Preparation of acellular scaffold for corneal tissue engineering by supercritical carbon dioxide extraction technology

In this study, we developed a novel method using supercritical carbon dioxide (SCCO₂) to prepare acellular porcine cornea (APC). Under gentle extraction conditions using SCCO₂ technology, hematoxylin and eosin staining showed that cells were completely lysed, and cell debris, including nuclei, was efficiently removed from the porcine cornea. The SCCO₂-treated corneas exhibited intact stromal structures and appropriate mechanical properties. Moreover, no immunological reactions and neovascularization were observed after lamellar keratoplasty in rabbits. All transplanted grafts and animals survived without complications. The transplanted APCs were opaque after the operation but became transparent within 2 weeks. Complete re-epithelialization of the transplanted APCs was observed within 4 weeks. In conclusion, APCs produced by SCCO₂ extraction technology could be an ideal and useful scaffold for corneal tissue engineering. Statement of significance We decellularized the porcine cornea using SCCO₂ extraction technology and investigated the characteristics, mechanical properties, and biocompatibility of the decellularized porcine cornea by lamellar keratoplasty in rabbits. To the best of our knowledge, this is the first report describing the use of SCCO₂ extraction technology for preparation of acellular corneal scaffold. We proved that the cellular components of porcine corneas had been efficiently removed, and the biomechanical properties of the scaffold were well preserved by SCCO₂ extraction technology. SCCO₂-treated corneas maintained optical transparency and exhibited appropriate strength to withstand surgical procedures. In vivo, the transplanted corneas showed no evidence of immunological reactions and exhibited good biocompatibility and long-term stability. Our results suggested that the APCs developed by SCCO₂ extraction technology could be an ideal and useful scaffold for corneal replacement and corneal tissue engineering.