Corneal Tissue Engineering by Using Peptide Hydrogel/Elastomer Membrane Lamellar Structures

Cornea, the outermost layer of the eye, is a transparent tissue composed of highly ordered collagen fibrils. Cornea provides pressure resistance and protection. Corneal cells residing in different corneal layers have unique properties to constitute this transparent, permeable, and durable tissue. Endothelial cells located on Descement’s membrane define fluid entrance to the cornea. Keratocytes give the unique structure of stroma and play an important role in light transmission. Epithelial cells constitute a protective barrier and regulate water, gas, and nutrient transport in and out of stroma. External chemical/mechanical damage, aging and diseases can directly affect these cells and thereby cause visual disturbances. Damaged cornea can be replaced through corneal transplantation, however only a limited number of patients can receive corneas due to limited tissue availability. A bioengineered corneal substitute can be a substantial solution for transplantable tissue shortage. An ideal bioengineered cornea is expected to be optically transparent, biocompatible, water and glucose permeable with mechanically stable. Various biomaterials including decellularized tissues, natural polymers, and peptide amphiphiles have been found investigated for corneal applications.<br/>Our aim is to sustain the growth of multiple cell types in three layers as co-cultures on peptide scaffolds to mimic natural cornea. Briefly, peptide hydrogel constituting the corneal stroma and polymer membrane being home to the epithelial and endothelial layers will be constructed. We initially isolated primary human corneal cells. Epithelial, stromal and limbal cells were characterized by immunocytochemistry and gene expression profiling. Keratocyte cells were immortalized and confirmed exhibiting cell specific phenotype until passage 15. Limbal cells were either immortalized or differentiated into distinct cells with corneal keratocyte or epithelial characteristics by using conditioning media. Endothelial cells proliferate only in initial culture and couldn’t be immortalized.<br/>Peptide hydrogels with different bioactive sequences to provide cell adhesion and anti-angiogenic properties were formed within serum free culture media. Refractive indexes of all hydrogels were found to be around 1,33, which is slightly lower than natural cornea making them suitable candidates for corneal tissue engineering. For all hydrogel formulations, storage and loss modulus were measured lower than elastic modulus of natural cornea. MAX8:MAX8-IKSAV and MAX8:MAX8-HRH exhibit higher mechanical strength relative to MAX8:MAX8-RGD. The hydrogels showed advanced biocompatibility to promote cell adhesion and proliferation for corneal keratocytes. Hydrogels’ transparency was decreased from 95% to only 85% following a two-week culture period.<br/>Elastomer membranes were prepared by curing pre-polymer at 120^oC under vacuum for 12-48 hours. Two methods were followed for membrane casting: (1) Dr. Blade and (2) polyHIPE. Membranes prepared by Dr. Blade exhibit amenable optical transmission, mechanical durability, and cellular biocompatibility for corneal epithelial cells. However, this membrane was lack of glucose permeability. Membranes prepared by polyHIPE method produced porous structures and exhibit glucose permeability with a diffusion coefficient of 3.4 x 10^-4 cm²/s. Decreasing the thickness of this membrane from 3 mm to 0.3 mm, we expect to decrease diffusion rate further.<br/><i>This work is funded by 2232 International Fellowship for Outstanding Researchers Program of TUBITAK (Grant number 118C371). The funder had no role in study design, data collection or analysis, the decision to publish, or the preparation of this abstract. </i>