Nano-Scale Stiffness and Collagen Fibril Deterioration: Probing the Cornea Following Enzymatic Degradation Using Peakforce-QNM AFM

Under physiological conditions, the cornea is exposed to various enzymes, some of them have digestive actions, such as amylase and collagenase that may change the ultrastructure (collagen morphology) and sequentially change the mechanical response of the cornea and distort vision, such as in keratoconus. This study investigates the ultrastructure and nanomechanical properties of porcine cornea following incubation with α-amylase and collagenase. Atomic force microscopy (AFM) was used to capture nanoscale topographical details of stromal collagen fibrils (diameter and D-periodicity) and calculate their elastic modulus. Samples were incubated with varying concentrations of α-amylase and collagenase (crude and purified). Dimethylmethylene blue (DMMB) assay was utilised to detect depleted glycosaminoglycans (GAGs) following incubation with amylase. Collagen fibril diameters were decreased following incubation with amylase, but not D-periodicity. Elastic modulus was gradually decreased with enzyme concentration in amylase-treated samples. Elastic modulus, diameter, and D-periodicity were greatly reduced in collagenase-treated samples. The effect of crude collagenase on corneal samples was more pronounced than purified collagenase. Amylase was found to deplete GAGs from the samples. This enzymatic treatment may help in answering some questions related to keratoconus, and possibly be used to build an empirical animal model of keratoconic corneas with different progression levels.

Effects of hydrogen peroxide solution on the biomechanics of porcine cornea

Objectives: To determine the biomechanical changes of porcine corneas after the application of hydrogen peroxide(H2O2) solution. Methods: Fifty-five porcine eyeballs with similar sizes were divided into 11 groups based on the H2O2 application. The eyeballs were treated with the following concentrations of H2O2 solution: 1 mol/L, 500 mmol/L, 250 mmol/L, 125 mmol/L, 62.5 mmol/L, 31.25 mmol/L, 15.63 mmol/L, 7.81 mmol/L, 3.91 mmol/L, 0.9% saline, or blank. The eyeballs were immersed into the solution for 30 min. The biomechanics of each cornea in the different groups was determined soon after the indentation and tensile tests. We calculated the average Young’s modulus of the different groups to determine the effects of H2O2 solution on porcine corneas. The comparison between the groups was conducted using ANOVA analysis. Moreover, the safety of each concentration of H2O2 solution on the corneal tissues was determined by histopathological examination. Results: The Young’s modulus was significantly different among all the groups ( p = 0.003). The modulus was the highest in the group treated with 3.91 mmol/L H2O2 and it was significantly different from that in the group treated with 0.9% saline or the blank group, for both the indentation and tensile tests. Histopathological examination showed that H2O2 at a concentration of ⩾62.5 mmol/L damaged the epithelium, stroma, or both, while H2O2 at a concentration ⩽31.25 mmol/L did not change the morphology of the epithelium or stroma. Conclusions: Treatment with 3.91 mmol/L H2O2 solution can safely and effectively increase the biomechanical strength of the cornea.

Modified acellular porcine corneal matrix in deep lamellar transplantation of rabbit cornea

This study presents to develop a modified acellular porcine corneal matrix (MAPCM) to maintain high transparency, stability and biocompatibility as a rabbit deep cornea replacement using 1-ethyl-3–(3-dimethylaminopropyl)-carbodiimide crosslinking and a mild decellularization technique. Scaffolds are translucent and remain higher amount of glycosaminoglycans after decellularization than acellular porcine corneal matrix (APCM). Enzymatic degradation kinetics and mechanical properties of scaffolds are regulated by 1-ethyl-3–(3-dimethylaminopropyl)-carbodiimide -crosslinking density. The porous structure and ultrastructure of collagenous lamellae are maintained, and the pore size of MAPCM crosslinked with 0.5% (w/v) 1-ethyl-3–(3-dimethylaminopropyl)-carbodiimide is 13.26 ± 1.65 µm, similar to that of normal porcine cornea. The transmittance of MAPCM gets 79.1 ± 0.45 to 92.7 ± 1.4% in the visible light range. Results from a CCK-8 assay indicate that MAPCM gets higher cell proliferation rate of rabbit corneal stroma cells than APCM. Since collagen fibres structural integrity and regularity of MAPCM are retained after crosslinking, the opacity and stability of MAPCM are better than those of APCM within 4 weeks of animal implantation. In addition, there is no indication of an immune response or neovascularization in or around the transplanted disc. These results reveal that MAPCM may be a more suitable scaffold for corneal substitute construction.

Development of a topical tissue cross-linking solution using sodium hydroxymethylglycinate (SMG): viscosity effect

Hyperviscosity agents are commonly used in ophthalmic formulations for improving corneal drug penetration by increasing tissue contact time. One such viscosity agent is hydroxypropyl methylcellulose (HPMC). HPMC has been used in riboflavin solutions for photochemical UVA cross-linking (CXL). Sodium hydroxymethylglycinate (SMG) is a small molecule formaldehyde releaser that can function as a therapeutic tissue cross-linker for corneal and scleral applications. The present study was undertaken in order to study formulation factors using HPMC and SMG that could positively influence the cross-linking effect in these ocular tissues. Formulations containing 10 mM SMG and 100 mM sodium bicarbonate were prepared with varying HPMC concentrations from 0 to 4.4%. Their cross-linking effects on porcine and rabbit eyes were measured using differential scanning calorimetry (DSC), expressed as the change/difference in melting temperature (ΔTm) compared with the control. SMG in 4.4% HPMC solution resulted in ΔTm of 6.3 ± 1.21, while other concentration showed no differences in Tm shift on porcine cornea. In ex vivo rabbit cornea, there was a trend toward an increasing cross-linking effect with higher viscosity albeit mild differences. While a significant Tm shift was observed in porcine and rabbit sclera, there was no difference in effect of cross-linking between four HPMC concentrations. Increasing the HPMC concentration does not negatively affect the cross-linking efficacy attributed by SMG and could still be a positive cross-linking enhancer by virtue of increasing tissue contact time in a dynamic biological system. This information will be useful for planning further animal and human studies.