Hydrogel-microcarrier composite systems for cell delivery in tissue engineering.
Lau, Ting Ting.
Date of Issue2014
School of Chemical and Biomedical Engineering
Amongst the diversity of scaffolding systems, hydrogel remains a popular choice for tissue engineering applications. However, good structural support and adequate biocompatibility are no longer the only emphasis in scaffold design; new generation of scaffolds are also expected to play a role in developmental guidance of the regenerated tissues. Thus, a simple hydrogel system could not satisfy the current demand. Instead of carrying out chemical modifications to hydrogel, we proposed the incorporation of microspheres into hydrogel to develop a hydrogel/microspheres (GS) composite system. By employing the microspheres as cell-affinitive interfaces for cell attachment or degradable moieties within hydrogels, the microspheres act as the bioresponsive entity within the composite model. Accordingly, this dissertation presents three distinct GS composite systems to deliver different cell types for cell-based therapy purposes. Based on the type of cell to be delivered, modification to GS system could be made respectively to suit the target cell development. Thermally stable microspheres were employed as permanent cellular attachment site within the GS system for delivery of typical anchorage-dependent cells (ADCs) like osteoblasts and mesenchymal stem cells (MSCs). For delivery of another type of model cell, HepG2 cell, partially crosslinked microspheres were used in the second GS system. Upon introduction of collagenase, microspheres in the GS system degrade, creating micro-cavities in the hydrogel bulk for further cell expansion and development. This design serves to use microsphere as a transient cell delivery vehicle and a porogen to establish a micro-cavitary hydrogel (MCG) construct. The dual functionality of the microspheres in the GS system enables direct cell delivery to the cavities of the hydrogel and also permits formation of controlled size cellular aggregates. In all previous set ups, cells were delivered on the microspheres while in this last GS system proposed, cells were delivered in the hydrogel phase of the composite system. As the cells would be encapsulated and maintained their rounded morphology, primary chondrocytes, a typical non-ADC was selected. This third GS composite is designed to be a thermally responsive system where microspheres dissolved upon exposure to physiological temperature without the need for collagenase. The beneficial effects of MCG are further demonstrated in this study where a macroscopic scaffold-free living hyaline cartilaginous graft (LhCG) consisting of only chondrocytes and ECM proteins could be derived. Given the high purity of LhCG construct, it served as an excellent in vitro cartilaginous template for mimicking endochondral ossification process. It is shown that the LhCG construct, rich in ECM protein, favours osteoblast and MSC attachment. Osteogenesis of the seeded cells on LhCG was achieved based on the positive expression of bone markers and calcification. Besides the ability to derive a scaffold-free LhCG construct, the thermally responsive GS system was also employed for delivery and differentiation of stem cells. Murine embryonic stem cells (mESCs) and induced pluripotent stem cells (iPSCs) were encapsulated respectively in this third GS system. Our results indicate that this system is able to facilitate embryoid bodies (EBs) formation and also allow differentiations to take place. Spontaneous formation of EBs and subsequent endodermal and hepatic differentiation could all occur in the continuous MCG system. In summary, the studies in this dissertation proved that GS composite system is an easily customizable platform for delivery of various cell types – model cell lines, primary cells, and pluripotent stem cells.