Construction of a collagen-based, split thickness cornea substitute

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2013
Acun, Aylin
Cornea is the transparent outermost layer of the eye. It is a thin (500 µm) multilayer tissue which performes around 75% of the total refraction in the eye. It also protects the inner layers against any type of damage. Since it is avascular, the three cellular layers of cornea always need transport of nutrients and other materials in and out of the tissue via diffusion. Any change in shape, transparency or thickness of cornea, or physical damages and infections, may cause serious defects. The conventional methods are satisfactory in the treatment of mild injuries but severe cases require the substitution of the tissue with an equivalent. Keratoprosthesis and donor corneas that are used as replacements do not completely meet requirements. Tissue engineering can be an alternative method for preparing a biocompatible and stable cornea equivalent. The ability to choose from a variety of materials and the ability to incorporate bioactive agents allow the researchers to tailor make the construct. The structure needs to be seeded with the patient’s own cells and cultured in vitro to yield an optimal corneal replacement. In this study a novel, split thickness cornea replacement is proposed to substitute the two upper cellular layers (epithelium and stroma) of the native cornea. The design includes a chondroitin sulfate impregnated collagen type I (isolated from rat tail) foam (CSXLF) produced by lyophilization carrying electrospun fibers of the same polymer collected directly on top of the foam, forming the bilayer structure (Fo-Fi). The fiber layer was intended to separate the epithelium and the stroma of the reconstructed cornea yet to allow material transfer in between. The foam layer (bottom) was crosslinked by N-ethyl-N-[3-dimethylaminopropyl] carbodiimide (EDC), and N-hydroxy succinimide and after fiber deposition the bilayer was further stabilized with physical crosslinking (DHT method). The physical characterization of the foam showed that their pore sizes (10-200 µm) and porosities (around 70%) were well within the desired range for typical tissue engineering applications. The cell free wet thicknesses of both single and bilayer constructs were close to that of the native stroma and light transmittance through these scaffolds was quite high (around 82% in the 500-700 nm range). The scaffolds were also tested for their stability and shown to be suitable for in vitro testing. In vitro studies were performed using retinal pigment epithelial cells (RPE, D407 cell line) and isolated human corneal keratocytes (HK) to reconstruct the epithelium and the stroma, respectively. Three types of constructs were prepared; only HK seeded Fo-Fi constructs, RPE-HK seeded CSXLFs, and RPE-HK seeded Fo-Fi constructs. All were shown to support cell attachment and promoted cell proliferation as was shown by the cells that covered the inner and outer spaces of the scaffolds. The fiber layer prevented the mixing of the two cell types, without hindering material exchange between them. Moreover, when co-cultured for 14 days, the keratocytes started to deposit collagen type I, a specific marker of these cells. In contrast, ECM deposition could not be observed in the single type cell seeded samples. The co-cultured bilayer construct was tested for suturability at the end of 31 days of in vitro incubation and it was shown that it could be successfully sutured without any major tears. Under the light of these results it was concluded that both the single layer and the bilayer constructs show promise for use as split thickness cornea replacements.

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Citation Formats
A. Acun, “Construction of a collagen-based, split thickness cornea substitute,” M.S. - Master of Science, Middle East Technical University, 2013.