A main focus in current biomaterial research is to develop materials capable of evoking a desired tissue response. Biodegradable biomaterials that stimulate generation of specific tissues are highly anticipated in surgery, as they could provide a breakthrough in reconstruction of lost organs or tissue. A strategy in developing appropriate bioactive properties is to integrate specific signals in the material, e.g. by adding cell signaling proteins which induce a physiological effect. Consequently much research effort is put into identifying potential signaling proteins and adapting their functionality to a biomedical device. Throughout the experimental work in this thesis a systematic step-by-step approach has been carried out with the final aim to develop a material suitable for guided bone formation. The starting point was the simplified model system of adsorption of relevant proteins on biomaterial surfaces. The progress required switching between synthesis and characterization of the increasingly complex material and testing on relevant biological systems.
The initial experiments included observation of protein adsorption on hydroxyapaptite (HA) with a gold surface used as a reference surface for comparison. The adsorption of two relevant proteins, bovine serum fibronectin and bovine milk osteopontin, was studied with nanotechnological methods (Quartz crystal microbalance with dissipation, Ellipsometry and atomic force microscopy). The techniques in combination allow for estimation of single protein physical characteristics such as foot print area, height, rigidity, associated water and exposure to the liquid phase. Both fibronectin and osteopontin appeared more spread out and exposed to the liquid phase on HA as compared to gold. These characteristics are considered compatible with a more cell active
configuration on HA.
Osteopontin from bovine milk is a relatively new protein in biomaterials research, and therefore found suitable for further studies. Two cell studies with human mesenchymal stem cells were setup to evaluate the activity of bovine milk OPN cell binding domains when preadsorbed on HA. Cell motility, cell spreading and focal adhesion spot formation was observed. Again gold was used as a reference surface and both HA and gold surfaces preadsorbed with serum proteins were included as controls. OPN was found to induce cell motility, cell spreading and formation of focal adhesion spots, but exclusively when adsorbed on HA. These results strongly indicate that OPN adsorption on HA favors presentation of cell binding domains. This conclusion is in line with results obtained from the protein adsorption studies.
Having established the adsorption characteristics and in vitro activity we setup two in vivo studies aimed at utilizing the HA/OPN effect in a biomaterial. The in vivo studies were setup in two stages: Initially the osteoconductivity of a 50/50 vol% composite of poly-D,L-Lactic-Acid (PDLLA) and 20-70 nm HA nanoparticles was compared to that of pure PDLLA. The materials were applied as thin coatings on experimental cylindrical titanium implants and tested in sheep with a 2 mm gap for 12 weeks. On 40% of the composite coated implants the coating was replaced with a layer of new bone, whereas no bone formation had followed resorption of pure PDLLA. In the second in vivo study, OPN was adsorbed at the HA nanoparticles before mixing with PDLLA. Two composites, one with OPN and one without OPN, were compared in a similar canine study with 4 weeks observation time. The osteoconductivity of the OPN/HA composite was close to 100% higher than the pure HA composite. Thus it was shown that OPN bulk functionalization of a composite can aid in providing osteoconductivity to materials intended for guided bone formation.
The work in this thesis emphasizes the strength of a combined nanoscience/in vitro/in vivo approach. It allows for deductive step-by-step development of a biomaterial tailor-made for the application.