Elastic modulus and collagen organization of the rabbit cornea: Epithelium to endothelium

doi:10.1016/j.actbio.2013.09.025

Acta Biomaterialia

Volume 10, Issue 2, February 2014, Pages 785-791

https://doi.org/10.1016/j.actbio.2013.09.025 Get rights and content

Abstract

The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet’s membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57 ± 0.29 kPa (mean ± SD); ABM, 4.5 ± 1.2 kPa, anterior stroma, 1.1 ± 0.6 kPa; posterior stroma, 0.38 ± 0.22 kPa; DM, 11.7 ± 7.4 kPa; and endothelium, 4.1 ± 1.7 kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.

Introduction

The cornea functions as a protective barrier while simultaneously transmitting and refracting light at the anterior aspect of the eye. In order to complete these diverse functions, the human cornea is composed of six different histological layers, each with a unique structure: the epithelium, anterior basement membrane, Bowman’s layer, stroma, Descemet’s membrane and endothelium. The regenerative, stratified, nonkeratinized anterior epithelium consists of a superficial squamous, polygonal wing and a single layer of columnar basal cells. The basal cells participate in the elaboration of an anterior basement membrane, a specialized extracellular matrix comprising specific proteins and possessing a complex three-dimensional architecture. Bowman’s layer is a thin, acellular zone of compacted collagen fibers that resides subjacent to the anterior epithelium and its associated basement membrane. It is absent in all non-primate laboratory animals, including rabbits. The bulk of the corneal thickness (approx. 90%) is attributable to the stroma, which is composed of sheet-like, fibrillar, parallel bundles of collagen and a sparse population of interconnected keratocytes located between the lamellae. Descemet’s membrane, similar to the anterior basement membrane, is a specialization of the extracellular matrix that connects the endothelial cells to the underlying stroma. The endothelium is composed of a single layer of hexagonal cells that participate in the production of Descemet’s membrane. In addition to each layer possessing a distinct structure and chemical composition, the biomechanical properties of each layer are also unique.

Our group has previously reported the local elastic modulus of the human corneal basement membranes, the anterior stroma and Bowman’s layer as determined by atomic force microscopy (AFM) [1], [2]. Interestingly, the elastic modulus for the more compact Descemet’s membrane was greater than the anterior basement membrane at 47 and 8 kPa, respectively [1]. A similar observation was found for the more compact Bowman’s layer in comparison to the anterior stroma, with elastic moduli of 110 and 33 kPa, respectively [2]. Despite the wide use of the rabbit to study corneal biology and wound healing, as well as in the development of ocular therapeutics and devices, the biophysical attributes of the rabbit cornea have not been fully characterized. Specifically, only the bulk elastic modulus of rabbit cornea has been reported prior to and following photodynamic cross-linking at 11 and 20 MPa, respectively, using tensile testing [3]. As indicated in a recent review article, elastic modulus values are strongly dependent on the testing method employed and values obtained by tensile testing do not directly correlate with values obtained using AFM [4]. Knowledge of the elastic modulus of the rabbit cornea would inform the design and fabrication of biomimetic substrates in order to improve the relevance of results obtained from in vitro investigations and provide critical data for development of keratoprosthetics with enhanced performance. Thus, the purposes of this study were: (i) to determine the elastic modulus of the epithelium, anterior basement membrane, anterior and posterior stroma, Descemet’s membrane and endothelium of the rabbit cornea; (ii) to characterize the collagen fiber organization of the rabbit cornea in three dimensions using nonlinear optical high-resolution macroscopy (NLO-HRMac) and correlate these finding with the biomechanical data; and (iii) to determine the influence of storage in Optisol, a commercially available preservative commonly used for human donor corneal tissue, on the elastic modulus of the rabbit corneal layers.

Section snippets

Animals

Sixteen Dutch Belted female rabbits (Covance, Princeton, NJ), with a mean ± SD body weight and age of 2.1 ± 0.2 kg and 1.1 ± 0.2 years, respectively, were utilized in this study. All aspects of the study were approved by the Institutional Animal Care and Use Committee of the University of California-Davis and were performed according to the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. A complete ophthalmic examination (slit lamp examination and

Results

All corneas appeared healthy and free of disease following ophthalmic examination and no corneas retained fluorescein stain, indicating an intact epithelium. The mean (±SD) intraocular pressure was 15.9 ± 4.8 and 16.6 ± 4.8 μm for the left and right eyes, respectively, and there was no significant difference (p = 0.38) between the two eyes. The mean (±SD) central corneal thickness was 364 ± 17 and 360 ± 14 μm for the left and right eyes, respectively, and there was no significant difference (p = 0.49)

Discussion

This study determined the local stiffness of six distinct spatial regions of the rabbit cornea using AFM nanoindentation. We chose to use a spherical AFM tip with a radius of approximately 5 microns to minimize the strain field and to ensure that the tip size was larger than that of topographic features observed in the corneal stroma and membranes. There was some variability in elastic modulus between individuals at all corneal locations despite the rabbits being of the same breed and gender

Conclusions

The elastic modulus of each layer examined in the rabbit cornea is unique and related to its intrinsic structure. The rabbit cornea is markedly softer than the human cornea primarily due to differences in collagen fiber arrangement and spacing. Keratoprosthetics that incorporate mechanical properties from one species may behave differently in another species with a different corneal elastic modulus. Knowledge of the mechanical properties of each corneal layer is a crucial step in the design of

Conflicts of interest

The authors have no conflicts of interest.

Acknowledgments

The authors thank Dr. Julie Last and JL Analytics, as well as Bradley Shibata, Rebecca Seraphin and Sarah DeRemer, for assisting with this study. This work was funded by the National Institutes of Health National Eye Institute KO8EY021142 (S.M.T.), R01EY019970 (C.J.M.), R01EY016134 (C.J.M.), P30EY12576 (UC Davis) and an unrestricted gift from Research to Prevent Blindness (UC Davis).

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There are more references available in the full text version of this article.

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Acta Biomaterialia

Abstract

Introduction

Section snippets

Animals

Results

Discussion

Conclusions

Conflicts of interest

Acknowledgments

References (21)

Determining the mechanical properties of human corneal basement membranes with atomic force microscopy

J Struct Biol

Compliance profile of the human cornea as measured by atomic force microscopy

Micron

The effect of biophysical attributes of the ocular trabecular meshwork associated with glaucoma on the cell response to therapeutic agents

Biomaterials

The role of substratum compliance of hydrogels on vascular endothelial cell behavior

Biomaterials

Atomic force microscopic study of the human cornea following excimer laser keratectomy

Exp Eye Res

The thermodynamic response of soft biological tissues to pulsed ultraviolet laser irradiation

Biophys J

Long-term biomechanical properties of rabbit cornea after photodynamic collagen crosslinking

Acta Ophthalmol

Indentation versus tensile measurements of Young’s modulus for soft biological tissues

Tissue Eng Part B Rev

Nanoscale topography of the corneal epithelial basement membrane and Descemet’s membrane of the human

Cornea

Comparative in vivo confocal microscopical study of the cornea anatomy of different laboratory animals

Curr Eye Res

Cited by (99)

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Efficient reduction of the scrolling of Descemet membrane endothelial keratoplasty grafts by engineering the medium

Successful rebubbling of spontaneous Descemet's membrane detachment eighteen years after penetrating keratoplasty for keratoconus

Effect of mechanical stretching and substrate stiffness on the morphology, cytoskeleton and nuclear shape of corneal endothelial cells

Mimicking the Natural Basement Membrane for Advanced Tissue Engineering

ECM stiffness modulates the proliferation but not the motility of primary corneal keratocytes in response to PDGF-BB