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C.R. McLaughlin, S.G. Vascotto, L. Liu, L. Kuffova, J.V. Forrester, M. Hassanlou, W. Hodge, M. Griffith; An Effective Method for Monitoring the Incorporation of Tissue Engineered Corneas Following Transplantation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5615.
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© ARVO (1962-2015); The Authors (2016-present)
Regenerative medicine in the cornea includes the integration and remodelling of a biomaterial upon transplantation into a host. Because of difficulties in long–term labelling of a biomaterial, tracking host–graft integration within the corneal stroma has yet to be effectively performed. Our objective is to develop a method to label our tissue engineered corneal implants and track tissue remodelling (if any) that occurs following implantation.
Tissue engineered collagen–based hydrogels for in vitro studies were fabricated from type 1 collagen and chondroitin sulphate C and crosslinked with glutaraldehyde. Biotin was either incorporated either pre– or post–crosslinking. Samples with or without human stromal cells were maintained in culture for up to 1 month and cell viability determined via live/dead staining. Persistance of biotin labelling was determined by staining explants or histological slices using HRP–conjugated streptavidin and DAB substrates. TE corneas for transplantation were fabricated from higher collagen concentrations, crosslinked with a synthetic crosslinker. These were first tested for mechanical strength and then for suturability. Formulations deemed suturable were biotinylated and transplanted into BALB/c mice by penetrating keratoplasty (PK). Eyes were sectioned at 7d, 14d, and 30d post transplantation, and stained for biotin.
Biotin was stably incorporated into the gels, both as a pre–treatment and post–treatment and persisted for at least 1 month in vitro. Biotinylated gels stained dramatically compared to non–biotinylated samples, allowing for distinct and simple visualization. When stromal keratocytes were included within the tissue engineered collagen matrix, there was minimal evidence of cytotoxicity and normal cell proliferation. Over longer periods of culture, there was evidence of collagen remodelling surrounding individual keratocytes. TE corneas when subjected to suturing showed that high concentrations of crosslinked collagen tolerated 16 sutures without any microshearing. Following PK, host remodelling of the biomaterial was observed at later time points by differential staining patterns of the biotin tracer.
Biotinylation of collagen within TE corneal matrices is an effective method for monitoring the integration of the artificial constructs into the host’s natural cornea.
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