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J. W. Ruberti, X. Q. Guo, S. A. Melotti, A. E. Hutcheon, V. Trinkaus-Randall, J. D. Zieske; Fibril Organization and Morphology in Stromal Constructs Synthesized by Primary Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1867. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the organization and morphology of extracellular matrix (ECM) fibrils in stromal constructs synthesized by primary human corneal fibroblasts.
Human corneal keratocytes were stimulated with stabilized Vitamin C in the presence of 10% FBS. The resulting fibroblasts were plated at high density onto transwell membranes and allowed to produce matrix for up to 12 weeks. The constructs were processed for TEM and stained for collagen at 4, 8 and 12 weeks. Quick Freeze Deep Etch (QFDE) imaging was performed on 4 week constructs to reveal cell and matrix details not elucidated by standard TEM. Indirect immunofluorescence was performed on 4 week constructs to determine whether collagen type V and VI were present.
By 4 weeks the fibroblasts stratified to 8 to 12 layers and produced a stromal construct comprising flattened cells interspersed in a matrix containing alternating arrays of large striated fibrils in stromal-like lamellae. Confluent layers of fibroblast cells bounded the construct at the transwell membrane and at the surface. The predominant ECM fibrils were striated and averaged 38 nm in diameter (estimated by TEM) but possessed a polydisperse diameter distribution (S.D. of 7.2 nm). The growing constructs bore a strong resemblance to the developing mammalian stroma in that they exhibited a high cell-to-matrix volume ratio, organized arrays of smaller microfibrils (<20 nm) and flattened cells parallel to the alternating "lamellae". QFDE Microscopy revealed a dense intercellular matrix and that the fibroblasts expressed arrays of regular surface structures which were observed binding the ECM fibrils. Type V and VI collagens were detected by indirect immunofluorescence and were distributed throughout the constructs. At 8 and 12 weeks, TEM revealed decreasing numbers of the larger striated fibrils (but many aligned, weakly staining fibrils).
The investigation demonstrated that human corneal fibroblasts are capable of synthesizing organized ECM similar to developing mammalian stroma by 4 weeks in culture. The system is thus likely a good model in which to study collagen fibrillogenesis and organization. However, continued culturing led to resorption of the large striated fibrils and indicates that the longevity of the system may be limited without modifications such as addition of protease inhibitors or application of mechanical loads.
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