| Cornea, a transparent tissue with a certain radius of curvature, locates in the forefront of the eye, which plays an irreplaceable important function in protecting human visual function and eyeball. Human cornea is composed of epithelium, Bowman’ layer, stroma. Descemet’s layer and endothelium. The epithelium is about 50μm in thickness which composed of six to eight corneal epithelium cell (HCEP cells) layers, the stroma with the thickness of 500 μm is composed of human stromal cells (HCS) and highly organized extracellular matrix made up by collagen, and the endothelium with the thickness of about 5μm is composed of single corneal endothelial cell (HCE) layer. Because the cornea is located in the forefront of the ocular surface and contacts with the outside world directly, it is vulnerable to trauma, burns and infections, which can cause corneal opacity, decreased vision and even blindness. At present, corneal transplantation is the only effective way of corneal disease treatment clinically. Unfortunately, the vast majority of patients are unable to restore visual function through corneal transplantation due to a serious shortage of donor cornea and the ageing of donor cornea. As equivalent alternative to donor cornea, tissue-engineered human cornea (TE-HC) is the only hope to solve the problem of insufficient donor and bring sight hope for corneal blind patients. It has become the hot and difficult topic that how to get sufficient amount of the ideal seed cells and biocompatibility materials as ideal carrier scaffolds. In the aspect of seed cells, the researchers have focused on the stem cells, transfected cell line cells and primary cells. However, the applications of those cells are limited because of culture conditions in vitro, ethic, potential tumorigenicity and other restrictions. So they still cannot meet requirements of seed cells in tissue-engineered cornea. In recent years, non-transfected and non-tumorigenic HCE, HCS and HCEP cell lines have been established in our laboratory and based on these cell lines, tissue-engineered human corneal endothelium, stroma and epithelium have also been successfully constructed respectively, which makes tissue-engineered cornea construction possible. In the aspect of ideal biocompatibility scaffolds, scholars mainly focus on natural biological materials, synthetic polymer and biological macromolecular composite material. Due to the natural biological material can be affected by the risks of carrying pathogen, the limitation of source, as well as the deficiencies in the biological compatibility of synthetic polymer materials, the scientists focuse on the research on the materials coming from biological macromolecules.In the research on biological macromolecules, collagen, as a kind of source extensive, evolutionarily conserved, low immunogenicity and nice biocompatibility of biological macromolecules, has been widely used in the fabrication of various scaffolds. Beside that, people has used human collagen composite scaffold successful to construct the tissue-engineered cornea. Unfortunately, collagen composite scaffolds has some drawbacks such as poor mechanical properties, overquick degradation in vivo and expensive preparation cost. In recent years, the fast development of highly effective non-toxic crosslinking agent,crosslinking technology and the preparative technique of non-enzymatic hydrolysis, complete molecular fish collagen proteins brings hope for the fabrication of tissue engineering corneal scaffold using collagen.On the basis of the previous research, we used firstly non-enzymatic hydrolysis, complete molecular fish collagen protein purificated from the skin of raja porosa in our laboratory to fabricate the fish-collagen scaffold, and then use fish-collagen scaffold as tissue-engineered corneal scaffold and use three kinds of non-tumorigenic cell lines as seed cells to construct issue engineered full-layer human cornea (TE-flHC) in vitro, aim to established the technology of the fabrication of the fish-collagen scaffold and the construction of TE-flHC in vitro, and acquire the TE-flHC which has normal organizational structure and expression of marker and functional protein.In order to obtain the scaffold has the ideal biocompatibility using fish collagen, we extracted and purified collagen protein from raja porosa skin, then crosslinked the collagen with 1-(3-dimethyl amino propyl)-3-ethyl-2-imine carbide/N-hydroxy succinimide (EDC/NHS) and home-made mold to fabricate fish-collagen scaffold and identify its characterization. Analysis results indicated the extracted collagen had high purity and molecular weight, and had basic characteristics of collagen such as single chain with molecular weight of 120KDa, a total of 32.34% Gly. So it can be used in the preparation of tissue-engineered corneal scaffold. After identifying the purified collagen, we used collagen as raw materials, used 1-(3-dimethyl amino propyl)-3-ethyl-2-imine carbide/N-hydroxy succinimide (EDC/NHS) as crosslinker to fabricate fish collagen scaffold in the home-made mold. The water content of collagen scaffold with concentration of 60 mg/mL,80 mg/mL,100 mg/mL was 90.53%±0.06%、 89.88%±0.03% and 86.34.%±0.11%, and the transparency and light transmittance percentage was 91.15±2.36%,89.77±2.30% and 86.09±2.31%, respectively. Mechanical test results showed that increasing the concentration of collagen can improve the tensile strength and elongation at break of the scaffold. HE staining of frozen sections and scanning electron microscopy (SEM) results showed 100 mg/mL collagen scaffolds had the best internal structure, with compact structure, continuous collagen fiber and uniform aperture size compared with 60 mg/mL and 80 mg/mL collagen scaffolds. These results suggested that the constructed fish-collagen scaffold has good optical properties, mechanical properties and organization structure. Hence, it is hopeful as scaffold in reconstruction of TE-flHC in vitro.In order to construct TE-flHC in vitro as alternatives of donor cornea, we detected the morphology, proliferation, chromosome characteristics and expression of functional proteins of non-transfected cell lines of human corneal tissue cultured in our lab. The results of morphology and growth curve showed that the passage 80 HCEP cells, batch 28 passage 62 of HCS cells and the passage 67 HCE cells still had epithelioid, typical fibroblast and polygon endothelial morphological characteristics, with a population doubling time of 38.5 h,37.6 h and 36.2 h, respectively. Chromosome identification results showed that these three types of cells the characteristic chromosome number of these three types of cells is 2n=46. Immunocytochemical staining results showed that HCEP, HCS and HCE cells still expressed marker protein (keratin-3/12). cell junction proteins (integrinβ1 and zonula occluden) and functional protein (aldehyde dehydrogenase 3A1 and sodium potassium-ATPase); HCS cells still expressed marker protein (vimentin). cell junction proteins (integrin β1 and connexin-43) and functional protein (aldehyde dehydrogenase and sodium potassium-ATPase):HCE cells still expressed marker protein (vascular endothelial cell receptor protein), cell junction proteins (integrin (35. zonula occluden and connexin-43) and functional protein (sodium potassium-ATPase). herefore, HCEP, HCE and HCS cells cultured in vitro stilled had the typical properties in morphology, proliferation, chromosome characteristics, the expression of function protein and had the potential ability to form cell junctions and transport membrane. So it can be used as seed cells in TE-flHC building in vitro.In order to evaluate the biocompatibility of constructed fish-collagen scaffold, after getting enough of the seeder cells, we MTT assay, RT-PCR and immunohistochemical methods was used to examine the cell toxicity of leaching solution of the scaffold and the expression of genes of growth and proliferation and functional protein in scaffolds. MTT assay results showed that leaching solution of the fish-collagen scaffold had no influence on the viability of HCEP, HCE and HCS cells(P>0.05). The result of RT-PCR indicated that the scaffold had no influence on the genes expression of growth and proliferation of this three kinds of cells. Immunohistochemical staining results showed that HCEP cells could express keratin 3/12, the marker protein of corneal epithelial cells, indicating these cells could maintain their cell propertis of species in the fish collagen scaffold after inoculation. The positive expression of cell junction proteins (integrin (31 and zonula occluden) and functional proteins (aldehyde dehydrogenase and sodium potassium-ATPase) demonstrated that HCEP cells on the carrier scaffold can still maintain the characteristics of human corneal epithelial cells. After cells inoculation. HCS cells can express its characteristic protein-vimentin. indicating the carrier does not make changes of cell propertis. The expression of connexin-43 proteins such as integrin β1, connexin-43 and function proteins such as aldehyde dehydrogenase and sodium potassium-ATPase showed that HCS cells seeded on the scaffold still remain characteristics of the human corneal stromal cells. After inoculated on the surface of the scaffold, HCE cells expressed marker proteins-vascular endothelial growth factor receptor, indicating that the scaffold would not cause the change in the properties of HCE cells. The expression of integrin, connexin-43 and sodium potassium-ATPase by HCE cells showed the potential functions of cell junction and membrane transport in HCE cells after inoculation. These results suggested that constructed fish-collagen scaffold had good biocompatibility. and could be used as the scaffold to construct the TE-flHC.In order to establish technical strategy of TE-flHC construction in vitro, we carry on the research about reconstruction of TE-flHC in vitro using ideal seed cells and fish-collagen scaffold. Firstly, passage 62 HCS cells, passage 67 HCE cells and passage 80 HCEP cells were seeded on/into fish-collagen scaffold by negative pressure and direct inoculation method and cultured under air-liquid interface culture condition or submerged culture condition to reconstructed the TE-flHC. Then, we identified its optical properties, histological structure and the protein expression of the cells by light transmitting test, HE staining of frozen sections, alizarin red staining, immunohistochemical staining, transmission electron microscopy and scanning electron microscopy. Results of Light transmitting test showed that the TE-flHC had good light transmittance and transparency, with high similarity to the normal cornea. Fluorescent marker detection and HE staining results showed that three human corneal cells had clear boundaries and HCEP cells formed stratified structure of 5-8 lays on the surface of the scaffold. HCS cells were evenly distributed in thecollagen stroma and HCE cells form a single intact cells layer, similar to the anatomy structure of normal cornea. Results of Alizarin red staining showed that the HCE cells could form a continuous cell monolayer with cell number of 2750±260/mm2, which is to be equivalent to the corneal endothelial cells number of the 30 years old adult. Ultrastructure observation results showed that the HCEP cells presented ovoid and connected with each other closely, existing rich microvilli in surface, similar to normal corneal epithelium, HCS cells presented spread form connecting with the fish-collagen scaffold, and HCE cells formed a complete flat single layer and compact structure, which is similar to the normal corneal endothelium.In summary, this paper used the collagen purified from the skin of raja porosa to fabricate fish-collagen scaffold with good optical properties, mechanical property and histological structure, and then used fish-collagen scaffold as tissue-engineered corneal scaffold, and used HCEP cells. HCS and HCE cells as seed cells and constructed successfully the TE-flHC with similarity to the normal structure and function of cornea. It has an important role in guiding the high-quality application of marine biological macromolecules in tissue engineering and brings the hope for the millions of corneal disease patients recover their sight using TE-flHC in our country and even the whole world!... |
PDF Full Text Request