title: Construction of a complete rabbit cornea substitute using a fibrin-agarose scaffold.
abstract: OBJECTIVE
  To construct a full-thickness biological substitute of the rabbit cornea by tissue engineering.
  
  
  METHODS
  Ten rabbit corneas were surgically excised, and the three main cell types of the cornea (epithelial, stromal, and endothelial cells) were cultured. Genetic profiling of the cultured cells was performed by RT-PCR for the genes COL8 and KRT12. To develop an organotypic rabbit cornea equivalent, we used a sequential culture technique on porous culture inserts. First, endothelial cells were seeded on the base of the inserts. Then, a stroma substitute made of cultured keratocytes entrapped in a gel of human fibrin and 0.1% agarose was developed. Finally, cultured corneal epithelial cells were grown on the surface of the scaffold. Stratification of the epithelial cell layer was promoted by using an air-liquid culture technique. Corneal substitutes were analyzed by light and electron microscopy.
  
  
  RESULTS
  All three types of corneal cells were efficiently cultured in the laboratory, expanded, and used to construct a full-thickness cornea substitute. Gene expression analyses confirmed that cultured endothelial cells expressed the COL8 gene, whereas epithelial cells expressed KRT12. Microscopic evaluation of the cornea substitutes demonstrated that epithelial cells tended to form a normal stratified layer and that stromal keratocytes proliferated rapidly in the stromal substitute. The endothelial monolayer exhibited a pattern similar to a normal corneal endothelium.
  
  
  CONCLUSIONS
  These findings suggest that development of a full-thickness rabbit cornea model is possible in the laboratory and may open new avenues for research.
authors.affiliation: Department of Histology, University of Granada, Spain. malaminos@histolii.ugr.es
authors.forename: Miguel
authors.initials: M
authors.lastname: Alaminos
authors.forename: María
authors.initials: M
authors.lastname: Del Carmen Sánchez-Quevedo
authors.forename: José Ignacio
authors.initials: JI
authors.lastname: Muñoz-Avila
authors.forename: Daniel
authors.initials: D
authors.lastname: Serrano
authors.forename: Santiago
authors.initials: S
authors.lastname: Medialdea
authors.forename: Ignacio
authors.initials: I
authors.lastname: Carreras
authors.forename: Antonio
authors.initials: A
authors.lastname: Campos
mesh_terms: D000818:Animals; D001187:Artificial Organs; D002478:Cells, Cultured; D024102:Collagen Type VIII / Q000235:genetics; D003315:Cornea / Q000166:cytology* / Q000502:physiology; D003319:Corneal Stroma / Q000166:cytology / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism; D005337:Fibrin; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation; D007124:Immunoenzyme Techniques; D053551:Keratin-12; D007633:Keratins / Q000235:genetics; D008567:Membranes, Artificial; D008855:Microscopy, Electron, Scanning; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012685:Sepharose; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D024102:Collagen Type VIII; C507494:KRT12 protein, human; D053551:Keratin-12; D008567:Membranes, Artificial; D012333:RNA, Messenger; D007633:Keratins; D005337:Fibrin; D012685:Sepharose