Application of benzonase in preparation of decellularized lamellar porcine corneal stroma for lamellar keratoplasty.

Abstract

This study was to develop anovel and efficient method using endonuclease (benzonase) to preparedecellularized lamellar porcine corneal stroma (DLPCS). The DLPCS was preparedfrom native lamellar porcine corneal stroma (NLPCS) and was treated with 1000 U/ml benzonase for 5hours. We conducted the following measurements and animal transplantation tocompare DLPCS and NLPCS. The residual DNA was decreased significantly from 367.13 ± 19.96 ng/mg to 15.41 ± 0.65 ng/mg after treatment of benzonase by the detection of fluorescentnucleic acid stain. The residual benzonase was also less than detection limit.There was no significant difference in light transmittance of DLPCS comparedwith NLPCS. The extracts of DLPCS did not inhibit cell proliferation of human cornealepithelial cells, mouse fibroblast (L‐929) and African green monkey kidney cell(Vero cell). The DLPCS was transplanted into the corneas of rabbit by lamellarkeratoplasty. There was no corneal melting and graft rejection been observedwithin 12 months. The images demonstrated that the repairment of corneal nervesand keratocytes of DLPCS were in indentical shape and reflection compared withnormal cornea, and no obvious inflammatory cells were observed postoperation, byin vivo confocal microscopy. We provided novel evidence that the application ofbenzonase may improve the quality of DLPCS.