DEVELOPMENT OF HYPOXIA-INDUCIBLE FACTOR-1 ALPHA AND GHRELIN-LOADEDCORE/SHELL FIBROUS SCAFFOLDS FOR IMPROVING VASCULARIZATION ANDOSTEOGENESIS

Date

2023-12-11

Author

Küçük, Eren

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The purpose of bone tissue engineering is to develop biocompatible materials, that have adjustable durability depending on the area to be applied, and promote cell adhesion and growth to improve the bone regeneration process. There are still some restrictions and risks in the clinical treatments used today. In this thesis, the focus is on increasing vascularization, one of the limitations of bone tissue engineering, by using a special protein and enhancing regeneration using a bone regenerative peptide. The basic approach is stimulating the necessary pathways by local controlled release of two proteins: Hypoxia Inducing Factor-1 α; HIF1α (a transcription factor) and ghrelin(a peptide hormone) to support vascularization and bone regeneration, respectively. To investigate the potential of these biological molecules in a local delivery system a new scaffold was developed with cellulose acetate (CA), polycaprolactone (PCL), and gelatin (GEL) polymers. Scaffolds of double-layered fibers (core/shell fibers) were formed with CA in the shell and PCL-Gel in the core using the coaxial wet electrospinning method. The shell layer of the scaffold was loaded with HIF1-α protein to provide early release of this protein from the scaffold to start angiogenesis earlier when applied in vivo. To support bone regeneration throughout the healing period Ghrelin was loaded to the inner (core) layer of the fibers (PCL-GEL) of the scaffold for more sustained and controlled release. Initial structural characterizations by SEM analysis and observations on the final solid form were used to select the best scaffold composition and production parameters. Upon optimization studies, the best composition for fibrous scaffold production was found as CA (12 %) and PCL: Gel (80:20 ratio) with 10 % total polymer concentration which presented homogenous fiber diameters and durable 3D form. For 14 days of degredation, the dry weight of the scaffold did not change. As a result of 14-day swelling analysis, 1200% swelling was observed. Release experiments with a single protein/peptide loaded to different layers of the fibers of scaffolds provided the fast release of HIF1 α (within 2 days) from scaffolds and slower release of Ghrelin (extending up to 10 days) as initially aimed. In vitro experiments with scaffolds were performed both with an endothelial cell line (HUVEC) and a bone osteoblast cell line (HFOB). In vitro, scaffold cell culture studies with HUVEC cells showed that HIF1-ɑ increases VEGF production. In vitro, scaffold cell culture studies with HFOB cells showed that ghrelin hormone increases cell viability. The effect on osteogenic differentiation investigated by Alkaline phosphatase enzyme (ALP) activity analysis showed no significant effect of ghrelin on the 7th-day results, but the ALP activity results of the cells exposed to ghrelin were approximately 2.5 times higher than the control group on the 14th day. The developed dual protein/peptide delivery system is found to have the potential for bone tissue regeneration and can be suggested for further in vivo studies.

Subject Keywords

Bone Tissue Engineering, Ghrelin, HYPOXIA-INDUCIBLE FACTOR-1 ALPHA, Angiogenesis, Co-axial Electrospinning

URI

https://hdl.handle.net/11511/107797

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Graduate School of Natural and Applied Sciences, Thesis