Preparation Of Acellular Porcine Corneal Stroma Scaffold And Its Biocompatibility Evaluation With Human Corneal Stromal Cell

Cornea is the front part of the eye, covering the iris, pupil and anterior chamber,which is transparent and elliptical, and can refracts light accounting for approximatelytwo-thirds of the eye’s total optical power. The human cornea includes cornealepithelium, Bowman’s layer, corneal stroma, Descemet’s membrane and cornealendothelium from the anterior to posterior. Among the five layers, corneal stroma iscomposed of about200flattened collegenous lamellae, which plays an important rolein maintaining transparency of cornea. And stroma occupies up to90%of wholecornea thickness. Corneal disease is the second major cause of blindness, secondlyonly to cataracts. There are about60million bind corneal patients all over the worldcaused by blind cornea, and the number of patients increase at the speed of1~1.5million per year. Most of the patients are lack of the partial corneal stroma. Cornealtransplantation is the only effective method for the treatment of corneal blindness, butthe serious shortage of donated corneas limited the transplantation. Hence, it is animportant way to search for corneal substitutes to solve the problem. With fastprogression of tissue engineering research, reconstruction of tissue-engineered corneain vitro which has a similar structure and functions with the normal cornea is possible.Tissue-engineered corneal stroma, as substitue of live corneal stroma, is a newfeasible method to overcome corneal material shortage.Scaffold plays a key role in construction of tissue-engineered cornea, whichprovides structural support for cells and guides tissue regeneration. So characteristicsand properties of scaffold determine the success or failure of tissue-engineeriedcornea production. This study planed to find a suitable decellular procedure, whichcould prepare ideal acellular porcine corneal stroma scaffold （aPCS）. And theuntransfected and no-tumorigenic human cornea stromal cell line established by ourlaboratory was used to detect the biocompatibility of the aPCS. This study providedan experimental basis for further study on the reconstruction of tissue-engineeredcorneal stroma and whole cornea.Sodium deoxycholate and DNase/RNase were used to decellularize porcinecornea stroma. And the aPCS scaffold was gained after air drying in the super-cleanworktable. HE staning and DAPI fluorescence stain were used to detect the removal of cells and HE staining was also used to evaluate the general histoarchitecture of thetreated cornea. Evaluation of arrangement of the collagen fibres in the aPCS had beenachieved by transmission electron microscopy （TEM）. Alcian Blue staining, acationic dye that binds to GAG was employed to visualize disruption of theextracellular matrix （ECM）. The results showed that there were no cellular debrisand DNA in the treated aPCS. Distribution of GAG in the aPCS had no differencewith the untreated cornea. And collagen fiber neatly arranged in the aPCS. All theseresults suggested that the aPCS scaffold maintained complete structure andcomposition of ECM. In addition, the scaffold had good transparency. HE stainingand immunohistochemical staining were used to determine the biocompatibility of theaPCS scaffold. The results showed that after culturing for1days,3days,5days and7days, respectively, the human cornea stromal cells injected into aPCS distributed inthe scaffold and showed a trend of spread. Immunohistochemical staining showed thatvimentin, integrin β1, connexin-43, aldehyde dehydrogenase3A1, Na+/K+-ATPase,Ca2+-ATPase, K-ras, STAT3, CDK2and CD34expressed positively in the seededhuman cornea stromal cells, which suggested good biocompatibility of the aPCSscaffold.In conclusion, aPCS scaffold were successfully prepared, which had no residualcell, neat arrangement of collagen fiber and normal distribution of GAG. Theimmunofluorescence staining showed that the human cornea stromal cells positivelyexpressed marker protein, connexin protein, function protein, growth-related protein,proliferation-related protein and differentiation-related protein after seeded into thescaffold. The results suggested that the aPCS scaffold had good biocompatibility,which established the foundation for the reconstruction of tissue-engineered cornealstroma and whole cornea.