Proliferation and differentiation of transplantable rabbit epithelial sheets engineered with or without an amniotic membrane carrier.
OBJECTIVE
To report a novel method of engineering transplantable, carrier-free corneal epithelial sheets by using a biodegradable fibrin sealant and to compare its characteristics with epithelial sheets cultivated on denuded amniotic membrane carriers.


METHODS
Stratified corneal epithelial sheets were prepared in culture dishes coated with biodegradable fibrin glue. Amniotic membrane (AM) carriers served as the control. The quality of cultivated sheets was compared by immunohistochemistry for cytokeratin (K)3, K12, K14, p63, occludin, and integrin beta1; electron microscopy; and colony-forming assays. K3 protein expression was compared by Western blot analysis. In a limbal-deficient rabbit transplantation model, postoperative adaptation and proliferation of BrdU-labeled cell sheets were examined by histology and anti-Ki67 staining.


RESULTS
Epithelial sheets were successfully engineered by using a biodegradable fibrin sealant. Cell sheets in both groups were multilayered, expressed K3, K12, and K14, and had functioning occludin(+) apical tight junctions as well as p63 and integrin beta1 staining in basal cells. The carrier-free sheets appeared to be more differentiated than the AM sheets, which was also demonstrated by the higher levels of K3 in the Western blots. The colony-forming efficiency of dissociated cells was similar in both groups, although larger colonies were observed on the AM sheets. AM sheets retained higher levels of BrdU-labeled cells and fewer Ki67(+) cells compared with carrier-free sheets after transplantation.


CONCLUSIONS
Tissue engineering with a commercially available fibrin sealant was an effective means of creating a carrier-free, transplantable corneal epithelial sheet. Carrier-free sheets were more differentiated compared with AM sheets, while retaining similar levels of colony-forming progenitor cells.
Higa
Kazunari
K
Department of Ophthalmology, Tokyo Dental College, Chiba, Japan.
Shimmura
Shigeto
S
Kato
Naoko
N
Kawakita
Tetsuya
T
Miyashita
Hideyuki
H
Itabashi
Yuji
Y
Fukuda
Keiichi
K
Shimazaki
Jun
J
Tsubota
Kazuo
K
D016475:3T3 Cells / Q000166:cytology; D000650:Amnion; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation; D017690:Cell Transplantation; D018920:Coculture Techniques; D003316:Corneal Diseases / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation*; D005260:Female; D005337:Fibrin / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D053547:Keratin-14 / Q000378:metabolism; D053558:Keratin-3 / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D051379:Mice; D011817:Rabbits; D013234:Stem Cells / Q000473:pathology; D023822:Tissue Engineering
D015415:Biomarkers; D053547:Keratin-14; D053558:Keratin-3; D019394:Ki-67 Antigen; D005337:Fibrin; D001973:Bromodeoxyuridine