CAPSULE ASSESSMENT WITH NANOINDENTATION
testing device (CSM Instruments). A reflective, aluminum-coated,
2 mm thick rectangular-shaped cantilever (Bruker Nano, Bruker
Corp.) was used with a standard Berkovich-type indenter, which
had a sharp diamond tip of 100 mm diagonal length (PR-CO10,
Bruker Corp.). The indenter geometry was an axisymmetric
cone with a q apical half angle. In this study, the mean sample
thickness was determined to be approximately 18 mm and the
depth of indentation was set to 400 nm at the deepest point.
The indentation depth was less than 3% of the sample thickness,
which prevented the underlying glass slides from affecting the
indentation measurements (Figure 3). The nanoindentation
method was characterized by recording the P-h values via com-
puter throughout the loading–unloading cycle. The P-h values
are the result of the penetration of a sharp indenter of a very
hard material, such as diamond, with known mechanical prop-
erties into a homogenous hard material with penetration loading
(P) and penetration depth (h) from the surface. The quality of
elastic–plastic transformation can be analyzed using the loading–
unloading curves obtained with the Oliver-Pharr method.9
The load was gradually increased until maximum loading was
achieved. Maximum indentation loading was applied at a constant
displacement rate to the inside of the lens capsule sample material
(loading phase). After achieving maximum indentation loading,
the sample was set aside for a specific time (holding phase). The
loading was then removed and the indenter was withdrawn
from the sample (loading-displacement phase). Forces and depths
(displacement) were measured and transferred to a P-h graph via
computer-based data collection and storage. When the material
reaches deformation at maximum depth, an elastic recovery
occurs during displacement of the loading: residual depth occurs
at this point. The elastic recovery that occurs at the region between
maximum depth and residual depth is the elastic work region.
The percentage of the total work region is the ratio of the
residual depth to the maximum depth.10
The deformation that occurs during the loading phase is elastic
and plastic in character. Elastic deformation during the loading
phase recovers during the loading-displacement phase. A trace
with a certain depth appears at the surface of the material at the
end of the experiment because there are characteristic deformation
differences between the loading and loading-displacement phases.
Using the elastic recovery that occurs during the initial unloading
phase and the projection contact area of the trace that occurs
under maximum indentation loading during the indentation
process, the elastic–plastic properties of the material can be
determined.9
Measurement of Elastic Modulus and Hardness
The elastic modulus (E) and microhardness (H) can be described
by loading and penetration depth data. To determine E and H,
Volume 43 Issue 8 August 2017
3 key parameters must be measured as follows: maximum load
(Pmax), indenter contact area at maximum depth (Ac), and loading
displacement initial contact rigidity (S Z dP/dh). As with conven-
tional microhardness testing, dynamic microindentation hardness
is also calculated by the division of indentation maximum load
(Pmax) by the trace area (Ac).11 As such, the hardness equation
is as follows:
Pmax
HZ
Ac
The Ac (contact area) can be stated as Ac Z F (hc) when the
indenter during the loading displacement phase is considered a
function of the distance between the material and the indenter.
If the indenter geometry is considered an axisymmetric cone
with a q apical half angle, the contact area equality can be
calculated using an indenter geometric formula.
Ac Z pðtan2 qÞh2c
The equation for the contact area for Berkovich transformed to
an equality is
Ac Z 26:43 h2c
The microhardness of a material dependent on the applied
indentation test load is known as the indentation size effect
and it typically exhibits a reduction in apparent microhardness
with increasing test loads (increasing indentation size).
This occurs as a result of numerous factors, including loading
initial plastic deformation throughout the indentation,
indentation elastic recovery, elastic–plastic deformation of
the material, and formation of dislocation loops throughout
the indentation cycle. Another material property that can be
obtained from indentation experiments is Young’s modulus.
Young’s modulus can be calculated using an equation that
uses the slope of the initial unloading phase of the P-h curve
(dP/dh) and the contact area under maximum load (Amax)
using the following formula:
!  
1
 1  v2 1  vin2 1 dP
E Z þ Z  pffiffiffiffiffiffiffiffiffi
E Ein c Amax dh
where E* is the decrease in elastic modulus of the indenter sample
system. The effect of the indenter with non-ideal rigidity to the
loading-depth action can be considered by the definition of this
modulus,10 where c* is 1.167 (Berkovich), v is the Poisson ratio,
E is Young’s modulus and defines the properties of the indenter,
and A is area (Figure 4). This study used Indentation software
(version 5.06, CSM Instruments). After the measurement of
E and H, an image of the surface was obtained using the atomic
Figure 3. A: Curves formed by 1 flex-
ibility measurement for an anterior
lens capsule based on the Oliver-
Pharr method. The x-coordinate indi-
cates loading and the y-coordinate
indicates depth. B: Graphic repre-
sentation of an anterior lens capsule
hardness measured via nanoinden-
tation and Martens hardness
method (CIT Z instrumented creed;
E*Z loss modulus; EIT Z instru-
mented elastic modulus; Er Z
reduced modulus; HIT Z instru-
mented hardness; HM Z Martens
hardness; RIT Z relaxation
instrumented).
 ANTERIOR LENS CAPSULE ASSESSMENT WITH NANOINDENTATION
Figure 4. A: Indentation section. B: Significant points on the
loading–unloading P-h curve. Values expressed using the Oliver-
Pharr method in the loading-displacement curve (A Z area; Ch2
Z depth function; dh Z change in depth; dPu Z change in unload;
h Z depth; hc Z contact depth; hmax Z maximum depth; hr Z re-
sidual depth; m Z an integer at function for calculating P).
force microscope of the nanoindentation testing device. The 3-
dimensional images were transferred to a computer, and the sur-
face of the anterior capsule and the changes made by the indenter
were observed in the images.
Evaluation of Lens Capsule Thickness
The lens capsule thickness was measured using an SEM. The
samples were fixed on stubs and coated with gold–palladium for
conductivity. Then, the samples were placed on a mobile platform
for the thickness measurement. The platform was moved along the
x- and y-coordinates, and each sample was examined under
100
and
20 magnification. The indenter application points were
attempted until a clear image was observed. Six measurements
were obtained from 5 points at the center and circumference
(Figure 5). The goal was to obtain the measurements as far from
the center as possible in patients from Group 2 to include the
region containing the most PXF material. Subsequently, the
platform was manually placed under the indenter and the related
program was run at the connected computer. Ten patients were
randomly selected from each group. The capsule thickness was
compared between the 3 groups and the correlation between age
and capsule thickness was evaluated (Figure 6).
Statistical Analysis
Data were analyzed using SPSS for Windows software (version
17.0, SPSS, Inc.). The Shapiro-Wilk test was used to determine
the normality of the data and the Levene test was used to deter-
mine the homogeneity of the group variances. A one-way analysis
of variance and the Tukey HSD (honest significant difference) test
were used to compare group means and correlations between
the variables, which were evaluated using Pearson correlation
coefficient. Descriptive data are shown as means G SD. The level
of statistical significance was set at a Z .05.
RESULTS
Patient Population
The study comprised 72 patients, who were divided into
3 groups. There were 24 patients in each group. Table 1
shows the demographic characteristics of the 3 patient
1057
Figure 5. The indenter application points were attempted until a
clear image was observed. A: Traces made by the nanoindenter
on a lens capsule (red arrows) seen via SEM. B: Optical microscopic
image of a lens capsule with PXF syndrome at
20 magnification
(bar Z 20 mm). C: Traces made by the nanoindenter on a lens
capsule at
100 magnification via optical microscopy (bar Z 4 mm).
groups, and Figure 7 shows the patient age distribution
for each group.
Anterior Capsule Thickness
There was no significant difference in age or sex distribution
between the groups (P Z .94). The mean capsule thickness
measured by SEM was 18.19 G 2.18 mm in Group 1,
18.41 G 2.64 mm in Group 2, and 18.36 G 1.95 mm in
Group 3. The differences were not statistically significant
(P Z .98). The correlation between age and capsule thickness
was evaluated in each group and in the overall study popula-
tion. There was a 90.1% linear correlation between age and
capsule thickness in the overall study population versus
92.1% in Group 1, 89.5% in Group 2, and 90.6% in Group 3.
Young’s Modulus Elasticity and Martens Hardness
The Young’s modulus values were as follows: Group 1:
7.53 G 1.07 GPa (range 5.42 to 10.05 GPa), Group 2:
6.01 G 1.25 GPa (range 3.72 to 8.91 GPa), and Group 3:
8.12 G 0.98 GPa (range 6.66 to 11.06 GPa). There was a sta-
tistically significant difference in the mean Young’s
modulus between Group 1 and Group 2 and between
Group 2 and Group 3 (both P ! .001). Elasticity was signif-
icantly higher in Group 2 than in Group 1 and Group 3
(P ! .001); however, there was no significant difference
between Group 1 and Group 3 (P Z .17).
The correlation between age and capsule elasticity was
evaluated in each group and in the overall study population.
Volume 43 Issue 8 August 2017
1058 ANTERIOR LENS CAPSULE ASSESSMENT WITH NANOINDENTATION

Figure 6. Measurement of the anterior lens

capsule thickness in the 3 groups using im-
ages acquired by the SEM. A: Patient with
no pathology other than cataract (
12 000
magnification). B: Patient with PXF syndrome
(
8000 magnification). C: Patients adminis-
tered with trypan blue 0.06% during cataract
surgery (
8000 magnification). D: Patient
with senile cataract (
8.000 magnification).

There was a significant linear correlation (66.0%) between
age and capsule elasticity in the overall study population
(P ! .001), versus 89.1% in Group 1 (P ! .001), 85.8%
in Group 2 (P ! .001), and 83.7% in Group 3 (P ! 0.01)
(the elasticity of a material decreases as the elasticity
coefficient increases).
The capsule hardness values were 326.41 G 98.40 MPa
(range 206.0 to 574.0 MPa) in Group 1, 210.5 G 52.32 MPa
(range 106.0 to 288.0 MPa) in Group 2, and
315.54 G 163.15 MPa (range 212.0 to 430.0 MPa) in Group 3.
There was a significant difference in mean hardness values be-
tween Group 1 and Group 2 (P Z .002) and Group 2 and
Group 3 (P Z .006). Hardness was significantly lower in
Group 2 than in Group 1 and Group 3 (P ! .001). However,
there was no significant difference between Group 1 and
Group 3 (P Z .94) (Figure 8).
There was a significant linear correlation (36.5%)
between age and hardness in the overall study population
versus 47.1% in Group 1, 57.7% in Group 2 (P ! .001),
and 53.9% in Group 3 (all P ! .001). When all the data
were evaluated between the capsule elasticity coefficient
and capsule hardness, the linear correlation was 49.3%
(P ! .001).
DISCUSSION
In the present study, anterior lens capsules in patients with
no ocular pathology other than cataract (Group 1), patients
with PXF (Group 2), and patients administered trypan blue
during cataract surgery (Group 3) were evaluated for
thickness and the correlation between age and thickness.
To our knowledge, the present study is the first to evaluate
anterior capsule Young’s modulus and hardness using a
conventional nanoindentation testing device.
Capsule thickness did not differ significantly between the
3 groups; however, there was a significant linear correlation
between age and capsule thickness. The correlation between

Table 1. Demographics in the 3 patient groups.

Sex Age (y)

Group Female Male Mean ± SD Range

1 (cataract only) 11 13 69.25 G 5.07 60, 80
2 (PXF syndrome) 12 12 69.33 G 5.63 60, 81
3 (trypan blue) 13 11 69.37 G 5.19 61, 83
Figure 7. Patient age distribution for each group (PXF Z
PXF Z pseudoexfoliation pseudoexfoliation).

Volume 43 Issue 8 August 2017

 ANTERIOR LENS CAPSULE ASSESSMENT WITH NANOINDENTATION
Figure 8. Elastic modulus values of the anterior lens capsule in rela-
tion to age and Martens hardness values of the anterior lens capsule
in relation to age in the 3 groups (PXF Z pseudoexfoliation).
lens capsule elasticity and hardness with age has been
measured using several methods.12 An earlier study12
concluded that although 10 factors caused an increase in
Young’s modulus with age, the increase in hardness with
age is not fully understood. Krag and Andreassen13 found
that lens capsule thickness increases with age and suggested
that this might be the cause of decreased elasticity and
increased hardness. Alternatively, changes to the microme-
chanical properties of the lens capsule can also be factors
associated with increased hardness. The mechanical
properties of tissues change with age, according to the
components of the tissue at the microlevel and to the
organization of the components on the macrolevel.14
Jardeleza et al.6 performed nanoindentation measurements
of the anterior capsule in diabetic and nondiabetic patients
before and after administering trypan blue and found a
significant decrease in capsule elasticity in both groups.
They suggested that trypan blue accumulation increased
capsule hardness by crosslinking with collagen when it was
exposed to photooxidative effects in the anterior lens capsule
basal membrane, which is composed of type IV collagen.
Wollensak and Pham15 administered trypan blue 0.1% to
pig eyes for 30 seconds, 1 minute, and 30 minutes, and
observed a significant increase in hardness and a decrease
in ultimate extensibility in eyes incubated for 1 minute and
30 minutes. They suggested that the effect might be caused
by changes in capsule elastic behavior resulting from
crosslinking of free oxygen radicals and collagen fibers,
which was associated with trypan blue’s sensitivity to light.
Haritoglou et al.16 administered brilliant blue 0.025%,
1059
indocyanine green 0.05%, and trypan blue 0.06% to dissected
lens capsules and measured their hardness using an atomic
force microscopy (AFM) nanoindentation method. That
study found a significant increase in hardness in the lens
capsules treated with brilliant blue, indocyanine green, and
trypan blue but not in the control samples. With regard to
anterior capsule thickness, Portes et al.17 found no difference
in thickness between capsules stained with trypan blue and a
control group.
In our study, there was no significant difference in capsule
elasticity between the cataract-only eyes (Group 1) and the
eyes treated with trypan blue (Group 3). Administering
trypan blue under in vivo conditions and maintaining the
samples in a balanced salt intraocular irrigation solution
might explain why there was no difference in capsule
elasticity between the other 2 groups. In accordance with
our findings, Jaber et al.7 did not observe a significant change
in capsule hardness or capsulorhexis tear resistance based
on biomechanical measurements performed after the
administration of trypan blue 0.06%. The researchers
suggested that these results were from using a different
measurement model and that measurements based on
prolongation data caused by elasticity at the capsulorhexis
could not provide a precise calculation.
Conventional nanoindentation testing is not common,
and the AFM nanoindentation method is more widely
used to measure the mechanical properties of biomaterials
in ophthalmology. An advantage of AFM over conventional
nanoindentation testing is that measurement of the contact
area and depth by the force-displacement curve is
accomplished with the same tip.18 Jee and Lee10 measured
the mechanical properties of different polymers and
evaluated the results using the Oliver-Pharr method and
image analysis. They found that the hardness and Young’s
modulus findings were compatible with each other via both
measurements. They also found that conventional nanoin-
dentation testing and the AFM nanoindentation method
yielded similar mechanical property values.
Nanoindentation measurement of biologic material is
more difficult than that of metals. In general, biologic
materials have a low Young’s modulus and their time-
dependent mechanical behavior changes. In addition,
hydration is an important factor that affects this behavior.19
A low Young’s modulus can make it difficult to position the
tip at a sample’s test location during the indentation
phase.20 Nanoindentation has been used to measure the
mechanical behavior of biologic materials such as enamel,21
blood vessels,22 and bone.23
Chaurasia et al.24 evaluated the elasticity and hardness of
Descemet membrane treated with fibrin glue by using AFM
nanoindentation. Biologic tissue glue (fibrin glue) is used
for numerous ophthalmologic indications such as corneal
perforation, conjunctival graft surgery, lamellar kerato-
plasty without sutures, and the prevention of leakage during
glaucoma surgery. That study found that the elasticity and
hardness of Descemet membrane treated with fibrin glue
increased as did the ability of Descemet membrane to
withstand kinking and bending. Dias and Ziebarth25
Volume 43 Issue 8 August 2017
1060 ANTERIOR LENS CAPSULE ASSESSMENT WITH NANOINDENTATION
compared the elasticity of the anterior corneal stroma with
the elasticity of the posterior corneal stroma using AFM
nanoindentation and found that the effective Young’s
modulus value in the anterior corneal stroma was
significantly higher than in the posterior corneal stroma.
According to the linear correlation between the anterior
and posterior stroma, the posterior stroma was 39.3%
harder than the anterior stroma, and the researchers
emphasized that the difference in elasticity and gradient
must be considered for diagnostic and treatment methods.
Grant et al.26 evaluated Young’s modulus in the scleral
stroma and episclera using AFM nanoindentation and
found that the episclera was significantly softer than the
stroma. Ziebarth et al.27 studied human and monkey
anterior lens capsules using AFM nanoindentation and
found that Young’s modulus was significantly higher in
human lenses. Young’s modulus elasticity was 20.1 to
131 kPa in capsule samples obtained from 18 donor eyes
via capsulorhexis.28 However, the monkeys’ ages were not
appropriate for comparative analysis.
Braunsmann et al.29 compared lamina cribrosa and
peripapillary sclera thickness between patients with and
without PXF. They found that Young’s modulus in the
lamina cribrosa was significantly lower in patients with
PXF and indicated that this was caused by impairment of
the organizational structure of the lamina cribrosa elastic
fiber system. Beginning during the early stages of PXF,
disorganization occurs as elastin, fibrillin-1, and fibrillin-4
lose their functionality in the elastic fiber tissue. In addition,
activity of the lysyl oxidase-like 1 (LOXL1) enzyme is also
reduced in the elastic fiber tissue.30 Lysyl oxidase-like 1 is
a member of the lysyl oxidase enzyme family and plays a
role in canalization of crosslinks between elastin and
collagen tissues.30 Lysyl oxidase-like 1 also assists in the
production and maintenance of elastic fibers by providing
for linkage between soluble tropoelastin and nonsoluble
tropoelastin via desmosine and isodesmosine bonds.30
A common nucleotide polymorphism in the LOXL1 gene
was identified as a genetic risk factor for PXF in multiple
ethnic groups.31 Earlier studies also confirmed that
LOXL1 is irregularly expressed in ocular tissues in eyes
with PXF.31
In our study, Young’s modulus and Martens hardness
were both lower in the anterior lens capsules of eyes with
PXF (Group 2). We propose that these low values were
caused by defects in LOXL1 that affected the biomechanical
properties of the anterior lens capsule. Reduced hardness in
eyes with PXF can cause ocular structures to become weak
and vulnerable to injury. We also found that anterior lens
capsule elasticity was higher and hardness was lower in
Group 2 than in Group 1 (cataract-only) and Group 3
(trypan blue–treated cataract). In addition, although
mean Young’s modulus and hardness values were higher
in Group 3 than in Group 1, the difference was not signif-
icant. Trypan blue was used during cataract surgery in our
study in Group 3; however, in other studies,15–17 trypan
blue was used subsequent to excision of the anterior lens
capsule. As such, we propose that trypan blue adherence
Volume 43 Issue 8 August 2017
to the lens was greater in those studies. Furthermore, elas-
ticity decreased with age in all 3 groups in the present study.
The Young’s modulus and Martens hardness values
found in this study are significantly different to those found
in other studies. Mechanical properties can change as a
result of the measuring instrument, measurement method,
and the type of equations used. There is no definitive elas-
ticity and hardness value for the anterior lens capsule and
therefore, the data obtained here were influenced by
the measurement technique. Nanoindentation testing is
becoming more common in ophthalmologic practice for
measurement of the biomechanical properties of live
tissues. The standard elastic values of numerous biomate-
rials remain unknown and can only be elucidated with
additional research. We propose that data obtained via
nanoindentation testing will be a substantial source for
advancing the development of cataract surgery techniques.
WHAT WAS KNOWN
 Trypan blue dye decreases anterior capsule elasticity and
leads to significant stiffening of the capsule.
 Patients with PXF suffer from capsule instability during
cataract surgery.
WHAT THIS PAPER ADDS
 Intracameral trypan blue application had no effect on
capsule elasticity and stiffness.
 Anterior lens capsules with PXF were more elastic and less stiff
than normal capsules and trypan blue–enhanced capsules.
REFERENCES
1. Danysh BP, Duncan MK. The lens capsule. Exp Eye Res 2009; 88:151–164.
Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2674021/pdf
/nihms-105801.pdf. Accessed June 20, 2017
2. Pedrigi RM, David G, Dziezyc J. Regional mechanical properties and stress
analysis of the human anterior lens capsule. Vision Res 2007; 47:1781–
1789. Available at: http://www.sciencedirect.com/science/article/pii
/S0042698907001204. Accessed June 20, 2017
3. Naumann GOH, Schlo €tzer-Schrehardt U, Ku
€chle M. Pseudoexfoliation syn-
drome for the comprehensive ophthalmologist; intraocular and systemic
manifestations. Ophthalmology 1998; 105:951–968
4. Shingleton BJ, Crandall AS, Ahmed K II. Pseudoexfoliation and the cataract
surgeon: preoperative, intraoperative, and postoperative issues related to
intraocular pressure, cataract, and intraocular lenses. J Cataract Refract
Surg 2009; 35:1101–1120
5. Kothari K, Jain SS, Shah NJ. Anterior capsular staining with trypan blue for
capsulorhexis in mature and hypermature cataracts; a preliminary study.
Indian J Ophthalmol 2001; 49:177–180. Available at: http://www.ijo.in
/article.asp?issnZ0301-4738;yearZ2001;volumeZ49;issueZ3;spage
Z177;epageZ180;aulastZKothari. Accessed June 20, 2017
6. Jardeleza MSR, Daly MK, Kaufman JD, Klapperich C, Legutko PA.
Effect of trypan blue staining on the elastic modulus of anterior lens
capsules of diabetic and nondiabetic patients. J Cataract Refract
Surg 2009; 35:318–323
7. Jaber R, Werner L, Fuller S, Kavoussi SC, McIntyre S, Burrow M,
Mamalis N. Comparison of capsulorhexis resistance to tearing with and
without trypan blue dye using a mechanized tensile strength model.
J Cataract Refract Surg 2012; 38:507–512
8. Lucca DA, Hermann K, Klopfstein MJ. Nanoindentation: measuring
methods and applications. CIRP Ann Manuf Technol 2010; 59:803–819
9. Oliver WC, Pharr GM. An improved technique for determining hardness and
elastic modulus using load and displacement sensing indentation experi-
ments. J Mater Res 1992; 7:1564–1583. Available at: http://web.mit
.edu/cortiz/www/3.052/3.052CourseReader/45_OliverPharr1992Nano
indentation.pdf. Accessed June 20, 2017
 ANTERIOR LENS CAPSULE ASSESSMENT WITH NANOINDENTATION
10. Jee A-Y, Lee M. Comparative analysis on the nanoindentation of polymers
using atomic force microscopy. Polym Test 2010; 29:95–99
11. Uzun O, Ko €lemen U, Çelebi S, Gu
ۍlu
€ N. Modulus and hardness evaluation
of polycrystalline superconductors by dynamic microindentation technique.
J Eur Ceram Soc 2005; 6:969–977
12. Fisher RF. Elastic constants of the human lens capsule. J Physiol 1969;
201:1–19. Available at: http://www.pubmedcentral.nih.gov/picrender
.fcgi?artidZ1351628&blobtypeZpdf. Accessed June 20, 2017
13. Krag S, Andreassen TT. Effect of freezing on lens capsule mechanical
behavior. Ophthalmic Res 1998; 30:280–285
14. Rho J-Y, Kuhn-Spearing L, Zioupos P. Mechanical properties and the
hierarchical structure of bone. Med Eng Phys 1998; 20:92–102
15. Wollensak G, Spo €rl E, Pham D-T. Biomechanical changes in the anterior
lens capsule after trypan blue staining. J Cataract Refract Surg 2004;
30:1526–1530
16. Haritoglou C, Mauell S, Schumann RG, Henrich PB, Wolf A, Kernt M,
Benoit M. Increase in lens capsule stiffness caused by vital dyes.
J Cataract Refract Surg 2013; 39:1749–1752
17. Portes ALF, Almeida AC, Allodi S, Monteiro MLR, Miguel NCO. Trypan blue
staining for capsulorhexis: ultrastructural effect on lens epithelial cells and
capsules. J Cataract Refract Surg 2010; 36:582–587
18. Thome  T, Fouchez S, Delalande S. Determination of silicone coating Young’s
modulus using atomic force microscopy. Physica B 2009; 404:22–25
19. Oyen M, Taylor D. Special issue on nanoindentation of biological materials.
[editorial]. J Mechan Behav Biomed Mater 2006; 2:311
20. Kaufman JD, Klapperich CM. Surface detection errors cause overestima-
tion of the modulus in nanoindentation on soft materials. J Mechan Behav
Biomed Mater 2009; 2:312–317
21. Men cík J, He LH, Swain MV. Determination of viscoelastic–plastic material
parameters of biomaterials by instrumented indentation. J Mechan Behav
Biomed Mater 2009; 2:318–325
22. Akhtar R, Schwarzer N, Sherratt MJ, Watson REB, Graham HK,
Trafford AW, Mummery PM, Derby B. Nanoindentation of histological spec-
imens: Mapping the elastic properties of soft tissues. J Mater Res 2009;
24:638–646. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles
/PMC2854807/pdf/ukmss-4436.pdf. Accessed June 20, 2017
23. Bouzakis K-D, Mitsi S, Michailidis N, Mirisidis I, Mesomeris G, Maliaris G,
Korlos A, Kapetanos G, Antonarakos P, Anagnostidis K. Loading simulation
of lumbar spine vertebrae during a compression test using the finite ele-
ments method and trabecular bone strength properties, determined by
means of nanoindentations. J Musculoskelet Neuronal Interact 2004;
4:152–158. Available at: http://www.ismni.org/jmni/pdf/16/11BOUZAKIS
.pdf. Accessed June 20, 2017
24. Chaurasia SS, Champakalakshmi R, Li A, Poh R, Tan XW,
Lakshminarayanan R, Lim CT, Tan DT, Mehta JS. Effect of fibrin glue on
the biomechanical properties of human Descemet’s membrane. PLoS
One 2012; 7:e37456. Available at: https://www.ncbi.nlm.nih.gov/pmc
/articles/PMC3360777/pdf/pone.0037456.pdf. Accessed June 20, 2017
1061
25. Dias JM, Ziebarth NM. Anterior and posterior corneal stroma elasticity as-
sessed using nanoindentation. Exp Eye Res 2013; 115:41–46. Available
at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795859/pdf/nihms
-499366.pdf. Accessed June 20, 2017
26. Grant CA, Thomson NH, Savag MD, Woon HW, Greig D. Surface charac-
terisation and biomechanical analysis of the sclera by atomic force micro-
scopy. J Mech Behav Biomed Mater 2011; 4:535–540
27. Ziebarth NM, Arrieta E, Feuer WJ, Moy VT, Manns F, Parel J-M. Primate lens
capsule elasticity assessed using Atomic Force Microscopy. Exp Eye Res
2011; 92:490–494. Available at: https://www.ncbi.nlm.nih.gov/pmc
/articles/PMC3104078/pdf/nihms283487.pdf. Accessed June 20, 2017
28. Courtois Y. The capsule of the crystalline lens. In: Stark L, Obrecht G, eds,
Presbyopia: Recent Research and Reviews from the Third International
Symposium on Presbyopia (3d: 1985: Haiti). New York, NY, Professional
Press, 1987; 45–52
29. Braunsmann C, Hammer CM, Rheinlaender J, Kruse FE, Sch€affer TE,
Schlo €tzer-Schrehardt U. Evaluation of lamina cribrosa and peripapillary sclera
stiffness in pseudoexfoliation and normal eyes by atomic force microscopy.
Invest Ophthalmol Vis Sci 2012; 53:2960–2967. Available at: http://iovs.arvo
journals.org/article.aspx?articleidZ2127893. Accessed June 20, 2017
30. Thorleifsson G, Magnusson KP, Sulem P, Walters GB, Gudbjartsson DF,
Stefansson H, Jonsson T, Jonasdottir A, Jonasdottir A, Stefansdottir G,
Masson G, Hardarson GA, Petursson H, Arnarsson A, Motallebipour M,
Wallerman O, Wadelius C, Gulcher JR, Thorsteinsdottir U, Kong A,
Jonasson F, Stefansson K. Common sequence variants in the LOXL1
gene confer susceptibility to exfoliation glaucoma. Science 2007;
317:1397–1400
31. Schlo €tzer-Schrehardt U, Pasutto F, Sommer P, Hornstra I, Kruse FE,
Naumann GOH, Reis A, Zenkel M. Genotype correlate expression of lysyl
oxidase-like 1 in ocular tissues of patients with pseudoexfoliation syn-
drome/glaucoma and normal patients. Am J Pathol 2008; 173:1724–1735.
Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2626384/pdf
/JPATH173001724.pdf. Accessed June 20, 2017
Disclosure: None of the authors has a financial or proprietary interest
in any material or method mentioned.
First author:
Cem Simsek, MD
Department of Ophthalmology, Keio
University School of Medicine, Tokyo,
Japan
Volume 43 Issue 8 August 2017