ABSTRACT
Background The select of supporter is critical for the construction of tissue engineering cornea.Many carrier carl be utilized in the construction of tissue engineering cornea,but de-cellular corneal matrix is known to be one of optimal supporters.Objective Present study was to investigate the characteristics of de-cellular corneal matrix of porcine of structure and biocompatibility for rabbit cornea stroma and limbal epithelial ceHs. Methods The porcine cornea was prepared as de-cellular corneal matrix of porcine by the application of detergent enzyme combined process.The corneal epithelial cells,keratocyte and endothelial cells of porcine were removed completely and stored in -20℃ refrigerator after sterilization.The morphology of de-cellular corneal matrix of porcine was examined by hematoxylin-eosin staining under the light microscope.The structure characteristics of de.cellular corneal matrix of porcine under the scan electron microscope,and its physics features were investigated by the evaluation of water content,strength,expansion and transparency.The de-cellular corneal matrix of porcine were implanted to cornea stroma of rabbit and co-cultured with rabbit corneal epithelial cells for 4 weeks in vitro to assess the keracyte compatibility. Results The epithelial cells,keratocyte and endothelial cells of porcine were removed completely by trypsogen digestion.The collagen fibril network and collagen plates paralleled to corneal surface under the light microscope.The water content,strength,expansion。Ratio of light transparency of de-cellular corneal matrix of porcine were similar to normal porcine cornea.After implantation of de.cellular comeal matrix of porcine into rabbits corneal stroma,the edema of tissue was found in one week,and edema disappeared on two weeks and became clear on four weeks after surgery.The de-cellular eorneal matrix attached to rabbit cornea stroma well.No inflammatory eell and new vessel were found after surgery.The co-cultured rabbit corneal epithelial cells differentiated and proliferated on the surfaee of de-cellular corneal matrix and showed positive response for CK3.No statistically significant differences were found in the water content,strength,expansion of de-cellular cornea matrix of porcine among the normal,before dehydration,2 and 4 hours after dehydration cornea matrix(P＞0.05).However,the transparency was much better in the corneal matrix with 2-hour,4-hour dehydration in comparison with non-dehydration one(P＜0.05). Conclusion The structure features of de-cellular cornea matrix of porcine are similar to normal porcine cornea.Good biocompatibility is proved for xenogenesis of rabbit cornea.
ABSTRACT
Background Researches demonstrated that corneal dystrophy is associated with the mutation of transforming growth factor beta induced gene(TGFBI)located at chromosome 5q31 domine.Recent study showed that the gene mutation location is in R124H of TGFBI gene. Objective This study was to identify the mutation characteristics of TGFBI gene in a Chinese family with Avellino corneal dystrophy. Methods This Chinese family with Avellino corneal dystrophy were determined and surveyed in Peking University Third Hospital.Periphery blood from 8 patients with Avellino corneal dystrophy and 2 unaffected subjects were collected from a Chinese family with corneal dystrophy for the extraction of DNA.Exons 4,11,12 of the TGFBI gene were amplified by polymerase chain reaction(PCR),and the amplified products were sequenced directly and compared the gene sequence with that of TGFBI in GenBank.Written informed consent was obtained from each Subject prior to any medieal process. Results This family included 27 members of consecutive 4 generation.The hereditary pattern W88 in accordance with the autosomal dominant inheritance.Directly sequencing of 8 affected members revealed a G tO A transition at codon 124 (CGC to CAC),producing R124H mutation of TGFBI gene.Two synonymous single nucleotide polymorphism(SNP)of TGFBI gene occurred in the family.including a C to T transition at eodon 472(CTC to CTT)in 8 members,and a T to C transition at codon 540(TTT>TTC)in 9 members,which wag unrelated with disease. Conclusion R124H mutation of the TGFBI gene is found in this Chinese family with Avellino corneal dystrophy.
ABSTRACT
PURPOSE: The morphologic characteristics and adhesion complex formation of cultured limbal epithelium of rabbit on amniotic membrane, in vitro and in vivo. METHODS: Rabbit limbal explants were cultured in vitro on amniotic membrane for 4 weeks. In the in vivo culture, the rabbit corneal epithelium was removed. Next, a tunnel was created at the limbus and, the edge of amniotic membrane was secured in the tunnel and cultured for 4 weeks. The proliferation of epithelium on the amniotic membrane was observed for 4 weeks at 1 week intervals. RESULTS: AE-5 immunohistochemical staining was positive and PAS staining was negative for cultured rabbit limbal epithelium, in vitro and in vivo. Hematoxylin and Eosin staining showed the morphologic characteristics of normal rabbit corneal epithelium in both groups. Transmission electron microscopy performed at an interval of 1 week showed adhesion complex by 3 weeks of in vitro culture, and no significant change was seen until week 4. The formation of the adhesion complex was shown starting at week 1 of in vivo culture and increased until week 4. CONCLUSIONS: The morphology of corneal limbal epithelium of rabbits cultured on amniotic membrane in vitro and in vivo, did not differ significantly compared with normal rabbit epithelium. In vivo culture resulted in more a normal-looking adhesion complex compared with the in vitro culture.
Subject(s)
Rabbits , Amnion , Eosine Yellowish-(YS) , Epithelium , Epithelium, Corneal , Hematoxylin , Microscopy, Electron, TransmissionABSTRACT
0.05).The morphological characteristics were similar and positive on HE staining and AE5 immunohistochemical staining.PAS staining were negative in two groups.Conclusion The cellu- lar biological activations of limbal epithelial cells are decreased in the ultra low temperature preservation condition,but the affect are limit and can't change the property of corneal limbal epithelial cells and application for ocular surface reconstruction.(Ophthalmol CHN,2008,17:108-112)