The growth in refractive surgeries and corneal replacements has fueled interest in the development of a tissue-engineered cornea. This study characterizes the microstructure and biomechanical properties of film-based corneal stroma equivalents over time in culture. The increased collagen density in the films was hypothesized to result in improved mechanical properties both initially and over time. The microstructure of the film-based stromal equivalent was examined using atomic force microscopy and scanning electron microscopy; the mechanical properties, relaxed modulus, and ultimate tensile strength were quantified using uniaxial tensile testing. The dense, film-based stromal equivalent had a lamellae-like microstructure, which was notably different than the porous structure of sponges used previously. Seeded human corneal stromal fibroblasts remained on the surface of the film rather than migrating into the film and produced fibers of extracellular matrix with diameters of 35-75 nm. After an initial decrease during hydration, the relaxed modulus and ultimate tensile strength for fully hydrated collagen films were 0.4 +/- 0.2 MPa and 0.3 +/- 0.1 MPa, respectively. The mechanical properties of cell-seeded films mimicked those of control films. While further studies are needed to quantify the optical properties, the dense, lamellae-like structure of collagen films is a feasible scaffold for the development of tissue-engineered stroma.

The overall objective of this investigation was to characterize the extracellular matrix deposited by the stromal fibroblasts as a function of time in culture and matrix microstructure. Stromal fibroblasts were seeded onto collagen matrices and cultured for up to 5 weeks. The collagen matrices contained collagen fibrils with an average diameter of 215 +/- 20 nm. When cultured on a collagen film, an average fibril diameter of 62 +/- 39 nm was observed for single layer films with only slight variations with time in culture, and after 1 week of culture between two film layers 67 +/- 47 nm fibrils were observed after 1 week. When the film surface was molded into 1 and 2 microm microgrooves, the initial average fibril diameter of the extracellular matrix was 73 +/- 21 and 73 +/- 31 nm respectively. When cultured on a collagen sponge, an average fibril diameter of 107 +/- 20 nm was initially observed and decreased to 47.5 +/- 17 nm after 1 week in culture. For cells cultured on a collagen sponge, Western blotting showed an increase in myofibroblast phenotype expression with time in culture. Shifts in phenotype were less distinct for cells cultured on collagen films. The microstructure, rather than geometry, of the matrix substrate appeared to influence the newly synthesized extracellular matrix and cell phenotype.

Interest in developing tissue-engineered cornea has increased with the decrease in the supply of donor tissue; however, the high strength and transparency of the cornea present a challenge. Both the collagen processing and crosslinking methods were hypothesized to influence the optical and biomechanical properties of collagen matrices, while regular surface topography was hypothesized to align stromal fibroblasts. Improved transparency and strength were observed when soluble tropocollagen was added to the insoluble collagen and when glucose-mediated ultraviolet (UV) crosslinking as opposed to dehydrothermal crosslinking was used. The fraction of transmittance of the collagen films fabricated from insoluble collagen and soluble tropocollagen and glucose-mediated UV crosslinking was initially 0.91 +/- 0.02 and 0.98 +/- 0.01 for the smooth films and 0.90 +/- 0.02 and 0.97 +/- 0.02 for the microgrooved films at 400 and 700 nm and was comparable to that of the native cornea, while the relaxed modulus and ultimate tensile strength ranged from 0.9 to 9.4 MPa and from 0.7 to 4.1 MPa, respectively, over the 3 weeks of culture and were initially at or below the range of values for the native cornea. These collagen scaffolds were significantly stronger and more transparent than previous scaffolds, and aligned stromal fibroblasts were observed on microgrooved surfaces.

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