| Cornea is the transparent fiber membrane in the forefront of the eye ball, it has a varietyof important functions such as defense and refractive. Human corneal stroma is composed ofhuman corneal stromal cell and more than200layers of collagen fiber lamellar. It accountsfor about90%of the thickness of the cornea and locates between the corneal epithelial andcorneal endothelium. Corneal stromal opacity caused by various diseases is not reversible，which is the main reason for blindness. The number of blindness caused by corneal stromalopacity is second to cataract. And a large number of blindness increase every year. Cornealtransplantation is the main effective method for the treatment of corneal blindness. Thecorneas that donated are lack, and can’t meet the demand. Reconstructed corneal tissue invitro by tissue engineering techniques has a similar structure and functions with the normalcornea. They can meet the demand of corneal transplant. TE-HCS as equivalent substitutesof the donor cornea is an effective way to resolve the lack of donor cornea. It also bringshope to the patients who have abnormal corneal stroma. It laid the foundation forreconstruction of full-thickness corneal tissue engineering in vitro too. So, it is urgent tocarry out reconstruction of TE-HCS in vitro.Tissue engineering of cornea is to reconstruction human cornea corneal tissue substitutewith tissue engineering technology and biological materials or synthetic materials in vitro.The substitute has the similar morphology and function with the normal human cornea.Seeder cells and biological materials are the two basic elements of the tissue engineeringcorneal stromal. The seeder cells that used in the tissue engineering corneal stromal areeither primary cells or transfected cells, which limit the application of tissue engineeringcorneal stromal. The untransfected and no-tumorigenic human cornea stromal cell lineestablished by our laboratory solve this problem. Collagen is the main component ofconnective tissue, its chemical and biological characteristics of different animal origin arevery similar. Collagen has low immunogenicity, low toxicity, low antigenicity, and have hasgood biocompatibility and biodegradability. Collagen as natural biological materials has longbeen used in tissue engineering. It is used as scaffolds to reconstruction of TE-HCS in thiswork. To ensure the features of the human corneal stromal cell line, the human cornealstromal cell line is conducted by using microscope observation, growth characteristics,chromosome morphological observation, immunocytochemistry analysis and tumorigenicitytest. According to the results of optical microscope observation, the human corneal stromalcells that cultured in vitro are dendritic at low density, and fibroblast-like with monolayer.Population doubling time of the human corneal stromal cells is41.44h indicated it keepstrong ability to cleavage. Chromosome analysis showed that the human corneal stromal celllines have their predominant chromosome number in46. The immunofluorescence showedthat human corneal stromal cell lines expressed the marker protein----vimentin, theconnection protein----integrin β1and connexin-43, the function protein----Aldehydedehydrogenase3A1, Na+/K+-ATPase and Ca2+-ATPase positively, which suggested that thehuman corneal stromal cells still had normal phenotypes and the potential to form normalhuman corneal stromal. Besides, it is safe to use in TE-HCS and clinical application as thecells had no tumorigenicity. So, the HCSC is suitable for TE-HCS.Human type I, type IV, type V, fibronectin and chondroitin sulfate are used to build3-D collagen scaffold. The crosslinker are EDC/NHS (1-Ethyl-3-(3-dim-ethyllaminopropyl)carbodiimide hydrochloride/N-Hydroxysuccinimide) in the3-D collagen scaffold formprogress. Paraffin section HE, immunohistochemical staining, SEM, light transmittance,water content were used to determine the characteristics of the collagen scaffold. The resultsshowed that: COL-CS-Gel1’s surface with certain apertures, and the internal structureorganization is loosely; COL-CS-Gel2’s surface shows scaly, and the internal structureorganization is uneven. COL-CS-Gel3’s surface structure is pyknotic, there is no aperturesexistence. COL-CS-Gel4’s surface structure is irregular, and the internal structureorganization is uniform. COL-CS-Gel5’s apertures are about100μm, and the internalstructure organization is uniform. COL-CS-Gel5has a good light transmission, thetransmittance is about90%, and water content is89.3%±4%. So, the-Gel is suitable forTE-HCS.Frozen section HE and immunohistochemical staining were used to determine the timeof human cornea stromal cells migrate into the COL-CS-Gel scaffold and thebiocompatibility of COL-CS-Gel scaffold. The results showed that: the cells distribution inthe form is uniform after culturing for3days,5days,7days and9days respective.Immunohistochemical staining showed that vimentin, integrin β1, connexin-43, Aldehydedehydrogenase3A1, Na+/K+-ATPase and Ca2+-ATPase expressed positively, whichsuggested the biocompatibility of collagen scaffold form is good, and it can be used in TE-HCS.Tissue engineering human corneal stromal was reconstructed in vitro by using passage100human cornea stromal cells as seeder cells and3D-collagen scaffold as scaffold carries,which was produced in DMEM/F12(1:1) medium containing10%fetal bovine serum at37°C with5%CO2. Frozen section HE, immunohistochemical staining, SEM and TEMwere used to identify the tissue engineering human corneal stromal morphology andpotential functions. The results showed that the cells distribution in the form is uniform afterculturing for3days. The cells spread on the collagen surface and link to collagen. There arepresent junctions between cells also. Immunohistochemical staining showed that vimentin,integrin β1, connexin-43, Aldehyde dehydrogenase3A1, Na+/K+-ATPase and Ca2+-ATPaseexpressed positively. All those suggested tissue engineering human corneal stromal has thesimilar morphology and potential function with normal corneal stromal. At last, the surgicaloperation of corneal transplant were carry out with New Zealand rabbit and tests the functionof TE-HCS.In conclusion, the cells have higher transparency and plump conformation，and have astrong ability to cleavage also. Karyotype analysis showed that the cells have a typicaldiploid karyotype. The immunofluorescence showed that the cell lines expressed the markerprotein, the connection protein and the function protein positively. The biocompatibility ofcollagen scaffold is good. At last, tissue engineering human corneal stromal wasreconstructed successfully in vitro. |
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