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J.W. Ruberti, X.Q. Guo, S.A. Melotti, A.E. Hutcheon, J.D. Zieske; Three Dimensional Organization of Cells and Matrix in Stromal Constructs Secreted by Human Primary Corneal Fibroblasts . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3948.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize, with a non–invasive imaging method, the 3–dimensional organization of the cellular population and extracellular matrix secreted by primary human corneal fibroblasts stimulated with 2–O– a –D–Glucopyranosyl–L–acorbic acid.
Primary human corneal fibroblasts (harvested from donor corneas) were cultured onto polyester transwells and stimulated to produce matrix with a stable form of ascorbic acid. The cell and matrix organization in the living tissue were characterized by differential interference contrast (DIC) imaging at four and eight weeks. Confocal microscopy on fresh constructs was performed to support the DIC. The total thickness of the constructs, the density and organization of the cells and the thickness of the organized "lamellae" within the constructs were recorded.
Confocal and DIC microscopy detected reasonably consistent 3–dimensional stratified cellular organization within the stromal constructs. Generally, there were 4 identifiable regions: 1) at the transwell membrane surface there was a thin, dense, aligned layer of spindle shaped cells. 2) The next layer was populated with fewer cells and in this region DIC revealed aligned thin "lamellar" sheets of matrix which alternated direction. 3) the next region comprised several layers of cells with a more random orientation. 4) Finally at the construct surface there was a layer of loosely attached cells. The construct thickness averaged 36 (sd 6.6) microns at four weeks and 50 (sd 17.7) microns thick at eight weeks (not significant difference, p=0.18). The layer of aligned lamellar matrix was 25 microns both at four weeks (sd 6.4) and at eight weeks (sd 12.4). There was variability within each construct group possibly because the cell sources were not age–matched.
DIC imaging revealed that the current series of stromal constructs demonstrate a reasonably consistent organizational stratification with varying amounts of lamellar matrix secretion. The constructs increase in total thickness over the period from four to eight weeks and the thickest region of aligned alternating "lamellae" was 35 microns in at least two of the eight week specimens. These results suggest that 15 to 20 constructs could be "stacked" to produce a full–thickness stroma comprising aligned lamellae for use as a scaffolding for tissue engineered corneas. Because of specimen variability, live DIC imaging may be used to screen candidate constructs for suitable matrix quantity and organization.
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