Kornea Doku Mühendisliği İçin Hibrit Biyomateryal Geliştirilmesi

Abstract

The cornea is a transparent structure that covers 1/6 of the eye, has a convex shape, has the task of focusing light and protecting the eye from external influences, and has a major role in vision. Corneal blindness; It occurs due to damage to the tissue due to corneal traumas, bacterial and viral infections, genetic disorders. Especially in severe trauma, the tissue may need to be replaced with a suitable equivalent. Such surgical applications are performed in the form of allogeneic cornea transplantation or replacement of the artificial cornea (keratoprosthesis) with damaged tissue. Researchers are trying to produce tissue equivalents for removing pathological corneal tissue from the patient and transplanting the damaged area with tissue conjugates, with innovative perspectives provided by tissue engineering applications. In recent years, corneal tissue engineering has been applying some strategies to solve the problems encountered in the treatment of corneal ailments and injuries. These strategies are to design biomimetic matrix systems for corneal tissue conjugates such as hydrogel technique, prefabricated matrices and decellularized corneal tissues. However, there are some limitations such as insufficient mechanical strength of hydrogel systems and difficulty of sustainability. Decellularized matrices cannot serve as an ideal corneal replacement without being supported with a suitable hydrogel, due to their insufficient mechanical strength. For this reason, it is necessary to use a polymer that will improve the mechanical strength and transparency properties of the decellularized matrix. Within the scope of this thesis, it is aimed to produce hybrid matrixes with superior mechanical strength and transparency by crosslinking deselularized matrices supported with Gelatin Methacrylate (GelMA) polymer at appropriate time and energy under UV. In the first stage of the study, a Sodium Dodecyl Sulfate (SDS) based decelularization protocol was applied following dissection of bovine corneal tissues. Characterization tests were performed to determine the success of the decellularization process. For this, Scanning Electron Microscopy analysis (SEM), Total Reflectance Reduced Fourier Transform Infrared Spectrophotometer analysis (ATR-FTIR) were used to comparatively study the structural properties of decelularized and natural tissue. Hematoxylin and eosin (HE) for the determination of decellularization success; Histological analyzes such as DAPI staining were performed to detect residual nuclei. PicoGreen test to numerically determine the amount of residual DNA remaining in the tissue after the decellularization process; dimethyl methylene blue test (DMMB) to numerically determine the amount of glycosaminoglycan remaining in tissue; Hydroxyproline test was performed to determine the amount of collagen in the structure. According to the PicoGreen test result, a successful decellularized matrix was obtained by removing 70.1% DNA from natural tissue. According to the DMMB test result, when compared with the natural cornea and the decellularized cornea was found to have a 40% decrease in sGAG content compared to the natural cornea. According to the hydroxyproline test result, the amount of hydroxyproline of natural tissue and decellularized tissue was determined as 0.65 ± 0.01 mg and 0.74 ± 0.01 mg. Comparing the findings with the literature knowledge, the superiority of the decelularization process has been revealed. In the second stage of the thesis, gelatin methacrylate polymer (GelMA) produced at 8% concentration with decellularized corneas, different energy under UV (3200 μJ/cm2, 6210 μJ/cm2 and 6900 μJ/cm2) and at different times (immediately, 5 minutes and 24 hours) is characterized by cross-linking. The diameter and thickness of the natural cornea and the hybrid and decellularized corneas obtained were measured FTIR was performed in order to examine the chemical structure of hybrid structures and GelMA polymer in detail. Hybrid matrix, decellularized matrix and natural corneal structure; tested for enzymatic degradation, water holding capacity and rate of degradation in phosphate buffer saline (PBS). According to the results of the swelling test, the water holding capacity of the natural cornea was found to be 85.7 ± 0.22%, while the water holding capacity of the decellularized cornea was 85.2 ± 3.09%, and the water holding capacity of the hybrid matrix was found to be 74.2 ± 1.03%. According to the results of the degradation test in PBS, which was maintained for 28 days, mass loss was 24.73 ± 6.55% in the natural cornea, 51.37 ± 4.29% in the decellularized cornea, and 50.9 ± 10.34% in the hybrid cornea. According to the results of the enzymatic degradation test performed with collagenase A, at the end of four hours, the hybrid structure lost 44.68 ± 5.99%, natural cornea 15.56 ± 2.84% and decelularized tissue 19.98 ± 2.56% mass. In the hybrid matrices subjected to mechanical testing, the group that was cross-linked with an energy of 6210 μJ/cm2 after 24 hours after impregnation of GelMA polymer came to the fore compared to the other groups. Finally, by the in vitro method, stromal keratocyte cells were encapsulated into the hybrid matrix obtained by cross-linking with GelMA, and finally proliferation analyzes were performed. Cells used for sowing were reproduced from bovine cornea by primary culture method. Appropriate number of cells were sowed in the experimental groups and the behavior of these groups in cell culture was observed over a period of 14 days. Cell viability tests were performed in cell culture studies. At the last stage of cell culture, cells were examined using the DAPI staining method and the living dead test. As a result of the study, results were obtained that both optical and mechanical properties were improved. Within the scope of the thesis, it is recommended as a promising approach for the successful development of hybrid matrix structures and corneal transplants.