A polymeric protein-loaded microsphere delivery system for use in bone tissue engineering

The bone graft procedure is the gold standard therapy for large bone defects and usually involves implanting autologous bone into the defect site. The need to find more patient-friendly alternatives to the bone graft procedure is a driving force behind recent advances in bone tissue engineering. Different synthetic and natural biomaterials are being investigated for their use in the repair of large bone defects. However, there is still a need for scaffold materials that are able to sinter in situ with the capability of delivering growth factors that promote the bone repair process with minimal invasion. In this study, our main aim was to develop a porous, protein-loaded microsphere delivery system with the capability of sintering in situ and with injectable potential. Poly (lactic-co-glycolic acid) (PLGA) was used for this formulation due to its biodegradability, biocompatibility, controllable mechanical properties and good processing capabilities. An optimised procedure for formulating porous and non-porous (protein-loaded) microspheres was established and the microspheres were extensively characterised. There was an inverse relationship between the molecular weight of the PLGA and both the protein release and compressive strength. An intermediate molecular weight (53 kDa) variety of PLGA was chosen for further work based on balancing the need for retaining sufficient compressive strength and a slower protein release profile. The burst release of protein was addressed by investigating various coatings. The combination of chitosan and PLGA to form composite PLGA/chitosan microspheres resulted in the desired reduction in the burst release. The protein-loaded and porous microspheres were combined as a paste and found to sinter at body temperature (37°C) into scaffolds. Whilst previous investigations have focused primarily on a single type of PLGA microsphere (with or without an additional biomaterial), in this study we combined both porous and protein-loaded microspheres into a single delivery system. Furthermore, successful sintering was confirmed when a suspension of the microspheres was injected through a 19 G needle. The biocompatibility and osteogenic potential of the scaffolds were investigated. The composite PLGA/chitosan scaffolds supported the growth of MG-63 osteosarcoma cells and a primary human stem cell line. Furthermore, the scaffolds also supported the osteogenesis of the stem cells, as demonstrated by the presence of the late protein marker of osteogenesis, osteocalcin, and positive von Kossa staining, which is indicative of mineralization. The composite PLGA/chitosan and porous microspheres combine both porosity and the ability to load and sustain the release of protein into one system. Moreover, their ability to sinter at body temperature when injected or applied as a paste demonstrates the dual functionality of the system. This represents a novel approach to delivering protein for tissue regeneration as presently, there has been no report of the combination of PLGA/chitosan microspheres with porous PLGA microspheres as a system that is able to sinter, both post-injection and when packed as a paste, at 37°C. Therefore, the formulation presented in this thesis shows potential for further in vitro and in vivo testing to determine its suitability for bone tissue engineering applications