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Biomedical subjects

D M Grant

Publications and source records attributed to D M Grant.

At least 19 recordsLinked to original sources
PubMed2006

The effect of anisotropic architecture on cell and tissue infiltration into tissue engineering scaffolds.

A common phenomenon in tissue engineering is rapid tissue formation on the outer edge of the scaffold which restricts cell penetration and nutrient exchange to the scaffold centre, resulting in a necrotic core. To address this problem, we generated scaffolds with both random and anisotropic open porous architectures to enhance cell and subsequent tissue infiltration throughout the scaffold for applications in bone and cartilage engineering. Hydroxyapatite (HA) and poly(D,L-lactic acid) (P(DL)LA) scaffolds with random open porosity were manufactured, using modified slip-casting and by supercritical fluid processing respectively, and subsequently characterised. An array of porous aligned channels (400 microm) was incorporated into both scaffold types and cell (human osteoblast sarcoma, for HA scaffolds; ovine meniscal fibrochondrocytes, for P(DL)LA scaffolds) and tissue infiltration into these modified scaffolds was assessed in vitro (cell penetration) and in vivo (tissue infiltration; HA scaffolds only). Scaffolds were shown to have an extensive random, open porous structure with an average porosity of 85%. Enhanced cell and tissue penetration was observed both in vitro and in vivo demonstrating that scaffold design alone can influence cell and tissue infiltration into the centre of tissue engineering scaffolds.

Animals↗
PubMed2006

In vivo and in vitro metabolism of arylamine procarcinogens in acetyltransferase-deficient mice.

Arylamine N-acetyltransferases (NATs) catalyze the biotransformation of a number of aromatic and heterocyclic amines, many of which are procarcinogenic agents. Interestingly, these enzymes are binary in nature, participating in both detoxification and activation reactions, and thus it is unclear what role NATs actually play in either preventing or enhancing toxic responses. The ultimate direction may be substrate-specific and dependent on its tissue-specific metabolism by competing, but genetically variable, drug-metabolizing enzymes. To investigate the effect of N-acetylation on the metabolism of some classical procarcinogenic arylamines, we have used our double knockout Nat1/2(-/-) mouse model to test both in vitro activity and the in vivo clearance of some of these agents. As expected, N-acetylation activity was undetectable in tissue cytosol preparations from Nat1/2(-/-) mice for 4-aminobiphenyl (ABP) and 2-aminofluorene (AF), whereas significant levels were measured in all wild-type tissue cytosols tested, indicating the widespread metabolism of these agents. Nat1/2(-/-) mice displayed a variable response with respect to in vivo pharmacokinetics. AF appeared to be most severely compromised, with a 3- to 4-fold increased area under the curve (AUC), whereas the clearance of ABP was found to be less dependent on N-acetylation, with no difference in ABP-AUC between wild-type and knockout animals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine was neither N-acetylated nor was its clearance affected by NAT genotype, signifying a dependence on other drug-metabolizing enzymes. The elucidation of the role that N-acetylation plays in the clearance of procarcinogenic agents is the first step in attempting to correlate metabolism by NATs to toxic outcome prevention or augmentation.

Amines↗
PubMed2005

Attentional biases in social anxiety and dysphoria: does comorbidity make a difference?

This study examined whether comorbid symptoms influence the attentional biases associated with social anxiety and dysphoria using the Emotional Stroop Task (EST). Participants were recruited into three groups: a Social Anxiety group, a Dysphoric group, and a Social Anxiety/Dysphoric group. Four types of stimulus words were used: social anxiety threat, depressive threat, neutral words, and positive words. It was hypothesized that the Social Anxiety group would display an attentional bias to emotionally threatening stimuli whereas neither the dysphoric nor the Social Anxiety/Dysphoric group would display an attentional bias. Results found that the Social Anxiety group took longer to color name social threat and depressive words, whereas neither the Dysphoric nor the Comorbid group displayed an attentional bias. These results are discussed in light of their implications for cognitive theories of social anxiety and depression.

Adult↗
PubMed2005

Determination of age and gender differences in biochemical processes affecting the disposition of 2-butoxyethanol and its metabolites in mice and rats to improve PBPK modeling.

2-Butoxyethanol (BE) is the most widely used glycol ether solvent. BEs major metabolite, butoxyacetic acid (BAA), causes hemolysis with significant species differences in sensitivity. Several PBPK models have been developed over the past two decades to describe the disposition of BE and BAA in male rats and humans to refine health risk assessments. More recent efforts by Lee et al. [Lee, K.M., Dill, J.A., Chou, B.J., Roycroft, J.H., 1998. Physiologically based pharmacokinetic model for chronic inhalation of 2-butoxyethanol. Toxicol. Appl. Pharmacol. 153, 211-226] to describe the kinetics of BE and BAA in the National Toxicology Program (NTP) chronic inhalation studies required the use of several assumptions to extrapolate model parameters from earlier PBPK models developed for young male rats to include female F344 and both sexes of B6C3F1 mice and the effects of aging. To replace these assumptions, studies were conducted to determine the impact of age, gender and species on the metabolism of BE, and the tissue partitioning, renal acid transport and plasma protein binding of BAA. In the current study, the Lee et al. PBPK model was updated and expanded to include the further metabolism of BAA and the salivary excretion of BE and BAA which may contribute to the forestomach irritation observed in mice in the NTP study. The revised model predicted that peak blood concentrations of BAA achieved following 6 h inhalation exposures are greatest in young adult female rats at concentrations up to 300 ppm. This is not the case predicted for old (> or =18 months) animals, where peak blood concentrations of BAA in male and female mice were similar to or greater than female rats. The revised model serves as a quantitative tool for integrating an extensive pharmacokinetic and mechanistic database into a format that can readily be used to compare internal dosimetry across dose, route of exposure and species.

Administration, Inhalation↗
PubMed2005

The influence of dispersant concentration on the pore morphology of hydroxyapatite ceramics for bone tissue engineering.

There is a clinical need for synthetic scaffolds that will promote bone regeneration. Important factors include obtaining an optimal porosity and size of interconnecting windows whilst maintaining scaffold mechanical strength, enabling complete penetration of cells and nutrients throughout the scaffold, preventing the formation of necrotic tissue in the centre of the scaffold. To address this we investigated varying slip deflocculation in order to control the resulting porosity, pore size and interconnecting window size whilst maintaining mechanical strength. Hydroxyapatite (HA) porous ceramics were prepared using a modified slip casting process. Rheological measurements of the HA slips were used to identify deflocculation conditions which resulted in changes in the cell and window sizes of the resulting ceramics. Sintered ceramics were characterised by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Pore and window size distribution was determined by SEM. XRD analysis confirmed that the crystal structure remained HA after the sintering process. SEM showed that HA porous ceramics presented a highly interconnected porous network with average pore sizes ranging from 391+/-39 to 495+/-25 microm. The average window size varied from 73+/-5 to 135+/-7 microm. Pore diameters obtained were controllable in the range 200-500 microm. Window sizes were in the range 30-250 microm. The use of dispersant concentration allows pore and window size to be modified whilst maintaining control over porosity demonstrated by a porosity of 85% for seven different dispersant concentrations. The advantage of this approach allows the correlation between the rheological conditions of the slip and the resultant sintered ceramic properties. In particular, optimising the ceramic strength by controlling the agglomeration during the casting process.

Bone Substitutes↗
PubMed2005

Thin-film theories for two-phase reactive flow models of active cell motion.

The aim of this paper is to develop a broadly-applicable and self-consistent thin-film biphasic modelling framework for the full crawling cycle of a single animal cell. A hierarchy of thin-film two-phase 'reactive flow' models is derived; between them these cover a wide range of biologically relevant parameter regimes. The mathematical properties and biological implications of the resulting systems of high-order nonlinear degenerate parabolic-elliptic evolution equations are investigated. Linear-stability arguments suggest the formation of highly localized regions of high or low network density associated with small irregular oscillations or 'ruffling' of the plasma membrane. Local analyses at the contact line identify the classes of admissible contact-line conditions, through which we study for the first time the effect on the cell-scale motion of the 'mesoscopic' contact-line physics, which consists of the chemical and mechanical mechanisms for protrusive and retractive force generation near the outer cell periphery. One of the formulations is used to develop a minimal model for cell body translocation over a thin pseudopod, which predicts that myosin-driven contraction is not essential for rapid translocation. An analytic prediction for the translocation speed is given in terms of the network viscosity and slip coefficient (a parameter measuring the adhesion strength), of the membrane tension and of the thicknesses of the pseudopod and actin cortex; this is in good agreement with the translocation speed of osteoblasts on biomaterial substrates commonly used for orthopaedic implants. Limitations of the modelling approach and directions for future work are outlined.

Actins↗
PubMed2004

The effect of surface chemistry and nanotopography of titanium nitride (TiN) films on primary hippocampal neurones.

The cell-substrate interaction of primary hippocampal neurones with thin films of TiN was studied in vitro. TiN films of different surface chemistries and topographies were deposited by pulsed DC reactive magnetron sputtering and closed field unbalanced magnetron sputter ion plating by Teer Coatings Ltd., Hartlebury, UK to result in TiN films with similar surface chemistries but different topographical features. TiN films were characterised using X-ray diffraction, X-ray photoelectron spectroscopy and atomic force microscopy. The neuron-substrate interaction was examined using environmental scanning electron microscopy (FEG-ESEM) for morphological information. Bromodeoxyuridine and TUNEL assays were used to identify proliferating neurones as well as apoptotic neurones. Fluorescent staining for MAP-2 was used to label neuronal network formation. Primary hippocampal neurones were found to attach and spread to all of the TiN film chemistries and topographies investigated. Neuronal network morphology appeared to be more preferential on the nitrogen rich TiN films and also with reduced nanotopographical features.

Animals↗
PubMed2003

The effect of surface chemistry and nanotopography of titanium nitride (TiN) films on 3T3-L1 fibroblasts.

The cell-material interaction of 3T3-L1 fibroblasts with TiN films was studied in vitro. TiN films were deposited onto glass substrates to thicknesses of 0.2 and 1.0 microm by pulsed dc reactive magnetron sputtering. For comparison TiN films were deposited by closed field unbalanced magnetron sputter ion plating by Teer Coatings Ltd. (Hartlebury, UK) to result in TiN films with similar surface chemistries but having increased topographical features. TiN films were characterized using X-ray diffraction, X-ray photoelectron spectroscopy, and atomic force microscopy. The cell-material interaction was examined morphologically by monitoring fibroblast attachment and growth and comparing to a control substrate. At early time points increased numbers of 3T3-L1 fibroblasts were found to preferentially attach to TiN films with an increase in the percentage of surface interstitial nitrogen and also with decreased topographical features. At later time points the presence of nanotopography appeared to play a greater role than the effects of surface chemistry and resulted in increased numbers of attached 3T3-L1 fibroblasts. The results show that by changing the deposition route and parameters to produce TiN films, the resultant films can be used to investigate the cellular response to surfaces of differing chemistry and topography.

3T3 Cells↗
PubMed2003

Properties of calcium phosphate coatings deposited and modified with lasers.

Physical, chemical and biological properties of calcium phosphate coatings fabricated by a pulse laser deposition method at room temperature (RT PLD) have been studied. In vitro evaluation of RT PLD coatings on bioresorbable polymers (Poly-epsilon-caprolactone and Poly-L-lactide) have been carried out. It was shown that both polymers support osteoblast growth, with increased cell activity, alkaline phosphatase activity and total protein content on those surfaces that have been coated. The advantages of RT PLD coatings in biomaterials surface optimization are discussed.

Journal Article↗
PubMed2002

The effect of surface chemistry and structure of titanium nitride (TiN) films on primary hippocampal cells.

Thin films of TiN were investigated as a candidate microelectrode material for multi-electrode arrays, which are used for recording from electrically active cells in culture. TiN films were deposited onto glass substrates by DC pulsed reactive magnetron sputtering. The structure, phase composition and surface chemistry were studied using X-ray diffraction (XRD), Atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The biocompatibility of the TiN films was examined morphologically by monitoring neuronal network formation and comparing this to a control substrate. Results indicate that neuronal cell adhesion and growth is influenced by the surface chemistry and associated crystal orientation of the TiN thin films.

Cells, Cultured↗
PubMed2001

Characterization of stereochemistry and molecular conformation using solid-state NMR tensors.

A solid-state NMR technique is described for establishing stereochemistry using the natural product terrein as a model compound. This method involves comparison of experimental (13)C tensor principal values with ab initio computed values for all possible computer-generated stereoisomers. In terrein the relative stereochemistry is confirmed by NMR to be 2R*,3S with high statistical probability (>99.5%). The proposed approach also simultaneously verifies the molecular conformation of the two hydroxy groups in terrein established by X-ray data. It is sufficient to use only shift tensor values at carbons 2 and 3, the stereocenters, to characterize both the stereochemistry and molecular conformations. The solid-state NMR method appears to be especially useful for determining relative stereochemistry of compounds or their derivatives that are difficult to crystallize.

Carbon Isotopes↗
PubMed2001

Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop.

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560-564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29-131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122-131 in NAT1 model and residues 122-133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

Acetyltransferases↗
PubMed2001

Carbonates, thiocarbonates, and the corresponding monoalkyl derivatives. 1. Their preparation and isotropic (13)C NMR chemical shifts.

Three series of potassium carbonate and thiocarbonate salts were synthesized, and the corresponding (13)C isotropic solid-state NMR and the aqueous solution (13)C and (1)H NMR data were collected. The series of compounds that were studied consists of (1) the parent compounds, i.e., potassium carbonate, K(2)CO(3), potassium hydrogen carbonate, KHCO(3), potassium monothiocarbonate, K(2)CO(2)S, potassium dithiocarbonate, K(2)COS(2), and potassium trithiocarbonate, K(2)CS(3), (2) the oxygen monoalkyl substituted derivatives of the parent compounds (OR series), i.e., three potassium O-alkylcarbonates, KO(2)COR, three potassium O-alkylmonothiocarbonates, KOSCOR, and three potassium O-alkyldithiocarbonates, KS(2)COR, all with R = CH(3), CH(2)CH(3), CH(CH(3))(2), and (3) the sulfur monoalkyl substituted derivatives of the parent compounds (SR series), i.e., two potassium S-alkylmonothiocarbonates, KO(2)CSR; two potassium S-alkyldithiocarbonates, KOSCSR, and two potassium S-alkyltrithiocarbonates, KS(2)CSR, all with R = CH(3) or CH(2)CH(3). The preparation and proper characterization of KO(2)CSR and KOSCSR with R = CH(3) and CH(2)CH(3) along with new IR and X-ray powder diffraction data for several other compounds in the series are reported for the first time in this study. Solution NMR data for KO(2)CSR (R = CH(3), CH(2)CH(3)) and KOSCSR (R = CH(3)) and solid-state NMR data for K(2)CO(2)S and K(2)COS(2) could not be obtained because they are unstable under the corresponding measurement conditions. The isotropic chemical shift values of the central carbon atoms obtained from solid-state MAS (magic angle spinning) NMR experiments deviate at most by 3 ppm from the corresponding solution values. Two major trends in the (13)C chemical shift values of the central carbon atoms were found. First, if an oxygen atom in a parent compound or in an alkyl-substituted derivative is replaced by a sulfur atom, a significantly higher chemical shift value is observed. This trend is discussed in terms of the paramagnetic contribution to the chemical shielding constant. Second, the size of the alkyl group in the monoalkyl derivatives has a very small effect on the chemical shift values of the central carbon atoms. This observation is explained using the concept of varying inductive effects produced by alkyl groups. The trends observed for the (13)C and (1)H chemical shift values of the alkyl groups follow common concepts on the structure dependency of chemical shifts.

Journal Article↗
PubMed2001

Carbonates, thiocarbonates, and the corresponding monoalkyl derivatives. 2. X-ray crystal structure of potassium methyltrithiocarbonate (KS(2)CSCH(3)).

This study presents the first crystal structure determination of a potassium S-alkylthiocarbonate, the title compound potassium methyltrithiocarbonate (KS(2)CSCH(3)) Single crystals of KS(2)CSCH(3) were obtained by the slow introduction of methylene chloride into a saturated solution of KS(2)CSCH(3) in a 1:1 mixture of methylene chloride and tetrahydrofuran at 0 degrees C in a dry N(2) atmosphere. The compound crystallizes in the monoclinic space group P2(1)/c containing Z = 4 K(+) cations and S(2)CSCH(3-) anions per unit cell. The unit cell dimensions are a = 7.6639(3) A, b = 6.5804(2) A, c = 12.8426(5) A, and beta = 91.565(2) degrees. The isomorphism to the structurally closely related compounds KO(2)COCH(3), KOSCOCH(3), and KS(2)COCH(3) is examined.

Journal Article↗
PubMed2001

Investigation of the polymorphs of dimethyl-3,6-dichloro-2,5-dihydroxyterephthalate by (13)C solid-state NMR spectroscopy.

Two of the three conformational polymorphs of dimethyl-3,6-dichloro-2,5-dihydroxyterephthalate are studied by solid-state NMR techniques. The structural differences between the polymorphs have previously been studied by X-ray. In these two polymorphs named white and yellow due to their color, the major structural difference is the torsional angle between the ester group and the aromatic ring. The yellow form has a dihedral angle of 4 degrees between the plane of the aromatic ring and the plane of the ester group, while the white form has two different molecules per unit cell with dihedral angles of 70 degrees and 85 degrees. This change greatly affects the conjugation in the pi-electronic system. In addition, there are differences in the hydrogen-bonding patterns, with the white form having intermolecular hydrogen bonds and the yellow form having intramolecular hydrogen bonds. In this work, the carbon isotropic chemical shift values and the chlorine electric field gradient (EFG) tensor information are extracted from the (13)C MAS spectra, and the principal values of the chemical shift tensors of the carbons are obtained from 2D FIREMAT experiments. Quantum chemical calculations of the chemical shift tensor data as well as the EFG tensor are performed at the HF and DFT levels of theory on individual molecules and on stacks of three molecules to account for the important intermolecular interactions in the white form. The differences between the spectral data on the two polymorphs are discussed in terms of the known electronic and structural differences.

Carbon Isotopes↗
PubMed2001

A novel dipolar dephasing method for the slow magic angle turning experiment.

Complete suppression of the resonances from protonated carbons in a slow magic angle spinning experiment can be achieved using five dipolar dephasing (Five-DD) periods distributed in one rotor period. This produces a spectrum containing only the spinning sidebands (SSB) from the nonprotonated carbons. It is shown that the SSB patterns corresponding to the nonprotonated carbons are not distorted over a wide range of dipolar dephasing times. Hence, this method can be used to obtain reliable principal values of the chemical shift tensors for each nonprotonated carbon. The Five-DD method can be readily incorporated into isotropic-anisotropic 2D experiments such as FIREMAT and 2D-PASS to facilitate the measurement of the (13)C chemical shift tensors in complex systems.

Carbon Isotopes↗