Development, characterisation and application of calcium phosphates nanocrystals aggregates in a collagen matrix to be used as biomaterial in bone regeneratio

Three-dimensional scaffolds and bone proteins have shown to be important for bone tissue engineering. A scaffold for bone tissue engineering might have several applications and advantages such as: supporting cells for bone regeneration; being part of macromolecule delivery system and act as haemostatic sponge. The goal of this thesis was to develop a biomaterial to be applied for bone regeneration, as a novel means to guide bone cells towards tissue regeneration and/or to allow for local release of therapeutic macromolecules depending on what is the most adequate treatment strategy for each case. In order to achieve this main goal, several stages were considered in this work. Production of nanometer-sized particles of hydroxyapatite at laboratory scale by chemical precipitation method. This method was selected after a thorough study of other nantechnology techniques for nanocrystals preparation and characterization. It was shown that CaP nanoparticles are similar to those present in human bone in morphology, dimensions and chemical composition. In collaboration with the Laboratory of Separations and Reaction Engineering - LSRE at Faculdade de Engenharia, Universidade do Porto, a patent on the production of nanohydroxyapatite in a novel mixed reactor was obtained. Once the nanohydroxyapatite production method in the laboratory was made reproducible, microspheres based on nanohydroxyapatite particles were produced as a potential vehicle for transport and release of enzymes or antibiotics. Thus, in vitro studies were carried out to evaluate: nanosized hydroxyapatite performance in microspheres construction, nanohydroxyapatite microspheres as delivery system for antibiotics in periodontitis treatment, and collagen/nanoHA/SPARC (Secreted Protein Acidic and Rich in Cysteine) scaffold biocompatibility as a potential alternative biomaterial for bone cells. Results indicated that SPARC played an important role in the preparation of this new type of scaffold for tissue engineering due to its calcium and collagen binding sites. Its in vitro performance suggests a remarkable influence of SPARC on cellular adhesion and proliferation during the process of osteogenesis. A collagen type I / III matrix supporting hydroxyapatite nanocrystals was synthesised, being a viable porous scaffold for cell proliferation. Additions of SPARC, (Secreted Protein Acidic and Rich in Cysteine) to this biomaterial is a novel approach in Bone Tissue Engineering. SPARC was used because it provides chemical binding between calcium phosphates and collagen fibers in the matrix, which make this biomaterial unique because the protein is not merely placed in the network but chemically attached, due to the SPARC 's calcium affinity domains. Finally, in vivo experiments in rats were conducted to evaluate osteoinductive and osteoconductive properties of the nanoHA/Col/SPARC scaffolds. It was concluded that this biomaterial presented a similar nanostructure to bone in which hydroxyaptite nanocrystals 80x20 nm in size aggregating into microsized clusters, were well dispersed and bound to a collagen matrix through the presence of SPARC. The composite was incorporated into the remodeling process of bone, resorbed by osteoclastic cells, and new bone was formed by osteoblasts after resorption, as if the composite were grafted into autologous bone. The highly-performing bioactivity of this composite might derive from its similarity in composition and nanostructure to bone. The nanoHA/Col/SPARC biomaterial showed to have the potential to be used as a highly demanding bioactive bone graft material.