Nanoengineered titanium as protein-releasing implants: a molecular adjunct to reduce craniofacial surgery

Abstract

Craniosynostosis is a developmental disorder characterised by the premature fusion of skull sutures in children, necessitating repetitive surgical interventions throughout infancy. A major goal of craniosynostosis research is to develop molecular adjunctive treatments to reduce the morbidity and complications associated with multiple craniofacial surgery. Recent progress in molecular biology has highlighted the regulatory effects of bone morphogenetic protein 2 (BMP2) antagonists, including glypicans (GPC1 and GPC3), on suture morphogenesis and cellular functions. Moreover, the availability of genetically-engineered murine models of human craniosynostosis and drug-delivery systems (DDS) has assisted towards investigation of the glypican-based therapeutics in vivo. However, the conventional DDS are limited by their uncontrolled release patterns and undesired pharmacokinetics. The development of clinically viable implantable DDS, prior to human trials, require preclinical studies to investigate their characterisation, efficacy, pharmacokinetics and toxicity both in vitro and in vivo (in animal models). Medical Titanium (Ti) implants nanoengineered with Titania nanotubes (TNTs) have been recognised as a superior delivery platform in complex bone therapies (i.e. orthopaedics, cancer etc.) to localise the release of therapeutics in a controlled and sustained manner. This thesis presents the use of therapeutic-releasing TNT/Ti implant technology in a murine model, to address a key clinical challenge of delaying post-operative sutural bone growth in craniosynostosis. This interdisciplinary project has three aspects and specific aims including: (i) engineering and in vitro study: to fabricate and optimise TNT/Ti implants to study glypican release in vitro and bioactivity in murine C2C12 cells, (ii) pre-in vivo cell study: to evaluate the biological response at TNT-cell interface of heterogeneous (human) suture mesenchymal cells (SMCs) and (iii) in vivo study: to assess in vivo implant biocompatibility and efficacy as a glypican delivery system in TNT/Ti implants with controllable nanotube dimensions were fabricated via electrochemical anodisation process, and their protein-releasing capability and protein functionality were tested spectrophotometrically in physiological buffer and transfected C2C12 cells (BMP reporter cells), respectively. A metabolic activity assay was performed to investigate human SMC behavior at TNT-cell interface. The in vivo performance was assessed using micro- CT and histology in a surgical cranial defect model to verify TNT/Ti implant biocompatibility and glypican release efficiency. A protein loaded, mechanically robust TNT/Ti implant (120 ± 10 nm pore-diameter) displayed a biphasic in vitro release profile, with high loading efficiencies and prolonged release durations, spanning across 1 to 4 weeks. The pharmacokinetic modelling, based on the protein release parameters, showed an anomalous burst release and a zero-ordered sustained release. GPC1 and GPC3 released from TNTs were biologically active and reduced the BMP2-osteogenic activity in C2C12 cells. A decrease in adhesion and proliferation of SMCs at the TNT-cell interface, rendered the implant nanotopography and surface chemistry suitable for craniosynostosis therapy. The murine studies confirmed the implant biocompatibility and reiterated the sustained delivery of glypicans in vivo, demonstrated by decreased bone volume and surface area in therapeutically-intervened cranial defects. These findings confirm the potential of the nanoengineered TNT/Ti implants as an effective glypican delivery system to delay rapid post-operative bone re-growth in a murine model. This approach may evolve into a non-surgical molecular adjunct to minimise the need for recurrent re-operations in human craniosynostosis management.