Construction of a collagen-based, split thickness cornea substitute

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.


Study of patterned. multilayered, collagen-based scaffolds designed toserve as a cornea stroma
Kılıç, Cemile; Hasırcı, Vasıf Nejat; Rodriguez-Cabello, Jose Carlos; Department of Biology (2013)
Cornea is the most exterior, avascular and transparent layer of the eye and is about 500 µm in thick. It protects the eye from external objects and it is the main optical element of the eye refracting 70 % of the incoming light. After cataract, corneal diseases and wounds are the second leading cause of the blindness that affects more than 4 million people worldwide. For the highly damaged corneas where the corrections with spectacles or contact lenses cannot be achieved, tissue replacement is the only choi...
Cornea engineering on biodegradable polyesters
Zorlutuna, Pınar; Hasırcı, Vasıf Nejat; Department of Biotechnology (2005)
Cornea is the outermost layer of the eye and has an important role in vision. Damage of cornea due to injuries or infections could lead to blindness lowering the quality of life of the patient severely. In such cases, transplantation or artificial corneas have been used for treatment but both had drawbacks. The novel approach for corneal replacements is the tissue engineering of the cornea, a promising method which would be free of these drawbacks, if successful. In this study, carriers for tissue engineeri...
Construction of a collagen-based, split-thickness cornea substitute
Acun, A.; Hasırcı, Vasıf Nejat (2014-08-01)
Tissue-engineered corneas may become a promising alternative to allografts in the treatment of serious cornea defects because of the tunable characteristics of the biomaterials, biomimetic designs, and incorporation of patient's own cells. In this study, collagen foam was coated with a fibrous mat to mimic the stromal layer and the Bowman's layer. The stromal layer substitute was made of N-ethyl-N-(3-dimethyl aminopropyl)carbodiimide/N-hydroxysuccinimide-cross-linked collagen-chondroitin sulfate foam and se...
Contact guidance enhances the quality of a tissue engineered corneal stroma
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Corneal stroma is a very complex structure, composed of 200 lamellae of oriented collagen fibers. This highly complex nature of cornea is known to be important for its transparency and mechanical integrity. Thus, an artificial cornea design has to take into account this complex structure. In this study, behavior of human corneal keratocytes on collagen films patterned with parallel channels was investigated. Keratocytes proliferated well on films and reached confluency after 7 days in the incubation medium....
An automated blood cell analysis and classification system
Ongun, G; Halıcı, Uğur; Leblebicioğlu, Mehmet Kemal; Atalay, Mehmet Volkan; Erkmen, Aydan Müşerref; Beksac, S; Beksac, M; Erol, A (1998-01-01)
Direct analysis of blood and bone marrow smear images obtained from microscope are not common in current trends of hematology. Current blood smear analysis methods heavily depend on flow cytometry based techniques in which the blood cells are flew through microtubes thus classified according to their flow characteristics and cell volumes. This method is an indirect way of measuring features hence is accurate at a certain level. In this work an automated blood cell classification system is presented includin...
Citation Formats
A. Acun, “Construction of a collagen-based, split thickness cornea substitute,” M.S. - Master of Science, Middle East Technical University, 2013.