| Since the number of original cells isolated from patients is limited, and it requires a supply of enough number of seed cells to meet the demand for treatment, hence achieving seed cells rapid expansion with their high bioactivity in vitro turns out to be the bottleneck of tissue engineering. In this thesis, my research is focused on designing and preparing the novel three-dimensional scaffolds for seed cells rapid expansion with high activity in vitro, and the biological mechanism of how these scaffolds regulating cells.In this thesis, porous carbon scaffolds were prepared through the foaming method and the template method, and porous carbon scaffolds were modified by hydroxyapatite(HA) coating to obtain HA/porous carbon composite scaffolds. The HA/porous carbon composite scaffolds possessed a high average porosity (83.5%) with three-dimensional interconnected porous network structure (pore size distribution of200μm-500μm). HA/porous carbon composite scaffolds showed good biocompatibility and were conducive to the cell adhesion and the formation of extracellular matrix. The HA coating could accelerate cell adhesion behavior, and improve cell proliferation as well. HA/porous carbon composite scaffolds could significantly improve the proliferation behavior and regulate cell proliferation rate by6.7times and10.1times, compared with the two-dimensional cell culture models (4.2times). With HA/porous carbon composite scaffolds, small-sized perfusion bioreactor were designed to set up models for three-dimensional cell culture in vitro and it was explored that the cell expansion efficiency of dynamical culture in the bioreactor was2.4times than that of the static culture after one week.In this thesis, icariin delivery porous PHBV scaffold (IDPPS) was prepared by the solvent casting and particle leaching method. Experimental data showed that the average porosity of IDPPSs was88.8%, and the interconnected porous diameter50-200μm. The compressive strength was0.27MPa, while the tensile strength was0.13MPa. Drug release curve testing showed that the icariin could be detected in PBS solution (concentration of approximately4.67mg/L) by the first day, and could be observed in the process of continuous release of icariin during the whole28day test period, without the burst release effect. It was also found that when the cells cultured directly contacting with the scaffold for5days, the cell proliferation rate was1.5times of the non-contact samples, indicating that the icariin prefers directly through the cell membrane into the cells. Furthermore, the cell proliferation data showed that compared with the cell culture plate, when the MG-63cells were cultured on IDPPSs, the cell proliferation rate increased by2.3folds, while MC3T3-E1cells were cultured on IDPPSs, the cell proliferation rate increased by1.7folds. By RT-PCR detection of the RNA transcripts of MG-63cells cultured with2D culture dishes,2D PHBV film,3D PPSs and3D IDPPSs, it was found that IDPPSs could up-regulate FN and TGF-β1gene transcription level of cells to enhance the formation of extracellular matrix and induce cells in the early proliferation behavior through some growth factors and extracellular matrix genes’transcription, including bone morphogenetic BMP2, BMP6, BMP7. Finally, transcription of extracellular matrix gene BGN was enhanced to improve the anchoring effect of cell growth factor in the extracellular matrix, and the three dimensional structure of the cell proliferation inhibiting the transcription of TGF-β1and Col-I genes. With icariin/PHBV porous scaffolds, medium-sized perfusion bioreactor were designed to construct three-dimensional cell culture in vitro models and it was explored that the cell expansion efficiency of dynamical culture model in the bioreactor was4folds than that of the static culture model.In this thesis, icariin delivery PHBV/porous carbon scaffolds (IDPCSs) were prepared using the solvent evaporation method. Experimental data showed that the average porosity of the IDPCSs declined to84.2%, but retained the three-dimensional interconnected porous network (porous diameter range of200μm-500μm). The compressive strength, flexural strength and tensile strength of IDPCSs were1.076MPa,0.819MPa and0.187MPa, respectively. The IDPCSs were also found a good performance of the drug controlled release, that the release of the drug concentration in PBS was0.727mg/L after7days, while the value reached1.207mg/L after28days. IDPCSs with normal cell attachment behavior, could significantly enhance cell proliferation and the proliferation rate of MG-63cells was up to10times after a week culture while2.5folds than that of the2D cell culture model.In summary, the preparation and characterization of porous carbon scaffolds and PHBV scaffolds were systematically investigated in this thesis, and data showed that the surface modification with HA, PHBV and icariin could significantly promote the proliferation of osteoblasts on these scaffolds, the biological mechanisms of which were further clarified to build the theoretical foundation for rapid expansion of seed cells for tissue engineering in vitro. |
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