Investigation Of Bacterial Cellulose For Reconstruction Of Tissue Engineering Cornea Stroma

Background Corneal disease is a major cause of blindness. Corneal transplantation is the most effective way to treat, but there are not enough donors for graft and, transplant rejection and other issues. Cornea tissue engineering research provides a new way to solve shortage of corneal transplantation for graft donor. To evaluate the biocompatibility of bacterial cellulose(BC) membrane in vitro, we cultured rabbit corneal epithelial cells and rabbit corneal stromal cells on BC membrane and observed the growth of corneal epithelial cells and corneal stroma cells. To provide the experimental basis for the clinical application of corneal tissue engineering, the transparency of bacterial cellulose and biocompatibility were observed through the animal experiments in vivo.Method 1. Rabbit corneal epithelial cells and rabbit corneal stromal cells were isolated and cultured on the plate. We identified them with CK-3 kit and Vimentin kit and observed their growth under the microscope. 2. The rabbit epithelial cells and corneal stromal cells were seeded on the bacterial cellulose membrane(BC) to construct the tissue engineering corneal epithelial and tissue engineering corneal stroma, and also LIVE-DEAD cell cytotoxicity assay kit was used to detecting cell activity. 3. The growth of corneal stromal cells on the BC membrane and BC membrane’s biocompatibility were obserbed by scanning electron microscope(SEM) and transmission electron microscope(TEM); The growth of corneal epithelial cells on the BC membrane was observed by SEM. 4. The BC membrane was grafted into the lamellar stromal pocket in the center of rabbit cornea. The transparency and biocompatibility of BC membrane were evaluated by slit lamp microscope 1 day, 1 week, 1 month, 3 months after operation. 5. The rabbit eyes were removed for hematoxylin-eosin(HE) staining 1 day, 1 week, 1 month, 3 months after operation.Results 1. The corneal epithelial cells and corneal stromal cells were successfully isolated and cultured. Also the identification results were positive, indicating that the cells were corneal epithelial cells and keratocytes. 2. Cell viability toxicity testing showed corneal epithelial cells and corneal stromal cells could survive on the BC film and basic survival rate was 100%, no cytotoxicity BC Film. 3. SEM and TEM results showed that corneal stromal cells grew well on BC membrane, cell activity, cell closely connected with BC membrane, and BC membrane biocompatibility; SEM result showed that corneal epithelial cells grew well on the BC membrane. 4. We successfully constructed rabbit corneal pocket model, and slit lamp observation showed graft transparent, no significant inflammation. 5. HE staining showed that around grafts ill-defined, merging with the surrounding tissue, no significant inflammatory cell infiltration.Conclusion In vitro experiments showed that corneal epithelial cells and stromal cells grew well on BC membrane, BC film no cytotoxicity, and good biocompatibility. In vivo experiments also demonstrated its good biocompatibility. BC film was expected to be used as a new biological material for the corneal stromal tissue engineering.