Anne M. Young

FTIR investigation of monomer polymerisation and polyacid neutralisation kinetics and mechanisms in various aesthetic dental restorative materials

Diamond ATR FTIR has been used to quantify light catalysed polymerisation and polyacid neutralisation rates in various glass ionomer cements (GIC), resin-modified GICs (RMGIC) and compomers. At 150s after the start of light exposure, levels of methacrylate polymerisation on the lower surfaces of 1mm thick specimens were 97% and 98% for the RMGIC, Vitremer and Fuji II LC and 47% and 37% for the compomers, Compoglass and Dyract. After light exposure, polymerisation rates for the compomers decreased linearly with inverse time. By 50,000s Compoglass and Dyract were 62% and 51% polymerised. Initial rate of polyacid neutralisation in the GIC Shofu HIFI was 0.32 times that of Fuji IX GIC. Those in Vitremer, Fuji II LC, Compoglass and Dyract were 0.16, 0.09, 0.004 and 0.004 times that of Fuji IX. Excluding short initial periods, log of neutralisation rates decreased linearly with log-time. Average gradients were -1.35 for the GIC, -0.80 for the RMGIC and -0.59 for the compomers. By 50,000s, polyacid salt concentrations for the RMGIC and compomers were 0.41 and 0.016 times that of the GIC. Reaction mechanisms have been discussed and used to help interpret material mechanical properties, fluoride release rates and adhesion to tooth structure.

Dense collagen matrix accelerates osteogenic differentiation and rescues the apoptotic response to MMP inhibition

Bone is distinguished from other tissues by its mechanical properties, in particular stiffness. However, we know little of how osteoblasts react to the stiffness of their microenvironment; in this study we describe their response to a dense (>10 wt.%) collagenous 3D environment. Primary pre-osteoblasts were seeded within a novel form of native collagen, dense collagen, and cultured for up to 14 days in the presence and absence of osteogenic supplements: analysis was via Q-PCR, histology, fluorescent in situ zymography, MMP loss-of-function and tensile testing. Differentiation as measured through the up-regulation of Bsp (247-fold), Alp (14.2-fold), Col1A1 (4.5-fold), Mmp-13 (8.0-fold) and Runx2 (1.2-fold) transcripts was greatly accelerated compared to 2D plastic at 7 and 14 days in the same medium. The scale of this enhancement was confirmed through the use of growth factor stimulation on 2D via the addition of BMP-6 and the Hedgehog agonist purmorphamine. In concert, these molecules were capable of the same level of osteo-induction (measured by Bsp and Alp expression) as the dense collagen alone. Mineralisation was initially localised to remodelled pericellular regions, but by 14 days embedded cells were discernible within regions of apatite (confirmed by MicroRaman). Tensile testing of the matrices showed that this had resulted in a significant increase in Youngs modulus at low strain values, consistent with a stiffening of the matrix. To determine the need for matrix remodelling in the mineralisation event the broad spectrum MMP Inhibitor Ilomastat was used. It was found that in its presence mineralisation could still occur (though serum-specific) and the apoptosis associated with MMP inhibition in hydrated collagen gels was abrogated. Analysis of gene expression indicated that this was due to the up-regulation of Mmp-13 in the presence of Ilomastat in dense collagen (400-fold), demonstrating a powerful feedback loop and a potential mechanism for the rescue from apoptosis. Osteoid-like matrix (dense collagen) is therefore a potent stimulant of osteoblast differentiation in vitro and provides an environment that enables survival and differentiation in the presence of MMP inhibition.

Use of Raman spectroscopy in the characterisation of the acid}base reaction in glass-ionomer cements

Raman spectra of various combinations of glass-ionomer cement components have been compared with those of the reactants and the salts of polyacrylic and tartaric acids. The components consisted of a fast-setting acid-degradable dental glass (containing, inter alia, oxides of Si, Al, Ca, Ba and Na), polyacrylic acid (PAA) and/or tartaric acid (TA). On the addition of water to the glass and tartaric acid, Raman spectroscopy indicated loss of acid and production of tartrate salts within seconds of mixing. Mixtures containing the glass, PAA and water in mass ratios 2:1:(0.1-4) reacted to form polyacrylate salts. The maximum fraction of unreacted PAA was found to decrease linearly with initial water/PAA mass ratio to a minimum of approximately 20% when this ratio exceeds 1.5. The data are consistent with 5.6 moles of water being required when each mole of acidic groups is neutralised. In newly prepared cements containing glass, water, polyacrylic and tartaric acids, polyacrylic acid and its salts, in both ionised and solid state form, can be detected. After about 1 h, however, Raman peaks associated with ionised species disappear.

FTIR-monitoring of a fast setting brushite bone cement: effect of intermediate phases

The setting reaction of an equimolar β-tricalcium phosphate/monocalcium phosphate monohydrate (β-TCP/MCPM) cement was monitored in real time with ATR-FTIR at 23 and 37 °C using powder to liquid ratios (PLRs) of 2.0 and 3.3 g ml−1 and aqueous retardant citric acid concentrations of 800, 1000 and 1500 mM. The final set products, for PLRs of 2.0 to 3.3 g ml−1 and citric acid concentrations of 300 to 1500 mM, were characterised with regard to phase composition, compressive strength, density and relative porosity. FTIR provided evidence for the formation of an intermediate dicalcium phosphate–citrate complex (DCPC). As the concentration of citric acid in solution increased so did the maximum level of citrate intermediate. Decreasing the PLR reduced the rate of citrate removal, but had no effect on its rate of formation or maximum level. FTIR also indicated a time delay before formation of any observable dicalcium phosphate (DCP) in solution. This delay increased as the citric acid solution concentration was raised or the temperature reduced, but was less affected by the PLR. There was then an additional delay between DCP formation in solution and its precipitation. Both Rietveld analysis of XRD patterns and density measurements 24 h after setting confirmed that the final product was primarily dicalcium phosphate dihydrate (DCPD or brushite) when the citric acid concentration was less than 1000 mM, irrespective of temperature or PLR. On the other hand, with 1500 mM citric acid significant levels of dicalcium phosphate anhydrous (DCPA or monetite) also present, this led to increased porosity and a dramatic decline in strength. As the levels of the intermediate phase increased, the final wet compressive strength of the resulting cements also deteriorated. It is therefore proposed that strength reduction may be due to formation of the intermediate at early stages of setting or DCPA formation in the final product, both causing increased material inhomogeneity. This study thereby illustrates that real time ATR-FTIR monitoring of a setting reaction clearly indicates that there is an upper limit to the use of citric acid as a setting retardant for a fast setting brushite-forming cement system, a limit that can also be expected for the use of other setting retardants, and that ATR-FTIR monitoring comprises a useful complement to the traditional before–after investigations.

Temperature dependent setting kinetics and mechanical properties of β-TCP–pyrophosphoric acid bone cement

Brushite (CaHPO4·2H2O) is the product of acidic calcium phosphate cement forming reactions. It has a higher solubility than hydroxyapatite under physiological conditions and is a promising resorbable bone cement candidate. It reacts extremely fast so dilute mixes are required to form cements having setting times with surgical utility, which in turn compromises strength and produces a highly porous material. Pyrophosphate ions have a strong affinity for calcium orthophosphate surfaces, can inhibit their mineralisation and are thought to hydrolyze, forming orthophosphate during bone formation. The effect of replacing the acid orthophosphate component of a brushite cement with pyrophosphoric acid on the cement setting reaction time at temperatures of between 24 °C and 70 °C was determined. The substitution of pyrophosphate for orthophosphate retarded setting and improved mechanical performance of the cement. Pyrophosphate did not inhibit the extent of reaction but did influence microstructure of the brushite crystals. Temperature was found to have a significant (p < 0.01) influence on mechanical performance, and this was attributed to the formation of monetite (CaHPO4) rather than brushite at temperatures of ≥55 °C.

Premature degradation of poly(α-hydroxyesters) during thermal processing of Bioglass®-containing composites

Bioactive, biodegradable composites are increasingly being explored as bone replacement materials and as scaffolds for tissue engineering. Their properties are not only dependent on the properties of the filler and matrix, but are also determined by their interaction. This study investigated the effect on poly(D,L-lactide) (PDLLA) matrix when processed at high-temperatures in the presence of Bioglass particulate filler. Composites with different filler contents were compounded at elevated temperatures by co-extrusion followed by compression moulding and compared with composites of similar composition prepared by thermally induced phase separation (TIPS), a low-temperature processing route. It was found that the inclusion of Bioglass in PDLLA under elevated temperatures resulted in the degradation of the matrix, leading to a reduction in the mechanical properties of the composites and in the molecular weight of the matrix. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy showed the presence of a peak at 1600 cm(-1) in the composite material, particularly when processed at elevated temperatures, whereas no peak at this wavelength was discernible for the pure PDLLA. Furthermore, time-based ATR-FTIR spectra taken at elevated temperatures on the TIPS-processed composites showed an increase in the intensity of the peak at 1600 cm(-1) and a concomitant reduction of the CO stretch peak at 1745 cm(-1) with time. This suggested the formation of a carboxylate salt by-products as a consequence of a reaction at the interface between the Bioglass filler and the PDLLA matrix. Therefore, the results confirmed that this degradation was not solely due to shear effects during the extrusion process. This work thereby supports the assertion that, in the presence of Bioglass filler particles, poly(alpha-hydroxyester)-based composites should not be processed at elevated temperatures.

Characterization of chlorhexidine-releasing, fast-setting, brushite bone cements

The effect of antibacterial chlorhexidine diacetate powder (CHX) on the setting kinetics of a brushite-forming beta-tricalcium phosphate/monocalcium phosphate monohydrate (beta-TCP/MCPM) cement was monitored using attenuated total reflection Fourier transform infrared spectroscopy. The final composition of the set cement with up to 12 wt.% CHX content before and after submersion in water for 24h, the kinetics of chlorhexidine release and the total sample mass change in water over four weeks was monitored using Raman mapping, UV spectroscopy and gravimetry, respectively. Below 9 wt.%, CHX content had no significant effect on brushite formation rate at 37 degrees C, but at 12 wt.% the half-life of the reaction decreased by one-third. Raman mapping confirmed that brushite was the main inorganic component of the set cements irrespective of CHX content, both before and after submersion in water. The CHX could be detected largely as discrete solid particles but could also be observed partially dispersed throughout the pores of the set cement. The percentage of CHX release was found to follow Ficks law of diffusion, being independent of its initial concentration, proportional to the square root of time and, with 1mm thick specimens, 60% was released at 24h. Total set cement mass loss rate was not significantly affected by CHX content. On average, cements exhibited a loss of 7 wt.% assigned largely to surface phosphate particle loss within the initial 8h followed by 0.36 wt.% per day.

Development of remineralizing, antibacterial dental materials

Light curable methacrylate dental monomers containing reactive calcium phosphate filler (monocalcium phosphate monohydrate (MCPM) with particle diameter of 29 or 90microm) and beta-tricalcium phosphate (beta-TCP) at 1:1 weight ratio in a powder:liquid ratio (PLR) of 1:1 or 3:1 and chlorhexidine diacetate (0 or 5 wt.%), were investigated. Upon light exposure, approximately 90% monomer conversion was gained irrespective of the formulation. Increasing the PLR promoted water sorption by the set material, induced expansion and enhanced calcium, phosphate and chlorhexidine release. Concomitantly, a decline in compressive and biaxial flexural strengths occurred. With a reduction in MCPM particle diameter, however, calcium and phosphate release was reduced and less deterioration in strength observed. After 24h, the remaining MCPM had reacted with water and beta-TCP, forming, within the set materials, brushite of lower solubility. This provided a novel means to control water sorption, component release and strength properties. Measurable chlorhexidine release was observed for 6weeks. Both diffusion rate and total percentage of chlorhexidine release decreased with lowering PLR or by adding buffer to the storage solutions. Higher chlorhexidine release was associated with reduced bacterial growth on agar plates and in a biofilm fermenter. In cell growth media, brushite and hydroxyapatite crystals precipitated on the composite material surfaces. Cells spread on both these crystals and the exposed polymer composite surfaces, indicating their cell compatibility. These formulations could be suitable antibacterial, biocompatible and remineralizing dental adhesives/liners.

FTIR investigation of polymerisation and polyacid neutralisation kinetics in resin-modified glass-ionomer dental cements

A new diamond ATR FTIR method has been developed to quantify the processes occurring in the resin-modified glass-ionomer cement (RMGIC). Fuji II LC (Improved), at 1 mm depth from the cement/water interface. With Fuji II LC (Improved) various changes in the spectra due to 90% monomer polymerisation were observed within 1 min after 20 s exposure to a dental light. Following polymerisation further different peak shifts with time were detected. Comparison with spectral changes seen during setting of the conventional glass-ionomer cement, Fuji IX, showed these could be assigned to water sorption and/or polyacid neutralisation. Any absorbance change due to the acid/glass reaction alone exhibited 2 linear regions when plotted against square root of time. Such behaviour suggests two separate diffusion mechanisms for acid neutralisation. The first faster one ceases at 30 or 150 min after mixing in Fuji IX and Fuji II LC (Improved), respectively. It was proposed that these were the times at which all the water (a required component of the reaction) in the original formulation is used up. The slower process was the same acid/glass reaction but initiated by water sorption. The initial rates of absorbance change due to acid neutralisation were 17 times faster for Fuji IX than Fuji II LC (Improved). By 4 days however, the total absorbance change due to acid neutralisation for Fuji IX was only 4 times that for Fuji II LC (Improved). Such results can help to explain changes in cement properties with time.

Hydroxyapatite, fluor-hydroxyapatite and fluorapatite produced via the sol-gel method. Optimisation, characterisation and rheology.

OBJECTIVES Currently, most titanium implant coatings are made using hydroxyapatite and a plasma spraying technique. There are however limitations associated with plasma spraying processes including poor adherence, high porosity and cost. An alternative method utilising the sol-gel technique offers many potential advantages but is currently lacking research data for this application. It was the objective of this study to characterise and optimise the production of Hydroxyapatite (HA), fluorhydroxyapatite (FHA) and fluorapatite (FA) using a sol-gel technique and assess the rheological properties of these materials. METHODS HA, FHA and FA were synthesised by a sol-gel method. Calcium nitrate and triethylphosphite were used as precursors under an ethanol-water based solution. Different amounts of ammonium fluoride (NH4F) were incorporated for the preparation of the sol-gel derived FHA and FA. Optimisation of the chemistry and subsequent characterisation of the sol-gel derived materials was carried out using X-ray Diffraction (XRD) and Differential Thermal Analysis (DTA). Rheology of the sol-gels was investigated using a viscometer and contact angle measurement. RESULTS A protocol was established that allowed synthesis of HA, FHA and FA that were at least 99% phase pure. The more fluoride incorporated into the apatite structure; the lower the crystallisation temperature, the smaller the unit cell size (changes in the a-axis), the higher the viscosity and contact angle of the sol-gel derived apatite. SIGNIFICANCE A technique has been developed for the production of HA, FHA and FA by the sol-gel technique. Increasing fluoride substitution in the apatite structure alters the potential coating properties.