Proliferation and differentiation of transplantable rabbit epithelial sheets engineered with or without an amniotic membrane carrier

Kazunari Higa, Shigeto Shimmura, Naoko Kato, Tetsuya Kawakita, Hideyuki Miyashita, Yuji Itabashi, Keiichi Fukuda, Jun Shimazaki, Kazuo Tsubota

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

PURPOSE. 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 β1; 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 β1 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.

Original languageEnglish
Pages (from-to)597-604
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume48
Issue number2
DOIs
Publication statusPublished - 2007 Feb

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Amnion
Fibrin Tissue Adhesive
Rabbits
Occludin
Bromodeoxyuridine
Integrins
Keratin-3
Transplantation
Western Blotting
Staining and Labeling
Tight Junctions
Tissue Engineering
Histology
Electron Microscopy
Stem Cells
Immunohistochemistry
Proteins

ASJC Scopus subject areas

  • Ophthalmology

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Proliferation and differentiation of transplantable rabbit epithelial sheets engineered with or without an amniotic membrane carrier. / Higa, Kazunari; Shimmura, Shigeto; Kato, Naoko; Kawakita, Tetsuya; Miyashita, Hideyuki; Itabashi, Yuji; Fukuda, Keiichi; Shimazaki, Jun; Tsubota, Kazuo.

In: Investigative Ophthalmology and Visual Science, Vol. 48, No. 2, 02.2007, p. 597-604.

Research output: Contribution to journalArticle

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abstract = "PURPOSE. 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 β1; 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 β1 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.",
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AU - Miyashita, Hideyuki

AU - Itabashi, Yuji

AU - Fukuda, Keiichi

AU - Shimazaki, Jun

AU - Tsubota, Kazuo

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N2 - PURPOSE. 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 β1; 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 β1 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.

AB - PURPOSE. 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 β1; 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 β1 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.

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