The local release of complexed siRNA from biomaterials enables targeted therapy of specific cells and tissues. This thesis focused on gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA) as a drug delivery system for siRNA. The siRNA-loaded cGM were aggregated with SaOS-2 cells or human mesenchymal stem cells (hMSC) to microtissues and stimulated with osteogenic supplements. Cell survival and tissue formation in microtissues could be improved by incorporating cGM in spheroid cultures. We observed hydroxyapatite deposition in the particles in dependence of medium and cell type. Osteogenic stimulation with BMP-2 and simultaneous silencing of BMP-2 antagonist chordin accelerated matrix mineralization of the microtissues. Higher cross-linking degree of cGM positively influenced chordin silencing and alkaline phosphatase (ALP) activity as a marker for osteogenic differentiation. These higher cross-linked cGM mineralized in an osteogenic medium within 8–9 days, in presence and absence of cells. The effects of pre-differentiated and chordin-silenced microtissues were investigated by simulation of in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). Increased ALP activity and osteoprotegerin (OPG) secretion were observed after 14 days compared to co-cultures with siRNA-free controls. These results indicate that the pre-differentiated and silenced microtissues can induce osteogenic differentiation of surrounding unstimulated cells. Using the microtissue approach with siRNA complexed with tyrosine-modified low molecular weight polyethyleneimine (P10Y/P5Y) as transfection reagent was not successful. The results of this thesis indicate that the pre-differentiation of microtissues with BMP-2 in combination with chordin silencing stimulates and enhances osteogenic differentiation of other stem cells. As a combination of biomaterial, RNAi, and autologous cells, microtissues could be a promising approach to regenerating bone defects.:Chapter I: Controlled release of siRNA for bone tissue engineering Chapter II: Microtissues from mesenchymal stem cells and siRNA-loaded cross-linked gelatin microparticles for bone regeneration Chapter III: Mineralizing gelatin microparticles as cell carrier and drug delivery system for siRNA for bone tissue engineering Chapter IV: Tyrosine-modified polyethylenimines for siRNA transfection in microtissues Chapter IV: Final discussion

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23339号 / 生博第457号 / 新制||生||61(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 今吉 格, 教授 鈴木 淳 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The local release of complexed siRNA from biomaterials opens precisely targeted therapeutic options. In this study, complexed siRNA was loaded to gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA). We aggregated these siRNA-loaded cGM with human mesenchymal stem cells (hMSC) to microtissues and stimulated them with osteogenic supplements. An efficient knockdown of chordin, a BMP-2 antagonist, caused a remarkably increased alkaline phosphatase (ALP) activity in the microtissues. cGM, as a component of microtissues, mineralized in a differentiation medium within 8–9 days, both in the presence and in the absence of cells. In order to investigate the effects of our pre-differentiated and chordin-silenced microtissues on bone homeostasis, we simulated in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). We found enhanced ALP activity and osteoprotegerin (OPG) secretion in the model system compared to control microtissues. Our results suggest osteoanabolic effects of pre-differentiated and chordin-silenced microtissues.