title: The anti-bacterial effect of zinc-doped phosphate-based glasses (Zn-PBG) and its role in the demineralisation and remineralisation process of bovine enamel
Creators: name: Rajadorai, Sindhuja
Publisher: name: University of Liverpool Repository
Year: 2019

description: The anti-bacterial effect of zinc-doped phosphate-based glasses (Zn-PBG) and its role in the remineralisation and demineralisation process of bovine enamel by Sindhuja Rajadorai Aims This is a laboratory-based experimental study that aims to assess the anti- bacterial effect of zinc-doped phosphate based glasses on the growth of Streptococcus mutans and the role of zinc in the demineralisation and remineralisation process of bovine enamel. Enamel demineralisation is highly prevalent during orthodontic treatment. Newly developed zinc doped phosphate-based glasses (Zn-PBGs) are controlled delivery agents for zinc ions that may be effective in reducing the incidence of white spot lesions. Materials and Methods Zinc doped phosphate-based glasses (3mol% zinc and varying calcium concentrations, denoted as C11, C12 and C13) and control glasses which are zinc free phosphate-based glasses (C-PBG) rods (5 x 2mm) were produced using a conventional melt quenching method, at 1100°C for 1 hour. The calcium in the glass serve to control and alter the degradation rate of the glasses which contributes to a controlled manner manner of ion delivery. The glass characteristics were assessed through glass degradation studies (0- 56h), which were carried out using a weight loss method in dH2O at 37 ± 10C and a starting pH of 7 ± 0.1. A pH analysis was conducted and calcium, zinc, sodium and phosphate concentrations remaining in solution were measured by inductively- coupled optical-emission spectrometry (ICP-OES). The anti-bacterial effects were assessed through disc diffusion assays and liquid broth analysis. Disc diffusion assays were conducted on isosensitest (IST) agar with standardised cultures of S.mutans NCTC10449. The plates were incubated for 24 hours aerobically at 37°C. The diameters of the zones of inhibition that developed around each of the glass samples were measured. A liquid broth assay was conducted in phosphate buffered saline (PBS) using S.mutans suspension to a standardised optical density (OD600=0.03) exposed to C11, C12 and C13 and controls (a positive control of 0.2% chlorhexidine and a negative control of C-PBG). At 2, 4, 6 and 24 hours, samples were removed, diluted appropriately in PBS, spread on BHI agar to assess viable colony-forming units (CFU) present. The effect of Zn-PBG on bovine enamel was investigated under pH cycling and utilising a constant depth film fermentor (CDFF) model. pH cycling of enamel blocks allocated to 1 of 5 conditions (Zn-PBG, C-PBG, ZnSO4, NaF and H2O) over 5 days of cyclical acidic challenge of immersion in demineralising solutions for 6h (2.0 mM CaCl2, 2.0 mM KH2PO4, 0.04 ppm NaF and 0.075 M acetic acid adjusted to pH 4.7 with 1.0 M NaOH;) and remineralising solutions for 18h (1.5 mM CaCl2, 0.9 mM KH2PO4, 150 mM KCl, 0.05 ppm NaF and 20 mM HEPES adjusted to pH 7.0 with 1.0 M NaOH) followed by 2 days in remineralising solution. Data obtained from the pH cycled samples were analysed using non- contact surface profilometry, (NCSP) (Proscan 2000) to assess surface roughness (Iso Ra) of the bovine enamel samples. Biofilms were grown in the CDFF model on bovine enamel discs using artificial saliva. On the 5th day and 12th day, the pans were removed aseptically from the CDFF system and the discs containing biofilms were removed, subjected to 10 minutes exposure of Zn-PBG (C11) compared with 0.2% diluted chlorhexidine, 0.05% sodium fluoride or water. Transverse micro-radiography (TMR), to quantify and assess mineral loss (ΔZ) from bovine enamel, was used for bovine enamel samples exposed to dissolving acids in the CDFF model and the pH cycling experiment. All tests were conducted in triplicate. Statistical Analysis Statistical analyses were conducted using the Prism GraphPad software (San Diego, California, USA). Paired T-tests, a one-way analysis of variance (ANOVA), and Tukey Kramer multiple comparison tests were used to compare values and p values &lt;0.05 were considered statistically significant. Results The average pH values over time were 6.95 ± 0.19 for C-PBG, 6.87 ± 0.17 for C11, 6.71 ±0.04 for C12 and 6.71 ± 0.06 for C13. pH analyses showed a statistically significant difference (p &lt;0.05) for C12 and C13 when compared with C-PBG. The degradation rates of the Zn-PBGs were significantly (p&lt;0.05) different (28.94μg mm-2 h-1 for C11, 25.56μg mm-2 h- 1 for C12 and 19.72μg mm-2 h-1 for C13) compared with C-PBG (10.17μg mm-2 h-1). Zinc ion release was decreased as calcium concentration increased in the glasses, which showed a strong correlation with the weight loss of the glasses. Zones of inhibition from disc diffusion assays were found to decrease (from 18± 1.7, 16± 1.6 to 15.0 ± 1.1 mm) as the calcium concentration in the glasses increased from 11, 12 and 13 mol% of calcium. Zones of inhibition were significantly increased for C11, C12 and C13 (p &lt; 0.01) when compared with C-PBG. Viable cells in the liquid broth assay were found to decrease as the calcium concentration in the glasses increased from 11, 12 and 13 mol% of calcium. Viable cell counts in the untreated group and C-PBG group were significantly increased (p&lt; 0.01) when compared with groups C11, C12 and C13. From the constant depth film fermentor (CDFF) model, there was statistically significant mineral loss (p &lt;0.05) in the bovine enamel samples that were exposed to C-PBG, NaF, Chx and enamel discs (ED) at day 12 compared with day 5. There was no statistically significant difference in mineral loss (p&gt;0.05) in the bovine enamel samples that were exposed to C11 between day 5 and day 12. NCSP analysis of the bovine enamel samples that were subjected to alternating remineralisation and demineralisation solutions through pH cycling was carried out. It was revealed that there were no significant differences in surface roughness (Iso Ra) of the bovine enamel samples between C11, C12, C13, C-PBG, ZnS04, NaFandH20. Transversemicroradiography(TMR)analysesshowedsignificant(p&lt;0.05) mineral loss in the pH-cycled H20 group when compared with pH-cycled C11, C12, C13, C- PBG, ZnS04 and NaF. Conclusion The results suggest that the controlled delivery of zinc ions from zinc-doped phosphate based glasses may have potential in oral applications due to the anti-bacterial effect of Zn-PBG demonstrated in the disc diffusion and liquid broth assay and the inhibition of demineralisation as shown in the CDFF model via TMR analysis. There is scope for further research in this area.