In Vitro Reconstruction And Characterization Of Tissue-engineered Human Complete Cornea Using Acellular Porcine Corneal Stroma And Its Function In Animal Transplantation

Cornea is a kind of the transparent connective tissue located in the front of eye, withbarrier and refractive function. For various diseases caused by the body’s internal andexternal factors such as poor development of congenital cornea, corneal scarring, infection orsevere corneal perforation, corneal thermal burns and corneal opacity affect the eye sightrestoring, penetrating keratoplasty is the only therapeutic approach. However, the number ofdonor cornea is currently a serious shortage of the necessary, can not meet the clinical needs.With the development of corneal tissue engineering, tissue-engineered human cornea(TE-HC) is expected to become a donor cornea equivalent alternatives to solve the problemof insufficient donor for corneal blind patients to bring sight hope.The core elements of reconstruction of TE-HC in vitro include two aspects. On the onehand, we are capable to get a huge amount of seed cells with great proliferative capacity andsecurity, on the other hand, we can get ideal scaffold with good biocompatibility. Currentlyseed cells for TE-HC are mainly embryonic stem cells (ESCs), adult stem cells (ASCs) andthe transfected cell line cells. Biological scaffold materials include natural biomaterials andsynthetic polymer biomaterials. The applications of ESCs lead to ethical controversy, thedifferentiation rates of ASCs are very low and the transfected cell line cells exist tumorigenicrisk, these factors limit the use of stem cells and cell lines transfected cells in the corneatissue engineering. Application of collagen as natural biomaterial scaffold with insufficientin terms of mechanical properties, as well as synthetic polymer biomaterials with poorbiocompatibility, these disadvantages restrict their applications as the corneal tissueengineering scaffold. Therefore, to find the ideal seed cells and scaffolds become crucialfactors in the reconstruction of TE-HC in vitro. In this thesis, human corneal epithelium (HCEP) cells, stromal (HCS) cells andendothelial (HCE) cells used in the experiment have the advantages of untransfected,non-tumorigenic, normal attribute and function. These seed cells have good proliferationability so that we can get large numbers of cells in a short time. Thus they maybe solve thequestion of source of seed cells in reconstructing TE-HC in vitro. The acellular porcinecorneal stroma (aPCS) with high transmittance, good biocompatibility, which can be used torestructed the TE-HC in vitro.In order to prove if HCEP cells, HCS cells and HCE cells are appropriate seed cells forcorneal tissue engineering, growth properties, chromosome morphological observation,immunocytochemistry and tumorigenesis assays were used to identify the properties,functions and latent risk of tumorigenicity of passage70HCEP cells, HCS cells and HCEcells. The results showed that the HCEP cells and HCE cells with the typical recoveryepithelioid cells and HCS cells with the typical fibroblast cell morphology. Both HCEP cellsand HCE cells are closely with each other and convergence, alignment presented "pavingstone" shape, with the ability to connect into pieces, and HCS cells arranged in streamlined.HCEP cells, HCE cells and HCS cell population doubling time were39.6h,38.6h and36.5h, their predominant chromosome number of the cells are46. The HCEP cells expresskeratin12positively, which is a specific marker of HCEP cells. In addition, The HCEP cellsalso expresse Na+/K+-ATPase, and integrin positively, which suggested that the HCEP cellsstill had normal phenotypes and the potential to form normal HCEP. The HCS cells expressvimentin positively, which is a specific marker of HCS cells. In addition, The HCS cells alsoexpresse glyoxylate dehydrogenase ALDH3A1and integrin positively, which suggested thatthe HCS cells still had normal phenotypes of HCS cells. The HCE cells expressNa+/K+-ATPase, zonula occludens1, and integrin positively, which suggested that the HCEcells still had normal phenotypes and the potential to form normal HCE. Tumorigenicity testresults show that these three corneal seed cells have good security, no tumorigenicity.Therefore, HCEP cells, HCS cells and HCE cells can be used as ideal seed cells forreconstructing the TE-HC in vitro.In order to obtain the ideal biomaterial scaffold, this thesis conducted preparingresearch on decellularization of porcine cornea.0.5%sodium deoxycholate and0.04% sodium orthovanadate and DNase/RNase were used to deal with porcine cornea. And theaPCS scaffold was gained after air drying in the super-clean worktable. In order to detect theremoval of cells, HE staning and DAPI fluorescence stain were applied. And HE stainingwas also used to evaluate the general histoarchitecture of the treated cornea. Evaluation ofarrangement of the collagen fibres in the aPCS had been achieved by transmission electronmicroscopy (TEM). Alcian Blue staining, a cationic dye that binds to GAG was employed tovisualize the extent of disruption to the extracellular matrix (ECM). The results showed thatthere were no cellular debris and DNA in the treated aPCS. Distribution of GAG in the aPCShad no difference with the untreated cornea. And collagen fiber neatly arranged in the aPCS.All these results suggested that the aPCS scaffold maintained complete structure andcomposition of ECM. In addition, the scaffold had good transparency. In order to detectaPCS biocompatibility, we prepared aPCS extracts and the cell viability and cytotoxicity ofextracts were detected on HCEP cells, HCS cells, and HCE cells, the results showed that theaPCS extracts had no effect on cell growth, proliferation, and poptosis. aPCS extracts haveno toxicity. Therefore, aPCS can be used as ideal scaffold for reconstructing TE-HC in vitro.In order to establish TE-HC-scale reconstruction technique in vitro conditions, inobtaining the ideal seed cells and scaffold carrier, the thesis conducted a reconstruction ofthe TE-HC in vitro. Firstly, by micro-injection the HCS cells were seeded into aPCS andcultured for24hours in10%FBS (fetal bovine serum, FBS) in DMEM/F12(1:1) medium at37℃with5%CO2. Secondly, the aPCS with HCS cells placed in a petri dish insert,Descemet membrane up, the HCE cells were seeded in Descemet membrane of aPCS andcultured for24hours; then reversed aPCS, Descemet down, the HCEP cells were seeded onthe surface of aPCS stroma in air-liquid interface culture system in DMEM/F12(1:1)mediumcontaining10%FBS at37°C with5%CO2for5days. Alizarin red staining showed that theHCE cells are able to form a continuous cell monolayer; HE staining showed that stromalcells within aPCS stand a good stretch, combined with the scaffold close, aPCS bracketmechanism layer formed on the surface layer of6-7stratified epithelium structure, similar tonormal corneal structure; immunohistochemical staining showed HCEP cells, HCS cells andHCE cells marker protein, functional protein expression were positive, with the phenotypeand function the same as normal cells. The above results show that maybe successfully reconstruct the TE-HC in vitro and have in vivo corneal similar organizational structure,suggesting it may have a biological function.In order to identify the biological function of TE-HC in vivo, New Zealand whiterabbits and Beagle were used for penetrating keratoplasty with TE-HC, and the results wereassessed through transparency, thickness and neovascularization. The results of rabbits showthat10days after surgery, corneal edema, high intraocular pressure; after20days, edemacondition reduced and the intraocular pressure returned the normal; after30days, there haspartly transparent portion of the cornea. Beagle transplantation experiments showed earlypostoperative, graft edema, increased intraocular pressure, but after30days, cornealre-epithelialization, intraocular pressure returned normal, but the corneal thickness still notreturned to normal corneal thickness, the observation has continune to270days and foundthat corneal neovascularization situation improved. Howerer, the corneal thickness stillhigher than normal cornea.In conclusion, the reconstruction of TE-HC in vitro by using passage70HCEP cells,HCS cells and HCE cells as seed cells and aPCS as scaffold and conducted New Zealandrabbits and Beagles transplantation experiments, although these animal results did not meetideal situation, but it also provides us with the parameters follow TE-HC reconstructed onthese basis, so as to achieve the desired clinical applicability of corneal transplantation offersavailable TE-HC.