Nog Gen İfadesi Susturulmuş Hücrelerin Oluşturulması ve İpek Doku İskeleleri Üzerinde Osteojenik Farklılaşmalarının İncelenmesi

Abstract

This study was funded by Hacettepe University Scientific Research Projects Coordination Unit (BAP) graduate project entitled "Generation of Nog Knockout Cells by CRISPR/Cas9 Mediated Genome Editing and Examination of Their Osteogenic Differentiation on Silk Scaffolds." (FYL-2018-17253). This thesis, aimed to investigate gene editing mechanisms (CRISPR-Cas9, siRNA) to inhibit the synthesis of Noggin protein, which is an antagonist of bone morphogenetic protein (BMP) signaling pathway to achieve molecular regulation in MC3T3-E1 cells. Then, it is aimed to increase the osteogenic differentiation of transfected MC3T3-E1 cells seeded on silk scaffolds. For these purposes, the thesis study was carried out in 4 steps. In the first step, a silk solution was obtained from Bombyx mori silk cocoons and the solvent casting-particulate leaching process was performed to produce silk scaffolds. SEM images showed that the scaffolds have a porous structure with internal connections and the porosity was calculated as 93.5%. The average diameter of large pores was found to be 448±76 μm, while the average diameter of small pores was 85±27 μm. It was determined that equilibrium water uptake capacity of silk scaffolds reached 1300% in 5 minutes. In the second step of the thesis, the transfection study of the control CRISPR plasmid was performed on MC3T3-E1 cells using Ultracruz® and Lipofectamin®3000 transfection agents. However, it was found that the transfection agents were not sufficient for efficient transfection. Our laboratory conditions were not able to use electroporation or virus-based transfection methods to ensure efficient transfection, therefore transfection studies were continued with siRNA. In the third step, transfection studies were carried out with siNog on MC3T3-E1 cells. Primarily, transfection parameters were optimized with red fluorescence-labeled control siRNA/Lipofectamine®3000. Subsequently, a transfection study with siNog/ Lipofectamin®3000 was performed on MC3T3-E1 cells in static culture conditions. Real-Time Polymerase Chain Reaction (RT-PCR) analysis showed that the Nog gene expression was significantly decreased in the siNog group compared to control and negative control groups (p <0.05*). In the final step, the siNog/Lipofectamin®3000 transfection studies were performed on MC3T3- E1 cells seeded silk scaffolds. The culture was continued for 21 days with an osteogenic differentiation medium containing 100 ng/mL of BMP2. To investigate the bone formation and the transfection efficiency, gene expression levels of Nog, Alp, Col1a1, Osp, and Ocn measured by RT-PCR. As a result of RT-PCR, it was determined that Nog gene expression was successfully suppressed in siNog group and Ocn gene expression on the 21st day of cell culture, increased approximately 5 times in the siNog group compared to the control group (p <0.05*). The proliferation behavior of the cells was analyzed with the MTT (3- [4,5-Dimethylazol-2-yl] -Diphenyltetrazolium Bromide) test and it was concluded that transfected cells did not adversely affect cell adhesion and proliferation. When the cell morphology was examined by SEM analysis, it was observed that the cells in the control and siNog groups reached the inner surface of the pores, successfully grown on the t scaffold, and mineral structures were observed in the siNog groups. When mineral structures were examined with EDS analysis, intense Calcium (Ca) and Phosphorus (P) elements were found clustered on cells in siNog groups. Histological staining showed that siNog group have a more intense mineralized area compared to the control group. As a result, siNog transfected MC3T3-E1 cells seeded on silk scaffolds showed a more efficient osteogenic differentiation compared to the control group. Ocn gene expression which is responsible for mineralization was higher than that of control group and intense mineralized areas were observed in the siNog group. It was concluded that due to the increased mineralization, the siNog transfected preosteoblastic cells on 3D silk scaffolds can be used to induce bone regeneration.