الفهرس 
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Abstract
Summary and conclusions 7.1. Summary Synthetic/natural polymers/bioceramics composite scaffolds are considered promising in hard tissue engineering. They combine the flexibility and ease of shaping of polymers with the higher strength, stuffiness, and bioactivity of the bioactive ceramics thus, achieving the best mechanical and biological performance of the tissue engineering scaffold.
This present study aimed to prepare polycaprolactone (PCL) composite scaffolds with high porosity and pore interconnectivity for bone tissue engineering applications. Natural-derived polymers from different sources (Zein derived from a plant source and gelatin derived from an animal source) were blended with the PCL in an attempt to decrease the intrinsic hydrophobicity and accelerate the degradation rate of the PCL. Additionally, inorganic bioactive glass powder (BG) was added to the PCL/natural polymers networks as reinforcement material and to enhance the prepared composite scaffold‟s mechanical properties and bioactivity.
Bioactive glass nanoparticles of the quaternary SiO2-CaO-Na2O-P2O5 system were successfully synthesized using the conventional sol-gel method. Chemical analysis using different characterization tools, XRD, FTIR SEM and EDX, were used to analyze the chemical constitution of the prepared BG powder. The XRD and FTIR results revealed the amorphous nature of the prepared BG powder and proved the existence of the main constituting chemical functional groups. Also, the SEM and EDX revealed that the prepared BG powder was in the nano-sized range and confirmed the existence of Kα radiation of Si, Ca, P, and Na peaks of the BG powder.
The bioactivity of the prepared BG powder was evaluated by immersion 10 mg of the BG powder in 50 ml of phosphate buffer saline (PBS) for 14 days. The XRD results showed the appearance of new peaks related to carbonated hydroxyapatite (CHA). Also, the FTIR confirmed the formation of a new apatite layer over the surfaces of the BG
powder via the appearance of CO32- and PO43- peaks in the IR spectrum of BG after immersion. Furthermore, the elemental analysis by EDX showed that the intensities of the Ca, P and C after immersion increased compared to the intensities before immersimm. Also, the intensity of the Si decreased after immersion than its intensity before immersion which indicated the bioactivity of the BG powder.
Using the solvent casting/particulate leaching technique, composite scaffolds of PCL with natural polymers (zein and gelatin) as well as PCL/natural polymers/BG were prepared for bone tissue engineering applications. Sodium chloride (NaCl), with a particle size of 315-500 μm, was used as a porogen (pores producing particles) at a PCL/ NaCl ratio of 1:5. To study the effects of the natural polymers (zein and gelatin) in the PCL scaffold properties, two scaffold groups (II and IV) were prepared, PCL/zein and PCL/gelatin, respectively. The bioactive glass power was added to the PCL/zein and PCL/gelatin in groups III and V, respectively. A control pure PCL scaffold (group I) was also prepared.
The chemical characterization using FTIR analysis suggested possible chemical interactions between the PCL and zein or gelatin and between PCL/natural polymers and BG powder. The physical (porosity and biodegradation rate), mechanical as well as biological characterization of the prepared scaffolds were also assessed.
The porosity of the scaffolds was investigated using the liquid displacement method (n=5) and SEM. The statistical results of the porosity% revealed that the porosity% decreased with incorporation of the BG powder in groups III and V than the other three groups. The SE micrographs showed that all the groups had a markedly open porous microstructure. In addition, the pores exhibited a high degree of interconnectivity.
The biodegradation test of the PCL scaffolds (n=5) was evaluated by comparing the scaffold’s dry weights before and after immersion in PBS for six months. The weight loss% was calculated as the difference between the initial and final weights divided by the initial weight. The weight loss of the scaffold samples in group I (pure PCL) showed the statistically significant lowest weight loss% of all groups. Composite scaffold
samples of groups II and IV containing PCL zein and gelatin, respectively, showed higher weight loss% than the pure PCL scaffold. On the other hand, the statistically significant highest weight loss% was shown in groups III and V combining PCL/natural polymer with BG powder, indicating the positive effects of the natural polymers and BG on the degradation behavior of the PCL composite scaffolds compared to the pure ones.
The compressive strength of the scaffolds was measured by compressing five cylindrical samples (n=5) from each group until they were compressed up to 75% of their original height using a universal testing machine. On the contrary, to the porosity results, groups III and V containing BG showed a statistically significantly higher compressive strength. Furthermore, group V showed a significantly higher Young‟s modulus than the other four groups, assuming that incorporating BG as a stiff filler within the polymer‟s matrix caused increases in the mechanical properties of the composite scaffolds compared to the unfilled scaffolds.
Mesenchymal stem cells derived from the bone marrow of femur bone of rat (RBM-MSCs) were used to evaluate the in-vitro biocompatibility of the prepared five scaffold groups. Scaffold samples (n=4) were seeded with the cells. At the predetermined time points (1, 3 and 7 days), cell viability was measured using MTT assay. The optical density of the formazan purple color (MTT product) was recorded using a microplate reader at wavelength 570 nm. Results showed that the ability of the MSCs to survive and proliferate on the pure PCL (group I), PCL/gelatin (group IV), and PCL/gelatin/BG (group V) during all time intervals. On the other hand, groups II and III containing PCL/zein and PCL/zein/BG showed decreased cell viability% and increased cell toxicity%. However, on day 7, the PCL/zein/BG (group IV) showed increased cell viability% and decreased cell toxicity%. However, group II exhibited the statistically significant lowest cell viability% and highest cell toxicity% among all groups at all time intervals.
ALP activity assay was used to measure the osteogenic differentiation of the MSCs cultured in an osteogenic culture medium on different scaffold groups by reading the
color absorbance at the predetermined time points (1, 3 and 7 days) (n=4). Results on day one showed that group V exhibited higher ALP absorbance, followed by group IV than the other groups. On day 7, group III containing BG showed statistically significant higher ALP absorbance. group II followed the same trend in the MTT assay showed the lowest ALP absorbance among all groups. The same results were obtained in Alizarin red staining (mineralization assay) that acts as an indicator of calcium formation measured at 3, 7 and 15 days (n=4). Groups IV and V showed the higher calcium absorbance among all groups as measured by a microplate reader at 405 nm.