This thesis contains two parts. The first part is to study the effect of a signalling pathway of protein kinase C (PKC) on human mesenchymal stem cells (hMSCs) commitment to osteogenesis (chapter 1-2). We observed different PKC isozymes working differently for osteogenic differentiation. Amongst the PKC isozymes, PKCδ inhibition diminished the osteogenesis of hMSCs, while inhibiting conventional PKC and PKCµ enhanced the alkaline phosphatase (ALP) activity and mineralization. Thus the positive role of PKCδ and negative role of conventional PKC and PKCµ are suggested in order to benefit bone TE. The second part is to apply optical imaging techniques to investigate the cellular nutrient level and cell fate both in vitro and in vivo (chapter 3-5). The current hurdles in 3-dimensional cultures is to efficiently provide nutrients to cells whose survival and distribution rely on nutrient including oxygen availability. In chapter 3 I have evaluated bioluminescent imaging (BLI) in investigating the cell response to surrounding oxygen levels in 3D constructs both in vitro and in vivo. A gene fragment of hypoxia responsive element (HRE) was used as a promoter driving luciferase gene in cells. The protein luciferase activity can be quantified by an enzymatic assay or a CCCD camera. With this reporter construct, limited oxygen availability was recognized in 3D constructs both in vitro culture and in vivo subcutaneous nude mouse model. Moreover, the molecular diffusion in biomaterials (e.g. hydrogels) was studied in chapter 4 using both BLI of promoter-luciferase cells and fluorescence recovery after photobleaching (FRAP) of fluorescent molecules. Furthermore, BLI technique was used to study in vivo animal models, in which luciferase gene was under control of promoters for bone specific markers. With bone specific collagen I as the promoter, the Col(I)-Luc mouse line expressed strong light in osseous tissues. We observed strong light emission during bone remodelling and ectopic bone formation. Finally, chapter 6 summarizes and discusses the results in this thesis and raises ideas for the future work. Again in conclusion, I have successfully demonstrated the application in bioluminescent imaging in bone TE from both cell and animal levels.
|Award date||22 Oct 2009|
|Place of Publication||Enschede|
|Publication status||Published - 22 Oct 2009|