Adverse tissue reactions to hip prostheses containing CoCr alloys have been widely reported, particularly for implants utilising a metal-on-metal bearing surface or, more recently, a modular taper junction and have been termed Adverse Response to Metal Debris (ARMD). Histological assessments of synovial tissues from patients at revision operation often demonstrate an extensive accumulation of macrophages and abundant tissue necrosis or fibrosis. The inflammatory response starts with the recruitment of immune cells and requires the egress of macrophages from the inflamed site for resolution of the reaction. Metal particles have previously been shown to affect cell migration but the effects of cobalt and chromium on macrophages' motility remain largely unknown. In vitro and in vivo macrophage migration during exposure to cobalt and chromium ions and nanoparticles were examined in this thesis. Cobalt, but not chromium, was found to significantly reduced macrophage motility (>50%). This was found to involve an increase in both cell spreading and the formation of intracellular podosome-type adhesion structures, as well as enhanced cell adhesion to the extracellular matrix (ECM). The formation of podosomes was also associated with the production and activation of matrix metalloproteinase-9 (MMP9) and enhanced ECM degradation. These effects were driven by the down-regulation of RhoA signalling through the generation of reactive oxygen species (ROS). The effect of the Co2+ and Cr3+ metal ions on tissue remodelling and pseudotumour formation which can lead to pain, swelling, limited range of joint movement and extensive tissue lesions, was explored using a multiscale approach. Both 2D and 3D in vitro culture systems were deplored to examine the effects of these ions on human fibroblast activation and mechanobiology. It was observed that Co2+ induced a fibrotic response characterised by cytoskeletal remodelling and enhanced collagen matrix contraction. This was associated with an increase in cell stiffness (~45%) and contractile forces (~80%) measured by atomic force microscopy and traction force microscopy, respectively. These effects were also triggered by the generation of ROS. Moreover, this fibrotic response was enhanced in the presence of macrophages, which increased the prevalence of α-SMA positive fibroblasts and collagen synthesis. These events were verified in vivo by examining the synovial fibroblasts and tissues from hips of patients with metal-on-metal hip implants and patients undergoing primary hip replacement. The findings revealed that fibroblasts isolated from patients undergoing MoM revision THA were more biomechanically active than the control group. Moreover, synovial tissues from patients undergoing MoM revision THA displayed evidence of extensive tissue remodelling and fibrosis. These findings revealed that cobalt leads to adverse tissue reactions via inducing macrophage retention, fibroblast-mediated matrix remodelling and modulating the interplay between macrophage and fibroblast. These distinctive effects can help us understand the pathogenesis of ARMD and the cellular response to cobalt-based alloys, which will inform biocompatibility test protocols and future implant designs.

A procedure was developed for dealing with two problems that have impeded the use of quantal parameters in studies of transmitter release. The first, involving temporal and spatial biasing in the estimates for the number of functional release sites (n̄) and probability of release (p̄), was addressed by reducing temporal variance experimentally and calculating the bias produced by spatial variance in p (var(s)p). The second, involving inaccuracies in the use of nerve-evoked endplate potentials (EPPs), was circumvented by using only miniature EPPs (MEPPs). Intracellular recordings were made from isolated frog cutaneous pectoris, after decapitation and pithing of the animals, and the concentration of K+ ([K+]) was raised to 10 mM to increase the level of transmitter release. The number of quanta released (m̄) by the EPP was replaced by the number of MEPPs in a fixed time interval (bin), and 500 sequential bins used for each quantal estimate. With the use of 50-ms bins, estimates for var(s)p were consistently negative. This was due to too large a bin (and introduction of undetected temporal variance) because the use of smaller bins (5 ms) produced positive estimates of var(s)p. Increases in m, n, and p but not var(s)p were found in response to increases in [K+] or [Ca2+]/[Co2+]. La3+ (20 μM) produced increases in m and n, which peaked after 20 min and declined toward zero. There were also large increases in p and var(s)p, which peaked and declined only to initial control values. The increase in var(s)p was presumed to reflect La3+-induced release of Ca2+ from intracellular organelles. The results suggest that this approach may be used to obtain unbiased estimates of n̄ and p̄ and that the estimates of var(s)p may be useful for studying Ca2+ release from intraterminal organelles.