LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans
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Yazar "Elmalı, Gizem" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans'a göz atma
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ÖgePreparation and characterization of PCL/lignin sponges for bone tissue engineering applications(Graduate School, 2022-05-09) Elmalı, Gizem ; Kök, Fatma Neşe ; 521181108 ; Molecular Biology-Genetics and BiotechnologyOrgans and tissues may be damaged by accident, congenital anomalies, diseases and similar reasons. Although living tissues have the capacity to heal these damages, large defects or severe tissue losses can only be repaired to a certain extent. Tissue engineering techniques have been developed to trigger, assist and accelerate the healing process, or directly replace the targeted tissue. In tissue engineering, the most suitable three-dimensional scaffold structure is produced to match the characteristics of the targeted tissue and provide the initial structural integrity and organizational backbone for cells to replace the damaged tissues and organs. Especially bone and its related diseases can cause a significant impact on a person's health and quality of life if they are not treated and healed properly. Millions of bone injuries have been occurring each year and they are not only affecting people's life but also cause to a serious burden on country's economies. Bone tissue engineering was presented as a solution to address these problems. In this thesis, a synthetic and natural polymer blend was used to create a scaffold that is suitable for bone tissue engineering. Polycaprolactone (PCL) was used as synthetic polymer, while alkaline lignin was used as natural polymer. Solvent casting, particulate leaching and freeze-drying methods were used to create porous sponge-like scaffolds. PCL and lignin samples were prepared at different lignin concentrations (0, 10, 15, and 20 mg/ml) while keeping the PCL concentration constant at 200 mg/ml, PCL200, PCL200/Lignin10, PCL200/Lignin15 and PCL200/Lignin20, respectively. A sample with PCL concentration of 100 mg/ml and lignin concentration of 20 mg/ml was also prepared to see the effect of PCL concentration (PCL100/Lignin20). For all scaffolds, except for the control sample, NaCl crystals were used as porogen. Lignin containing scaffolds were appeared more brownish than pure PCL scaffolds. Since the structural integrity of PCL100/Lignin20 sample was very poor, and PCL200/Lignin20 without the salt addition cannot be removed from the mold surface, they were not used in further experiments. Even though, mechanical properties of the scaffolds have not been studied in this thesis, based on preliminary visual and manual analysis rest of the PCL/Lignin type scaffolds were observed to be more flexible than PCL constructs. Water uptake analysis showed that there is no significant difference between the samples with different lignin concentrations. The water uptake behavior of the scaffold, however, was dramatically increased with the lignin addition. While the water uptake for PCL200 was 44%, it was 380-400% for the samples with lignin after 24 h. In Fourier-Transform Infrared Spectroscopy (FTIR) analysis, typical peaks for PCL are identified for all scaffold types. Even though special peaks for lignin could not be observed, a reduction in the intensity of a peak, which represent –OH groups, was determined in all PCL/Lignin scaffolds compare to the PCL200. This can be evidence of reactions and new bonds between PCL and lignin polymers. Since water uptake and FTIR analysis showed that there is no significant difference between different lignin concentrations, it was decided to continue with only one sample type, PCL200/Lignin10, for further experiments. The porosities of pure PCL200 and PCL200/Lignin10 determined as 73% and 76%, respectively using liquid displacement technique. Pore sizes were investigated with ImageJ software on SEM images. For PCL200 scaffold, 31% and 58% of the pores on the surface were in the range of 100-300 µm and 300-600 µm, respectively. For PCL200/Lignin10, 30% and 43% of the pores were in the range of 100-300 µm and 300-600 µm, respectively. In addition, many pores that are smaller than 20 µm were observed for both sponges. The melting temperature of PCL200/Lignin10 scaffold was %5.4 higher than PCL200 scaffold as determined by Differential Scanning Calorimetry. The increased melting point might be the result of a new bond between PCL and lignin. Hydrolytic degradation was not seen within 7 days for both samples, but enzymatic degradation in lipase presence was observed for both sponges. After 7 days, only 43% weight loss was recorded for PCL200/Lignin10, while PCL200 sponge was completely degraded after the fifth day. Lignin's natural resistance to enzymatic degradation or the modifications to form an alkaline lignin might be the reason of this unexpected decrease in degradation. Biomineralization process was triggered by soaking scaffolds to modified-simulated body fluid solution. The mineralization rate was analyzed by SEM. SEM micrographs revealed significant mineral accumulations for both scaffold types at the end of the 7th day. When the 7th day samples were compared, it was seen that the hydroxyapatite-like structures were mostly deposited on top of each other in the PCL200 sample, while the aggregations were more evenly spread over the surface on the PCL200/Lignin10 sample. Furthermore, even though an intense accumulation was observed on the scaffold surface of the PCL200, the inside of the pores as appeared to be smooth. Lignin containing samples, on the other hand, had more mineral deposition within the pores. The elemental analysis of the formed hydroxyapatite-like structures was analyzed by EDX. It was observed that the Ca/P ratio of the accumulations on the PCL200/Lignin10 samples at all determined times was closer to the ideal hydroxyapatite Ca/P ratio (1.67). Finally, cell proliferation assay was performed with hFOB cells to examine the effects of lignin addition on cell adherence and proliferation on the surface. The proliferation of hFOB was significantly higher on PCL200/Lignin10 scaffolds compared to PCL200. The proliferation on PCL200/Lignin10 sponge was found to be 212% and 50% higher after day 3 and 7, respectively. Further studies are necessary in order to evaluate the usability of PCL200/Lignin10 sponge scaffolds in bone tissue engineering. However, the findings obtained within this thesis suggest that this blend is promising candidate for bone tissue engineering.