Reconstructed corneas: effect of three-dimensional culture, epithelium, and tetracycline hydrochloride on newly synthesized extracellular matrix.
OBJECTIVE
To evaluate the influence of the 3-dimensional collagen-glycosaminogycan-chitosan (CGC 3D) scaffold, epithelialization, and the addition of tetracycline hydrochloride on the ultrastructural organization, measured by the diameter and spacing of newly synthesized collagen I fibrils.


METHODS
Little is known about the role of interactions between epithelial cells and fibroblasts in controlling the extracellular matrix of the cornea. We developed a hemicornea from a CGC 3D matrix cocultured with keratocytes and human epithelial cells. The keratocytes colonized this substrate, proliferated, and synthesized the extracellular matrix, reproducing a living stroma equivalent.


RESULTS
Without a 3D scaffold, the collagen fibrils produced had an average diameter that was 42.7 nm and sigma = 16.9 nm. In the CGC 3D scaffold, the fibrils had an average diameter of 33.4 nm, with little dispersion (sigma = 6.7 nm), suggesting a greater regulation. The epithelium permitted a significant reduction in fibril diameter and interfibrillar spacing. Tetracycline hydrochloride had no effect on spacing but did have a significant effect on fibril diameter. We found positive interactions between the epithelium and tetracycline hydrochloride on the regulation of collagen fibrils but not on spacing. The presence of epithelium led to the increased formation of collagens I and V in the subepithelial area of the newly formed matrix. Type VI collagen was localized around the keratocytes throughout the matrix.


CONCLUSIONS
Epithelialization and the 3D scaffold had a great influence on the diameter of collagen I fibrils.
Banque de Corneés des Hospices Civils de Lyon, Lyon, France.
Nicolas
N
Builles
Virginie
V
Justin
Valerie
V
André
Carole
C
Burillon
Odile
O
Damour
D018929:Cell Culture Techniques; D049109:Cell Proliferation; D048271:Chitosan / Q000378:metabolism; D018920:Coculture Techniques; D024042:Collagen Type I / Q000378:metabolism* / Q000648:ultrastructure; D003315:Cornea / Q000502:physiology*; D019573:Epithelium, Corneal / Q000166:cytology* / Q000648:ultrastructure; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D046529:Microscopy, Electron, Transmission; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D013752:Tetracycline / Q000494:pharmacology*; D023822:Tissue Engineering
D024042:Collagen Type I; D006025:Glycosaminoglycans; D011500:Protein Synthesis Inhibitors; D048271:Chitosan; D013752:Tetracycline