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Morphologic Characterization of Organized Extracellular Matrix Deposition by Ascorbic Acid–Stimulated Human Corneal Fibroblasts

¹From the Schepens Eye Research Institute and ²Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; the ³Department of Mechanical and Industrial Engineering, Northeastern University, Boston, Massachusetts; and the ⁴Departments of Biochemistry and ⁵Ophthalmology, Boston University School of Medicine, Boston University, Boston, Massachusetts.

PURPOSE. To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures.

METHODS. Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to 5 weeks. Constructs were assessed by light (phase-contrast and differential interference-contrast) and transmission (standard and quick freeze/deep etch) microscopy.

RESULTS. Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27–51 nm) that often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct, whereas quick-freeze, deep-etch micrographs showed the details of the matrix interaction with fibroblasts through arrays of membrane surface structures.

CONCLUSIONS. Human keratocytes, cultured in a stable vitamin C derivative, are capable of assembling extracellular matrix, which comprises parallel arrays of ECM fibrils. The resultant constructs, which are highly cellular, are morphologically similar to the developing mammalian stroma, where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggests a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes that govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue engineering a biomimetic stroma.