Regulating Gene Expression to Promote Osteoblastic Differentiation of Stem Cells

Sammanfattning: Bone is a tissue that heals by itself, unless the defect is too large (critical size). Today, novel regenerative medicine approaches have emerged as an alternative to treat such defects. This thesis explores alternative therapeutic strategies for bone tissue engineering which are biocompatible and clinically translatable. Many types of scaffolds that can act as reservoirs for growth factors such as rh-BMP-2 have been developed for bone tissue engineering in the past. However, the role of cross-linking chemistries that are employed to make hydrogels on the integrity and function of the loaded growth factors is not well understood. In this thesis, we have explored the influence of cross-linking chemistry on rh-BMP-2 integrity and bioactivity both in-vitro and in-vivo. These studies have demonstrated that thiol-Michael addition cross-linking chemistry greatly affects the integrity and bio-functionality of the loaded protein BMP-2 and leads to poor bone formation in an in-vivo rat model. On the other hand, hydrogels employing hydrazone chemistry did not significantly affect the integrity and bioactivity of BMP-2, which lead to a superior bone formation in-vivo. Since the high dose of rh-BMP-2 is known to confer many side effects, alternative ex-vivo strategies involving transient transfection of BMP-2 expressing plasmid DNA and silencing of anti-osteogenic genes using siRNA are developed. Our optimized method involves rapid transfection of hMSCs in suspension (5 minutes) with plasmid DNA followed by centrifugation and encapsulation in a hydrogel not only reduced cytotoxicity but also lead to efficient osteoblast differentiation of stem cells. Furthermore, this thesis presents the role of ECM-derived polymer HA in interacting with siRNA and trafficking across the plasma membrane, presumably through CD44 receptors and successfully silencing the target gene in-vitro. We explored the potential of such a non-cationic transfection method to deliver functional siRNA (anti-Pleckho-1 siRNA) in MSCs and compared it with commercially available cationic lipid LipofectamineTMRNAiMAX, using our optimized suspension transfection method. Our novel ex-vivo strategy employing HA hydrogels enabled efficient silencing of BMP-2 signaling pathway antagonist Pleckho-1 while avoiding the cytotoxicity issues in 3D, which further qualifies them for potential clinical application for cell-based therapies.