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

Abstract

The ability to accurately determine the elastic modulus of each layer of the human cornea is a crucial step in the design of better corneal prosthetics. In addition, knowledge of the elastic modulus will allow design of substrates with relevant mechanical properties for in vitro investigations of cellular behavior. Previously, we have reported elastic modulus values for the anterior basement membrane and Descemet’s membrane of the human cornea, the surfaces in contact with the epithelial and endothelial cells, respectively. We have completed the compliance profile of the stromal elements of the human cornea by obtaining elastic modulus values for Bowman’s layer and the anterior stroma. Atomic force microscopy (AFM) was used to determine the elastic modulus, which is a measure of the tissue stiffness and is inversely proportional to the compliance. The elastic response of the tissue allows analysis with the Hertz equation, a model that provides a relationship between the indentation force and depth and is a function of the tip radius and the modulus of the substrate. The elastic modulus values for each layer of the cornea are: 7.5 ± 4.2 kPa (anterior basement membrane), 109.8 ± 13.2 kPa (Bowman’s layer), 33.1 ± 6.1 kPa (anterior stroma), and 50 ± 17.8 kPa (Descemet’s membrane). These results indicate that the biophysical properties, including elastic modulus, of each layer of the human cornea are unique and may play a role in the maintenance of homeostasis as well as in the response to therapeutic agents and disease states. The data will also inform the design and fabrication of improved corneal prosthetics.

Impact of Hydration Media on Ex Vivo Corneal Elasticity Measurements

“…For this reason, researchers use various media to maintain corneal hydration during mechanical testing; such media include saline solutions like PBS or HBSS 9, 23, 28, 29, 33 , Dextran solutions 30–32, 40–42 , oils 14, 36 , as well as commercial ophthalmic solutions 22, 24 . Although numerous studies have been conducted to investigate the effect of different media on corneal thickness and swelling changes over time 40–46 , only a few studies looking into the impact of corneal hydration solutions on corneal biomechanical response have been published 27, 47 .…”

Robust and Artifact-Free Mounting of Tissue Samples for Atomic Force Microscopy

“…After mounting, force versus indentation curves were obtained as previously described (11,13,14) using an MFP-3D Bio AFM system (Asylum Research, Goleta, CA) and silicon nitride cantilevers (PNP-TR-50, NanoWorld, Neuchâtel, Switzerland) with attached borosilicate microspheres (radius = 4–6 μm, Thermo-Fisher Scientific). Each sample was measured at 4–6 locations with 4–7 curves collected at each location.…”

Cellular Stiffness as a Novel Stemness Marker in the Corneal Limbus

“…Corneal epithelial cells grown on low-stiffness substrates exhibited the early-differentiation marker cytokeratin 19, whereas cells grown on high-stiffness substrates expressed the late-differentiation markers cytokeratins 3 and 12 (39,40). Furthermore, previous studies indicated that the basement membrane is softer than the apical Bowman's layer (41) and that limbal tissue is softer than central cornea tissue (42). Since cells are known to modulate F-actin organization and thereby alter their stiffness in response to the stiffness of the underlying substrate (43), the finding that the limbal niche is softer than the central cornea niche supports the contention that the Young's modulus results reflect differences between the in vivo mechanical environments of the stem-like and differentiated cell types.…”