Quaternary ammonium silane-functionalized, methacrylate resin composition with antimicrobial activities and self-repair potential

Abstract

The design of antimicrobial polymers to address healthcare issues and minimize environmental problems is an important endeavor with both fundamental and practical implications. Quaternary ammonium silane-functionalized methacrylate (QAMS) represents an example of antimicrobial macromonomers synthesized by a sol-gel chemical route; these compounds possess flexible Si-O-Si bonds. In present work, a partially hydrolyzed QAMS co-polymerized with 2,2-[4(2-hydroxy 3-methacryloxypropoxy)-phenyl]propane is introduced. This methacrylate resin was shown to possess desirable mechanical properties with both a high degree of conversion and minimal polymerization shrinkage. The kill-on-contact microbiocidal activities of this resin were demonstrated using single-species biofilms of Streptococcus mutans (ATCC 36558), Actinomyces naeslundii (ATCC 12104) and Candida albicans (ATCC 90028). Improved mechanical properties after hydration provided the proof-of-concept that QAMS-incorporated resin exhibits self-repair potential via water-induced condensation of organic modified silicate (ormosil) phases within the polymerized resin matrix.

Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Figures

Figure 1

Reaction scheme for synthesizing QAMS using tetra-alkoxysilane as anchoring unit. Depending on the molar ratio of the two trialkoxysilanes, macromonomers with monofunctional (QAMS-1), bifunctional (QAMS-2) or trifunctional methacryloxy functionalities (QAMS-3) may be produced.

Figure 2

Hydrolysis and condensation kinetics of QAMS-3 monitored by ATR-FTIR. A. Infrared spectra of the monomer mix for preparing QAMS-3, after partial hydrolysis of the monomer mix at pH 2.5 and after condensation at pH 7.4. B. Hydrolysis kinetics of completely- and partially-hydrolyzed QAMS-3 at pH 2.5. Plots represent changes in peak heights of the Si-O-C band at 1082 cm−1 and Si-OH band at 914 cm−1. C. Condensation kinetics of completely- and partially-hydrolyzed QAMS-3 at pH 7.4. Plots represent changes in peak heights of the Si-O-Si band at 1117 cm−1 and Si-OH band at 914 cm−1.

Figure 3

Kinetics of monomer conversion. A. Conversion values increased as the concentration of TEGDMA diluent was reduced and replaced by equivilent amounts of QAMS-3PH. Dotted vertical line at 60 seconds represents termination of light curing. B. Conversion values at specific time points among the five comonomer blends. This figure shows the increase in degree of monomer conversion at each time point with respect to increasing QAMS-3PH concentration. From the cure rate (C), it can be seen that there were two distinct phases in the polymerization process: an auto-accleration phase and an auto-deceleration phase. Curing of the 5 comonomer blends peaks at a maximum rate from 9–11% per second. This peaks occurs very eary in the conversion process, that is, within the first 3–4 seconds. D. Degree of conversion at maximum cure rate. Groups that are labeled with the same upper case letters are not statistically significant (p > 0.05). A clear trend of decreasing conversion at maximal cure rate with respect to increasing QAMS-3PH concentration is evident for the materials tested.

Figure 4

Kinetics of polymerization shirnkage. A. The five bis-GMA comonomer blends all demonstrated polymerization shrinkage when exposed to visible light. With the exception of an initial expansion caused by the heat generated during light exposure (arrow), polymerization shrinkage continued to increase until a maximum shrinkage value was achieved for each rspective resin. B. Maximum volumetric shrinkage among the five comonomer blends. Groups that are labeled with the same low case letters are not statistically significant (p > 0.05). C. Changes in shrinkage rate with time. Similar to the conversion kinetcs in S-4A, two distinct phases in the polymerization process are evident, with the highest shrinkage rate occurring between 3–5 seconds before auto-decleration occurred. D. A powder regression model provides an excellent fit (adjusted R2 = 0.986; p < 0.01) for the relation between the maximum shrinkage rate and increases in QAMS-3PH concentration.

Figure 5

Antimicrobial activities of polymerized resin containing QAMS-3PH. A. Confocal laser scanning microscopy images (2-D overlay projections) of BacLight-stained 48-hour microbial biofilms (live-green; dead-red) grown on resin disks with bis-GMA:TEGDMA:QAMS-3PH mass ratios of 70:30:0, 70:10:20 and 70:0:30 (bar=20 μm). QAMS-3PH: methanol-solvated, partially-hydrolyzed QAMS-3. B. Percentage of live microbes within the respective single-species biofilms. For each species, groups identified with the same designator are not statistically significant (p>0.05). C. Three-dimensional reconstruction of a z-stack of images taken from a BacLight-stained A. naeslundii biofilm grown on a resin disk with BisGMA:TEGDMA:QAMS-3PH mass ratio of 70:0:30. Bacteria in contact with resin surface were non-viable (red), while those within the body of the biofilm remained viable (green).

Figure 6

Mitochondrial succinic dehydrogenase activities (normalized to Teflon negative control – 100%) of MDPC-23 cells after exposure to A) polymerized resin disks prepared from the 5 comonomer blends and the polymethacrylate (PMMA) positive control at two time-periods, and B) eluents from the resin disks (1:10 dilution) at the two time-periods. Initially cytotoxicity was identified from the resin disks at the first week but not at the second week, after extraction of toxic components from the polymerized resins. By contrast, the eluents derived from these resins were considerably more cytotoxic. After the second week cycle, eluents derived from polymerized resins with bis-GMA:TEGDMA:QAMS-3PH mass ratios of 70:5:25 and 70:0:30 were as biocompatible as the Teflon negative control.

Figure 7

A. Distribution of early and late apoptotic MDPC-23 cells, after exposure to i) Teflon negative control; ii) Polymethyl methacrylate (PMMA) positive control; iii) Polymerized resin composed of bis-GMA:TEGDMA:QAMS-3PH with a mass ratio of 70:30:0; iv) Polymerized resin composed of bis-GMA:TEGDMA:QAMS-3PH with a mass ratio of 70:10:20; and v) Polymerized resin composed of bis-GMA:TEGDMA:QAMS-3PH with a mass ratio of 70:0:30. The presence of later apoptotic cells even in the negative control group may be attributed to membrane damage to some of the cells (hence, stainable with Annexin-V) when trypsin was employed for cell detachment from the culture plates. Despite this limitation, comparison of the percentage of early and late apoptotic cells shows that the 70:0:30 polymerized resin group has the lowest % of apoptotic cells among the three resin groups. B-F. Two-photon laser fluorescence microscopy imaging of MDPC-23 cells after exposure to eluents derived from 5 groups. The nuclei of healthy cells were stained positively with Hoescht 33342 (stains DNA of both vital and non-vital cells; blue fluorescence) only. The nuclei of dead cells were stained also positively with ethidium homodimer-III (Etd-III, non-vital DNA marker; red fluorescence). Merging of the channels results in pink nuclei due to combination of the blue and red fluorescence. The cytoplasm of apoptotoic cells were stained positively with FITC-Annexin V (stains cytoplasmic phosphatidylserine; green fluorescence). B. Only healthy cells (stained by Hoechst only, not by FITC-Annexin V and EtD-III) are seen in the Teflon negative control (absence of apoptosis or necrosis). C. Cells were stained blue, green and red in the PMMA positive control, which is indicative of late apoptotic cells or dead cells progressing from the apoptotic cell populations. D. Similar dead cells were seen in the experimental resin group with bis-GMA/TEGDMA/QAMS-3PH mass ratio of 70:30:0. E. Early apoptotic cells (stained both blue and green) observed in the in the experimental resin group with bis-GMA/TEGDMA/QAMS-3PH mass ratio of 70:10:20. F. Most of the cells are healthy (stained blue only) in the experimental resin group with bis-GMA/TEGDMA/QAMS-3PH mass ratio of 70:0:30. Some early apoptotic cells are also present.

Figure 8

Dynamic mechanical behavior (complex modulus, loss modulus and storage modulus) under dehydrated and hydrated conditions. A–B. Representative mechanical property maps of polymerized resins prepared from control bis-GMA/TEGDMA and experimental bis-GMA/QAMS-3PH (both 70:30 mass%) before (A) and after (B) hydration. Note difference in scale between the control and experimental resins in B. QAMS-3PH: methanol-solvated, partially-hydrolyzed QAMS-3. C–E. Complex modulus (C), loss modulus (D) and storage modulus (E) of the control and experimental groups. Group designations along the horizontal axis - #:bisGMA/TEGDMA (70:30 mass%); *:bisGMA/QAMS-3 monomer mix (70:30 mass%); **: bis-GMA/QAMS-3PH (70:30 mass%); ***: bis-GMA/QAMS-3PH (60:40 mass%). For each material property, same lower-case designations in “before hydration” groups denote no significant difference. Same upper-case designations in “after hydration” denote no significant difference. For each “resin composition”, horizontal bar over the two “hydration modes” denotes no-significant difference. F. Left: Transmission electron microscopy of phosphotungstic acid-stained, stressed-and-hydrated experimental resin (bis-GMA/QAMS-3PH, mass ratio:70:30) showing electron-dense phases (asterisks) deposited around a crack-tip (arrow). Right: High magnification of an asterisked region revealing an interconnecting network of probable organic modified silicate condensation products (arrowhead).