Effects of prior freezing or drying on the swelling behaviour of the bovine cornea.

Background: Frozen or dried corneal grafts are commonly used for stromal transplantation such as lamellar keratoplasty (full or partial thickness), keratophakia, epikeratophakia. Structural properties are important for the final optical results of these surgeries but the effects of freezing/thawing and drying/rehydration on the properties of the stroma are known little compared with the corneal endothelium, mainly because of lack of non-invasive technique to evaluate the stromal structure. This study aimed to investigate the swelling and structural properties of the bovine corneal stroma following freezing or drying by X-ray diffraction which was a non-invasive technique and could give ultra-structural information in hydrated tissues.

Methods: Bovine corneas were either frozen at -40 degrees C or dried to constant weight in a dessicator over silica gel. Swelling was carried out by placing the corneas into dialysis tubing and equilibrating them against various concentrations of polyethylene glycol (PEG) to obtain a range of tissue hydrations. This method minimises the loss of soluble tissue components during the swelling process. Synchrotron X-ray diffraction was used to measure the average intermolecular spacing, the interfibrillar spacing and the fibril diameter as a function of hydration. Changes in light scattering were detected using a microdensitometer.

Results: Freezing and thawing of the cornea caused an increase in light scattering by 63.9% at tissue hydration (H) = 3.4, and by 50.0% at H = 4.9. Repeated freezing and thawing causes further increased by 38.9% at the second time and another 36.0% at the third time (P < 0.05). There was a tendency for both the frozen and the dried corneas to lose some swelling ability, achieving hydrations respectively of 10% and 18% below those of fresh corneas at 0 PEG. There were no changes in the fibril diameters, interfibrillar or intermolecular spacings as measured by X-ray diffraction in the equilibrated fresh, pre-frozen and pre-dried corneas.

Conclusions: The increase in light scattering and the loss of swelling ability after freezing and thawing probably results from structural changes following the close association of the collagen molecules and fibrils whilst the tissue is in the dry or frozen state. Some unknown changes in the extracellular matrix between the collagen fibrils may also play a role in the light scattering. The equilibration technique may improve the quality of rehydrated corneal graft or lenticules used for corneal surgeries.