Abstract
This study was financially supported by Turkish Scientific and Research Council (Tübitak) Project no: 214M100. In the present thesis study, it was aimed to investigate the potential of proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in perfusion bioreactors.
In the first step of this study, chitosan-hydroxyapatite superporous hydrogel (chitosan-HA SPHC) scaffolds were prepared by using sodium bicarbonate (NaHCO3) as a foaming agent and glyoxal as a cross-linking agent. The microwave assisted gas foaming technique has produced tissue scaffolds that are faster to obtain, in high yield and higher reproducibility in vitro studies.
In the second step of the experimental study, the installation of the perfusion bioreactor system, in which leakage and diffusion problems can be solved and which provides sustainable sterility through long-term dynamic culture studies has been completed. At the next stage of the study, dynamic cell culture studies were carried out for 21 days using hMSCs at different flow velocities and tissue scaffolds of different sizes (P3D-6: 0.1 mL/min, P3D-6:0.2 mL/min; P3D-10: 0.27 mL/min) and media changes were made on certain days (days 3, 6, 9, 12, 15 and 18) of the culture. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) analysis was performed to observe viability and proliferation of cells on certain days of cell culture studies. SEM (Scanning Electron Microscopy) analysis was performed to observe cell morphologies and penetrations. At the end of the cell culture studies, RT-PCR (Real Time Polymerase Chain Reaction) analyzes were performed to determine the expression levels of Collagen1 (Col1), Runt Associated Transcription Factor 2 (RUNX2), Osteocalcin (OCN) and Osteopontin (OPN) genes in hMSCs.
In the last part of the thesis study, flow and mass transfer simulation studies were carried out in the perfusion bioreactor using COMSOL software. The accuracy of the model was tested with models developed for low and high flow rates in a bioreactor without a tissue scaffold and in the presence of a non-porous tissue scaffold. Affterwards the flow model and mass transfer model in perfusion bioreactor in the presence of a permeable tissue scaffold took place. The results obtained from the Computational Fluid Dynamics (CFD) modeling studies conducted within the scope of the thesis seem to support the experimental findings.
In the light of all these analyzes and findings, it has been shown that the dynamic culture approach performed with P3D-6 scaffolds at 0.1 mL/min and 0.2 mL/min flow rates support the osteogenic differentiation of hMSCs in the perfusion bioreactor. In addition, it can be seen that the CFD approach is the decisive factor in achieving successful results in vitro production of engineered bone grafts when different operating parameters are considered
This study was financially supported by Turkish Scientific and Research Council (Tübitak) Project no: 214M100. In the present thesis study, it was aimed to investigate the potential of proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in perfusion bioreactors. In the first step of this study, chitosan-hydroxyapatite superporous hydrogel (chitosan-HA SPHC) scaffolds were prepared by using sodium bicarbonate (NaHCO3) as a foaming agent and glyoxal as a cross-linking agent. The microwave assisted gas foaming technique has produced tissue scaffolds that are faster to obtain, in high yield and higher reproducibility in vitro studies. In the second step of the experimental study, the installation of the perfusion bioreactor system, in which leakage and diffusion problems can be solved and which provides sustainable sterility through long-term dynamic culture studies has been completed. At the next stage of the study, dynamic cell culture studies were carried out for 21 days using hMSCs at different flow velocities and tissue scaffolds of different sizes (P3D-6: 0.1 mL/min, P3D-6:0.2 mL/min; P3D-10: 0.27 mL/min) and media changes were made on certain days (days 3, 6, 9, 12, 15 and 18) of the culture. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) analysis was performed to observe viability and proliferation of cells on certain days of cell culture studies. SEM (Scanning Electron Microscopy) analysis was performed to observe cell morphologies and penetrations. At the end of the cell culture studies, RT-PCR (Real Time Polymerase Chain Reaction) analyzes were performed to determine the expression levels of Collagen1 (Col1), Runt Associated Transcription Factor 2 (RUNX2), Osteocalcin (OCN) and Osteopontin (OPN) genes in hMSCs. In the last part of the thesis study, flow and mass transfer simulation studies were carried out in the perfusion bioreactor using COMSOL software. The accuracy of the model was tested with models developed for low and high flow rates in a bioreactor without a tissue scaffold and in the presence of a non-porous tissue scaffold. Affterwards the flow model and mass transfer model in perfusion bioreactor in the presence of a permeable tissue scaffold took place. The results obtained from the Computational Fluid Dynamics (CFD) modeling studies conducted within the scope of the thesis seem to support the experimental findings. In the light of all these analyzes and findings, it has been shown that the dynamic culture approach performed with P3D-6 scaffolds at 0.1 mL/min and 0.2 mL/min flow rates support the osteogenic differentiation of hMSCs in the perfusion bioreactor. In addition, it can be seen that the CFD approach is the decisive factor in achieving successful results in vitro production of engineered bone grafts when different operating parameters are considered
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