Browsing by Author "Foster, LJR"
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- ItemApplication of polyethylene glycol to promote cellular biocompatibility of polyhydroxybutyrate films(Hindawi Publishing Corporation, 2011-08-22) Chan, RTH; Marçal, H; Russell, RA; Holden, PJ; Foster, LJRPolyhydroxybutyrate (PHB) is a biomaterial with potential for applications in biomedical and tissue engineering; however, its brittle nature and high crystallinity limit its potential. Blending PHB with a variety of PEGs produced natural-synthetic composite films composed of FDA-approved polymers with significant reductions in crystallinity, from 70.1% for PHB films to 41.5% for its composite with a 30% (w/w) loading of PEG2000. Blending also enabled manipulation of the material properties, increasing film flexibility with an extension to break of 2.49±1.01% for PHB films and 8.32±1.06% for films containing 30% (w/w) PEG106. Significant changes in the film surface properties, as measured by porosity, contact angles, and water uptake, were also determined as a consequence of the blending process, and these supported greater adhesion and proliferation of neural-associated olfactory ensheathing cells (OECs). A growth rate of 7.2×105 cells per day for PHB films with 30% (w/w) PEG2000 loading compared to 2.5×105 for PHB films was observed. Furthermore, while cytotoxicity of the films as measured by lactate dehydrogenase release was unaffected, biocompatibility, as measured by mitochondrial activity, was found to increase. It is anticipated that fine control of PEG composition in PHB-based composite biomaterials can be utilised to support their applications in medicinal and tissue engineering applications. Copyright © 2011 Rodman T. H. Chan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
- ItemBioPEGylation of polyhydroxybutyrate promotes nerve cell health and migration(ACS Publications, 2013-12-03) Chan, RTH; Russell, RA; Marçal, H; Lee, TH; Holden, PJ; Foster, LJRThis study reports on the superior suitability of Polyhydroxybutyrate-polyethylene glycol hybrid polymers biosynthesised by Cupriavidus necator over PHB as biomaterials for tissue engineering. Incorporation of PEG106 (DEG) during PHB biosynthesis reduced crystallinity, molecular weight, and hydrophobicity while improving mechanical properties. In vitro olfactory ensheathing cell (OEC) proliferation was enhanced by cultivation on PHB-b-DEG films. Cultivation on PHB and PHB-b-DEG films showed no cytotoxic responses and cell viability and membrane integrity was sustained. PHB-b-DEG films promoted OECs entering into the DNA replication (S) phase and mitotic (G2-M) phase during the cell growth cycle and apoptosis was low. This study also confirmed an association between the level of neurite-outgrowth inhibitory protein (Nogo) and receptor pair Ig-like receptor B (PirB) expression and cell proliferation, both being down-regulated in cells grown on hybrid films when compared with PHB and asynchronous growth. Thus, DEG-terminated PHB-based biomaterials have great potential as biological scaffolds supporting nerve repair. © 2013 American Chemical Society.
- ItemBiopolymer deuteration for neutron scattering and other isotope-sensitive techniques(Elsevier, 2015) Russell, RA; Garvey, CJ; Darwish, TA; Foster, LJR; Holden, PJThe use of microbial biosynthesis to produced deuterated recombinant proteins is a well-established practice in investigations of the relationship between molecular structure and function using neutron scattering and nuclear magnetic resonance spectroscopy. However, there have been few reports of using microbial synthetic capacity to produce labeled native biopolymers. Here, we describe methods for the production of deuterated polyhydroxyalkanoate biopolyesters in bacteria, the polysaccharide chitosan in the yeast Pichia pastoris, and cellulose in the bacterium Gluconacetobacter xylinus. The resulting molecules offer not only multiple options in creating structural contrast in polymer blends and composites in structural studies but also insight into the biosynthetic pathways themselves. © 2015 Elsevier Inc.
- ItemBiosynthesis and characterization of deuterated polyhydroxyoctanoate(American Chemical Society, 2006-04) Foster, LJR; Russell, RA; Sanguanchaipaiwong, V; Stone, DJM; Hook, JM; Holden, PJThe synthesis of a polyhydroxyalkanoate with medium chain length alkyl substituents by Pseudomonas oleovorans was investigated using protonated and deuterated forms of octanoic acid in a minimal salts medium. Cultivation with deuterated octanoic acid resulted in a reduced rate of polymer accumulation compared to that with its protonated counterpart (107 and 207 mg of polymer L-1 of medium h-1 of cultivation, respectively). Nuclear magnetic resonance and gas chromatography coupled mass spectrometry of the derivatized polymer was used to establish the extent and distribution of deuterium in the biopolymer. A partially deuterated heteropolymer with 3-hydroxyoctanoic acid as the main constituent was produced. Deuteration is an important tool for contrast variation studies using neutron scattering, but predicates that the deuterated polymer is otherwise comparable in its physiochemical and material properties to its protonated counterpart. In studies reported here, the deuterated biopolymer exhibited an additional diffraction maximum at 7.55 Å and slight differences in its melting point (60 and 55 °C) and glass transition temperature (−39 and −36 °C) when compared to its protonated equivalent. While significant differences between the protonated and deuterated biopolymers were determined, our results support the use of this deuterated polyhydroxyalkanoate in its application in investigations using analytical neutron scattering techniques. © 2006, American Chemical Society
- ItemCarbon nanotube mediated miscibility of polyhydroxyalkanoate blends and chemical imaging using deuterium-labelled poly(3-hydroxyoctanoate)(Elsevier, 2018-08) Russell, RA; Foster, LJR; Holden, PJBiopolymers have potential as scaffolds supporting regrowth of damaged tissues, however their material properties may limit the range of applications. Blending polymers with different thermomechanical properties has been demonstrated to extend the range of possible applications for polyhydroxyalkanoate (PHA) polymers, while the addition of nanoparticles can be used to modulate miscibility which influences strength and flexibility of the blend. Here we report on the blending of Poly(3-hydroxybutyrate) and Poly(3-hydroxyoctanoate) which possess different thermomechanical properties, and the effect of single wall carbon nanotubes (SWCNT) on their miscibility, electrical conductivity and thermomechanical performance. The apparent perturbation of phase boundaries in nanocomposite films observed by Scanning Electron Microscopy (SEM) was complemented by chemical mapping of film cross sections containing a deuterium-labelled poly(3-hydroxyoctanoate) phase in the blend using Infrared Microspectroscopy (IRM), suggesting increased miscibility due to nanoparticle addition. The electrical percolation threshold in nanocomposite films was observed between 0.5 and 1 wt% SWCNT, where the surface resistivity was reduced by eight orders of magnitude compared to the insulating polymer blend. Addition of SWCNT did not impact significantly on mechanical properties of films containing up to 2.5 wt% SWCNT. A solvent cast bionanocomposite film containing optimally 1 wt% SWCNT yielded a material with improved electrical conductivity compared to the SWCNT-free blend and which supported growth of Olfactory Ensheathing Cells, providing a basis for developing biopolymer scaffolds capable of conducting electrical stimulation. Crown Copyright © 2018. Published by Elsevier Ltd.
- ItemEffect of sol-gel encapsulation on lipase structure and function: a small angle neutron scattering study(Springer, 2005-01) Rodgers, LE; Holden, PJ; Knott, RB; Finnie, KS; Bartlett, JR; Foster, LJRThe application of small angle neutron scattering (SANS) to the characterisation of sol–gel hosts containing biomolecules offers the opportunity to explore the relationship between gel structure and catalyst. A model system involving the immobilisation of Candida antarctica lipase B (CALB) was investigated. Gels were produced by fluoride-catalysed hydrolysis of fixed ratios of tetramethylorthosilicate (TMOS) and methyltrimethoxysilane (MTMS). Phase separation between the enzyme and the evolving sol–gel matrix was minimised by incorporating glycerol into the sol–gel precursor solution. The potential stabilising effect of the NaF catalyst upon the enzyme was also investigated. Scattering studies were conducted on both immobilised lipase, and lipase in free solution. Scattering studies on free enzyme provided evidence of multiple populations of enzyme aggregates and showed that choice of solvent affected the degree of aggregation. Both NaF and glycerol affected neutron scattering, indicating changes in lipase conformation. Increasing glycerol concentration increased the degree of aggregation and produced differences in solvent packing on the surface of protein molecules. Initial evidence from SANS data indicated that the presence of the enzyme during gel formation conferred structural changes on the gel matrix. Modelling the effect of sol–gel encapsulation on lipase requires comparison of data from free enzyme to the immobilised form. Removal of the enzyme from the sol–gel structure, post gelation, is necessary to better characterise the modified matrix. This methodological problem will be the subject of future investigations. © 2005, Springer.
- ItemIn vivo deuteration of a native bacterial biopolymer for structural elucidation using SANS(Elsevier, 2004-07-15) Holden, PJ; Russell, RA; Stone, DJM; Garvey, CJ; Foster, LJRIn order to facilitate future structural studies, biodeuteration of bacterial polyhydroxyalkanoates (PHAs) was investigated. We report here the in vivo deuteration of poly 3-hydroxyoctanoate (PHO) produced by its native host, the bacterium Pseudomonas oleovorans. Bacterial biomass was produced in bioreactor studies by growth on hydrogenated substrates and PHO was subsequently produced intracellularly (10–20% w/w) during batch fed growth on deuterated octanoic acid under oxygen limitation. GC-MS analyses of the PHO demonstrated that 13 of the 15 hydrogen atoms had been replaced with deuterium (except in position 3), the remaining two hydrogen presumably being derived from water. A SANS contrast variation study was conducted on whole cells and the results indicate the potential to discriminate inclusion bodies formed from deuterated precursor from an otherwise hydrogenated background. © 2004, Elsevier Ltd.
- ItemIn vivo deuteration strategies for neutron scattering analysis of bacterial polyhydroxyoctanoate(Springer Nature, 2008-05-15) Russell, RA; Holden, PJ; Wilde, KL; Garvey, CJ; Hammerton, KM; Foster, LJRThe cultivation of microorganisms on deuterated substrates has allowed us to control deuterium incorporation into biopolymer systems which is important for characterisation using neutron scattering techniques. Bacterial polyhydroxyoctanoate (PHO) is a polyester formed within inclusions inside bacterial cells and was deuterated in vivo under various conditions to characterise the formation of these inclusions by neutron scattering. Manipulation of deuterated media during microbial growth and PHO production phases resulted in polymer with partial or complete substitution of hydrogen by deuterium, as shown by gas chromatography. Sequential feeding of hydrogenated and deuterated forms of the same precursor was used to demonstrate that neutron scattering analysis could be used to differentiate between chemically similar phases in these polymer inclusions. © 2008 Crown Copyright
- ItemInvestigation of the phase morphology of bacterial PHA inclusion bodies by contrast variation SANS(Elsevier, 2006-11-15) Russell, RA; Holden, PJ; Garvey, CJ; Wilde, KL; Hammerton, KM; Foster, LJRUnder growth-limiting conditions, many bacteria are able to metabolise excess organic acids into polyhydroxyalkanoates (PHA) and store these polymers as intracellular inclusions until the return of favourable conditions. Various models have been proposed for the macromolecular organisation of the boundary layer Surrounding the polymer, and contrast-variation small-angle neutron scattering (SANS) was used to study its organisation. Inclusions formed by Pseudomonas oleovorans under hydrogenating conditions showed lowest scattering intensity at ca. 20% D2O. The inclusions consist of protein and membrane lipids in the boundary layer and polyhydroxyoctanoate (lipid) in the inclusion body. At 20% D2O the contributions of lipids were contrast matched with the solvent, indicating that lipids contributed the bulk of the scattering intensity observed at other D2O/H2O ratios. These results are inconsistent with a model of the boundary layer which proposed outer and inner layers of crystalline protein lattice sandwiching a membrane lipid membrane layer [E.S. Stuart, R.W. Lenz, R.C. Fuller, Can J Microbiol 41(Suppl 1) (1995) 84 93], and is more consistent with a model consisting of a lipid monolayer containing embedded proteins [U. Pieper-furst, M.H. Madkour, F. Mayer, A. Steinbuchel, J. Bacteriol. 176 (1994) 4328-4337.] By altering the H/D content of the precursors, we were able to collect SANS data from preparations of both deuterated and H/D copolymer inclusions, where initial PHA produced was hydrogenated followed by deuteration. Deuterated inclusions showed minimum intensity above 90% D2O/H2O whereas the sequentially produced copolymer (assumed to be in a core/shell arrangement) displayed minimum scattering some 20% lower, which is consistent with the increased hydrogenation of the boundary layer expected from its synthesis during supply of hydrogenated followed by deuterated precursors. © 2006, Elsevier Ltd
- ItemManipulation of polyhydroxybutyrate properties through blending with ethyl-cellulose for a composite biomaterial(Hindawi Publishing Corporation, 2011-05-30) Chan, RTH; Garvey, CJ; Marçal, H; Russell, RA; Holden, PJ; Foster, LJRPolyhydroxybutyrate (PHB) is widely used as a biomaterial in medical and tissue-engineering applications, a relatively high crystallinity limits its application. Blending PHB with ethyl-cellulose (EtC) was readily achieved to reduce PHB crystallinity and promote its degradation under physiological conditions without undue influence on biocompatibility. Material strength of composite films remained unchanged at 6.5 ± 0.6 MPa with 40% (w/w) EtC loadings. Phase separation between the two biopolymers was determined with PHB crystallinity decreasing from 63% to 47% for films with the same loading. This reduction in crystallinity supported an increase in the degradation rates of composite films from 0.39 to 0.81% wk−1 for PHB and its composite, respectively. No significant change in morphology and proliferation of olfactory ensheathing cells were observed with the composites despite significant increases in average surface roughness (Ra) of the films from 2.90 to 3.65 μm for PHB and blends with 80% (w/w) EtC, respectively. Copyright © 2011 Rodman T. H. Chan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
- ItemPoly(ethylene glycol)-modulated cellular biocompatibility of polyhydroxyalkanoate films(Wiley-Blackwell, 2013-06-01) Chan, RTH; Marçal, H; Ahmed, T; Russell, RA; Holden, PJ; Foster, LJRPolyhydroxybutyrate (PHB) and its copolymer with hydroxyvalerate, P(HB-co-HV), are widely used biomaterials. In this study, improvements of their biological properties of degradability and compatibility were achieved by blending with low-molecular-weight poly(ethylene glycol) (PEG106) approved for medical use. Surface morphology and chemistry are known to support cell attachment. Attachment and proliferation of neural olfactory ensheathing cells increased by 17.0 and 32.2% for PHB and P(HB-co-HV) composite films. Cell attachment was facilitated by increases in surface hydrophilicity, water contact angles decreased by 26 ± 2° and water uptake increased by 23.3% depending upon biopolymer and PEG loading. Cells maintained high viability (>95%) on the composite films with no evidence of cytotoxic effects. Assays of mitochondrial function and cell leakage showed improved cell health as a consequence of PEG loading. The PEG component was readily solubilised from composite films, allowing control of degradation profiles in the cell growth medium. Promotion of biopolymer compatibility and degradability was not at the expense of material properties, with the extension to break of the composites increasing by 5.83 ± 1.06%. Similarly, crystallinity decreased by 36%. The results show that blending of common polyhydroxyalkanoate biomaterials with low-molecular-weight PEG can be used to promote biocompatibility and manipulate physiochemical and material properties as well as degradation. © 2013, Wiley-Blackwell.
- ItemPolyhydroxyalkanoate-based natural–synthetic hybrid copolymer films: a small-angle neutron scattering study(Elsevier B. V., 2006-11-15) Foster, LJR; Knott, RB; Sanguanchaipaiwong, V; Holden, PJPolyhydroxyalkanoates have attracted attention as biodegradable alternatives to conventional thermoplastics and as biomaterials. Through modification of their biosynthesis using Pseudomonas oleovorans, we have manipulated the material properties of these biopolyesters and produced a natural–synthetic hybrid copolymer of polyhydroxyoctanoate-block-diethylene glycol (PHO-b-DEG). A mixture of PHO and PHO–DEG were solvent cast from analytical grade chloroform and analysed using small-angle neutron scattering. A scattering pattern, easily distinguished above the background, was displayed by the films with a diffraction ring at q∼0.12 Å−1. This narrow ring of intensity is suggestive of a highly ordered system. Analysis of the diffraction pattern supported this concept and showed a d-spacing of approximately 50 Å. In addition, conformation of the hybrid polymer chains can be manipulated to support their self-assembly into ordered microporous films. Copyright © 2006 Elsevier B.V.
- ItemPolyhydroxyalkanoate-based natural–synthetic hybrid copolymer films: a small-angle neutron scattering study(The Bragg Institute, Australian Nuclear Science and Technology Organisation, 2005-11-27) Foster, LJR; Knott, RB; Sanguanchaipaiwong, V; Holden, PJPHAs have attracted attention as biodegradable alternatives to conventional thermoplastics and as biomaterials. By modifying their bioprocessesing we have manipulated their PHA material properties and produced a a natural–synthetic hybrid copolymer of polyhydroxyoctanoate-block-diethylene glycol (PHO--DEG). While the molecular weight ratio of these hybrids is approximately 900:1, the polymer displays amphipillic properties, A mixture of PHO and PHO–DEG were solvent cast from analytical grade chloroform and analysed using small-angle neutron scattering. A scattering pattern, easily distinguished above the background, was displayed by the films with a diffraction ring at q∼0.12 Å−1. This narrow ring of intensity is suggestive of a highly ordered system. with d-spacing of approximately 50 Å. Once can speculate that the hydrophilic synthetic blocks of the polymer chains may group together when the polymer is dissolved in organic solvent. This speculative model is supported by the formation of microporous films by these hybrids when processed by sold solvent evaporation under a humid flow (92% rH). © The Authors
- ItemProduction and use of deuterated polyhydroxyoctanoate in structural studies of PHO inclusions(Elsevier, 2007-11-01) Russell, RA; Holden, PJ; Wilde, KL; Hammerton, KM; Foster, LJRThis work reports on the biosynthesis of polyhydroxyalkanoates with medium chain length alkyl substituents in the side chain by Pseudomonas oleovorans using hydrogenated and deuterated substrates. These investigations aimed to obtain polyhydroxyalkanoates with varying degrees of deuterium substitution, and establish whether they are suitable analogues for structural investigation. In order to understand the formation and structure of inclusions in their native state, whole inclusions were isolated from microbial cells and were analysed using Small Angle Neutron Scattering. A contrast variation study was conducted on hydrogenated and deuterated inclusions of polyhydroxyoctanoate, as well as inclusions resulting from co-feeding or sequentially feeding different precursors. The data indicated a core/shell structure resulting from feeding hydrogenated followed by perdeuterated PHO precursor, and demonstrated the utility of this analysis for characterising chemically similar systems. © 2007, Elsevier Ltd.
- ItemSmall angle neutron scattering study of the interface between ethylcellulose/polyhydroxybutyrate blends during annealing(Australian Institute of Physics, 2009-02-05) Garvey, CJ; Russell, RA; Garamus, VM; Boué, F; Foster, LJR; Holden, PJNot available
- ItemStructural evolution and stability of sol-gel biocatalysts(Elsevier, 2006-11-15) Rodgers, LE; Knott, RB; Holden, PJ; Pike, KJ; Hanna, JV; Foster, LJR; Bartlett, JRImmobilisation strategies for catalytic enzymes are important as they allow recovery and reuse of the biocatalysts. In this work, sol-gel matrices have been used to immobilise Candida antarctica lipase B (CALB), a commonly used industrial enzyme. The sol-gel bioencapsulate is produced through fluoride-catalysed hydrolysis of mixtures of tetramethylorthosilicate (TMOS) and methyltrimethoxysilane (MTMS) in the presence of CALB, yielding materials with controlled pore sizes and surface chemistries. Sol-gel matrices prolong the catalytic life and enhance the activity of CALB, although the molecular basis for this effect has yet to be elucidated due to the limitations of analytical techniques applied to date. Small angle neutron scattering (SANS) allows such multi-component systems to be characterised through contrast matching. In the sol-gel bioencapsulate system at the contrast match point for silica, residual scattering intensity is due to the CALB and density fluctuations in the matrix. A SANS contrast variation series found the match point for the silica matrix, both with and without enzyme present, to be around 35%. The model presented here proposes a mechanism for the interaction between CALB and the surrounding sol-gel matrix, and the observed improvement in enzyme activity and matrix strength. Essentially, the inclusion of CALB modulates silicate speciation during evolution of the inorganic network, leading to associated variations in SANS contrast. The SANS protocol developed here may be applied more generally to other encapsulated enzyme systems. © 2006, Elsevier Ltd.
- ItemStructural evolution and stability of sol-gel biocatalysts(The Bragg Institute, Australian Nuclear Science and Technology Organisation, 2005-11-27) Rodgers, LE; Holden, PJ; Knott, RB; Foster, LJR; Bartlett, JRImmobilisation strategies for catalytic enzymes are important as they allow reuse of the biocatalysts. Sol-gel materials have been used to immobilise Candida antarctica lipase B (CALB), a commonly used industrial enzyme with a known crystal structure. The sol-gel bioencapsulate is produced through the condensation of suitable metal alkoxides in the presence of CALB, yielding materials with controlled pore sizes, volume and surface chemistry. Sol-gel matrices have been shown to prolong the catalytic life and enhance the activity of CALB, although the molecular basis for this effect has yet to be elucidated due to the limitations of analysis techniques applied to date. Small angle neutron scattering (SANS) allows such multicomponent systems to be characterised through contrast matching. In the sol-gel bioencapsulate system, at the contrast match point for silica, residual scattering intensity is due to the CALB and density fluctuations in the matrix. A SANS contrast variation series found the match point for the silica matrix, both with and without enzyme present, to be around 35 percent. The model presented here proposes a mechanism for the interaction between CALB and the surrounding sol-gel matrix, and the observed improvement in enzyme activity and matrix strength. The SANS protocol developed here may be applied more generally to bioencapsulates. © The Authors
- ItemUsing humidity to control the morphology and properties of electrospun bioPEGylated polyhydroxybutyrate scaffolds(American Chemical Society (ACS), 2020-10-05) Foster, LJR; Chan, RTH; Russell, RA; Holden, PJElectrospinning produces nanofibrous scaffolds with potential for tissue engineering and wound repair. Spinning parameters control scaffold morphology and properties. BioPEGylation of polyhydroxybutyrate (PHB) introduces terminal hydrophilic groups into the hydrophobic chain, making this natural-synthetic hybrid copolymer more susceptible to humidity. Varying the humidity from 10 to 50% RH during electrospinning had a relatively little effect on polyhydroxybutyrate (PHB) average fiber and pore diameters, which remained around 3.0 and 8.7 μm, respectively. In contrast, fiber and pore diameters for electrospun bioPEGylated PHB scaffolds varied significantly with humidity, peaking at 30% RH (5.5 and 14.1 μm, respectively). While scaffolds showed little change, hydrophobicity decreased linearly with humidity during electrospinning. Compared to solvent-cast films, electrospun scaffolds showed significantly greater average cell spread. A 108% increase for olfactory ensheathing cells (OECs) cultivated on bioPEGylated PHB scaffolds was proportionally greater than their counterparts on electrospun PHB scaffolds, (70%). OECS grown on bioPEGylated PHB scaffolds were over twice the size, 260 ± 20 μm diameter, than those on PHB electrospun scaffolds, 110 ± 18 μm diameter. Electrospun scaffolds also promoted cell health compared to their solvent-cast counterparts, with increases in the mitochondrial activity of 165 ± 13 and 196 ± 13% for PHB and bioPEGylated PHB, respectively. OECS cultivated on electrospun scaffolds of bioPEGylated PHB had significantly better membrane integrities compared to their counterparts on solvent-cast films, 47 ± 5% reducing to 17 ± 6%. The combination of bioPEGylation and humidity during electrospinning permitted significant controllable changes to scaffold morphology and properties. These changes resulted in the significantly greater promotion of cell growth on electrospun bioPEGylated PHB scaffolds compared to their solvent-cast counterparts and electrospun PHB. © 2020 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.