Tissue Engineering Corneal Stroma With Accellular Porcine Corneal Stroma Sheet In A Rabbit Model

Objective: To investigate the feasibility of preparing acellular porcine corneal stroma sheet by Na Cl solution treatment and frozen section, and the feasibility of engineering corneal stroma by seeding rabbit keratocytes on the acellular sheets. The therapeutic potential of engineered corneal stroma with acellular sheets was evaluated in a rabbit model.Methods: Porcine corneal stroma was treated by Na Cl solution, and the decellularization efficiency was detected by histological analysis. Acellular porcine cornea was cut into thin sheets at a thickness of 20 μm by frozen section. The sheets were sterilized by ultra violet illumination. Rabbit keratocytes were cultured by tissue cultivation method. Cells at passage 3 were used as seed cells. Cells were seeded on acellular stroma sheet and stacked 6 layers together as a sandwich to make a cell-scaffold construct. After 2 weeks of culture in vitro, histological examination was performed to test the tissue formation. To repair the rabbit corneal stroma defects in vivo, keratocytes were seeded layer by layer in the defects for 5 layers in each defect. The defects without treatment, with thick acellular stroma, with acellular stroma sheets but not cells, were served as controls. Photos are taken at 1 month, 3 months, 6 months post-operation. Anterior segment optical coherence tomography(AS OCT), corneal thickness measurement, biomechanical tests, histological examination, electromicroscopy examination, and corneal transparency analysis were performed to evaluate the efficiency of each treatment.Results: NaCl treatment could effectively decellularize porcine corneal stroma with maintenance of stroma collagen arrangement. Acellular stroma sheets were easily prepared by frozen section. The sheets possess good biocompatibility that rabbit keratocytes grow well on the sheets, which were demonstrated by scanning electron microscope examination. Animal study showed that in the defect without treatment group, the cornea was thin, corneal macula could be seen; In thick acellular stroma transplantation group, the corneal transparency was fine, but corneal nebula presented in the central area of pupil; In the acellular sheet group, no scars were observed but the transparency was poor. In acellular stroma sheet with keratocytes group, the cornea was transparent, and iris texture was clear. OCT examination showed that in sheet + cell group, central corneal thickness was similar to normal cornea. The mechanical properties of corneas in this group were also similar to normal cornea. Histological examinations showed that in thick acellular stroma transplantation group and acellular sheet group, no keratocytes were observed in the grafts. While in the experiment group, keratocytes were distributed evenly in the grafts. Collagen fibers in the experiment group were well orientated as normal cornea.Conclusions: It is feasible and easy to construct acellular porcine corneal stroma sheet by Na Cl solution treatment and frozen section. The acellular stroma sheets could be used to repair corneal stroma defect together with keratocytes. This study provides a new approach for regenerating corneal stroma in vivo.