Abstract
Purpose: :
We examined clarity, function and biocompatibility of the porcine collagen based tissue engineered (TE) cornea substitutes in mice. We also evaluated the local and systemic immune response of mice to the biomaterial.
Methods: :
Orthotopic TE corneas (80 µm thick and 2mm in diam.) were fabricated from porcine collagen cross–linked with EDC/NHS. They were transplanted into BALB/c mice using the full thickness keratoplasty procedure with running sutures. Mice were examined under an operating microscope twice weekly for corneal opacification. At different time points (6h, 2d, 6d, 12d, 24d, 48d, 60d, 90d and 120d) after grafting, cryostat sections of the grafted eyes were immunostained with a panel of monoclonal antibodies (CD4, CD8, CD11b, F4/80 and Gr–1). Cells from submandibular draining lymph nodes (SMDLN) of grafted mice were labelled with anti CD3, CD4, CD8, CD28, CD11c, CD40, CD86 and MHC class II antibodies and analysed by flow cytometry.
Results: :
The collagen–EDC/NHS TE corneas remained clear for 10–12 days, but became progressively opaque from 13–20 days post graft, in association with a significant inflammatory response. After 3 weeks, the grafted TE corneas became less opaque and corneal neovascularization receded gradually. By 120d considerable clearing of the corneal opacification had occurred. Gr–1, CD11b and F4/80 positive cells were detected in recipient cornea from 6h, but no CD4+ and CD8+ T cells. The infiltrating cells number increased with the time, forming a retro–corneal membrane, and peaked at 12d. The collagen polymer was surrounded by CD11b+ and Gr–1+ cells and some degradation of the graft was observed. At 24d, the retro–corneal membrane receded and there were less infiltrating cells compared to 12d. At 60d and 90d, infiltrating cells were confined to the wound edge and around the sutures. Only a few infiltrating cells were present in the cornea after 120d and most were CD11b+ cells. No significant T and B cell activation was observed in SMDLN cells by flow cytometry.
Conclusions: :
Porcine collagen–EDC/NHS TE corneas were tolerated well in murine recipients, causing mainly a self–limiting local innate immune response. Furthermore, flow cytometry analysis did not show any significant T and B cell sensitization for these TE corneas.
Keywords: cornea: basic science • transplantation • immune tolerance/privilege