Papers by Author: Neil Rushton

Abstract: Dense and porous HA and Si-HA discs and granules with varying percentages of silicon substitution have been produced and physically and chemically characterised using scanning electron microscopy, surface area analysis, porosimetry, density measurement, image analysis, Xray diffraction, X-ray fluorescence, FT-infrared spectroscopy and in-vitro and in-vivo testing. Results have shown that cell adhesion in-vitro and bone apposition in-vivo are enhanced by the presence of silicon substitution in the hydroxyapatite structure. The biological response to the materials appears to indicate an optimum outcome for levels of silicon substitution of 0.8wt%.

Abstract: Fine nanoapatite relics were deposited on glass substrates by electrohydrodynamic atomisation, using nanohydroxyapatite (nHA), nano-carbonated hydroxyapatite (nCHA) and nanosilicon- substituted hydroxyapatite (nSiHA) suspensions. These electrosprayed nanoapatites were evaluated in-vitro using simulated body fluid (SBF) and human osteoblast (HOB) cells. The SBF study revealed that newly-formed apatite layers were observed on the surface of the relics. Furthermore, enhanced HOB cell growth was observed on each of the nanoapatites at all time points. Hence, this work demonstrated that electrosprayed nanoapatites offer considerable potential as biomaterials.

Abstract: In this study, a biocomposite comprising nanostructured α-tricalcium phosphate (α-TCP) in a poly(D,L-lactic-co-glycolic acid) (PLGA) matrix was fabricated by a modified solution evaporation method. As a potential temporary bone fixation and substitution material, its bioactivity was evaluated by its ability to form bone-like apatite layer in simulated body fluid (SBF). Owing to the increased surface area covered by the osteoconductive bioceramic of α-TCP, rapid apatite formation was observed. After 7 days of immersion, enhanced nucleation of apatite was observed on the nanocomposite. At day 14, dense lamellar-like apatite was formed on the nanocomposite whilst apatite nucleation had only just started to develop on the surface of pure PLGA. At the same time, a preliminary in-vitro cell culture study was conducted using human osteoblast-like (HOB) cells. A significant increase in cell number with culturing time was observed for the nanocomposite. After 9 days incubation, a confluent lamellar-like apatite layer was formed on the composite surface. This apatite layer was also shown beneath the proliferating HOB cells at Day 16.

Abstract: Computerized X-ray microtomography (micro CT) is a powerful technique for imaging and quantification in biomedical materials research. Mapping of various tissue/implants in 3-dimension, which is not achievable with traditional histological slices, is particularly desirable in ex-vivo implant analysis. However, due to a variety of artifacts during image formation, especially the blurred interfaces due to the machine contrast transfer function and the partial volume effect, quantification based on traditional histograms is not accurate. In this work we propose a new 2-dimensional histogram and its application in the analysis of micro CT data of ex-vivo implants, which significantly improve the clarity of the definition of different phases and drastically reduced the artifacts of segmentation compared to conventional methods.

Abstract: Nano-sized HA (nHA) was applied to the surface of glass and titanium substrates using electrostatic atomisation spray (EAS) deposition. The phase purity of nHA was confirmed by X-ray diffraction. The nHA suspension consisted of rod–like particles 20-30nm in width and 50-100nm in length. The viscosity and conductivity of nHA suspension were 321 mPa s and 5.6 x 10-4 S/m, respectively. EAS of nHA in cone-jet mode was achieved at flow rate of 10-9 m3s-1 with the applied voltage between the needle and the ring-shaped ground electrode of ~6 kV. Micrometer- to submicrometerscaled nHA islands were successfully deposited on the substrate surface. Image analysis showed that the area percentage of nHA increased with deposition time, it covered 50% of the surface area after 10s of spraying. Partial dissolution of nHA was observed after immersion in deionised water for 1 month, particularly on the submicrometer sized nHA islands. Formation of a bone-like apatite layer was found after incubation in simulated body fluid (SBF K9) for 5 days, indicating the high bioactivity of the nHA deposits. In vitro culture with human osteoblast cells showed that the nHA islands were able to support the growth of HOB cells during 7 days of culture; the HOB cell activity increased with culture time as well as EAS deposition time. Immunofluorescence study showed that HOB cells expressed well-organised actin stress fibres on nHA deposited surfaces after 3 days of culture. The result indicated that nHA deposition provided more favourable surfaces for cell attachment. Therefore, electrostatic atomization spray deposition of nHA offers great potential for the creation of bioactive surfaces on bioinert implant surface to provide improved interfacial bonding with host tissues.

Abstract: The mechanism by which carbonate substitution within the hydroxyapatite (HA) lattice improves osteoconduction is unclear. Discs of dense, sintered, phase-pure HA and carbonate substituted hydroxyapatite (CHA) were cultured with human CD14+ cells in the presence of macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of nuclear factor (NF)-κB (sRANKL), during which time osteoclasts developed and resorbed the ceramic surface. Discs were then seeded with human osteoblasts (HOBs), and proliferation and collagen synthesis measured. Proliferation was increased on resorbed compared to control (unresorbed) surfaces on both materials. Collagen synthesis was increased on CHA compared to HA, an increase accelerated on a previously resorbed surface. The results suggest that osteoclasts can condition synthetic bioceramic surfaces and alter the responses of osteoblasts which subsequently populate them. Carbonate substitution may enhance osteoconduction via effects on enhanced bioresorption.

Abstract: Human osteoclasts derived from CD14+ precursors were cultured on discs of stoichiometric hydroxyapatite (HA) and carbonate substituted hydroxyapatite (CHA) of varying carbonate contents. Resorption of the ceramic increased with increasing carbonate content up to 2.35 wt. %. Development of osteoclasts is qualitatively different on ceramics compared to dentine, occurring in discrete, confluent subpopulations, which suggests local cell signalling may be important in the process. Resorption appears to drive further development of osteoclasts. Controlling carbonate content may be one way of controlling the rate of resorption of synthetic HA ceramics.

Abstract: The object of this study was to investigate the effect of the concentration of orthosilicic acid (0, 0.5, 1, 5 and 10µM) on gene expression in human osteoblast cells isolated from trabecular bone. This was measured using reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify messenger RNA (mRNA) levels for collagen type I, alkaline phosphatase and osteocalcin. Results showed that while collagen type I mRNA expression was increased by the addition of up to 10µM orthosilicic acid, ALP message was suppressed over time and osteocalcin levels were decreased.

Abstract: The object of this study was to investigate methods of seeding cells onto porous hydroxyapatite granules with the aim of optimising cell attachment. Two cells types were used; an osteosarcoma cell line, MG63, and human osteoblasts (HOBs) isolated from trabecular bone. Several conditions were investigated to determine their effect on cell attachment. These included varying the initial seeding concentration, pre-adsorption of the granules with the adhesion protein fibronectin and the use of mechanical agitation. Human osteoblasts and MG63 osteosarcoma cells attached to both dense and porous HA granules but with a low seeding efficiency while seeding was not significantly improved by pre-coating scaffolds with fibronectin or by introducing fluid flow.

Abstract: The adhesion of bovine collagen type I, bovine serum albumin, bovine IgG, 1 % and 10 % (v/v) human serum to hydroxyapatite (HA), silicon-substituted hydroxyapatite (Si-HA) and tissue culture plastic were studied. The materials were incubated at 37 °C for 30 minutes, after which the protein solution was removed and analyzed. The adsorbed protein was evaluated by electrophoresis and immunoassay after extraction from the materials. The degree of adhesion was higher for collagen, followed by IgG and albumin on all materials. However there was no difference in the amount of collagen adsorbed onto the surface of each material and this was also the finding with albumin and IgG. These results suggest that the increased bioactivity seen with Si-HA is not due to the degree of protein adhesion, but may possibly be due to changes in the conformation of the bound proteins.