Janice Dias

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-μm diameter) and nanoconical (10-nm radius of curvature) indenters at 2-μm/s and 15-μm/s indentation rates. Youngs 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 μm. At the tissue level, Youngs modulus (ES) showed a steep decrease at approximately 140-μm 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, Youngs modulus (EC) was significantly greater than at the tissue level; EC 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.

Impact of Hydration Media on Ex Vivo Corneal Elasticity Measurements

Objectives: To determine the effect of hydration media on ex vivo corneal elasticity. Methods: Experiments were conducted on 40 porcine eyes retrieved from an abattoir (10 eyes each for phosphate-buffered saline (PBS), balanced salt solution, Optisol, 15% dextran). The epithelium was removed, and the cornea was excised with an intact scleral rim and placed in 20% dextran overnight to restore its physiological thickness. For each hydration media, corneas were evenly divided into two groups: one with an intact scleral rim and the other without. Corneas were mounted onto a custom chamber and immersed in a hydration medium for elasticity testing. Although in each medium, corneal elasticity measurements were performed for 2 hr: at 5-min intervals for the first 30 min and then 15-min intervals for the remaining 90 min. Elasticity testing was performed using nanoindentation with spherical indenters, and Young modulus was calculated using the Hertz model. Thickness measurements were taken before and after elasticity testing. Results: The percentage change in corneal thickness and elasticity was calculated for each hydration media group. Balanced salt solution, PBS, and Optisol showed an increase in thickness and Young moduli for corneas with and without an intact scleral rim. Fifteen percent dextran exhibited a dehydrating effect on corneal thickness and provided stable maintenance of corneal elasticity for both groups. Conclusions: Hydration media affects the stability of corneal thickness and elasticity measurements over time. Fifteen percent dextran was most effective in maintaining corneal hydration and elasticity, followed by Optisol.

The effect of nicotine on the mechanical properties of mesenchymal stem cells

PURPOSE: To measure the elasticity of the nucleus and cytoplasm of human mesenchymal stem cells (MSCs) as well as changes brought about by exposure to nicotine in vitro. METHODS: MSCs were synchronized to the G(0) stage of the cell cycle through serum deprivation techniques. The cells were then treated with medium containing nicotine (0.1 µM, 0.5 µM, and 1 µM). Atomic force microscopy was then used to measure the Youngs modulus of both the nucleus and cytoplasm of these cells. RESULTS: For both unsynchronized and synchronized cells, the nucleus was softer than the cytoplasm, although this difference was not found to be statistically significant. The nucleus of cells treated with nicotine was significantly stiffer than the control for all concentrations. The cytoplasm was significantly stiffer in nicotine-treated cells than in control cells for the 0.5 µM and 1.0 µM concentrations only. CONCLUSIONS: The results of this study could suggest that nicotine affects the biophysical properties of human MSCs in a dose-dependent manner, which may render the cells less responsive to mechanoinduction and other physical stimuli.

Quality of corneal lamellar cuts quantified using atomic force microscopy

Purpose To quantify the cut quality of lamellar dissections made with the femtosecond laser using atomic force microscopy (AFM). Setting Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA. Design Experimental study. Methods Experiments were performed on 3 pairs of human cadaver eyes. The cornea was thinned to physiologic levels by placing the globe, cornea side down, in 25% dextran for 24 hours. The eyes were reinflated to normal pressures by injecting a balanced salt solution into the vitreous cavity. The eyes were placed in a holder, the epithelium was removed, and the eyes were cut with a Visumax femtosecond laser. The energy level was 180 nJ for the right eye and 340 nJ for the left eye of each pair. The cut depths were 200 μm, 300 μm, and 400 μm, with the cut depth maintained for both eyes of each pair. A 12.0 mm trephination was then performed. The anterior portion of the lamellar surface was placed in a balanced salt solution and imaged with AFM. As a control, the posterior surface was placed in 2% formalin and imaged with environmental scanning electron microscopy (SEM). Four quantitative parameters (root‐mean‐square deviation, average deviation, skewness, kurtosis) were calculated from the AFM images. Results From AFM, the 300 μm low‐energy cuts were the smoothest. Similar results were seen qualitatively in the environmental SEM images. Conclusion Atomic force microscopy provided quantitative information on the quality of lamellar dissections made using a femtosecond laser, which is useful in optimizing patient outcomes in refractive and lamellar keratoplasty surgeries. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.