| Cornea is kind of hyaline connective tissue located in the front of eye, as animportant part of the refraction system of eye. The injury of cornea from infection,mechanical damage and chemical burn, can cause corneal opacity, even blindness.Among them, deep scald wound and chemical burn of cornea can usually lead to lossof corneal epithelium function and corneal stroma deep damage. Perforationkeratoplasty has a bigger risk than plate keratoplasty in immune rejection andneovascularization. Considering no injury of corneal endothelium, lamellarkeratoplasty has an advantage in keeping the original corneal endothelium and soundpart of corneal stroma. But for replacing the damaged corneal epithelium and stroma,twice operations are needed, which could induce neovascularization and affectexpectation. If tissue-engineered human hemicornea (TE-hHC) be reconstructed bythe method of tissue engineer, the risk of twice operations can be avoided. In addition,the short supply of cornea donator has been an international tough problem puzzledcornea transplantation class. TE-hHC will be a kind of appropriate real substitute forhemicornea, and satisfy the need of cornea hemicornea transplant. Seed cells andscaffolds are both elements of tissue engineer. Seed cells for tissue engineer couldcome from primary cell culture, stem cells and transfected cell lines; but theirapplications was limited because of safety and uncertainty of them. Scaffolds fortissue engineer include natural polymer and synthetic polymer. Natural polymer hassome defects, such as easy degradation and weak mechanical properties, as well assynthetic polymer probably has bad biological compatibility. Both human cornealepithelium (HCEP) cells and human corneal stroma (HCS) cells used in theexperiment have the advantages of untransfected, non-tumorigenic, normal attributeand function. Acellular porcine corneal stroma (aPCS) has good transparency andbiological compatibility. In order to prove if HCEP cells and HCS cells are appropriate seed cells fortissue engineer, the characters of both cells were identified. The result showed that,passage90HCEP cells with high transparency and cobblestone appearance in shape,proliferated actively and constantly with a population doubling time of39.2hours.Predominant chromosome number of the cells is46. The HCEP cells express keratin3/12positively, which is a specific marker of HCEP cells. This proves their HCEPorigin. Results of immunocytochemistry showed that HCEP cells expressed zonulaoccludens1, E-cadherin, connexin-43, and integrin β1positively, which suggestedthat the HCEP cells still had normal phenotypes and the potential to form normalHCEP. Passage25HCS cells have cultured in vitro are fibroblast-like with monolayer.Population doubling time of the human corneal stromal cells is43.3h indicated itkeep strong ability to cleavage. Chromosome analysis showed that the human cornealstromal cell lines have their predominant chromosome number of46. Theimmunofluorescence showed that human corneal stromal cell lines expressed themarker protein----vimentin, the connection protein----integrin β1and connexin-43, thefunction protein----Aldehyde dehydrogenase3A1, Na+/K+-ATPase and Ca2+-ATPasepositively, which suggested that the human corneal stromal cells still had normalphenotypes and the potential to form normal human corneal stromal. Besides, both ofthe cells had no tumorigenicity.The aPCS with bowman’s membrane was made with high transparency and goodmechanical properties as scaffold. By micro-injection the HCS cells were seeded intoaPCS and cultured for24hours in10%fetal bovine serum DMEM/F12（1:1）mediumat37℃with5%CO2. Then the HCEP cells were seeded on the surface of aPCS inair-liquid interface culture system in DMEM/F12（3:1）medium containing10%fetalbovine serum at37°C with5%CO2for6days. Light microscope observation,paraffin section HE, immunohistochemical staining, SEM and TEM were used toidentify the TE-hHC morphology and potential functions. The result showed that,TE-hHC has high transparency, and the HCS cells stretched well, forming closeintegration with aPCS. The HCEP cells on bowman’s membrane of aPCS were rich inmicrovilli, and constructed numerous intercellular cell junctions includingdesmosomes and cell-aPCS hemidesmosomes. Results of immunohistochemistryshowed that the seeder cells of TE-hHC still had natutal cell classical properties, andcould potentially form tight junction, anchoring junction and communicating junction. In order to identify the biological function of aPCS in vivo, beagle and macaquewere used for anterior lamellar keratoplasty with aPCS, and the results were assessedthrough transparency, thickness and neovascularization. The result of beagletransplantation showed that the edema and inflammation reaction of graft weregradually subsiding with30days, the whole cornea recovered transparencycompletely in90th day. The result of macaque transplantation showed that the edemaand inflammation reaction of graft were gradually subsiding with time and thetransparency of the graft recovered in30th day.In conclusion, TE-hHC was reconstructed in vitro by using passage90HCEPcells and passage25HCS cells as seeder cells and aPCS as scaffold carries in thisthesis. The TE-hHC with normal structure and function can make cornea of thetransplanted eyes transparent for a long time, which can be used promisingly asequivalents for clinical hemicornea transplantation. Therefore, successful in vitroreconstruction of TE-hHC and their future clinical applications have not only greattheoretical significance, but also very important social and economic benefits. |
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