Biomechanical and Microstructural Characteristics of a Collagen Film-Based Corneal Stroma Equivalent

Abstract

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 influence of various rehydration protocols on biomechanical properties of different acellular tissue matrices

“…Our tension results indicate good agreement with previous studies that demonstrated a significant decrease in mechanical properties Representative DSC thermograms of MD (a) and DM (b) in either the dry or rehydrated state of collagen matrices going from the dry to the wet state [18,19]. The present study did not determine the specific mechanism for the change in mechanical properties following rehydration of the different collagen matrices.…”

Microstructural Characteristics of Extracellular Matrix Produced by Stromal Fibroblasts

“…The initial relaxed modulus was 0.03 MPa for collagen sponges and 2.1 MPa for single collagen films. 12 In contrast, the modulus of collagen gels is in the range of 1.4-24.3 kPa and dependent on the concentration of collagen during at the start of gelation which was from 0.3 to 3 mg/mL of collagen in studies by Roeder et al 47 The modulus of the collagen sponge used in this investigation is lower than that of the collagen films. The difference in fibril diameter synthesized on the collagen sponge may reflect the influence of both compaction and microstructure.…”

“…Collagen sponges formed from freeze-drying and crosslinking a dispersion of insoluble collagen had both a modulus and ultimate tensile strength (UTS) of 0.03 MPa, and modification of the matrix manufacturing method resulted in the formation of a collagen film with a relaxed modulus and UTS of 2.1 MPa and 1.6 MPa respectively. 12 These mechanical properties are considerably higher than those observed in a collagen gel (modulus 1.4-24.3 kPa for 0.3-3 mg/mL collagen 47 ). The properties of the matrix can change with time in culture as seen with a decline in the modulus for both collagen sponges 5,40 and collagen films.…”

“…12 Briefly, for films the 1% w/v collagen dispersions in 0.001 M HCl were blended and deaerated as described above, cast 2-3 mm thick in plastic petri dishes, allowed to dry uncovered in a fume hood overnight, cut with a 14 mm diameter punch, and dehydrothermally crosslinked in a Fisher Scientific Isotemp vacuum oven at 110°C and 2.5 torr for 5 days. During drying the collagen settled to the bottom of the dish creating films approximately 60 lm thick once dry.…”

“…The properties of the matrix can change with time in culture as seen with a decline in the modulus for both collagen sponges 5,40 and collagen films. 12 Additional gene expression studies have shown that seeding stromal cells into a three-dimensional (versus two-dimensional) matrix upregulates both matrix synthesis and matrix degradation. 5 Together, these studies suggest that the cell-matrix interactions play a critical role in the long-term properties of a tissue-engineered corneal replacement.…”