Development of a reconstructed cornea from collagen-chondroitin sulfate foams and human cell cultures.
OBJECTIVE
To develop an artificial cornea, the ability to coculture the different cell types present in the cornea is essential. Here the goal was to develop a full-thickness artificial cornea using an optimized collagen-chondroitin sulfate foam, with a thickness close to that of human cornea, by coculturing human corneal epithelial and stromal cells and transfected human endothelial cells.


METHODS
Corneal extracellular matrix was simulated by a porous collagen/glycosaminoglycan-based scaffold seeded with stromal keratocytes and then, successively, epithelial and endothelial cells. Scaffolds were characterized for bulk porosity and pore size distribution. The performance of the three-dimensional construct was studied by histology, immunofluorescence, and immunohistochemistry.


RESULTS
The scaffold had 85% porosity and an average pore size of 62.1 microm. Keratocytes populated the scaffold and produced a newly synthesized extracellular matrix as characterized by immunohistochemistry. Even though the keratocytes lost their CD34 phenotype marker, the absence of smooth muscle actin fibers showed that these cells had not differentiated into myofibroblasts. The epithelial cells formed a stratified epithelium and began basement membrane deposition. An endothelial cell monolayer beneath the foam was also apparent.


CONCLUSIONS
These results demonstrate that collagen-chondroitin sulfate scaffolds are good substrates for artificial cornea construction with good resilience, long-term culture capability, and handling properties.
Vrana
N Engin
NE
METU, BIOMAT, Department of Biological Sciences, Biotechnology Research Unit, Ankara, Turkey.
Builles
Nicolas
N
Justin
Virginie
V
Bednarz
Jurgen
J
Pellegrini
Graziella
G
Ferrari
Barbara
B
Damour
Odile
O
Hulmes
David J S
DJ
Hasirci
Vasif
V
D000199:Actins / Q000378:metabolism; D001187:Artificial Organs; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002809:Chondroitin Sulfates / Q000378:metabolism*; D018920:Coculture Techniques; D003094:Collagen / Q000378:metabolism*; D003315:Cornea; D003319:Corneal Stroma / Q000166:cytology*; D004728:Endothelium, Corneal / Q000166:cytology*; D019573:Epithelium, Corneal / Q000166:cytology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007150:Immunohistochemistry; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D016062:Porosity
C558396:Acta2 protein, mouse; D000199:Actins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D002809:Chondroitin Sulfates; D003094:Collagen