title: A dual-layer silk fibroin scaffold for reconstructing the human corneal limbus.
abstract: Membranes prepared from Bombyx mori silk fibroin have shown potential as a substrate for human limbal epithelial (L-EC) and stromal cell cultivation. Here we present fibroin as a dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. We have compared the growth and phenotype of L-EC on non-porous versus porous fibroin membranes. Furthermore, we have compared the growth of limbal mesenchymal stromal cells (L-MSC) in either serum-supplemented medium or the MesenCult-XF(®) culture system within fibroin fibrous mats. The co-culture of L-EC and L-MSC in fibroin dual-layer constructs was also examined. L-EC on porous membranes displayed a squamous monolayer; in contrast, L-EC on non-porous fibroin appeared cuboidal and stratified. Both constructs maintained evidence of corneal phenotype (cytokeratin 3/12) and distribution of ΔNp63(+) progenitor cells. L-MSC cultivated within fibroin fibrous mats in serum-supplemented medium contained less than 64% of cells expressing the characteristic MSC phenotype of CD73(+)CD90(+)CD105(+) after two weeks, compared with over 81% in MesenCult-XF(®) medium. Dual-layer fibroin scaffolds consisting of L-EC and L-MSC maintained a similar phenotype as on the separate layers. These results support the feasibility of a 3D engineered limbus constructed from B. mori silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration.
authors.affiliation: Queensland Eye Institute, South Brisbane, Queensland 4101, Australia. laura.bray@qut.edu.au
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Bray
authors.forename: Karina A
authors.initials: KA
authors.lastname: George
authors.forename: Dietmar W
authors.initials: DW
authors.lastname: Hutmacher
authors.forename: Traian V
authors.initials: TV
authors.lastname: Chirila
authors.forename: Damien G
authors.initials: DG
authors.lastname: Harkin
mesh_terms: D000818:Animals; D012831:Bombyx; D002448:Cell Adhesion / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D018920:Coculture Techniques; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure; D005349:Fibroins / Q000378:metabolism* / Q000494:pharmacology / Q000648:ultrastructure; D006801:Humans; D016130:Immunophenotyping; D016850:Limbus Corneae / Q000187:drug effects / Q000502:physiology*; D008567:Membranes, Artificial; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure; D016062:Porosity / Q000187:drug effects; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D008567:Membranes, Artificial; D005349:Fibroins