Biomedical subjects

S Downes

Publications and source records attributed to S Downes.

At least 37 records · Page 2Linked to original sources

PubMed1999

Effects of surface-treated cpTi and Ti6Al4V alloy on the initial attachment of human osteoblast cells.

This study concerns the effect of simple surface treatments on the nature of the oxide layer, of commercially pure titanium (cpTi) and Ti6Al4V alloy substrates and their effect on human osteoblast cells (HOBS). After treatment the surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) in order to identify the surface groups responsible for the cell attachment process. The assessment of cell attachment was monitored by the Alamar blue assay (AB), measuring cell activity, in three types of media: phosphate-buffered saline (PBS), serum containing and serum-free Dulbecco's modified Eagle's cell culture medium (SER+ and SERF respectively). XPS analysis of the treated surfaces revealed consistent peaks representative of TiO2 on all surfaces and Ti(0) and Ti2O3 on the non-heat-treated surfaces. The cell activity assays indicated that there were no significant differences in cellular activity caused by surface treatments, but the cellular activity compared between the three types of medium was greatest in the PBS over the initial stages of attachment.

Journal Article↗

PubMed1999

Regulation of cell surface tissue transglutaminase: effects on matrix storage of latent transforming growth factor-beta binding protein-1.

Using a cytochemical approach, we examined the role of tissue transglutaminase (tTgase, Type II) in the incorporation of latent TGF-beta binding protein-1 (LTBP-1) in the extracellular matrix of Swiss 3T3 fibroblasts in which tTgase expression can be modulated through a tetracycline-controlled promoter. Increased tTgase expression led to an increased rate of LTBP-1 deposition in the matrix, which was accompanied by an increased pool of deoxycholate-insoluble fibronectin. Matrix deposition of LTBP-1 could also be reduced by the competitive amine substrate putrescine. Immunolocalization at the fluorescence and electron microscopic level showed that extracellular tTgase is located at the basal and apical surfaces of cells and at cell-cell contacts, and that LTBP-1 is co-distributed with cell surface tTgase, suggesting an early contribution of tTgase to the binding of LTBP-1 to matrix proteins. LTPB-1 was also found to co-localize with both intracellular and extracellular fibronectin, and increased immunoreactivity for LTBP-1 and fibronectin was found in large molecular weight polymers in the deoxycholate-insoluble matrix of fibroblasts overexpressing tTgase. We conclude that regulation of tTgase expression is important for controlling matrix storage of latent TGF-beta1 complexes and that fibronectin may be one extracellular component to which LTBP-1 is crosslinked when LTBP-1 and tTgase interact at the cell surface. (J Histochem Cytochem 47:1417-1432, 1999)

3T3 Cells↗

PubMed1998

Growth of human osteoblast-like cells on alkanethiol on gold self-assembled monolayers: the effect of surface chemistry.

Primary human osteoblasts were cultured on self-assembled monolayers (SAMs) of alkylthiols on gold with carboxylic acid and methyl termini, and the kinetics of cell attachment and proliferation were measured. Over 90 min approximately twice as many cells attached to carboxylic-acid-terminated monolayers as attached to methyl-terminated monolayers. After 24 h the number of cells attached to carboxylic-acid-terminated monolayers was ten times that attached to the methyl-terminated monolayers. Cell morphology and cytoskeletal actin organization also were found to be different. Osteoblasts were cultured on SAMs that were patterned by photolithographic techniques. Cells attached almost exclusively to carboxylic-acid-functionalized areas of the patterned surfaces, leaving methyl-functionalized regions bare. The patterns strongly influenced the morphology of the attached cells. After 24 h, cells were observed to bridge between carboxylic-acid-terminated regions separated by 75 microns, methyl-terminated regions but not those separated by 150 microns methyl-terminated regions. After 6 days in culture osteoblasts formed multilayers on the carboxylic-acid-terminated regions of the pattern.

Actins↗

PubMed1998

Thyroid dose in children undergoing prophylactic cranial irradiation.

PURPOSE: To determine the radiation dose received by the thyroid gland as a result of prophylactic cranial irradiation (PCI) in childhood leukemia and the factors influencing that dose. METHODS AND MATERIALS: The dose to the thyroid resulting from simulated cranial irradiation with parallel opposed lateral fields of an adult anthropomorphic (ART) phantom with both 6 MV X-rays and Cobalt-60 gamma-rays was measured using thermoluminescent dosimeters (TLDs). The dependence of thyroid dose on the distance of the field from the thyroid and the proportions of thyroid dose from stray radiation (leakage, scatter from jaws, etc.) and tissue scattered radiation were measured. The effects of a shadow tray and shielding blocks were also determined. Calculation of thyroid dose using the Clarkson scatter integration method was performed for 6 MV X-rays to compare with the measured doses. In vivo thyroid dose estimates were made using TLD measurements for three children receiving PCI with 6 MV X-rays. RESULTS: Using open, unshielded fields, the thyroid region of the phantom received 1.2-1.4% of the prescribed cranial dose for 6 MV X-rays and 1.5-1.7% for Cobalt-60. For both treatment units, stray radiation accounted for approximately two thirds of the thyroid dose and tissue scatter accounted for the remaining one third. The thyroid dose increased as the field moved closer to the thyroid, with an increasing proportion of the dose due to tissue scatter. Placement of a thyroid shielding block on a shadow tray reduced the thyroid dose by only 20% compared with the open, unshielded setup. Thyroid dose from 6 MV using open fields was affected by the orientation of the collimator. When the inferior field edge was defined by the lower jaw, the dose was reduced by 27% compared with the upper jaw. Good correlation of dose to the thyroid region was obtained between phantom measured doses, in vivo measured doses and calculation of dose using the Clarkson method. CONCLUSION: For PCI doses of 1800 or 2400 cGy in the adult phantom, the dose to the thyroid was 20-40 cGy (1-2%). For small children this could rise to approximately 5% of the prescribed dose, of which half was due to stray radiation. As the thyroid in children is very sensitive to radiation and the dose-response curve for thyroid tumor induction is linear, attempts to shield the thyroid during cranial irradiation are mandatory. Cobalt-60 units should not be used, as the thyroid dose was higher than using 6 MV X-rays. Collimator orientation and the use of shadow trays and shielding were important factors in determining thyroid dose.

Adult↗

PubMed1998

Higher-order suppression in diffraction-grating monochromators using thin films.

Although a continuously tuneable source of photons is a very desirable feature of synchrotron radiation it has one main drawback: the contamination of the photon beam by higher-order diffracted light. Several elements have absorption edges which lie between 10 and 200 eV, a range prone to high second- and third-order content in XUV monochromators. They can, therefore, be used as transmission filters to reduce this higher-order content. This paper describes the use of thin filters to reduce the higher-order content in diffraction-grating monochromators. Their suppression efficiency, transmission and ageing have been characterized using photoelectron spectroscopy and compared with calculated values. The effect of oxide contamination on their performance has been assessed. Filters are now installed on eight XUV beamlines and have been in routine use for several years.

Journal Article↗

PubMed1998

Release of bioactive human growth hormone from a biodegradable material: poly(epsilon-caprolactone).

We have characterized the biodegradable material poly(epsilon-caprolactone) (PCL) as a delivery system for recombinant human growth hormone (hGH). Two contrasting methods for the manufacture of the biomaterial were investigated: namely, solvent casting and solvent casting particulate leaching; the latter yielded porous PCL discs. The degree of porosity, which was assessed by scanning electron microscopy, could be controlled by incorporating selected concentrations of particulate sodium chloride during the manufacturing process. Bioactive hGH released from the PCL preparations was quantified with a highly sensitive and precise bioassay which was based upon hGH activation of rat lymphoma Nb2 cells. Eluates obtained from control discs of PCL which had not been loaded with hGH proved to be nontoxic when tested on these cells. The release of bioactive hGH from hormone-loaded nonporous discs of PCL was found to be a direct function of the initial hormone loading dose. Increased porosity of the discs manufactured by solvent casting particulate leaching increased the delivery of hGH from discs which had been immersion loaded. However, hGH release after surface loading was independent of porosity. Hormone concentrations were also assessed by immunoassay so that the ratios of bio- to immunoactivity (B:I ratio) of the hormone release could be determined. We found that the B:I ratio of the hormone after release from unstored discs was identical to that of the hormone prior to its incorporation into the PCL, demonstrating that the mild incorporation procedures utilized had not adversely affected the structural integrity of the hormone. However, if the hormone-loaded discs were stored at 37 degrees C prior to elution, the B:I ratios of the hGH released decreased indicating that this compromised the bioactive site.

Animals↗

PubMed1998

Sedentary snakes and gullible geckos: predator-prey coevolution in nocturnal rock-dwelling reptiles.

We investigated (1) the importance of chemical cues for predator detection by the nocturnal, rock-dwelling velvet gecko, Oedura lesueurii, and (2) how the lizards' responses to snake odour may have exerted selection on the foraging behaviours of a nocturnal elapid snake. This snake species (broadheaded snake, Hoplocephalus bungaroides) feeds primarily on velvet geckos, and does so by means of a distinctive foraging behaviour: the snakes remain sedentary in rock crevices for days or weeks, waiting to ambush geckos. Behavioural assays showed that geckos that are sympatric with this sedentary 'ambush' predator can detect and respond to the scent of the snake. Retreat-site selection experiments showed that geckos are less likely to enter crevices if the snake's scent is distributed over the entire rock surface, rather than localized to a central portion. Together, these data support the notion that the 'ambush' predator benefits by remaining sedentary within a retreat-site for long periods, because it thereby minimizes the extent to which it spreads its scent over the rocks forming the crevice. Geckos from a population sympatric with the 'ambush' predator responded strongly to the snake scent, but those from an allopatric population did not. Additionally, geckos from sympatric populations were able to detect the scent of a nocturnal snake that does not eat geckos (small-eyed snake, Rhinoplocephalus nigrescens), but did not modify their retreat-site selection or locomotory behaviours in response to this cue. Lizards from allopatric populations apparently did not detect the scent of small-eyed snakes. Collectively, our findings support an interpretation of predator-prey coevolution in the present system, and emphasize the importance of chemosensory cues to these rock-dwelling reptiles. Copyright 1998 The Association for the Study of Animal Behaviour. Copyright 1998 The Association for the Study of Animal Behaviour.

Journal Article↗

PubMed1998

Heat, safety or solitude? Using habitat selection experiments to identify a lizard's priorities.

Laboratory experiments with a rock-dwelling nocturnal gecko, Oedura lesueurii, showed that retreat-site selection (and other behaviours) are affected by the interplay between thermal benefits, social advantages and avoidance of predators. Velvet geckos were highly selective in habitat choice: they preferred artificial retreat-sites that mimic the thermal properties of natural rocks in full sun rather than those that mimic rocks in full shade; mature male geckos rarely shared retreat-sites with other adult males; and these lizards strongly avoided retreat-sites covered with the scent of a natural predator (the broadheaded snake, Hoplocephalus bungaroides). After documenting these preferences, we carried out additional trials in which two or more of these factors co-occurred, as is often the case in nature. Social dominance interacted with thermal benefits in determining retreat-site selection, with smaller (subordinate) males forced to use cooler retreat-sites when larger (dominant) males were present. Avoidance of predators was a higher priority than thermoregulation: the lizards would forego a warmer retreat-site with predator scent in favour of a cooler, unscented one. There was also an interplay between social dominance and predator scent: smaller males were forced to use either predator-scented retreat-sites or no retreat-site when larger males were present. General activity levels, and the frequencies of specific behavioural acts, also shifted in response to social and predator-scent cues. Our study emphasizes the complexity of habitat-selection behaviour in these lizards, and clarifies the criteria used in retreat-site selection when (as is commonly the case) the animal must choose between conflicting priorities. Copyright 1998 The Association for the Study of Animal Behaviour. Copyright 1998 The Association for the Study of Animal Behaviour.

Journal Article↗

PubMed1998

Interactions of chondrocytes with methacrylate copolymers.

Copolymers of poly(ethylmethacrylate) (PEMA) and tetrahydrofurfurylmethacrylate (THFMA) have been shown to exhibit potential as a biomaterial for use in cartilage repair. However, the interactions of chondrocytes with the polymer surface is not well understood. A series of novel methacrylate copolymers containing PEMA, THFMA and hydroxyethylmethacrylate (HEMA) were prepared and the ability of these various copolymers to support chondrocytes attachment in vitro has been assessed by the Alamar blue assay for cell number and environmental scanning electron microscopy (ESEM). As the mole fraction of HEMA in PEMA/THFMA/HEMA copolymers increased, chondrocyte attachment to the polymer surface in 24 h decreased. Chondrocytes maintained a rounded morphology and were strongly attached on the THFMA/PEMA polymer surface, but as the mole fraction of HEMA increased the cells present became much smaller with fewer cell to cell interactions. The effect of pre-adsorbing fibronectin on to the polymer surface on cell attachment was assessed both in the presence and absence of serum. Chondrocyte attachment was significantly reduced in serum-free medium. Pre-adsorption of fibronectin on to the copolymer surface substantially increased cell attachment in all cases. In conclusion, chondrocyte attachment and proliferation on these copolymers may be controlled by changes in the polymer surface chemistry and is highly sensitive to the presence of proteins either in the culture media or pre-adsorbed on to the copolymer surface.

Journal Article↗

PubMed1998

Evaluation of the osteoblast response to a silica gel in vitro.

Many bioactive glasses and glass ceramics contain silica, yet the effect of silica on the osteoblast is not well understood. The osteoblast cell response to a silica surface, without the interference of the other ions present in glasses and glass ceramics has been investigated. A silica sol-gel was prepared which gave a molar ratio of 1:4:4 tetraethyl orthosilicate (TEOS): ethanol:acidified water 0.2 M HCl) and spin cast on to thermanox discs. The gel was characterized in terms of bioactivity and release of silicic acid. Primary human osteoblasts (HOBs) were seeded on the surface of upright or inverted silica discs. Cell activity (alamar blue reduction), number (DNA content) and differentiation (alkaline phosphatase activity, nodule formation and mineralization) were measured. There was no apparant difference in cell number, activity or alkaline phosphatase activity between silica discs and controls. Nodules formed much earlier on the silica surfaces and these eventually mineralized. Nodule formation was reproducibly enhanced on the silica surface and less markedly on the inverted discs. It is likely that both the surface characteristics of the silica gel and silicic acid release from the disc affect osteoblast behaviour.

Journal Article↗

PubMed1997

Investigation into the release of bioactive recombinant human growth hormone from normal and low-viscosity poly(methylmethacrylate) bone cements.

Previous studies showed that recombinant human growth hormone (hGH) released from hormone-loaded poly(methylmethacrylate) (PMMA) cement stimulated osteoid formation in a rabbit model. Local delivery of hGH from cemented hip arthroplasties may thereby provide a means of reducing the problem of aseptic loosening. We have investigated two different formulations of PMMA as delivery systems for bioactive hGH. The bioactivity of the hormone release in vitro was monitored with an eluted stain assay (ESTA). The hGH was also measured by an immunoassay, which provides an alternative assessment of structural integrity of the hormone released. In addition, we adapted the ESTA bioassay to assess the in vitro cytotoxicity of the cements. Using unloaded cements, the undiluted eluates from both types of PMMA proved cytotoxic. This cytotoxicity could be diluted out, and the procedure allowed us to measure the bioactivity of hGH in the eluates from hormone-loaded cements independent of their cytotoxicity. The major fraction of the bioactivity was released from both of the PMMA cements during the first 24 h, but the hormone remained detectable in eluates collected after 36 days of elution. Comparison of the bio- and immunoactivity of the hGH released showed that the ratio of these two activities (i.e., the B:I ratio) was constant over this time period. However in parallel studies in which hormone-loaded discs were stored under dry conditions prior to elution, we found that the B:I ratio then declined markedly. This suggests that fully hydrated conditions, such as when the discs are bathed in assay medium, are necessary to maintain the bioactivity of the hGH. Both cements released only approximately 1% of the hormone originally incorporated, but the hGH concentration which accumulated in the eluates were high in physiologic terms (approximately 1000 mU/L).

Animals↗

PubMed1997

Heterocyclic methacrylates for clinical applications-further studies of water sorption.

The room temperature polymerizing system comprising poly(ethyl methacrylate)-tetra hydrofurfuryl methacrylate (PEM/THFMA) has potential in orthopaedic and dental applications, and earlier work has shown it to have unusual water absorption characteristics. This aspect has been studied in further detail, by studying the water absorption behaviour from some biological solutions, and the effect of the addition of an antibiotic (gentamicin). For comparison purposes, a parallel system whereby tetrahydrofuryl methacrylate was replaced by hydroxyethyl methacrylate (PEM/HEMA), was studied. In the case of PEM/THFMA, water uptake was substantially reduced when absorption was carried out from solutions (from about 30% in water to about 1.5% in solutions of higher concentrations), and the corresponding diffusion coefficient increased (by a factor of several hundred). The addition of gentamicin increased uptake, but the extent of increase also decreased in solutions. It was concluded that uptake was related to the osmolarity of the external solution, and also on the presence of osmotic sites within the polymer; hence the uptake process appears to be governed by chemical potential considerations. At the higher uptakes, there was evidence of water clusters. In marked contrast, the uptake by the PEM/HEMA system was independent of the osmolarity of the external solutions, presumably due to the hydrophilic nature of HEMA.

Journal Article↗

PubMed1997

The dependence of osteoblastic response on variations in the chemical composition and physical properties of hydroxyapatite.

Two synthetic hydroxyapatite powders (A and B), supplied by different manufacturers, were physically and chemically characterized before being die pressed and sintered at 1250 degrees C. The powders were characterized using X-ray diffraction (XRD), infrared spectroscopy (IRS), X-ray fluorescence, surface area analysis (BET), particle size analysis and scanning electron microscopy (SEM). The materials were then pressed and sintered to produce hydroxyapatite discs of similar densities and grain sizes for in vitro evaluation. The ceramics were seeded with osteoblastic cells and after 15 days in culture the cell morphology was assessed using scanning electron microscopy (SEM), the ultrastructure of the cells was studied using transmission electron microscopy (TEM) with EDAX, and the rate of cell growth was assessed using biochemical techniques. The results clearly showed that the rate of cell proliferation but not the rate of alkaline phosphatase production, was highly dependent on the composition of the hydroxyapatite powders that were used to make the ceramic discs. The ultrastructural studies confirmed the relative viabilities of the cells and the nature of the ceramic interface indicating visually the marked differences in the performance of the two materials.

Journal Article↗

PubMed1996

Growth hormone-responsive DT-diaphorase-mediated bioreduction of tetrazolium salts.

Microculture tetrazolium assays (MTAs) rely upon the bioreduction of tetrazolium salts to their intensely coloured formazans. Although these assays are being extensively used, the intracellular mechanisms responsible for the formazan production are not known. MTAs currently provide the basis for uniquely precise in vitro bioassays for human growth hormone (hGH) which use the Nb2 cells. We have compared two contrasting tetrazolium salts, namely 3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-++ +sulfophenyl) tetrazolium, inner salt (MTS), in this system. An intermediate electron acceptor (IEA) is obligatory for the MTS- but not the MTT-bioassay. We report that inhibitors of DT-diaphorase abolished MTS- but not MTT-formazan production. We conclude that substitution of MTT with MTS/menadione resulted in formazan production via a different electron transfer pathway which is exclusively mediated by DT-diaphorase.

Animals↗

PubMed1996

Effect of co-monomer composition on the integrity of bioactive growth hormone released from novel PEMA based polymers.

The release of human growth hormone (hGH) from hormone loaded bone cement was previously shown to enhance osteoid formation. hGH is a complex protein and its incorporation into such cements may compromise its bioactivity. We therefore characterized the release of hGH from a series of methacrylate systems based upon poly(ethylmethacrylate) (PEMA). Different mixtures of two monomers, hydroxyethylmethacrylate (HEMA) and n-butylmethacrylate (n-BM) were used to provide polymers with graded water uptakes. Exclusive use of only one of the monomers resulted in enhanced cytotoxicity and also reduced release of the bioactive hormone. Combinations of the monomers improved the recovery of bioactivity from the polymers and reduced their cytotoxicity. hGH released from the polymer with the lowest water uptake (100% n-BM, 0% HEMA) had an exceptionally low bioactivity: immunoactivity ratio, suggesting that the bioactive site of the hormone is particularly susceptible to disruption when it is incorporated into this matrix.

Animals↗

PubMed1996

The use of intermediate electron acceptors to enhance MTT bioreduction in a microculture tetrazolium assay for human growth hormone.

We contrast the effects of three intermediate electron acceptors (IEAs) on the highly quantitative ESTA bioassay for human growth hormone. This is a microculture tetrazolium assay based upon the in vitro reduction of the tetrazolium salt MTT, by Nb2 cells which have been activated with hGH. Each of the IEAs influenced MTT-formazan production in a distinctive manner. The two quinonoids, namely menadione and co-enzyme Q0 markedly increased the MTT-formazan produced by hormone activated Nb2 cells and thereby amplified the response of our bioassay for human growth hormone (hGH). The exceptionally low bioassay baseline which is characteristic of the unstimulated Nb2 cells when only MTT is added was retained in the presence of CoQ0, but was greatly increased by menadione. Phenazine methosulphate, which is the most widely used redox intermediary in microculture tetrazolium assays, also increased the baseline, but had only a minimal additional effect on MTT reduction by activated Nb2 cells. We conclude that CoQ0 is the preferred IEA for this ESTA bioassay for hGH.

Animals↗

PubMed1995

Resonant photoemission from complex cuprates and nickelates.

Synchrotron-excited resonant-photoemission measurements at rare-earth 4d --> 4f and transition-metal 3p --> 3d thresholds have been carried out using a variety of complex cuprates and nickelates on stations 6.1 (grazing-incidence monochromator) and 6.2 (toroidal-grating monochromator) at the SRS CLRC Daresbury Laboratory. The systems studied are Nd(2)Ni(1 - x)Cu(x)O(4), La(2 - x)Sr(x)Ni(1- y)Fe(y)O(4 + delta) and Bi(2)Sr(2)Ca(1 - x)Y(x)Cu(2)O(8 + delta). A combination of EDC and constant-initial-state data is used to examine the 4f and 3d contributions to the valence-band density of states and their binding-energy positions relative to the Fermi energy. This allows the study of the valence states of the transition-metal ions and their modulation on doping. For La(2 - x)Sr(x)Ni(1 - y)Fe(y)O(4 + delta), this approach is used to infer a valence state of >/= 3.0 for Fe. In the case of Bi(2)Sr(2)Ca(1 - x)Y(x)Cu(2)O(8 + delta), the effect of Cu valence modulation on the 3p resonance is observed as x is varied. This is discussed in the light of controversy surrounding shifts in core-level photoemission with doping for this system.

Journal Article↗

PubMed1995

Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS.

Microculture tetrazolium assays are being widely exploited to investigate the mechanisms of both cell activation and cell damage. They are colorimetric assays which are based upon the bioreduction of a tetrazolium salt to an intensely coloured formazan. We contrast the responses obtainable with two new tetrazolium salts, MTS and XTT, when used on the rat lymphoma cell line (Nb2 cells), which has been activated by human growth hormone. These tetrazolium salts, unlike the more commonly used MTT, form soluble formazans upon bioreduction by the activated cells. This has the advantage that it eliminates the error-prone solubilisation step which is required for the microculture tetrazolium assays which employ MTT. Bioreduction of XTT and MTS usually requires addition of an intermediate electron acceptor, phenazine methosulphate (PMS). We found that the XTT/PMS, but not the MTS/PMS, reagent mixture was unstable. Nucleation and crystal formation in the XTT/PMS reagent mixture, prepared in DPBS, could occur within 1-3 min. This resulted in a decline in XTT-formazan production and manifested itself in the microculture tetrazolium assay as both poor within-assay precision and serious assay drift. Several features of the system suggested that the formation of charge-transfer complexes between XTT and PMS accounted for this instability. No such instability was encountered when MTS and PMS were mixed. We demonstrate that MTS/PMS provides microculture tetrazolium assays for hGH which are free from these serious artefacts and which are uniquely precise. In conclusion we therefore advocate the use of MTS in preference to XTT for the new generation of microculture tetrazolium assays.

Animals↗