Biomedical subjects

N E Bishop

Publications and source records attributed to N E Bishop.

13 recordsLinked to original sources

PubMed2006

Artificial composite bone as a model of human trabecular bone: the implant-bone interface.

The use of artificial bones in implant testing has become popular due to their low variability and ready availability. However, friction coefficients, which are critical to load transfer in uncemented implants, have rarely been compared between human and artificial bone, particularly for wet and dry conditions. In this study, the static and dynamic friction coefficients for four commercially used titanium surfaces (polished, Al(2)O(3) blasted, plasma sprayed, beaded) acting on the trabecular component of artificial bones (Sawbones) were compared to those for human trabecular bone. Artificial bones were tested in dry and wet conditions and normal interface stress was varied (0.25, 0.5, 1.0MPa). Friction coefficients were mostly lower for artificial bones than real bone. In particular, static friction coefficients for the dry polished surface were 20% of those for real bone and 42-61% for the dry beaded surface, with statistical significance (alpha<0.05). Less marked differences were observed for dynamic friction coefficients. Significant but non-systematic effects of normal stress or wet/dry condition on friction coefficients were observed within each surface type. These results indicate that the use of artificial bone models for pre-clinical implant testing that rely on interface load transfer with trabecular bone for mechanical integrity can be particularly sensitive to surface finish and lubrication conditions.

Biomechanical Phenomena↗

PubMed2006

Shear does not necessarily inhibit bone healing.

Interfragmentary shear has been perceived as inhibitory to bone healing. We think this is because of inadequate balance between stimulatory and disruptive interfragmentary displacement magnitudes in the shear direction. We hypothesized that pure shear is not necessarily detrimental to bone healing. This was investigated by comparing bone healing under interfragmentary torsional shear, axial compression, and no applied motion. Applied motion was controlled carefully with similar interfragmentary principal strain magnitudes found to stimulate healing under axial compression. The observation period was 8 weeks. Torsional rotation stimulated intercortical mineralized callus formation with greater area than the group without applied motion, and led to a stiffness and rate of bony bridging similar to that of the no motion group. Axial compression stimulated less intercortical mineralized callus of a lower density than the no motion group, and there also was little bridging. These results support the hypothesis that interfragmentary shear does not necessarily inhibit bone healing.

Animals↗

PubMed2003

An experimental two degrees-of-freedom actuated external fixator for in vivo investigation of fracture healing.

A novel two degrees-of-freedom external fixator that can accurately control interfragmentary mechanical conditions over an ovine tibial osteotomy is presented. The device can apply either axial or torsional motion, to investigate mechano-transduction mechanisms in fracture healing. The device was bench-tested over a range of gap stiffnesses, for its ability to apply pure axial or torsional motions, control interfragmentary strain magnitudes and to measure stiffness over the gap. The experiments reveal very little cross-coupling between axial and torsional displacements, although some transverse displacement occurs for both degrees-of-freedom. The mean interfragmentary strain can also be accurately controlled, but with some variation over the gap (due to coupled transverse displacement error). Interfragmentary stiffness measurements are accurate up to gap stiffnesses corresponding to bony bridging. This characterisation study has shown that the system is adequate to control and measure the mechanical conditions in a fracture healing experiment.

Animals↗

PubMed2000

Uncoating kinetics of hepatitis A virus virions and provirions.

When the growth kinetics of immature hepatitis A virus provirions and mature virions were monitored, distinct eclipse phases were noted for both types of particles. Strikingly, uncoating of virions occurred around 4 h postinfection, while uncoating of provirions occurred predominantly between 8 and 10 h postinfection. It is proposed that the heterogeneous mixture of infectious hepatitis A virus particles (virions and provirions) typically present in inocula is responsible for the normally asynchronous nature of hepatitis A virus uncoating kinetics.

Hepatovirus↗

PubMed2000

Hepatitis A virus replication: an intermediate in the uncoating process.

Dense, RNase-sensitive, VP2-containing, non-infectious hepatitis A virus (HAV) particles were found to be formed at early times after the infection of cultured cells. These particles formed with kinetics mirroring those reported for HAV uncoating. The kinetics of the formation of dense HAV particles corresponded to a decrease in detectable, mature input virions, as detected by RNA dot blot hybridization of CsCl density gradient fractions. The dense HAV particles did not appear to have altered sedimentation coefficients, and as the fate of small capsid protein VP4 is not yet known, these particles cannot yet be termed 'A particles' or 'infectosomes', as have the uncoating intermediates in some picornavirus-cell systems.

Animals↗

PubMed1999

Conformational changes in the hepatitis A virus capsid in response to acidic conditions.

Low pH values encountered during uptake of viruses by receptor-mediated endocytosis have been shown to expose hydrophobic residues of many viruses and result in viral conformational changes leading to uncoating of the viral genome. An assay for hydrophobicity utilising the non-ionic detergent Triton X-114 was established, making use of metabolically-labelled hepatitis A virus (HAV). In this assay, hydrophilic proteins interact with the aqueous (buffer) phase, while hydrophobic proteins interact with the Triton (detergent) phase. HAV particles interact with the aqueous phase at neutral pH, whereas, under acidic conditions, HAV was found predominantly in the detergent phase. This indicates that the capsid of HAV undergoes conformational changes rendering the particle more hydrophobic under acidic conditions. A further two conformational changes were found in HAV on exposure to low pH, as detected by changes in buoyant density in CsCl gradients. These were maturation of provirions to virions and the formation of dense particles. These results may have implications for uncoating of the HAV RNA genome, and these conformational changes could represent intermediates in the viral uncoating process.

Acids↗

PubMed1999

Effect of low pH on the hepatitis A virus maturation cleavage.

Cleavage of VP0 to VP2 via intramolecular scission is known as the viral maturation cleavage, as VP0 is found in immature particles, whilst VP2 is found in mature particles. The effect of low pH on the kinetics of hepatitis A virus (HAV) capsid protein VP0 cleavage in provirions was examined by Western blot analysis. VP0 scission was found to be dramatically enhanced under acidic conditions, similar to those encountered on entry of virus particles into the cell via endocytosis. The cleavage of VP0 to VP2 led to an increase in the specific infectivity of viral particles, indicating that mature virions are more infectious than immature provirions. The data are consistent with a model where conformational changes induced by low pH aid scission of VP0, and the increase in kinetics of VP0 cleavage may have relevance for viral uncoating, as only mature HAV particles are thought capable of uncoating within the host cell.

Animals↗

PubMed1998

Examination of potential inhibitors of hepatitis A virus uncoating.

Hepatitis A virus (HAV) replication in BS-C-1 cells was studied in the presence of ten potential uncoating inhibitors. Strong inhibition of HAV replication was only observed in the presence of the phenothiazine compound chlorpromazine and the lysosomotropic agent chloroquine, but not by other lysosomotropic agents. Chlorpromazine and chloroquine were found to prevent virus uncoating. Chlorpromazine is known to inhibit endocytosis of non- clathrin-coated vesicles. Chloroquine is a weak base amine, and thought to inhibit virus replication by preventing endosomal acidification. These results therefore suggest that entry of HAV in BS-C-1 cells does not depend on the low pH encountered in the clathrin-coated endocytic entry pathway. A possible role of calcium ions in mediating viral uncoating is discussed, as calcium ions were found to destabilize HAV particles in vitro.

Animals↗

PubMed1997

Hepatitis A virus subviral particles: purification, accumulation, and relative infectivity of virions, provirions and procapsids.

Virus-specific particles were isolated from hepatitis A virus (HAV)-infected cells and the role of each particle type in the replicative cycle assessed. Mature virions, provirions (immature virions) and empty capsids (procapsids) were detected in cell lysates, and both virions and provirions were found in the culture supernatant. Particle types were separated by isopycnic caesium chloride gradient or linear sucrose density gradient-ultracentrifugation, and their capsid proteins characterised. Virions, provirions and procapsids containing both VP1 and varying levels of the VP1 precursor protein PX were found, suggesting that trimming of PX is not essential for particle formation. Provirions (containing VP0) and virions (containing VP2) could not be clearly separated with these techniques, but sucrose gradients allowed greater separation of particle pools with distinct VP0 contents and specific infectivities which could be used for further studies of the biological role of VP0 cleavage. Virions, with a higher sedimentation coefficient and buoyant density presumably reflecting a more compact structure, had a higher relative infectivity when compared to provirions. HAV-infected cells therefore contain a heterogenous mixture of RNA-containing viral particles with characteristics between those of true provirions and virions, but all such particles are released from the cell and can participate in further rounds of infection.

Animals↗

PubMed1997

Early interactions of hepatitis A virus with cultured cells: viral elution and the effect of pH and calcium ions.

Hepatitis A virus (HAV) is less well-characterized than other picornaviruses due to its slow and inefficient replication. In order to gain a greater understanding of HAV-receptor interactions we have used the recovery of cell-bound, infectious virus particles to measure the effects of temperature, pH and divalent cations on the binding of HAV to susceptible cells. Viral attachment to cultured cells proceeded at similar rates between 4 degrees C and 37 degrees C, with a slight increase in the total amount of virus attached at 4 degrees C. In contrast, both acidic pH and the presence of calcium ions independently caused greater than 20-fold increases in the cell attachment of infectious HAV diluted in buffered sodium chloride solutions, to a level approaching that of binding in culture medium, whereas magnesium led to a slight enhancement and zinc had no effect. The increased levels of binding observed with low temperature, low pH and the presence of calcium coincided with reduced rates of virus elution under similar conditions, suggesting that these conditions lead to a strengthening of the virus-receptor binding. The addition of calcium to highly purified HAV in buffered sodium chloride reduced the stability of virus during protracted incubation at 37 degrees C, as measured by immunoblotting of capsid proteins. The results suggest that the major effect of calcium in promoting HAV-receptor interactions is through a direct effect on the conformation of the viral capsid.

Animals↗

PubMed1996

Porosity reduction in bone cement at the cement-stem interface.

The fatigue failure of bone cement, leading to loosening of the stem, is likely to be one mode of failure of cemented total hip replacements. There is strong evidence that cracks in the cement are initiated at voids which act as stress risers, particularly at the cement-stem interface. The preferential formation of voids at this site results from shrinkage during polymerisation and the initiation of this process at the warmer cement-bone interface, which causes bone cement to shrink away from the stem. A reversal of the direction of polymerisation would shrink the cement on to the stem and reduce or eliminate the formation of voids at this interface. We have investigated this by implanting hip prostheses, at room temperature or preheated to 44 degrees C, into human cadaver femora kept at 37 degrees C. Two types of bone cement were either hand-mixed or vacuum-mixed before implantation. We found that the area of porosity at the cement-stem interface was dramatically reduced by preheating the stem and that the preheating temperature of 44 degrees C determined by computer analysis of transient heat transfer was the minimum required to induce initial polymerisation at the cement-stem interface. Temperature measurements taken during these experiments in vitro showed that preheating of the stem caused a negligible increase in the temperature of the bone. Reduction of porosity at the cement-stem interface could significantly increase the life of hip arthroplasties.

Bone Cements↗

PubMed1994

Rapid and efficient purification of hepatitis A virus from cell culture.

Hepatitis A virus (HAV) characteristically remains strongly cell-associated when grown in culture, with only small yields in the culture supernatant. Cell factories (6000 cm2) of BS-C-1 cells infected with the cytopathic HM175A.Z strain of HAV for 3, 4 or 7 days were harvested using trypsin to disperse the infected cell monolayer, and cells were collected by low speed centrifugation. More than 70% of the yield of virus and viral antigen can thus be obtained in the packed cell pellet. Packed cell pellets were resuspended in 5 volumes of isotonic buffer and cell membranes lysed by the addition of a non-ionic detergent. After removal of nuclei by centrifugation, ionic detergent was added to the clarified cytoplasmic extract. Under these conditions, HAV particles (virions and empty capsids) are the only particulate material remaining in the sample, and were recovered in a single ultracentrifugation step through discontinuous sucrose/glycerol density gradients. In one day, this method yields viral antigen with minimal cellular contaminants, in a concentrated volume suitable for subsequent biochemical, vaccine or diagnostic uses. The yield of viral antigen over numerous batches varied from 200 to 1600 vaccine-equivalent doses per cell factory, with a titre of up to 1 x 10(10) infectious particles per ml.

Animals↗

PubMed1993

RNA-dependent cleavage of VP0 capsid protein in provirions of hepatitis A virus.

Stable provirions of hepatitis A virus containing up to 62% VP0 were purified from infected BS-C-1 cells by sucrose density gradient ultracentrifugation, and conversion of these provirions to virions through maturation cleavage of VP0 capsid protein was demonstrated. VP0 cleavage was slow but linear over 7 days at 37 degrees, with mature virions containing between 3 and 7 copies of VP0 in separate experiments. Cleavage of approximately 25% of VP0 molecules (15 copies) was accompanied by a twofold increase in specific infectivity. Particles with reduced levels of VP0 were observed to sediment more rapidly in sucrose than VP0-rich provirions, reflecting conformational changes in the particles. The kinetics and temperature-dependence of VP0 cleavage further suggest that such conformational changes accompanying VP0 cleavage are necessary for the formation of subsequent catalytic sites.

Animals↗