Development and characterization of silk fibroin and citrus pectin based scaffolds for bone tissue engineering

Ataol, Sibel
Current strategies of tissue engineering aim to design and develop biologically, physicochemically and mechanically proper scaffolds. Natural polymers are gaining interest in applications since they have already many roles in biochemical pathways and have proper mechanical properties. The objective of this thesis is to develop natural silk fibroin (SF) and citrus pectin (PEC) based three-dimensional porous scaffolds for bone tissue engineering applications. Additionally, we aimed to synthesize nano calcium phosphates with different Ca/P ratios using Flame Spray Pyrolysis method for preparing polymer/bioceramic composite scaffolds for future studies. Human urine derived stem cells were used for testing the in vitro biocompatibility of scaffolds and synthesized particles. The scaffolds were obtained by freeze-drying of chemically crosslinked scaffolds which prepared by three different methods; namely click reactions, carbodiimide reactions and imine formation reactions. The materials were chemically modified for these crosslinking processes. Further physical modifications were conducted by immersing into methanol and CaCl2. FTIR-ATR analyses confirmed the chemical modifications. Degradation profiles of scaffolds were assessed to compare the crosslinking efficiency of the different crosslinking agents and in vivo stability of the vi scaffolds. Silk fibroin-amidated pectin (SF:A.PEC) scaffolds crosslinked with EDC and oxidized pectin, amidated pectin and silk fibroin (O.PEC:A-PEC:SF) scaffolds pregelated with 0.015 M had lower weight loss after 1 week incubation and highest water adsorption. SEM analyses revealed that scaffolds had porous internal structures, with a polymer skin layer. To reinforce scaffolds with bioactive agents, nano sized calcium phosphates were synthesized. Nanoparticles were produced at different calcium to phosphorus ratios (1.20-2.19). X-ray diffraction (XRD) patterns, high resolution transmission electron microscopy (HRTEM), and selected area electron diffraction (SAED) analyses revealed the amorphous nature of the particles at low Ca/P ratios. An increase in the specific surface area and crystallinity were observed with increasing Ca/P ratios. In vitro cytotoxicity studies using human derived stem cells showed that there was no composition and dose dependent cytotoxic effect of nanoparticles on stem cells (5-50 μg/ml). Cells treated with prepared nanoparticles had higher alkaline phosphatase (ALP) enzyme activity than the control cells, indicating positive effect on osteogenic differentiation of the cells. Future studies are suggested to be investigated on use of urine derived stem cells seeded silk fibroin and citrus pectin based scaffolds reinforced with calcium phosphate nanoparticles as a potential engineered construct for regeneration of bone.