Cross-linked gelatin microparticles as drug-delivery-system for siRNA in bone tissue engineering

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 discussio