3D hydrogel/ bioactive glass scaffolds in bone tissue engineering: Status and future opportunities.

Repairing significant bone defects remains a critical challenge, raising the clinical demand to design novel bone biomaterials that incorporate osteogenic and angiogenic properties to support the regeneration of vascularized bone. Bioactive glass scaffolds can stimulate angiogenesis and osteogenesis. In addition, natural or synthetic polymers exhibit structural similarity with extracellular matrix (ECM) components and have superior biocompatibility and biodegradability. Thus, there is a need to prepare composite scaffolds of hydrogels for vascularized bone, which incorporate to improve the mechanical properties and bioactivity of natural polymers. In addition, those composites' 3-dimensional (3D) form offer regenerative benefits such as direct doping of the scaffold with ions. This review presents a comprehensive discussion of composite scaffolds incorporated with BaG, focusing on their effects on osteo-inductivity and angiogenic properties. Moreover, the adaptation of the ion-doped hydrogel composite scaffold into a 3D scaffold for the generation of vascularized bone tissue is exposed. Finally, we highlight the challenges and future of manufacturing such biomaterials.

Effects of temperature and solvent condition on phase separation induced molecular fractionation of gum arabic/hyaluronan aqueous mixtures.

Effects of temperature and solvent condition on phase separation-induced molecular fractionation of gum arabic/hyaluronan (GA/HA) mixed solutions were investigated. Two gum arabic samples (EM10 and STD) with different molecular weights and polydispersity indices were used. Phase diagrams, including cloud and binodal curves, were established by visual observation and GPC-RI methods. The molecular parameters of control and fractionated GA, from upper and bottom phases, were measured by GPC-MALLS. Fractionation of GA increased the content of arabinogalactan-protein complex (AGP) from ca. 11% to 18% in STD/HA system and 28% to 55% in EM10/HA system. The phase separation-induced molecular fractionation was further studied as a function of temperature and solvent condition (varying ionic strength and ethanol content). Increasing salt concentration (from 0.5 to 5 mol/L) greatly reduced the extent of phase separation-induced fractionation. This effect may be ascribed to changes in the degree of ionization and shielding of the acid groups. Increasing temperature (from 4 °C to 80 °C) also exerted a significant influence on phase separation-induced fractionation. The best temperature for GA/HA mixture system was 40 °C while higher temperature negatively affected the fractionation due to denaturation and possibly degradation in mixed solutions. Increasing the ethanol content up to 30% showed almost no effect on the phase separation induced fractionation.

Visualisation of xanthan conformation by atomic force microscopy.

Direct visual evidence obtained by atomic force microscopy demonstrates that when xanthan is adsorbed from aqueous solution onto the heterogeneously charged substrate mica, its helical conformation is distorted. Following adsorption it requires annealing for several hours to restore its ordered helical state. Once the helix state reforms, the AFM images obtained showed clear resolution of the periodicity with a value of 4.7nm consistent with the previously predicted models. In addition, the images also reveal evidence that the helix is formed by a double strand, a clarification of an ambiguity of the xanthan ultrastructure that has been outstanding for many years.

Efficiencies of fragmentation of glycosaminoglycan chloramides of the extracellular matrix by oxidizing and reducing radicals: potential site-specific targets in inflammation?

Hypochlorous acid and its conjugate base, hypochlorite ions, produced under inflammatory conditions, may produce chloramides of glycosaminoglycans, these being significant components of the extracellular matrix (ECM). This may occur through the binding of myeloperoxidase directly to the glycosaminoglycans. The N-Cl group in the chloramides is a potential selective target for both reducing and oxidizing radicals, leading possibly to more efficient and damaging fragmentation of these biopolymers relative to the parent glycosaminoglycans. To investigate the effect of the N-Cl group, we used ionizing radiation to produce quantifiable concentrations of the reducing radicals, hydrated electron and superoxide radical, and also of the oxidizing radicals, hydroxyl, carbonate, and nitrogen dioxide, all of which were reacted with hyaluronan and heparin and their chloramides in this study. PAGE gels calibrated for molecular weight allowed the consequent fragmentation efficiencies of these radicals to be calculated. Hydrated electrons were shown to produce fragmentation efficiencies of 100 and 25% for hyaluronan chloramide (HACl) and heparin chloramide (HepCl), respectively. The role of the sulfate group in heparin in the reduction of fragmentation can be rationalized using mechanisms proposed by M.D. Rees et al. (J. Am. Chem. Soc.125:13719-13733; 2003), in which the initial formation of an amidyl radical leads rapidly to a C-2 radical on the glucosamine moiety. This is 100% efficient at causing glycosidic bond breakage in HACl but only 25% efficient in HepCl, the role of the sulfate group being to favor the nonfragmentary routes for the C-2 radical. The weaker reducing agent, the superoxide radical, did not cause fragmentation of either HACl or HepCl although kinetic reactivity had been demonstrated in earlier studies. Experiments using the oxidizing radicals, hydroxyl and carbonate, both potential in vivo species, showed significant increases in fragmentation efficiencies for both HACl and HepCl, relative to the parent molecules. The carbonate radical was shown to be involved in site-specific reactions at the N-Cl groups, reacting via abstraction of Cl, to produce the same amidyl radical produced by one-electron reductants such as the hydrated electron. As for the hydrated electrons, the data support fragmentation efficiencies of 100 and 29% for reaction of carbonate radicals at N-Cl for HACl and HepCl, respectively. For the weaker oxidant, nitrogen dioxide, no fragmentation was observed, probably because of a low kinetic reactivity and low reduction potential. It seems likely therefore that the N-Cl group can direct damage to extracellular matrix glycosaminoglycan chloramides, which may be produced under inflammatory conditions. The in vivo species, the carbonate radical, is also much more likely to be site-specific in its reactions with such components of the ECM than the hydroxyl radical.

Characterisation of commercial LM-pectin in aqueous solution.

Fourteen commercial LM-pectin samples were investigated in this study. The degree of esterification (DE) varied from 9.6% to 42.0% and the degree of amidation (DA) from 0% to 25%. Chemical and structural characteristics were examined using atomic absorption (AA), dilute solution capillary viscometry, GPC-MALLS and dynamic light scattering. The intrinsic calcium was in the range of 47-1388 ppm, the intrinsic viscosity varied from 2.9 to 4.9 (dL/g) and the weight average molecular weight (M(w)) from 113 to 290 (kDa). Most of the samples had Huggins constants of ~0.5. However, for samples having acidic pH, Huggins constant values greater than 1, which can be as an indication of aggregation, were obtained. The high Huggins constant value could be reduced to ~0.5 by the addition of 3M urea, indicating that the aggregation was stabilised by hydrogen bonding. Shear flow viscosity revealed three types of rheological behaviour. Type A showed pronounced shear thinning behaviour, which was reduced by the addition of hydrogen bonding breaking agent urea. Type B with intrinsic Ca of 1mM and pH ~4 showed two shear thinning regions, with significantly enhanced shear viscosity upon addition of calcium. Type C showed the least aggregation due to its pH ~4 and low intrinsic Ca, but could be converted to type B upon addition of Ca. The effect of Ca on the rheological behaviour of types B and C was further confirmed by CaCl(2) and Ca-chelating agent (EDTA). Temperature affected the molecular conformation of all types and most significantly type A by eliminating the hydrogen bonding.

Improved sugar beet pectin-stabilized emulsions through complexation with sodium caseinate.

The study investigates the complexes formed between sodium caseinate (SC) and sugar beet pectin (SBP) and to harness them to stabilize SBP emulsions. We find that both hydrophobic and electrostatic interactions are involved in the complexation. In SC/SBP mixed solution, soluble SC/SBP complexes first form on acidification and then aggregate into insoluble complexes, which disassociate into soluble polymers upon further decreasing pH. The critical pH's for the formation of soluble and insoluble complexes and disappearance of insoluble complexes are designated as pH(c), pH(φ), and pH(d), respectively. These critical pH values define four regions in the phase diagram of complexation, and SC/SBP emulsions were prepared in these regions. The results show that the stability of SBP-stabilized emulsion is greatly improved at low SC/SBP ratios and acidic pH's. This enhancement can be attributed to an increase in the amount of adsorbed SBP as a result of cooperative adsorption to sodium caseinate. Using a low ratio of SC/SBP ensured that all caseinate molecules are completely covered by adsorbed SBP chains, which eliminates possible instability induced by thermal aggregation of caseinate molecules resulting from stress acceleration at elevated temperatures. A mechanistic model for the behavior is proposed.

Competitive adsorption between sugar beet pectin (SBP) and hydroxypropyl methylcellulose (HPMC) at the oil/water interface.

The emulsification performance, stability and competitive adsorption of two natural food emulsifiers, sugar beet pectin (SBP) and hydroxypropyl methylcellulose (HPMC) have been investigated. Both can reduce the surface tension and emulsify oil in water. However, due to their different structure and conformation they operate via different mechanisms. Using 15% middle chain triglycerides (MCTs) oil, the amounts of SBP and HPMC adsorbed in emulsions made with these individually and in mixtures were determined. The interfacial concentration (Γ) for SBP stabilized emulsion was ∼1.25mg/m(2) and for HPMC 3.5mg/m(2). The higher adsorption of HPMC was due to multilayer adsorption, whereas SBP adsorbed as a monolayer. Competitive adsorption between SBP and HPMC was also investigated. When the HPMC concentration approached that of adsorbed SBP, the effect of HPMC became dominant and at 1.5wt.% controlled the behavior of the mixed emulsions, which were then almost independent of SBP. The minor role of SBP was mainly to decrease the proportion of large droplets in the emulsion. A model to describe the competitive adsorption between SBP and HPMC is proposed.

Complexation of bovine serum albumin and sugar beet pectin: stabilising oil-in-water emulsions.

In a previous study (Langmuir 28 (2012) 10164-10176.), we investigated the complexation of bovine serum albumin (BSA) with sugar beet pectin (SBP). A pH-composition phase diagram was established and structural transitions in relation to the phase diagram during complexation were identified. The present study examines the implications of these interactions on the emulsifying performance of BSA/SBP mixtures. Middle-chain triglycerides (MCTs) in water emulsions were prepared using conditions corresponding to different regions of the phase diagram. At high pHs and in the stable region of mixed individual soluble polymers where complexation is absent, there is no improved emulsifying performance, compared with the individual protein and polysaccharide. For these mixtures, the emulsion characteristics are controlled by the major component in the solutions, as determined by the competitive adsorption of the two components at the oil-water interface. At low pHs and low BSA/SBP ratios, and so mainly within the stable region of intramolecular soluble complexes, BSA/SBP mixtures greatly improve the stability of emulsions. Here, stabilisation is controlled by the cooperative adsorption of the two components at the oil-water interface. Through electrostatic complexation BSA promotes the adsorption of SBP on to interfaces to form a thick steric layer around emulsion droplets and thus providing better stability. At low pHs and high BSA/SBP ratios, that is, mainly within the unstable region of intermolecular insoluble complexes, emulsions prepared are extremely unstable due to bridging flocculation between emulsion droplets.

Complexation of bovine serum albumin and sugar beet pectin: structural transitions and phase diagram.

The complexation between bovine serum albumin (BSA) and sugar beet pectin (SBP) was studied in situ by coupling glucono-δ-lactone (GDL) induced acidification with dynamic light scattering and turbidity measurements. Individual measurements at specific pHs and mixing ratios were also carried out using zeta potentiometry, gel permeation chromatography-multiangle laser light scattering (GPC-MALLS), and isothermal titration calorimetry (ITC). These investigations together enabled the establishment of a phase diagram of BSA/SBP and the identification of the molecular events during protein/polysaccharide complexation in relation to the phase diagram, which showed five regions: (I) a stable region of mixed individual soluble polymers, (II) a stable region of intramolecular soluble complexes, (III) a quasi-stable region of intermolecular soluble complexes, (IV) an unstable region of intermolecular insoluble complexes, and (V) a second stable region of mixed individual soluble polymers, on lowering pH. We found for the first time that the complexation could take place well above the critical pH(c), the value that most previous studies had regarded as the onset occurrence of complexation. A model of structural transitions between the regions was proposed. The borderline between region II and region III represents the BSA/SBP stoichiometry for intramolecular soluble complex at a specific pH, while that between region III and region IV identifies the composition of the intermolecular insoluble complex. Also studied was the effect of NaCl and CaCl(2) on the phase diagram and structural transitions.

Interaction of gum arabic with fatty acid studied using electron paramagnetic resonance.

Electron paramagnetic resonance (EPR) is here used to study the interaction between gum arabic and a fatty acid. The EPR spectra of 5-doxyl stearic acid (5-DSA), a spin-labeled fatty acid analog, displayed increasingly anisotropic line features upon addition of gum arabic, indicating a strong immobilization of the nitroxyl moiety when the fatty acid is bound to gum arabic. To understand the nature of the interaction, EPR measurements were carried out at different pHs and using two fractions of gum arabic separated by hydrophobic interaction chromatography (HIC). 5-DSA bound favorably to the hydrophobic fraction, which contains mainly glycoprotein, and a small amount of high molecular weight arabinogalactan protein (AGP). Binding occurred to a less extent to the hydrophilic fraction, which contains essentially arabinogalactan (AG). Such a hydrophobic binding mechanism is further supported by a sharp drop in the binding when pH is raised above the pK(a) value of 5-DSA (approximately pH 5). This is because the ionization of carboxylic groups would lead to increased polarity and hydrophilicity of the fatty acid. A secondary effect involving the formation of ionic hydrogen bonds between carboxylic groups in fatty acid and lysine residues in gum arabic might also contribute. This is consistent with the reduction in binding ability when the pH was elevated above the pK(a) value of lysine residue (approximately pH 10). The biological significance of these findings is considered.

Molecular structures of gellan gum imaged with atomic force microscopy in relation to the rheological behavior in aqueous systems in the presence or absence of various cations.

Aqueous solutions of gellan gum with comparable molecular masses but with different acyl contents were investigated by atomic force microscopy and rheological measurements in the presence or absence of various cations. For a high-acyl sample, no continuous network structures were identified microscopically, except in the presence of Ca (2+), where structural inhomogeneity was the highest in terms of the height distribution of molecular assemblies. Rheological thermal hysteresis between sol-gel transitions was detected in the presence of K (+) and Ca (2+), particularly Ca (2+). The storage modulus at 20 degrees C was larger in the order Na (+) < Ca (2+) < K (+). For a low-acyl sample, continuous network structures were identified in the presence of each cation, involving greater thermal hysteresis than the corresponding data for the high-acyl sample. Structural homogeneity was the highest in the presence of K (+). Thermal hysteresis and elasticity of the system were discussed in terms of continuousness and homogeneity of network structures.

Origin and thermodynamic properties of the instability of synthetic azo colorants in gum arabic solutions.

The instability of some industrially important synthetic azo colorants, including sunset yellow, azorubine, and allura red, toward gum arabic in aqueous solution has been a long-standing problem for the beverage and confectionery industries. Precipitation of these colorants causes the deterioration of product appearance and properties. This work examines the origin and nature of the problem by analysis of the precipitate and thermodynamic studies of gum arabic-colorant interactions using isothermal titration calorimetry (ITC). The presence of divalent alkaline earth metals in gum arabic samples, that is, calcium and magnesium, is shown to be responsible for the precipitation of the azo colorants. There is no direct interaction between gum arabic and the colorant molecules, and the precipitate is formed likely due to the mediation/bridging by the divalent cations. The thermodynamic knowledge gained from the ITC studies, for example, binding affinity, stoichiometry, and enthalpy, enables interpretation of many industrial observations.

Multiple steps and critical behaviors of the binding of calcium to alginate.

Previous research on the binding and gelation of calcium/alginate in aqueous solution were mostly conducted in the (semi-)concentrated regime, and it did not provide details of the binding process and the formation of egg-box junctions. In the present investigation, the binding of calcium to alginate, of low and high molecular weight and different guluronate/mannuronate ratios, was investigated in dilute solutions using isothermal titration calorimetry (ITC), Ca2+-selective potentiometry, and viscometry techniques. The results reveal three distinct and successive steps in the binding of calcium to alginate with increased concentration of Ca ions. They were assigned to (i) interaction of Ca2+ with a single guluronate unit forming monocomplexes; (ii) propagation and formation of egg-box dimers via pairing of these monocomplexes; and (iii) lateral association of the egg-box dimers, generating multimers. The third step has different association modes depending on the molecular weight of alginate. The boundaries between these steps are reasonably critical, and they closely correlate with the Ca/guluronate stoichiometry expected for egg-box dimers and multimers with 2/1 helical chains. The formation of egg-box dimers and their subsequent association are thermodynamically equivalent processes and can be fitted by a model of independent binding sites. The binding of Ca to alginates of different guluronate contents is controlled by a balance between enthalpy and entropy.

Hyaluronan and proximal tubular cell migration.

BACKGROUND: The ubiquitous polysaccharide hyaluronan has been associated with both acute renal injury and progressive renal disease. The aim of this study was to examine the effect of hyaluronan on proximal tubular cell migration. METHODS: The proximal tubular cell line, HK-2 cells, were grown in monolayer culture, and cell migration following addition of hyaluronan characterized in an in vitro model of injury that we have previously developed and characterized. RESULTS: Addition of well-defined preparations of exogenous hyaluronan increased cell migration; however, optimum enhancement of migration was seen with hyaluronan of high molecular weight. Activation of the mitogen-activated protein kinase (MAPK) signaling cascade, as assessed by increased expression of the dually phosphorylated active form of MAPK, could be demonstrated following addition of hyaluronan. This was blocked by the addition of a specific antibody to the hyaluronan receptor, CD44. Hyaluronan-dependent enhanced migration was also abrogated by addition the CD44 blocking antibody, and by inhibition of MAPK kinase (MEK) activity. Generation of a denuded area also led to increased synthesis of endogenous hyaluronan and activation of MAPK, and blockage of either CD44 or MAPK activation inhibited proximal tubule cell (PTC) migration and re-epithelialization under nonstimulated conditions. CONCLUSION: We have demonstrated that hyaluronan activation of the MAPK pathway through binding to its receptor CD44, enhances proximal tubule cell (PTC) migration. In addition, the results suggest that mechanical injury of PTC stimulated hyaluronan generation. These observations may have implications for both recovery from acute tubular injury and progressive renal fibrosis.

Molecular weight, tertiary structure, water binding and colon behaviour of ispaghula husk fibre.

Molecular variables, using aqueous and alkaline extracts, of the polysaccharide from ispaghula husk (IH) were examined using gel-permeation chromatography linked to multi-angle laser light scattering. Progressive extraction can yield a component with a molecular weight (MW)value up to about 7 x 106 Da, and gels, which accompany the extraction, have MW ranging from 10-20 x 106 Da. To mimic the polysaccharide degradation, particularly in the colon, the solid IH was degraded progressively using ionising radiation. A chain break occurs every 7.5 kGy in NaOH and every 15 kGy in water. The solid-state matrix is opened by the radiation to yield increased visco-elasticity of the aqueous extracts at critical radiation doses, before further degradation occurs after about 12 kGy. Differential scanning calorimetry is used to study the mechanism of interaction of water with IH. The first water to be taken up is non-freezing water and represents about twelve water molecules/disaccharide unit of the polysaccharide. As the water content is increased, the water becomes bound to the polysaccharide and freezes and melts at a temperature different from free water. This water is thermodynamically distinguishable from free water. It forms amorphous ice on cooling which crystallises exothermically and subsequently melts endothermically. Saturation occurs at a water content of 2-3 g water/g polymer, showing that about 60% of the water in the system is 'bound'. The most surprising conclusion is that despite the fact that the IH swells in water to form a solid and stiff gel, the greater part of that water in the gel is still free and behaves like liquid water.

Asymmetrical-flow field-flow fractionation with on-line multiangle light scattering detection. 1. Application to wormlike chain analysis of weakly stiff polymer chains.

Four samples of hyaluronan in the sodium form, ranging in weight-average molecular weight, M(w), from 6.67 x 10(5) to 4.23 x 10(6) were investigated by asymmetrical-flow field-flow fractionation coupled to multiangle light scattering (FlFFF-MALS) in 0.2 M aqueous NaCl at 25 degrees C. M(w) and z-average radii of gyration, R(G)(z)(), obtained via FlFFF-MALS showed a good agreement with the results obtained by conventional static light scattering. Furthermore, the molecular weight dependence of the radius of gyration for sodium hyaluronan obtained via FlFFF-MALS was analyzed on the basis of the Kratky-Porod model for unperturbed wormlike chains combined with the Yamakawa theory for radius expansion factor, and a sufficiently good agreement was observed between the theoretical prediction and experimental data. These results show the potential usage of FlFFF-MALS regarding size separation and molecular characterization even for weakly stiff chains.