Trikalsiyum Fosfata Bağlanmış Mezenkimal Kök Hücre Eksozomlarının Kemik Yenilenmesine

Abstract

The structure and functions of bone may be impaired with metabolic-diseases such as osteoporosis, necrotic or traumatic damages, although it is a dynamic tissue that can renew itself under physiological conditions. Critical bone losses need to be supported by osteoinductive growth factors, stem/precursor cells, as well as osteoconductive calcium phosphate compounds. Mesenchymal stem cell exosomes could be preferred instead of mesenchymal stem cells because they contain molecules that have regenerative effects in bone, be easily taken into the target cell and do not create an immune response. In this study, it was hypothesized that the proliferation, matrix synthesis and mineralization of osteoblasts can be stimulated with bioengineered formulation formed by binding annexin V to calcium deficient hydroxyapatite and human bone marrow derived mesenchymal stem cell exosomes. In order to test this hypothesis, an in vitro study consisting of an experiment and control group was designed. The objectives of the thesis are ; (1) production and characterization of calcium deficient hydroxyapatite, (2) isolation and characterization of human bone marrow-derived mesenchymal stem cell exosomes, (3) production and characterization of new formulation that contain calcium deficient hydroxyapatite, human bone marrow-derived mesenchymal stem cell exosomes, and binding material annexin V, and (4) evaluating the effect of a newly formulated material on proliferation and mineralization potentials of osteoblasts. In line with these goals, calcium deficient hydroxyapatite was synthesized using a chemical precipitation method and characterized by X-ray diffraction, Raman spectroscopy, and transmission electron microscopy. Human bone marrow derived mesenchymal stem cells were characterized by morphology, attachment to the culture plate, specific surface markers, osteogenic and chondrogenic differentiation analysis methods, and then exosome isolation was performed by ultracentrifugation and MACS methods. Exosomes were observed in the transmission electron microscope, protein concentrations were determined by BCA analysis and particle analysis with NTA. Exosomes have been shown by flow cytometry to specifically have all three markers (CD9, CD63, CD81). Calcium deficient hydroxyapatite has linked human bone marrow derived mesenchymal stem cell exosomes via annexin V and has been characterized by Raman spectroscopy and SDS-PAGE. The release study was carried out by BCA analysis. The exosomes are released from the hydroxyapatite in 12 hours. The proliferative dose of calcium-deficient hydroxyapatite and human bone marrow-derived mesenchymal stem cell exosomes were determined by real-time cell proliferation analysis 0.1 µg/ml, and 25 µg/ml respectively. The material prepared with effective doses was applied to the osteoblasts. The proliferative potential and matrix mineralization induction effect was reported by using real time cell proliferation and ALP activity analyzes, respectively. As a result, with this thesis study, an original and innovative, high-tech, personalized and targeted formulation was developed by a newly formulated material that contains calcium deficient hydroxyapatite and human bone marrow derived mesenchymal stem cell exosomes with annexinV binding molecule for the first time, and its regenerative effect on human osteoblasts in vitro was demonstrated. Following experimental validation of this new formulation in in vivo disease models, it has the potential to be involved in the repair of clinical metabolic or traumatic bone injuries as an allogeneic or autogenous personalized treatment agent. The new bioengineering-based treatment product produced within the scope of the thesis meets our country's goal of increasing the competitiveness in the international market in the treatment of orthopedic diseases. A patent application was made to Hacettepe Technopolis Technology Transfer Center Patent Office with the new formulation obtained from the thesis. (Application number: 2021/002221).