Biomechanical and microstructural characteristics of a collagen film-based corneal stroma equivalent

Rachael A.B. Crabb, M.S.[1]; Eric P. Chau[1]; Michael C. Evans, B.S.[2]; Victor H. Barocas, Ph.D.[1]; Allison Hubel, Ph.D.[3]

[1]Department of Biomedical Engineering; University of Minnesota; Minneapolis, Minnesota.
[2]Department of Chemical Engineering and Materials Science; University of Minnesota; Minneapolis, Minnesota.
[3]Department of Mechanical Engineering; University of Minnesota; Minneapolis, Minnesota.

The cornea is the most commonly transplanted tissue in the United States; with over 32,000 transplants every year, supply is unable to meet the demand, leading research towards tissue-engineered implants. The majority of the cornea’s focusing power is from the corneal stroma, which consists of numerous layers of organized collagen lamellae. Because of this, a corneal implant, called a keratoprosthesis, must replace the corneal stroma and mimic its properties.

Cornea engineering has already been performed by seeding human stromal fibroblasts onto collagen sponges, resulting in cells that behave similarly to their native counterparts in respect to their layers and shape. In vivo, the modulus of the cornea ranges from 3 to 13 MPa; in comparison, the modulus of the sponge ranges from 364 ± 41 Pa to 177 ± 75 Pa. In order to improve the biomechanical properties of these stroma replacements, these sponges must be strengthened or an entirely different material is needed. One potential alternative is the use of collagen films as a scaffold for engineered stroma.

Before seeding with fibroblasts, the surface of the collagen film was packed with fibers that exhibited no organization (Fig. 1). Two hours after seeding, a cross-section revealed these densely-packed collagen fibers were parallel to the film surface. The fibroblasts seeded on this film produced extracellular matrix (ECM) fibers with diameters between 35 to 75 nm, which is comparable to the fibers produced in the native cornea, where the fibril diameter is 70.5 ± 6.3 nm. In addition, human stromal fibroblasts that were seeded on these collagen films exhibited the same shape as native fibroblasts.

Fig. 1: A) Surface of an unseeded collagen film. B) Cross-section of collagen film 2 hours after seeding.

Using atomic force microscopy (AFM) and scanning electron microscopy (SEM), the microstructure of the film-derived stroma replacement was determined to be lamellae-like with interweaving cell layers. By stacking layers of collagen film, researchers believe they can replicate the layers of collagen lamellae in the human cornea.

The biomechanical properties of the film-based equivalent were also compared to those of the native stroma of the cornea. In vivo, the stroma exhibits a tensile strength of 3.8 MPa and a modulus that ranges from 3 to 13 MPa. The relaxed modulus of the film varied until the film was fully hydrated with media, at which point it stayed fairly constant at 0.3 ± 0.1 MPa. Upon complete hydration, the ultimate tensile strength of the film-based stromal equivalent was measured to be 0.4 ± 0.2 MPa. The research group does not understand the exact mechanism that links hydration to weakening, but they speculate the hydration period may coincide with unbound or soluble collagen leaving the matrix, thus weakening the material. While these properties are an improvement over the collagen sponge technique, both of values are significantly less than those of the native cornea; the paper did not explore how these differences could affect the performance of the implant.

While collagen films show potential for use as scaffolds for tissue-engineered stroma, researchers must perform additional studies to characterize the optical properties of the films. This research is promising because the films can be seeded with human stromal fibroblasts to create a multilayered implant, thus reducing the need for donated corneas.

Cornea implants are prevalent due to the insufficient supply of donor tissue for transplants. One particularly interesting aspect of the field of keratoprosthesis is the division into tissue engineering and biomaterials approaches. While the tissue-engineered implant is advantageous because it implements stromal fibroblasts to generate a new cornea, it still requires cell cultures for proper execution. On the other hand, the biomaterials approach does not require any donor tissue, but forces researchers to engineer a stroma replacement using different materials than the native cornea, like polymers or hydrogels. There is also potential for the artificial keratoprosthesis to incorporate various tools to improve its biological performance. It will be interesting to see which method emerges as the definitive solution.