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Corinna Petsch, Ursula Schlotzer-Schrehardt, Markus Frey, Friedrich E. Kruse, Björn Bachmann; Cross-linked Variants Of A Novel Semi-synthetic Collagen Substitute For The Reconstruction Of The Surface. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6037.
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To prove the suitability and biocompatibility of defined cross linked collagen membranes in comparison to non-cross linked variants for corneal surface reconstruction.
Cross linked variants of a type I collagen membrane (RESORBA, Germany) were generated by exposure to Riboflavin (2 different concentrations) and UV-light. Isolated human oral mucosa and limbal epithelial stem cells were clonally enriched on 3T3 feeder cells and subcultivated on both types of membranes. The cell cultures were examined concerning cell adhesion, proliferation, cellular phenotype and differentiation by light and electron microscopy and immunohistochemistry. Biocompatibility was evaluated following superficial or intrastromal corneal transplantation in New Zealand White rabbits. Corneas were excised at various time points. Cell cultures and excised corneas were analyzed by light and electron microscopy and immunohistochemistry.
All collagen membranes supported adhesion, proliferation and differentiation of oral mucosa and limbal stem cells and formation of multilayered epithelial cell sheets. Cell sheets on riboflavin containing membranes were thicker and more regular. Independent of the riboflavin concentration, cultured cells expressed the differentiation markers K3/12 and K4 throughout all cell layers, the stem cell markers p63 and Integrinβ1 were immunolocalized to basal cells. After superficial implantation closure of the epithelium was achieved within 7-10 days. Corneal transparency was obtained after 3-4 weeks. Cross linked membranes showed a lower rate of degradation. Histological analysis exposed overgrowth by corneal epithelium, degradation and repopulation by stromal keratocytes. After stromal implantation all membranes showed weak clinical signs of degradation during the first month which was confirmed on the histological and ultrastructural level.
Cross linked variants of a collagen membrane are suitable for the cultivation of stem cells and show high biocompatibility. For the reconstruction of the corneal surface the collagen membranes may represent a standardized alternative to amniotic membrane.
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