The Study On Acellular Corneal Stroma And Platelet Plasma Applied To Tissue Engineering Cornea

Objective:(1) To develop a method using pancreatic phospholipase A2 (PLA2) combining liquid N2 to prepare acellular porcine corneal stroma (APCS) and to investigate the feasibility of APCS as a scaffold of tissue engineering cornea. (2) To study the effect of autologous platelet plasma on the biological function of keratocytes. (3) To reaserch the feasibility of platelet-rich gel for the reconstruction of tissue engineering corneal stroma.Methods:(1) Acellular porcine corneal stroma (APCS) was prepared by using phospholipase A2 combining with liquid N2 to make the native procine cornea (NPC) hypoxia and frozen. The physical and biological characteristics in both of native procine corneas and APCS, including morphology, thickness, water-absorption, light transmittance and biomechanics were evaluated. (2) Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were isolated from the rabbit blood by two-step centrifugation method. CCK-8 assay was used to analyze the influences of PRP with various concentrations to the proliferation of rabbit keratocytes. (3) Platelet-rich gel was prepared by combining PRP with thrombin activators in proportion of 9:1. The features of morphology, influences to the keratocytes, adhesive properties and biocompatibilities were evaluated.Results:(1) The combination of PLA2 and liquid N2 to prepare APCS could remove almost all of the xenogenetic cells, which had little adverse effect on collagen components, and retained the fine ultrastructure of native corneal stroma. There was no significant difference in corneal thickness, light transmittance, and biomechanics variation between APCS group and NPC group (P>0.05) except for the water-absorption variation. (2) The proliferation of rabbit keratocytes was stimulated with PRP in the concentration of 2.5%～10.0%, and 5.0% PRP showed the best effect on cellular proliferation (P<0.05). (3) Prepared platelet-rich gel showed good transparency and the microanatomy revealed polyporous orderly structure similar to the native cornea. Phase contrast microscope examination and HE staining displayed that the keratocytes could adhere, grow, proliferate and differentiate in the platelet-rich gel very well. Besides, Platelet-rich gel used as an adhesive was able to attach the porcine corneal stroma closely and the tight junction could be observed with HE staining.Conclusion:(1) The APCS prepared by using pancreatic phospholipase A2 (PLA2) combining liquid N2, possesse favorable physical and biological properties which is similar to the normal cornea and thus APCS is a suitable scaffold for corneal tissue engineering. (2) PRP can promote the proliferation and activation of keratocytes. It may be favorable to rapid healing of corneal wound and prevention from serious complications. (3) The platelet-rich gel, prepared by combining PRP with activators in suitable proportion, has the nature of high transparency, orderly organization structure, favorable biocompatibility, undetectable immunogenicity, effective adhesiveness. These properties indicate that the platelet-rich gel is favorable for the reconstruction of tissue engineering corneal stroma.