Studies Of The Reconstruction And Transplantation Of Tissue-engineered Human Corneal Stroma By Using Acellular Procine Corneal Stroma

As the frontier and first barrier of the eyeball, cornea is vulnerable and frequentlysubjected to injury. Such injury, when occurred, can result in the maculae cornea andeventually corneal blind. There are approx. five million blind patients in China whilemore than six billion all over the world caused by corneal trauma. Surprisingly, thepopulation is still increasing fast. The only drastic method to solve this problem iscornea transplantation. However, amount of the donated cornea cannot be settled forthe requirement in cornea transplantation for the blind. Tissue-engineered humancorneal stroma, which was invented and rapidly developed in the last decade as asubstitute of human corneal stroma, holds great promise not only for the release ofdonor deficiency but also the sight restoration via graft operation.Plenty of seeders cells and scaffolds possessing ideal bio-compatibility are twoprerequisites to the reconstruction of tissue-engineered human corneal stroma. We haveestablished several non-transfected and non-tumorigenic human corneal stromal celllines that can be utilized as seeder cells in cornea reconstruction. In this study, weplanned to use the acelluar porcine corneal stroma as the scaffold to reconstruct thetissue-engineered corneal stroma after inoculating seeder cells into the acelluar porcinecorneal stroma. A series of animal transplantation and observation were carried outsubsequently. This study provides a new method for large scale scaffold preparationand also offers data for preclinical experiments.The optimisation of acelluar porcinecorneal stroma preparation method is the first milestone in this study. Firstly, weadopted deoxycholic acidsodium salt and sodium orthovanadate mixture to disposefresh porcine corneal stroma. The subsequent repeated freeze-thaw and combinedDNase-RNase digestion removed porcine cells in porcine corneal stroma efficiently.No visible intact cells were detected in porcine corneal stroma after decellularisedprocess via HE and fluoroscent staining. A series of phycial-chemical properties wereperformed. The results demonstrated that after decellularization the collagen fibrils of the acelluar porcine corneal stroma ranged regularly and no sign of disruption ordegradation of collagen fibril were observed, and the physical properties showed thatacelluar porcine corneal stroma was similar with the normal cornea, which could serveas a scaffold for tissue-engineered human corneal stroma.Before the biocompatibility test, human corneal stromal cells at passage15wasexamined by using growth curve, karyotye observation, and immunocytochemistryanalysis. Accordling, the population doubling time of human corneal stromal cells atpassage15is43.57h indicated it keep strong ability to cleavage. Karyotype analysisshowed that human corneal stromal cells have their predominant chromosome numberin46. The immunofluorescence showed that human corneal stromal cells expressed thetheir marker protein vimentin, connection protein i.e. integrin β1and connexin-43andvariousfunctional proteinincluding aldehyde dehydrogenase, Na+-K+-ATPase andCa2+-ATPase positively, which suggested that human corneal stromal cells maitainednormal phenotypes and possessed the potential to produce normal human cornealstroma. Herein we verified the human corneal stromal lines established by us couldserve as seed cell of tissue engineering human corneal stroma reconstructed in vitro.We employed microsinge technology to inoculate human corneal stromal cells atpassage15into the acelluar porcine corneal stroma and test its biocompability byusing MTT and and immunofluorescent stainningce. The result revealed that it was notcytotoxic to the human corneal stromal cells. We also performed immunofluorescentstainning to examine marker protein and connection proteins expression in humancorneal stromal cells1~5d after inoculation, respectively. The expression ofconnexin-43(an intercellular gap junction-associated proteins) and integrin β1(acell-matrix anchoring junction-associated protein) showed that the human cornealstromal cells had the capability to perform their specific biological functions.To study the effect of reconstructed tissue-engineered human corneal stroma invitro, the corneal transplantation onto lab animal was conducted. After20d oftransplantation on New Zealand rabbit, the implant was transparent according to slitlamp examination. The implant and plant bed healed well by HE staining. Cells intissue-engineered human corneal stroma, according to DiI labeling, were originatedfrom theinoculated human corneal stromal cells. The expression of vimentin,connexin-43, integrin β1, Na+/K+-ATPase, Ca2+-ATPase, and aldehyde dehydrogenasewere also visualized using immunofluorescent staining, indicating that the humancorneal stromal cells had the capability to perform their biological functions in the tissue-engineered human corneal stroma.In the early stage of reconstructed tissue-engineered human corneal stroma graftonto the beagles, the implant swelled slightly. The implanted cornea becametransparent gradually1month after graft.After120d of transplantation, the cornea wastransparent, and we still follow the tracks of the transparency. The observation ofimplant transparence is still ongoing. Results from lab animals transplantationdemonstrates that our reconstructed tissue-engineered human corneal stroma canperform normal functions, which prove its utilisation in the therapy for cornealstromal blind as a substitute to donated human cornea. The success in animaltransplantation experiment provides a solid base for further clinical study.In conclusion, we successfully prepared acelluar porcine corneal stroma viadecellularization as a scaffold, reconstructed tissue-engineered human corneal stromawith this scaffold and previously obtained non-transfected, non-tumorigenic humancorneal stromal cells and finally conducted lab animal transplantation. Results provesthe implanted tissue-engineered human corneal stroma performed similar biologicalfunctions as native cornea in lab animals. This study plays a vital role on large scalereconstruction in vitro and preclinical evaluation, which sets the stage for sightrestoration of the blind patients.