| BackgroundA healthy ocular surface with a steady cornea lacrimal film is the histologic and anatomic cornerstone to maintain the cornea physiological function. The integrity and regular function of the corneal epithelium are vital for maintaining useful vision. the kainogenesis of corneal epithelium relies on a sound physiological function of cornea stem cells, as these stem cells take places of dead and ablated cells through differentiation and proliferation. As the source of proliferation of corneal epithelium, the limbal stem cells maintain the stabilization of ocular surface and provide with transparency of cornea. Inversely, opacity even corneal-blind will display when the function of limbal stem cells is deficient. Some common corneal diseases, such as chemical and thermal injuries, aniridia, Stevens-Johnson syndrome (SJS), often induce persistent epithelial defects, chronic inflammation, opacity of the cornea, and neovascularization, resulting in loss of visual acuity severely. For severe ocular surface diseases, the most recent treatment involves the use of allograft and autograft transplantation of stem cells. However, the autograft stem cell material is of exiguity, it also holds potential risk to the other healthy eye, and it is not suitable to use in both eyes involved. Meanwhile, allograft often leads to fail because of graft rejection. So, it is the key point for resolving this problem to find a kind of cells easy to get and with sufficient quantity to replace the corneal limbal stem cells for application of transplantation.AimThe aim of the present experiment is to use bone marrow mesenchymal tissue stem cells as seed cells to induce these cells to differentiate to the corneal limbal stem cells, to construct a corneal epithelial graft with amniotic membrane (AM). and to observe the therapeutic effect after transplantation of the corneal epithelial graft on an animal model of alkali chemical injury in rats.MethodsPart 1:Construction of a corneal epithelial graft with induced bone marrow mesenchymal stem cells and amnion(1) MSCs of adult rats were isolated and purified by density gradient centrifugation combined with an attachment culture method. The morphology of MSCs cultured in vitro were observed and detected by immunofiuorescence stainings and scanning electronic microscopy.(2) The transdifferentiation of MSCs into corneal epithelial cells was induced in vitro by co-cultured with corneal stromal fibroblasts and the cells were observed with morphology and scanning electronic microscopy. The expression of CK12, a marker for corneal epithelial cells, was identified by immunofluorescent staining.(3) Amnion preparation: The amnion was obtained from newly healthy delivery and treated with trypsin.(4) The corneal epithelial graft was constructed by the induced MSC cells seeding and growing on the amnion and examined with H.E. and immunofiuorescence staining.Part 2:Application of the corneal epithelial graft in the ocular surface reconstruction(1) A rat model of corneal alkali burn injury was established.(2) All rats (n=36) were randomly assigned to three groups: control group, amniotic group and corneal epithelial graft group, 12 each. Transplantation with a corneal epithelial graft or only amnion on the burned cornea was performed 2 weeks after injury. The corneal status was evaluated by slit lamp examination at various time after transplantation. Using a scale system the difference of corneal status among 3 groups at 4 and 10 weeks after transplantation was compared with statistical analysis. The cornea was obtained and observed with H.E. staining, immunohistochemical staining at 2, 4 and 10 weeks and con-focal laser microscopy at 10 weeks after transplantation.ResultsPart 1:Construction of a corneal epithelial graft with induced bone marrow mesenchymal stem cells and amnion(1) Immunofluorescent staining and scanning electronic microscopy indicated that the cultured cells were MSC cells.(2) The co-cultured MSC cells induced by corneal stromal fibroblasts expressed positive staining to CK12, and scanning electronic microscopy showed the cells with characteristics of corneal epithelial cells.(3) The induced MSC cells attached to and grew well on the amnion treated with trypsin. The characteristics of the cells maintained unchanged 7 d after seeding on the amnion when examined with immunocytochemical staining and morphology.Part 2:Application of the corneal epithelial graft in the ocular surface reconstruction(1) A rat model of corneal alkali burn injury was well established.(2) The area of fluorescence staining, severity of opacity, and area of corneal neovascularization of the corneas were all better in the corneal epithelial graft group than those in amniotic group or control group by clinical examination. H.E. staining showed that there was an epithelium covered on the cornea in the amnion group and control group. But it was the conjunctival epithelial cells instead of corneal epithelium as showed by immunochemical staining. The epithelium in corneal epithelial graft group expressed specific antibody of corneal epithelium, indicating that the cornea in the group had normal epithelium. The result of con-focal microscopy showed the same as above examination. The epithelial cells in the graft group at 10 weeks after transplantation appeared polygon and flat-shaped with clear boundary. While the epithelium showed irregular arrangement in the amnion group and lack in the center area and irregular in the periphery in control group. ConclusionBone marrow mesenchymal stem cells could transdifferentiate well into the corneal epithelial cells when induced by corneal stromal fibroblasts in vitro. Amnion could serve as a suitable substrate for seeding cells. A corneal epithelial graft constructed with induced bone marrow mesenchymal stem cells and amnion could successfully transplant for reconstruction of ocular surface in a rat model of corneal alkali burn injury. |
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