Doku İskelesi-Destekli Perfüzyon Biyoreaktör İşletim Parametrelerinin Preosteoblastların Üreme ve Osteojenik Farklılaşma Potansiyelleri Üzerine Etkilerinin Araştırılması

Abstract

ABSTRACT INVESTIGATION OF EFFECTS OF SCAFFOLDSUPPORTED PERFUSION BIOREACTORS PROCESS PARAMETERS ON THE PROLIFERATION AND OSTEOBLASTIC DIFFERENTIATION OF PREOSTEOBLASTS Gizem AYDIN Master of Science, Department of Bioengineering Supervisor: Prof.Dr. Menemşe GÜMÜŞDERELİOĞLU September 2015, 121 pages This study was financially supported by Hacettepe University, Scientific Research Project number: 014 D02 602 001-509 and Turkish Scientific and Research Council (Tübitak) Project no: 214M100. The aim of this study is to investigate the growth and osteogenic differentiation of MC3T3-E1 preosteoblasts in perfusion bioreactor performed with dynamic cell seeding and following cell culture studies. At the first step of this study chitosan-hydroxyapatite (HA) superporous hydrogel composites (SPHCs) were synthesized by combining microwave irradiation and gas foaming techniques in the presence of crosslinking agent, glyoxal, and gas blowing agent, sodium bicarbonate (NaHCO3). Thus, composites hydrogels, showing better performance in vitro studies, were produced rapidly and efficiently. iv In the second step of this study, for using in long term in vitro dynamic culture installation of a perfusion bioreactor system that provides sustainable sterility, prevents leak and solves nutrient/waste mass transport problems were completed. In the next step, MC3T3-E1 cells were dynamically seeded in perfusion bioreactor under different flow ways and flow rates (one-way flow, 0.1 mL/min, one-way flow, 1 mL/min; two-way flow, 0.1 mL/min, two-way flow, 1 mL/min) in comparison with static conditions. At the end of the cell seeding studies, cellular activity and cell proliferation were followed by MTT (3-[4,5-dimethylthiazol-2-il]-2,5 diphenyltetrazolium bromide) assay, cell morphology and penetration was monitorized by scanning electron microscope (SEM) analysis and the results demonstrated that the most efficient cell seeding was realized at static conditions. As a result of findings obtained from the cell seeding and dynamic cell culture studies carried out in perfusion bioreactor (two-way flow, 0.1 mL/min), cultures were performed for 21 days in comparison with static conditions and cell culture media were refreshed on certain days (7th and 14th). In certain days of cell culture studies (1st, 7th, 14th and 21st days) cell viability and proliferation were analyzed by MTT and morphological examination was done with SEM. At the end of the culture studies Collagen 1 (Col1), Runt Related Transcription Factor 2 (RUNX2), Osteocalcin (OCN) and Osteopontin (OPN) expression levels were determined using real time-reverse transcriptase polymerase chain reaction (RT-PCR). As a result, it was understood that, the highest cell density and osteogenic differentiation were observed in static cell seeding-dynamic cell culture (two-flow way, 0.1 mL/min) system. In the light of these studies, it was concluded that dynamic cell culture which carried out in perfusion bioreactor for 21 days after static cell seeding could be evaluated to use in bone tissue engineering.