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C. J. Connon, N. Koizumi, M. Liles, N. Okumura, B. Caterson, S. Kinoshita; Cryopreservation of Corneal Tissue Combined With Alginate Encapsulation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1970. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Cryopreserved corneal tissue has been widely used for lamellar keratoplasty to replace opaque corneal stroma with a healthy lamellar graft. Visual recovery after surgery is greatly influenced by the preservation of a structurally normal corneal stroma, which relies on the controlled organisation of collagen fibrils and their association with corneal proteoglycans (PGs). This study was conducted to investigate whether or not the encapsulation of corneal tissue in alginate prior to cryopreservation affects the ultrastructure of the preserved corneal stroma.
Fresh rabbit corneas were encapsulated in 1.2% sodium alginate and frozen at -20°C for 28 days in OptisolGS ®. As controls, corneas frozen in OptisolGS® without alginate treatment, as well as fresh corneas without freezing, were used. After the cryopreservation period samples were defrosted and the collagen fibril organisation was studied by transmission electron microscopy (TEM) with Cupromeronic blue used to visualise PG-staining complexes.
Pre-treatment of the corneal tissues in alginate had no effect on collagen fibril diameter (alginate =32nm ±3.4, untreated = 32nm ±3.3), fibril spacing or orientation as evidenced by TEM. Differences in PG precipitates in the stroma were observed, however, with the alginate-treated samples possessing PGs in between the collagen fibrils (49.7nm ±9.9), whereas the frozen control tissues displayed larger and longer PG staining complexes (106.5nm ±24.9, with a significance of p< 0.001 (Students t-test)).
Pre-treatment of corneal buttons by alginate encapsulation before freezing can significantly reduce structural disruption to the extracellular matrix in terms of PG staining patterns. As PGs are known to play an important role in corneal fine structure and transparency we hypothesise that by better preserving the normal PG composition in corneal buttons prior to transplantation a quicker visual recovery might be possible. This technique to retain corneal matrix structure during preservation might be also applicable to cryopreserved corneas for tissue engineering.
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