Functionalisation of calcium phosphates with osteogenic and antimicrobial peptides for bone tissue regeneration

The demand for biomaterials for bone tissue regeneration that give consistent clinical results, especially in elderly or compromised patients, is not being met by currently available alloplastic materials. Biologically active peptides that enhance bone healing have been identified, however their use has been limited due to issues with their delivery, dosage, and risk of off-target effects. The aim of this research was to develop a medical device based on a synthetic calcium phosphate biomaterial with enhanced osteogenic and antimicrobial potential by the chemical immobilisation of bioactive peptides. Carboxylic acid functional groups were introduced onto ReproBone® calcium phosphate granules and discs via the plasma polymerisation of acrylic acid. This was confirmed by toluidine blue O titration, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). ReproBone® was further characterised using X-ray diffraction, mercury intrusion porosimetry and micro-computed tomography. Peptide candidates GFOGER, KR-12, HHC-36 and KRSR-FITC were successfully attached to calcium phosphate surfaces using carbodiimide chemistry. This was confirmed using FTIR, XPS, a peptide hydrolysis assay, a fluorescently labelled peptide and high-performance liquid chromatography (HPLC). Results of the HPLC and peptide hydrolysis assay showed significant peptide retention on the substrate surfaces when the full treatment was applied and were consistent with covalent coupling. The in vitro culture of rat mesenchymal stem cells over 28 days on peptide-enhanced ReproBone® granules showed no signs of any cytotoxicity, and that GFOGER and KR-12 peptides enhanced the expression of osteogenic differentiation markers. When Staphylococcus aureus and Pseudomonas aeruginosa were cultured in contact with peptide-enhanced ReproBone® discs, HHC-36 and KR-12 achieved 95 % or greater reductions in viable bacteria adhering to the disc surfaces. This thesis demonstrates the preparation of peptide-enhanced calcium phosphates with beneficial in vitro biological responses to mammalian cells and inhibition of bacteria. This research has the potential to underpin the manufacture of a new generation of biofunctional bone graft substitutes to address unmet clinical needs in dental and orthopaedic surgery.