In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma |
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Authors: | Xiao-Yong Liu Jian Chen Qing Zhou Jing Wu Xiao-Ling Zhang Li Wang and Xiao-Yan Qin |
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Institution: | Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China;Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China;Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China;Laboratory of Ophthalmology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong Province, China;Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China;Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China;Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China |
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Abstract: | AIM:To reconstruct the lamellar cornea using human amniotic epithelial (HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology.METHODS: Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells, and the cells were cultured to proliferate. Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets. The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days, then transferred to an air-liquid interface environment to culture for 2 weeks. Tissue engineered lamellar cornea (TELC) morphology was observed by Hematoxylin-eosin (HE) staining; its ultrastructure was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM); corneal epithelial cell-specific keratin 3 and keratin 12 were detected with immunofluorescence microscopy.RESULTS:HAE cells grew on the rabbit corneal stroma, forming a monolayer after 1-2 days. About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation, a result similar to normal corneal epithelium. Rabbit corneal stromal cells were significantly reduced after one week, then almost completely disappeared after 2 weeks. TEM showed desmosomes between the epithelial cells; hemidesmosomes formed between the epithelial cells and the basement membrane. SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli. The tissue-engineered cornea expressed keratin 3 and keratin 12, as detected by immunofluorescence assay.CONCLUSION: Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma. |
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Keywords: | amniotic epithelial cells cornea tissue engineering keratin |
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