Eksozom Yüklü Sıkıştırılabilir Kemik Greftleri

Abstract

This study was financially supported by Hacettepe University Scientific Research Projects Coordination Unit with the project entitled “Compressible Bone Gafts Loaded By Exosomes” (FHD-2017-16332). The goal of this thesis is to investigate the effect of enhanced bioactive, biocompatible, improved physical and mechanical properties of compressible scaffolds on bone repair and regeneration with three-dimensional (3D) in vitro culture studies. Within the scope of the thesis purpose, firstly, hydroxyapatite (HA) particles were precipitated from synthetic body fluid (SBF) in a microwave reactor under 600 Watt microwave irradiation for 9 times 30 s. The obtained HA particles were added to the poly(lactic-co-glycolic acid) (PLGA, 50:50) copolymer dissolved in 1,1,1,3,3,3 hexafluoro-2 propanol (HFIP) 7% by mass, and the 3D PLGA / HA composite scaffolds were fabricated by 3D electrospinning method. In order to improve the wettability of the fabricated 3D PLGA / HA composite tissue scaffolds, alkali treatment with NaOH was applied and the physicochemical characterization of the surface modified scaffolds were done by various methods. The effect of the applied surface modification on cell viability were evaluated with in vitro studies and it was seen that the samples treated with NaOH at 0.1M concentration increased the cell proliferation as compared to the control group. Afterwards, the isolation of MC3T3-E1 pre-osteoblasts derived exosomes was performed by ultracentrifugation method and by applying various characterization studies (TEM, Bradford Protein Test, Nanodrop A280 Protein Test, Flow cytometry, size and diameter analysis), it was proved that the isolated structures were exoxomes. The effect of exosomes on cell proliferation was examined by in vitro cell culture studies carried out with MC3T3-E1 cell line. The results showed that exosomes has no toxic effect on cells. In the last stage, the effect of exosome loaded compressible bone grafts on bone tissue and repair was investigated by loading pre-osteoblast derived exosomes on 3D PLGA/HA scaffolds which surface properties were improved by alkali surface treatment. In vitro culture studies showed that 3D fibrous PLGA / HA composite scaffolds lost approximately 80% of their volume when scaffolds were immersed into the cell culture medium at 37°C for 2 h. Due to this significant change in size and pore structures, it was concluded that the cells did not attach to the scaffold and did not penetrate into the scaffold. The absence of any cellular activity in the control and experimental groups in 3D in vitro cell viability assays performed with exosomes supported this result. In order to prevent the shrinkage of scaffolds, they were treated by surfactant Pluronic®. In the light of the data obtained within the scope of this thesis, fibrous 3D PLGA scaffolds with increased biocompatibility via alkali surface modification have been successfully produced. The shrinkage problem that was seen in the pore structure and size of the scaffolds during cell culture studies were eliminated. It was decided that the exosome loading and culture studies are to be performed on scaffolds treated with Pluronic® in the outgoing studies.