Development of a 3D Perfusion System to Monitor Response of Osteoblast-like Cells Incubated on Synthetic Bone Graft Substitute Granules with Varied Hierarchical Porosities under Dynamic Conditions

Abstract

The development of multi-level porous scaffolds with an optimised chemistry of 0.8wt% silicon substituted hydroxyapatite (SA) is an example of the successful contribution of material science to the medical world. The key to the development of next generation synthetic bone graft substitutes (BGS) may be through understanding of the mechanisms behind the structure of a BGS modulating cell response perhaps through alteration of the local environment within the graft material. The aim of this thesis was to develop an in vitro system with which to characterise ionic exchange and monitor cell response to real granular synthetic bone graft substitute materials within a truly 3D environment. The purpose of the system being to more closely mimic the 3D environment in vivo to enable a more systematic investigation of how scaffold structure can impact on the local physiological environment and subsequent cell behaviour. Initially, experiments were performed with small volumes of porous scaffold granules (identical in form to those used clinically) exposed to a range of volumes of continuously recirculating cell culture media, without serum protein supplementation. The results obtained demonstrated that the capacity of hydroxyapatite (HA) based porous granules to rapidly deplete calcium and phosphate ions from the local aqueous environment necessitated the use of a fully perfused flow to waste system. Subsequent experiments were performed to uniformly introduce osteoblast-like cells to the granules and to monitor the behaviour of the cells in situ on the porous granules within the perfused 3D environment for periods of up to 7 days. The work conducted in this thesis has successfully identified that porous apatite BGS granules interact significantly with the local aqueous environment. A 3D perfusion system has been developed that enables clinically relevant BGS to be seeded with osteoblast-like cells that can then be monitored as they colonise and respond to the various BGS types. With the use of osteoblast-like cells this system corroborates in vivo findings within orthotopic defect sites, with respect to a clear response to BGS granule chemistry, but mixed sensitivity to BGS structure.