DOI: 10.5061/dryad.z8w9ghx9f
title: Inflation test on eye globe for: Experimental evaluation of stiffening effect induced by UVA/riboflavin corneal cross-linking using intact porcine eye globes

description: UVA/riboflavin corneal cross-linking (CXL) is a common used approach to
 treat progressive keratoconus. This study aims to investigate the
 alteration of corneal stiffness following CXL by mimicking the inflation
 of the eye under the in vivo loading conditions. Seven paired porcine eye
 globes were involved in the inflation test to examine the corneal
 behaviour. Cornea-only model was constructed using the finite element
 method, without considering the deformation contribution from sclera and
 limbus. Inverse analysis was conducted to calibrate the non-linear
 material behaviours in order to reproduce the inflation test. The corneal
 stress and strain values were then extracted from the finite element
 models and tangent modulus was calculated under stress level at 0.03 MPa.
 UVA/riboflavin cross-linked corneas displayed a significant increase in
 the material stiffness. At the IOP of 27.25 mmHg, the average
 displacements of corneal apex were 307 ± 65 μm and 437 ± 63 μm (p = 0.02)
 in CXL and PBS corneas, respectively. Comparisons performed on tangent
 modulus ratios at a stress of 0.03 MPa, the tangent modulus measured in
 the corneas treated with the CXL was 2.48 ± 0.69, with a 43±24% increase
 comparing to its PBS control. The data supported that corneal material
 properties can be well-described using this inflation methods following
 CXL. The inflation test is valuable for investigating the mechanical
 response of the intact human cornea within physiological IOP ranges,
 providing benchmarks against which the numerical developments can be
 translated to clinic.    
description: Fresh porcine eyes were obtained from a local abattoir (Morphets,
 Tan house farm, Widnes) and tested within 6-9 hours after death. Soft
 muscular tissue was removed with surgical scissors. The superior direction
 was marked and the eye globe was placed in a customized compartment for
 accurate needle insertion through the posterior pole. The internal eye
 components were removed through the posterior pole using a 14G needle. The
 needle was then lightly glued around the posterior pole and the
 intra-ocular cavity was washed with 5 to 6 ml PBS (Sigma, Dorset, United
 Kingdom). The outer surface of the globe was continually kept hydrated by
 applying PBS every 2-5 minutes. Random speckles were applied on the globe
 by lightly spraying a waterproof and fast drying black paint to facilitate
 deformation tracking in post-analysis. The prepared specimen was then
 placed into a custom-designed eye chamber filled with PBS, and transferred
 onto the inflation rig. The inflation test rig provides
 full-field observation of ocular response to uniform intraocular pressure
 (IOP) changes. The physical test equipment is fully bespoke having been
 designed and built in-house. The equipment features closed loop control
 software written in LabVIEW (version 10.0.1, RRID:SCR_014325) to regulate
 IOP while collecting real-time data by triggering cameras to take pictures
 of the globe. The obtained images are used for measurement of deformation
 across the globe. The specimen was clamped in a horizontally placed eye
 chamber with high precision real-time laser (LK-2001, Keyence, UK)
 pointing towards the apical displacement. An array of six high resolution
 digital cameras (18.0 megapixels, 550D, Canon, Tokyo, Japan) surrounding
 the eye chamber and a pressure adjusting tank was placed vertically to
 inflate the eye while taking synchronous images. The camera setup allows
 an angle of 25° within each pair and an angle of 120° between each
 set. A custom-built LabVIEW software was
 used to tightly control the pressure. The experiments started by 3
 pre-conditioning cycles. The pre-conditioning cycles were to ensure the
 eye was sitting comfortably on the needle, and the tissue behavior was
 repeatable (15). An initial pressure of 2.5 mmHg was used to balance the
 external pressure applied by PBS in the pressure chamber, and was
 therefore considered a zero-pressure point for the inflation test.
 Specimens were loaded to a maximum internal load at a medium rate of 0.55
 mmHg/s for each cycle. During each cycle the eye was allowed to relax for
 a period of 2 minutes which was obtained experimentally to allow tissue to
 fully recover to its relaxation state. The behavior of specimen in the
 final loading cycle was used for post-analysis.
 After the experiment was completed, the eye was
 removed from the test rig and dissected into anterior and posterior parts.
 Eight meridian profiles of discrete thickness measurements were selected.
 The thickness at each desired point on each meridian line was determined
 using an in-house developed Thickness Measurement Device (TMD) (LTA-HS,
 Newport, Oxfordshire, UK) which was developed by the Biomechanical
 Engineering group to measure the thickness of biological tissue. A
 vertical measurement probe was located at a height of about 30 mm above
 the centre point of the support. The probe moved down with a controlled
 velocity until it reached the surface of the tissue. By precisely knowing
 the original distance between the initial position of probe and the
 surface of support, the measured value was recorded as the thickness of
 the tissue. To
 decrease the geometrical complexity and understand the effect of CXL
 treatment on corneas where the application of interest is, we built up a
 corneal-only model by excluding the sclera part from a whole globe model.
 In this corneal model, the orphan mesh of geometry was constructed with
 Abaqus 6.13 (Dassault Systèmes Simulia Corp., Rhode Island, USA) using
 bespoke software. The 2592 elements with 8611 total nodes adopted the
 hybrid and quadratic type with triangular cross-section (C3D15H), which
 were arranged in 12 rings across the cornea surface and 3 layers through
 the thickness. Corneal apex was restrained against displacement in X- and
 Y-directions, whereas limbus was restrained in the X-, Y-, and
 Z-direction. The intraocular pressure was distributed on the posterior
 surface of the cornea. The apical displacement of the entire cornea was
 extracted by the displacement of corneal apex minus the average
 displacement of limbus in the anterior-posterior
 direction. The
 image profiles obtained were analyzed using a 2D DIC method named Particle
 Image Velocimetry (PIV) to obtain deformations on the surface of the eye
 (Figure 3) (21, 22). PIV compares an un-deformed and deformed image pairs
 of specimen surface which was speckled to present the local displacements
 within the selected subsets. Three discrete locations including corneal
 apex and limbus were measured from each camera. As only cornea was
 considered in the study, the cornea deformation was calculated by
 subtracting the average displacement of limbus in the anterior-posterior
 direction from the displacement of corneal apex.
 An in-house built software that uses Particle Swarm Optimization
 (PSO) as an optimization strategy was developed in Matlab
 (RRID:SCR_001622) to conduct the inverse analysis optimization due to its
 success in the engineering applications. PSO evaluates the fitness of the
 apical displacement between simulation and experiment and iterates over
 the different values of material parameters to decrease the error until
 the best fitness appears. The material constitutive model chosen to
 demonstrate the material behavior of ocular tissue during loading was
 Ogden model, utilized in a number of previous studies on soft
 tissues. The Ogden material model order one relies on
 two parameters of μ (shear modulus) and α (strain hardening exponent) to
 define the non-linear material behavior. The use of first order material
 model (N=1) reduced the complexity of optimization and thus the
 computational cost as a result of less variables. The values of material
 parameters α and μ represented the output of the inverse modelling process
 that resulted in the highest fitness of simulation against inflation
 experiment. The design optimization process adjusts the value of μ and α
 within the constitutive model while setting a wide lower and upper
 boundary range (lower boundary = [0.005, 50]; upper boundary = [0.2,
 200]). The error limit of RMS was set as 10%, which terminated the
 optimization once the error is lower than the limit. With these
 parameters, stress and strain could then be extracted from the numerical
 modelling results. The uniaxial-mode stress was calculated through
 obtained μ and α in Table 2, based on the previously described
 method<i> </i>and then tangent modulus was calculated
 numerically from the gradient of the resulting stress-strain
 curve.