title: Corneal recovery in a rabbit limbal stem cell deficiency model by autologous grafts of tertiary outgrowths from cultivated limbal biopsy explants.
abstract: OBJECTIVE
  To determine the corneal regenerative capacity of sequentially generated primary, secondary, and tertiary limbal explant outgrowths in a limbal stem cell deficiency (LSCD) surgical model.
  
  
  METHODS
  Two-millimeter-long limbal shallow biopsies were surgically excised from the upper quadrant of the right eye of rabbits and set on preserved amniotic membrane for explant culture. After the generation of primary outgrowth, the biopsies were sequentially transferred to new amniotic membrane to generate secondary and then tertiary outgrowths. Eighteen rabbits were subjected to a 360° limbal peritomy extending into the scleral zone and combined with superficial keratectomy of the corneal periphery and thorough mechanical debridement of the central cornea in their left eye. Right eye outgrowths, six of each generation, were engrafted on the ocular surface. Clinical outcomes (neovascularization, corneal clarity, and corneal fluorescein staining) were graded after 6 months. Post-mortem corneas were compared with histology, immunochemistry for p63 and Krt3, ABCG2-dependent dye exclusion, and capacity for outgrowths in explant culture.
  
  
  RESULTS
  Immunohistology and western blot of the outgrowths for p63 and Krt3 indicated no differences in expression between the primary and tertiary outgrowths for these two markers of growth and differentiation. Clinically, all rabbits treated with amniotic membrane alone developed severe LSCD. Most rabbits grafted with cell outgrowths from all three outgrowth generations achieved stable (>6 months) recovery of the ocular surface. There were partial failures of grafts performed with two secondary and tertiary outgrowths. However, Kruskal-Wallis statistical analysis of the clinical scores yielded no significant difference between the three groups (p=0.524). Histology showed full anatomic recovery of grafts made with primary and tertiary outgrowths. Krt3 and p63 expression throughout the whole limbal corneal epithelium with primary or tertiary outgrowths was not distinguishable from each other. The percentage of dye-excluding cells present within this zone and the capacity of the explant epithelial outgrowth of the regenerated peripheral corneal zone were also on par with those of the donor corneas. The Krt3-negative cells that characterize the basal epithelial layer of the normal limbus could not be found in any regenerated cornea from the primary to tertiary outgrowths.
  
  
  CONCLUSIONS
  Our results demonstrate that in rabbits post-primary explant outgrowths retain the capacity for LSCD recovery found in primary explants.
authors.affiliation: Department of Ophtalmology, Dokuz Eylul University School of Medicine, Izmir, Turkey.
authors.forename: Ozlem Barut
authors.initials: OB
authors.lastname: Selver
authors.affiliation: Department of Ophtalmology, Dokuz Eylul University School of Medicine, Izmir, Turkey.
authors.forename: Ismet
authors.initials: I
authors.lastname: Durak
authors.affiliation: Department of Biochemistry, Dokuz Eylul University School of Medicine, Izmir, Turkey.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Gürdal
authors.affiliation: Department of Biochemistry, Dokuz Eylul University School of Medicine, Izmir, Turkey.
authors.forename: Kemal
authors.initials: K
authors.lastname: Baysal
authors.affiliation: Department of Hematology, Dokuz Eylul University School of Medicine, Izmir, Turkey.
authors.forename: Halil
authors.initials: H
authors.lastname: Ates
authors.affiliation: Department of Ophtalmology, Dokuz Eylul University School of Medicine, Izmir, Turkey.
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Ozbek
authors.affiliation: Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY.
authors.forename: Albert
authors.initials: A
authors.lastname: Wu
authors.affiliation: Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY.
authors.forename: J Mario
authors.initials: JM
authors.lastname: Wolosin
mesh_terms: D000650:Amnion; D000818:Animals; D001706:Biopsy; D015153:Blotting, Western; D018929:Cell Culture Techniques; D003315:Cornea / Q000502:physiology*; D003316:Corneal Diseases / Q000503:physiopathology / Q000628:therapy*; D004195:Disease Models, Animal; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D053558:Keratin-3 / Q000378:metabolism; D016850:Limbus Corneae / Q000473:pathology*; D011817:Rabbits; D020127:Recovery of Function / Q000502:physiology; D012038:Regeneration / Q000502:physiology; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000473:pathology*; D054457:Tissue Scaffolds; D014157:Transcription Factors / Q000378:metabolism; D014182:Transplantation, Autologous
chemical_list: D053558:Keratin-3; D014157:Transcription Factors