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Multiscale Integrative Approach for Corneal Biomechanics to Assess Corneal Crosslinking

Periodic Reporting for period 1 - MIMetiCO (Multiscale Integrative Approach for Corneal Biomechanics to Assess Corneal Crosslinking)

Reporting period: 2018-09-01 to 2020-08-31

Degradation or loss of vision greatly impacts quality of life. Almost 250 million people worldwide are visually impaired, which in 2009 had an estimated economic impact of $268.8B. The shape and optical properties of the cornea are determined by the mechanical balance between intraocular pressure and the internal stresses of corneal tissue. Interventions such as refractive surgery and pathologies such as keratoconus (KC) can alter this balance and compromise visual acuity. Mechanical imbalance can be caused by the ablation of corneal tissue in laser refractive surgery, or by the loss of the organization of corneal collagen in incurable KC disease. In both cases, a corneal protrusion, ectasia, can occur due to the mechanical weakening of the tissue.

Corneal crosslinking (CXL) is a widespread clinical treatment that affects corneal tissue without inducing mechanical weakening. In CXL, ultraviolet (UV) light activates a photosensitizer (riboflavin) generating oxygen species that induce covalent bonds (crosslinks) between corneal collagen fibrils and within the proteoglycan coating surrounding them. Due to this micromechanical modification, overall mechanical stiffness increases. At present, CXL is used to halt KC’s progression, and is regarded as the future of noninvasive refractive surgery. However, CXL micro/nano-mechanisms are still not completely understood. Treatments often rely on statistical and experimental nomograms (look-up tables), and little patient-specific information is taken into account. Not surprisingly, outcomes such as under/over-correction of visual acuity, or the progression of KC can ensue.

MIMetiCO aims at obtaining a better understanding and characterization of corneal biomechanics before and after CXL, which includes morphological, biochemical, and mechanical information at different scales. Based on this characterization, MIMetiCO will align with H2020’s societal challenges regarding in silico and personalized medicine by developing an experiment-based, computational model to test potential CXL strategies.
WORK PERFORMED
A) Mechanical and microstructural characterization of the corneal tissue
- Uniaxial testing
The broad idea behind mechanical testing is to cut strips of biological soft tissue in order to test their mechanical reaction when one of the ends is fixed in a grip and the other is pulled from. After the CXL treatment, samples present a higher stress for the same displacement. Meaning that the CXL treatment is increasing the bounds between collagen fibers and, thus, stiffening the mechanical response of the sample.
- Nanoindentation
Nanoindentation tests use a compressive load to characterize the mechanical response of a material. In order to determine a meaningful set of mechanical properties, it is necessary to create a computational model that is able to reproduce the real experiment and to run an optimization procedure. Our results suggest that corneas present a natural heterogeneous stiffness, being stiffer at the center and softer towards the periphery. After crosslinking, this heterogeneity is amplified by increasing up to three times the corneal stiffness at the corneal center.
- Two-photon confocal microscopy (SHG) and image processing
Two-photon confocal microscopy was used in order to determine the microstructural organization of the collagen network. In particular, the direction of the fibers and their density was studied in a reference volume. Preliminary data in porcine corneas was analyzed using OrientationJ, a free plugin for ImageJ. Data showed that fibers are ‘shorter’ and more densely packed close to the anterior surface while they are ‘longer’ and less packed close to the posterior surface.

B) Biochemical characterization of the corneal tissue
The more homogeneous the sample is before the reduction, the more precise the results will be since no crosslinks are lost during hydrolysis which were not reduced. By using the digestive effects of enzymes, it was tested whether or not a homogeneous sample mixture can be achieved. Unfortunately, the detection of crosslinks was not achieved with this approach.

C) Multiscale in silico modelling of the corneal tissue
Collagen fibers at the microstructure were modelled using the beam’s theory. The main advantage of this approach is that the simulation of the collagen fibers at the microstructural level is reduced to a semianalytical problem which is faster to solve. To model the interaction between the fibers, we assumed a perfect rigid connection between both fibers once they are in contact.
Once the microstructural model for the collagen fibers was developed, it was necessary to use a macroscopic material model for the corneal tissue in which to embed the microstructural behavior of the collagen fibers (multiscale approach). Numerical material models used to simulate biological tissues assume that the network of collagen fibers is embedded in an extracellular matrix. In this regard, we chose two well-known material models to work with: Holzapfel-Gasser-Ogden and the Markert. Both material behaviors were coded in a Fortran user subroutine for Abaqus and properly validated

EXPLOITATION AND DISSEMINATION
- 12 conference proceedings at the European Society of Biomechanics (ESB), the Swiss Society of biomedical engineering (SSBE) and ARVO of which 6 were cancelled due to COVID-19 outbreak
- One gold-acess scientific publication (https://doi.org/10.1167/tvst.9.11.26)
- Tag der offenen Tür (Open day at SITEM, Friday 30th August 2019). open day at the institute with more than 2,000 visitors
- Elevator pitch at the course Innosuisse Foundationand at the master of business administration of the University of Zürich
- Project’s webpage (http://mimetico-eu.com) which will be active at least until September 2022.
- Dissemination in the institute’s social medial account (LinkedIn)
There are some clear contributions that should be outlined:
1. Nanoindentation experiments: the use of finite element simulations and optimization procedures in order to determine the mechanical properties of the corneal tissue hadn’t been done before. This combination of advance computational simulations and nanoindentation experiments is key in order to characterize the mechanical properties of soft tissues as it is not currently possible to do it otherwise.
2. Biochemical analysis: it is one of the few studies done in this regard since the 70s. Due to the lack of experimental information or protocols, we had to develop a protocol from scratch (high investment of time and resources).
3. Mathematical modelling of 2 coupled fibers at the microscale level: it is the first time to be developed. This is of high interest for the community as tissues in Ophthalmology and Cardiovascular research present 2 families of fibers. This will open a new research line in this regard.

As it is the first time of performing this kind of research in Ophthalmology (multiscale simulations) is difficult to gauge how they will be accepted by the community or its potential economical and societal impact. Moreover, due to the COVID-19 outbreak, it was difficult to tackle all the tasks of this project (e.g. the use of human samples). Nevertheless, all the remaining tasks are ongoing and we are confident we will cover all the objectives proposed even after the finalization of the project.