- Email: [email protected]
Comparison of the mechanical properties of the anterior lens capsule in senile cataract, senile cataract with trypan blue application, and pseudoexfoliation syndrome
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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 Ophthalmology, Ankara, Turkey. Design: Prospective comparative study.
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).
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.
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 interchange 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 pseudolayers 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: [email protected]. Q 2017 ASCRS and ESCRS Published by Elsevier Inc.
0886-3350/$ - see frontmatter 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 surface 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 administration 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.
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PATIENTS AND METHODS
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 evaluated. 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.
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
Indentation Technique Young’s modulus, hardness, and the morphologic properties of the lens capsule surface were evaluated using a nanoindentation
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.
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 magnification. C: 6000 magnification. D: 20 000 magnification level.
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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 computer 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 properties 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,
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 conventional 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: HZ
Pmax 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 flexibility measurement for an anterior lens capsule based on the OliverPharr method. The x-coordinate indicates loading and the y-coordinate indicates depth. B: Graphic representation of an anterior lens capsule hardness measured via nanoindentation and Martens hardness method (CIT Z instrumented creed; E*Z loss modulus; EIT Z instrumented elastic modulus; Er Z reduced modulus; HIT Z instrumented hardness; HM Z Martens hardness; RIT Z relaxation instrumented).
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Figure 4. A: Indentation section. B: Significant points on the loading–unloading P-h curve. Values expressed using the OliverPharr 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 residual depth; m Z an integer at function for calculating P).
force microscope of the nanoindentation testing device. The 3dimensional images were transferred to a computer, and the surface 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 determine 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
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 population. 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 statistically 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 significantly 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
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Figure 6. Measurement of the anterior lens capsule thickness in the 3 groups using images acquired by the SEM. A: Patient with no pathology other than cataract (12 000 magnification). B: Patient with PXF syndrome (8000 magnification). C: Patients administered 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 between 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 Group
Age (y)
Female
Male
Mean ± SD
Range
11 12 13
13 12 11
69.25 G 5.07 69.33 G 5.63 69.37 G 5.19
60, 80 60, 81 61, 83
1 (cataract only) 2 (PXF syndrome) 3 (trypan blue) PXF Z pseudoexfoliation
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Figure 7. Patient age distribution for each group (PXF Z pseudoexfoliation).
ANTERIOR LENS CAPSULE ASSESSMENT WITH NANOINDENTATION
Figure 8. Elastic modulus values of the anterior lens capsule in relation 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 micromechanical 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%,
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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 nanoindentation 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 timedependent 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 keratoplasty 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
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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 significant. 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, elasticity 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 elasticity 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 biomaterials 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.
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10. Jee A-Y, Lee M. Comparative analysis on the nanoindentation of polymers using atomic force microscopy. Polym Test 2010; 29:95–99 €lemen U, C¸elebi S, Gu €c¸lu € N. Modulus and hardness evaluation 11. Uzun O, Ko 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 €rl E, Pham D-T. Biomechanical changes in the anterior 15. Wollensak G, Spo 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 T, Fouchez S, Delalande S. Determination of silicone coating Young’s 18. Thome 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 overestimation 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 specimens: 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 elements 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
25. Dias JM, Ziebarth NM. Anterior and posterior corneal stroma elasticity assessed 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 characterisation and biomechanical analysis of the sclera by atomic force microscopy. 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, €tzer-Schrehardt U. Evaluation of lamina cribrosa and peripapillary sclera Schlo 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 €tzer-Schrehardt U, Pasutto F, Sommer P, Hornstra I, Kruse FE, 31. Schlo Naumann GOH, Reis A, Zenkel M. Genotype correlate expression of lysyl oxidase-like 1 in ocular tissues of patients with pseudoexfoliation syndrome/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
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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 Ophthalmology, Ankara, Turkey. Design: Prospective comparative study.
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).
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.
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 interchange 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 pseudolayers 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: [email protected]. Q 2017 ASCRS and ESCRS Published by Elsevier Inc.
0886-3350/$ - see frontmatter 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 surface 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 administration 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.
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PATIENTS AND METHODS
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 evaluated. 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.
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
Indentation Technique Young’s modulus, hardness, and the morphologic properties of the lens capsule surface were evaluated using a nanoindentation
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.
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 magnification. C: 6000 magnification. D: 20 000 magnification level.
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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 computer 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 properties 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,
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 conventional 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: HZ
Pmax 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 flexibility measurement for an anterior lens capsule based on the OliverPharr method. The x-coordinate indicates loading and the y-coordinate indicates depth. B: Graphic representation of an anterior lens capsule hardness measured via nanoindentation and Martens hardness method (CIT Z instrumented creed; E*Z loss modulus; EIT Z instrumented elastic modulus; Er Z reduced modulus; HIT Z instrumented hardness; HM Z Martens hardness; RIT Z relaxation instrumented).
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Figure 4. A: Indentation section. B: Significant points on the loading–unloading P-h curve. Values expressed using the OliverPharr 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 residual depth; m Z an integer at function for calculating P).
force microscope of the nanoindentation testing device. The 3dimensional images were transferred to a computer, and the surface 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 determine 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
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 population. 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 statistically 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 significantly 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
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Figure 6. Measurement of the anterior lens capsule thickness in the 3 groups using images acquired by the SEM. A: Patient with no pathology other than cataract (12 000 magnification). B: Patient with PXF syndrome (8000 magnification). C: Patients administered 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 between 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 Group
Age (y)
Female
Male
Mean ± SD
Range
11 12 13
13 12 11
69.25 G 5.07 69.33 G 5.63 69.37 G 5.19
60, 80 60, 81 61, 83
1 (cataract only) 2 (PXF syndrome) 3 (trypan blue) PXF Z pseudoexfoliation
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Figure 7. Patient age distribution for each group (PXF Z pseudoexfoliation).
ANTERIOR LENS CAPSULE ASSESSMENT WITH NANOINDENTATION
Figure 8. Elastic modulus values of the anterior lens capsule in relation 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 micromechanical 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%,
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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 nanoindentation 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 timedependent 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 keratoplasty 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
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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 significant. 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, elasticity 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 elasticity 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 biomaterials 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.
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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