37
glass system as the aluminum (Al3+) plays a critical role in the setting of the materials39, however, neurotoxic concerns and claims of imparting defective mineralization has limited its use for GPC based bone adhesives40-42. When the glass component is mixed with Polyacrylic acid (PAA) and water, the surface of the glass partially degrades and releases cations that serve to crosslink the ionized PAA chains43. This results in a hard cement like material that can bond to both HaP in bone and tooth, in addition to bonding to surgical metals44. This acid-base setting reaction is non-exothermic and with the degradation of the glass, ionic species are liberated into the polysalt matrix43, 45-47. This study investigates the replacement of Si4+ with Cu2+ as Cu is cited as forming strong bonds with carboxylic acid groups on the PAA chains during setting48. This study investigates the effect that Cu has on the glass structure and solubility, characterizing important physical properties such as rheological and mechanical trends, in addition to determining the antibacterial effects49, 50 of Cu-GPCs in a range of relevant bacteria species. Bioactive Cu-GPCs could potentially be a safer and more practical alternative to antibiotic doped bone cements23, as the evolution of antibiotic resistant microbes such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Staphylococcus aureus (VRSA) are a persistent cause for concern when developing implantable bone cements.
3.3 Materials & Methods
38
Table 3.1 Glass compositions (Mol. %) where SiO2 is substituted by CuO.
SiO2 CuO ZnO CaO SrO P2O5
Control 48 0 36 6 8 2
CuG6 42 6 36 6 8 2
CuG12 36 12 36 6 8 2
3.3.2 X-Ray Diffraction (XRD)
Diffraction patterns were collected using a Phaser D2 X-ray Diffraction Unit (Bruker AXS Inc., WI, USA). Glass powder samples were packed into zero background sample holders. A generator voltage of 40 kV and a tube current of 30 mA was employed.
Scattering patterns were collected in the range 10˚<2θ<80˚, at a scan step size 0.02˚ and a step time of 10 s.
3.3.3 Differential Thermal Analysis (DTA)
A SDT Q600 Simultaneous Thermal Gravimetric Analyser-Differential Scanning Calorimetry (TGA-DSC) (TA Instruments, DW, USA) was used to obtain a thermal profile of each glass, specifically the glass transition temperature (Tg) and crystallization temperatures. A heating rate of 10°C/min was employed in an air atmosphere using alumina as a reference in a matched platinum crucible. Sample measurements were carried out every 0.5 s between 30˚C and 1200˚C. TA Universal Analysis software (TA Instruments, DW, USA) was used to plot and obtain the temperatures of interest.
3.3.4 Scanning Electron Microscopy & Energy Dispersive X-ray Analysis (SEM/EDS)
Imaging was carried out with an FEI Co. Quanta 200F Environmental Scanning Electron Microscope. Additional compositional analysis was performed with an EDAX Genesis Energy-Dispersive Spectrometer (EDS). All EDS spectra were collected at 20 kV using a beam current of 26 nA. Quantitative EDS spectra were subsequently converted into relative concentration data.
3.3.5 Advanced Surface Area and Porosity (ASAP)
In order to determine the surface area of each glass, Advanced Surface Area and Porosimetry, Micromeritics ASAP 2020 (Micrometrics Instrument Corporation, Norcross,
39
USA) was employed. Approximately 60 mg of each glass was analyzed and the specific surface area was calculated using the Brunauer-Emmett-Teller (BET) method.
3.3.6 Particle Size Analysis (PSA)
Particle size was analyzed using a Multisizer 4 Particle Size Analyzer (Beckman Coulter, CA, USA). Glass powder samples (n = 3) were loaded into 20°C Isoton II Diluent at a concentration <10%. Samples were evaluated using a 280 μm standard to a final count of 30000.
3.3.7 X-Ray Photoelectron Spectroscopy (XPS)
Prior to surface analysis, samples were kept in a vacuum desiccator. The chemical bonding environment of each glass was characterized by X-ray photoelectron spectroscopy carried out with a Quantera (Physical Electronics, Minnesota, US), using a monochromatic Al kα radiation (hv=1486.6 eV) at an output of 25.5 watts. All survey spectra were recorded at constant pass energy of 140 eV and step size of 0.5 eV. Na1s, O1s, Ca2p, C1s and Si2p high resolution scans were collected with a pass energy of 26 eV, step size of 0.05 eV, and beam dwell time of ~ 300 ms to yield a signal to noise ratio > 100:1. Analysis area for each sample is ~ 2 to 3 mm in diameter using a 100 μm beam. Spectra analysis was performed on CasaXPS (Casa Software Ltd.). Peak positions were calibrated through normalization of the C1s peak to 284.6 eV.
3.3.8 Magic Angle Spinning-Nuclear Magnetic Resonance (MAS-NMR)
Glass powdered samples were tested using a MAS-NMR Bruker Avance III 600 coupled with an Ultrasheild Plus solid state NMR magnet with a 4mm diameter probe. The
29Si and proton channels had a frequency of 600.20 MHz and 119.29 MHz respectively. Tetrakis (trimethylsiyl)-silane was used for reference with the chemical shift at -9.843 ppm. Each sample underwent low power decoupling and was spun at 5.0 kHz for 300 scans. The relaxation time was 15 s and the pulse length 75°. Additionally, the spectrum reference frequency was 498.23 Hz.
40 3.3.9 Ion Release Profiles
Each glass (Control, CuG6 and CuG12, where n=3) was immersed in sterile de- ionised H2O for 1, 10, 100 and 1000 hours. Approximately 1.0 m2 surface area of glass powder was submerged in 10 ml of de-ionised H2O and rotated on an oscillating platform at 37ºC. The ion release profile of each glass was measured using Inductively Coupled Plasma – Atomic Emission Spectroscopy (ICP – AES) on a Perkin-Elmer Optima 5300UV (Perkin Elmer, MA, USA). ICP – AES calibration standards for Ca, Si, Sr, Zn, P and Cu ions were prepared from a stock solution on a gravimetric basis. Three target calibration standards were prepared for each ion and de-ionized water was used as a control.
3.3.10 pH Measurements
Changes in pH of solutions were monitored using a Corning 430 pH meter. Prior to testing, the pH meter was calibrated using pH buffer solution 4.00 ±0.02 and 7.00±0.02 (Fisher Scientific, Pittsburgh, PA). Sample solutions were prepared by exposing 1m2 surface area of each glass (where n=3) in 10ml de-ionized water. Measurements were recorded over t = 1, 10 and 100 and 1000 hours. De-ionized water was used as a control and was measured at each time period.
3.3.11 Glass Polyalkeonate Cements Formulation
GPCs were prepared by thoroughly mixing the glass powders (<45 µm) with polyacrylic acid (E11 PAA—Mw, 210,000, <90 m, Advanced Healthcare Limited, Kent, UK) and deionized water on a glass plate. The cements were formulated with a powder to liquid (P:L) ratio of 2:1.5 with 40%, 50%, and 60 wt% additions of PAA. Complete mixing was undertaken within 20 s.
3.3.12 Rheological Evaluation
The setting times (Ts) of the cement series were tested in accordance with ISO991751 which specifies the standard for dental water-based cements. Ts was measured by lowering a 400 g mass attached to a Gilmore needle into a cement filed mold measuring 8x9x10mm internal diameter. Cements were stored at 37oC during setting and the Ts was taken as the time the needle failed to make a complete indent in the cement surface, (where n = 3). The working time (Tw) of the cements was measured under standard laboratory
41
conditions (Ambient Temp, 25oC), and was defined as the period of time from the start of mixing during which it was possible to manipulate the material without having an adverse effect on its properties. Each sample (where n = 3), was measured using a stopwatch on a clean glass plate with a sterile spatula. Each measurement was conducted under the same mixing conditions to ensure reproducibility.
3.3.13 Compressive Strength
The compressive strengths (σc) of the cements (6x4mm, where n = 5) were evaluated in accordance with ISO991751. Cylindrical samples were tested after 7 days’
incubation in de-ionized water. Samples were stored in sterile de-ionized water in an incubator at 37oC. After the incubation time has expired the cements were removed and tested while wet on an Instron 4082 Universal Testing Machine (Instron Ltd., High Wycombe, Bucks, UK) using a 5 kN load cell at a crosshead speed of 1 mm/min. The σc was calculated using Equation 1. where q is the maximum applied load (N) and is the diameter of sample (mm).
𝐶 =
4𝜌𝜋𝑑2
(1)
3.3.14 Shear Bond Strength Test
Shear bond strength testing was conducted according to ISO 2902252. Briefly, cylindrical samples (6x4ømm, where n = 3) were mounted on hydroxyapatite (HaP) plates which were pre-mounted in resin and ground using 125 m grit size. Cylindrical Cu-GPC samples were bonded to the HaP surface and left for 1 hour to set (Figure 3.1). The split ring molds were then removed, and the bonded samples were incubated in de-ionized water for 24 hours at 37oC before testing. The test was conducted at a crosshead speed of 1.0mm/min until failure. The bond strength was calculated using Equation. 2. Where σ is stress expressed in MPa, F is the force, expressed in N and Ab is the bonding area expressed in mm2.
𝜎 =
𝐹𝐴𝑏
(2)
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Figure 3.1 Bonded Cu6C cylinders to hydroxyapatite substrates for shear bond strength testing
3.3.15 Antibacterial Testing
The antibacterial activity of the GPCs (8x2ømm, where n = 5) were evaluated using E. coli strain ATCC 8739 (LB agar and broth), S. epidermidis strain ATCC 14990 (BHI agar and broth), S. aureus (TSB agar and broth) and Vancomycin Resistant S. aureus (TSB agar and broth) using the agar diffusion method. Each cement sample was placed in inoculated plates and the plates were cultured for 24 hours at 37oC. Each microbe was initially grown aerobically in liquid broth at 37oC for 24 hours. Preparation of the agar disc diffusion plates involved seeding agar plates with a sterile swab dipped in a 1/50 dilution of the appropriate 24-hour culture of bacteria. The agar diffusion test was performed under standard laboratory sterile conditions in a fumigation hood using sterile swabs for inoculation of bacteria. Symbiosis protocol 3 colony counter was used for imaging of the bacterial plates. Calipers were used to measure zones of inhibition where each sample was analyzed in triplicate and mean zone sizes ± standard deviations were calculated.
Inhibition Zone (mm) =Halo∅ − Disc∅
2
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