Construction of a complete rabbit cornea substitute using a fibrin-agarose scaffold.
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.
Alaminos
Miguel
M
Department of Histology, University of Granada, Spain. malaminos@histolii.ugr.es
Del Carmen Sánchez-Quevedo
María
M
Muñoz-Avila
José Ignacio
JI
Serrano
Daniel
D
Medialdea
Santiago
S
Carreras
Ignacio
I
Campos
Antonio
A
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*
D024102:Collagen Type VIII; C507494:KRT12 protein, human; D053551:Keratin-12; D008567:Membranes, Artificial; D012333:RNA, Messenger; D007633:Keratins; D005337:Fibrin; D012685:Sepharose