Multiscale investigation of the depth-dependent mechanical anisotropy of the human corneal stroma

Purpose. To investigate the depth-dependent mechanical anisotropy of the human corneal stroma at the tissue (stroma) and molecular (collagen) level by using atomic force microscopy (AFM). Methods. Eleven human donor corneas were dissected at different stromal depths by using a microkeratome. Mechanical measurements were performed in 15% dextran on the surface of the exposed stroma of each sample by using a custom-built AFM in force spectroscopy mode using both microspherical (38-?m diameter) and nanoconical (10-nm radius of curvature) indenters at 2-?m/s and 15-?m/s indentation rates. Young's modulus was determined by fitting force curve data using the Hertz and Hertz-Sneddon models for a spherical and a conical indenter, respectively. The depth-dependent anisotropy of stromal elasticity was correlated with images of the corneal stroma acquired by two-photon microscopy. Results. The force curves were obtained at stromal depths ranging from 59 to 218 ?m. At the tissue level, Young's modulus (E<inf>S</inf>) showed a steep decrease at approximately 140-?m stromal depth (from 0.8 MPa to 0.3 MPa; P = 0.03) and then was stable in the posterior stroma. At the molecular level, Young's modulus (E<inf>C</inf>) was significantly greater than at the tissue level; E<inf>C</inf> decreased nonlinearly with increasing stromal depth from 3.9 to 2.6 MPa (P = 0.04). The variation of microstructure through the thickness correlated highly with a nonconstant profile of the mechanical properties in the stroma. Conclusions. The corneal stroma exhibits unique anisotropic elastic behavior at the tissue and molecular levels. This knowledge may benefit modeling of corneal behavior and help in the development of biomimetic materials.