| Bone healing requires a complex series of coordinated, spatial and temporal cues from a variety of sources. Bone tissue engineering involves the use of scaffolds, cells, and signals and provides the healing environment with these cues to initiate and promote the formation of bone. Degradable matrices such as polymer microspheres may be used to deliver proteins and growth factors to enhance the growth-promoting environment in many tissues. However, in order to determine the desired release kinetics of these microspheres, the microsphere processing techniques must be evaluated and well-controlled. The host immune response to the polymer microspheres must also be minimal to control for any significant growth-inhibiting cues. Data presented in the first part of this thesis shows that microsphere size and morphology can be controlled by varying the processing conditions. In addition, these microspheres do not elicit an immune response and are shown to promote neurite growth following injection into the rat spinal cord.;Bone morphogenetic protein (BMP) is one of the most potent signals for bone and cartilage formation and has been well studied and documented for use in bone healing. The signaling pathway of BMP-2 involves a series of complex events and many different proteins may bind to BMP-2 and antagonize or enhance its activity. In particular, it has been found that Marshall Urist's original BMP is actually a fragment of secreted phosphoprotein-24 (spp-24), and it has been shown to interact with rhBMP-2. A synthetic peptide, based on 19 amino acids of spp-24, called BMP binding peptide, or BBP, has been shown to enhance rhBMP-2-mediated bone formationin vivo. The in vitro effects of BBP are investigated in Chapter 3 to determine if it enhances BMP-2-mediated osteogenesis. Results presented in this chapter did not show the enhancing effects of BBP in vitro however further optimization of the 3D culture system may be necessary to improve the outcome of the results.;Chapters 4 and 5 investigate the effects of the parental protein of BBP, spp-24, in a mouse ectopic bone forming model and rat spine fusion model. Previous work has shown the proteolytic susceptibility of spp-24 and thus various recombinant isoforms of this protein are also examined in these studies. The full-length recombinant isoform of spp-24 strikingly inhibits rhBMP-2-mediate bone formation while the truncated isoforms vary in their activity, but may suggest a mild inhibition to rhBMP-2. These results show that an obvious structure:function relationship exists between the different isoforms of spp-24 and rhBMP-2. Chapter 6 makes an attempt to elucidate the mechanism behind this mechanism of interaction by using the technology of surface plasmon resonance (SPR). The results in this final chapter show that the different isoforms do bind to rhBMP-2, but the significance behind the binding have been deemed inconclusive. However, further optimization of SPR will reveal significant information regarding the binding kinetics of these two proteins. Chapter 6 discusses these future experiments as well as the general conclusions of the thesis. |
