Highlights
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Primary study on synthesis of novel spherical chlorhexidine spheres incorporated in a dental resin.
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Novel chlorhexidine spheres incorporated in a dental resin demonstrated lower and responsive chlorhexidine release.
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Ultrasonics gave a responsive controlled chlorhexidine release, which is useful to treat severe or persistent infections.
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Chlorhexidine spheres produced by co-precipitation may be useful for the treatment of Periodontitis and Peri-implantitis.
Abstract
Objective
Establish the release kinetics of new chlorhexidine particles incorporated in a dental resin, and with the application of ultrasound.
Methods
Spherical chlorhexidine particles (SCP) were synthesized (5 wt%), freeze dried and incorporated into UDMA–HEMA resins. Chlorhexidine diacetate (CDP) (5 wt%) was similarly incorporated in separate resins. Resin discs were immersed in deionized water, and a release profile established (650 h). Ultrasound was used to trigger chlorhexidine (CHX) release from the resin discs at specific durations (10–30 s) and time intervals (1–425 h). Chlorhexidine content was determined by UV–vis absorption. The chlorhexidine particles/polymer composites were characterized using TGA, SEM, and confocal microscopy.
Results
SCP exhibited structures with high chlorhexidine content (90–95%), and a Mean (SD) diameter of 17.2 (2.5) μm which was significantly (p < 0.001) smaller than the CDP crystals at 53.6 (33.7) μm. The SCP discs had a lower (7.7%) CHX release compared to the CDP group (16.2%). Ultrasonication of the resin discs with increasing durations (10–30 s) resulted in higher drug release rates. CDP release rates (CHX) over 650 h were: 23.5% (10 s), 42.6% (20 s), 51.2% (30 s), and for SCP (CHX) were; 9.8% (10 s), 12.3% (20 s), and 14.0% (30 s). SEM/confocal microscopy revealed CDP discs exhibited dissolution associated with the particle surface and SCP from the interior.
Significance
Chlorhexidine spheres incorporated in a dental resin demonstrated a responsive and lower CHX release. Ultrasound enhanced CHX release and is useful in clinical situations where the drug is required on demand to treat severe or persistent infections.
1
Introduction
Since being introduced to Dentistry in the 1950s, dental composites have undergone considerable changes such as; the modification of fillers to enhance the mechanical properties and the optimisation of the polymeric matrix to improve their biocompatibility . Currently available dental composites have exceptional aesthetics and comparable mechanical properties (flexural strength, fracture toughness and tensile strength) when compared with porcelain and amalgam, and superior to that of glass ionomers . This allows their wide use for anterior and posterior tooth restorations, as well as pit and fissure sealants . Dental composites are cured by light and chemically initiated free radical polymerization which results in 2–5.63 vol.% shrinkage of the material . This can lead to the formation of gaps between the restoration and the tooth . Bacterial microleakage through these gaps can cause marginal discoloration, sensitivity, inflammation, recurrent caries and apical periodontitis . Furthermore, accumulation of bacterial biofilms on the surface of dental composites can lead to degradation of the polymer matrix, resulting in the weakening of the restoration. Dental composites with antimicrobial properties are therefore highly desirable to overcome these problems.
Chlorhexidine is a bis-biguanide antiseptic and disinfectant that has a bactericidal and bacteriostatic action against a wide range of gram-positive and gram-negative bacteria , and has been incorporated in a variety of resin composites . Chlorhexidine also stabilizes the resin–dentin bond by inhibiting matrix metalloproteinases (MMP) which are responsible for the breakdown of the bond at the dentin-restoration interface . Inclusion of chlorhexidine into dental composites has been achieved by mixing the chlorhexidine diacetate with monomers . This composite however suffers from an uncontrollable release of chlorhexidine, due to its rapid diffusion from the methacrylate based resin. To overcome these drawbacks efforts have been made to develop new chlorhexidine formulations and investigate novel drug carriers , to control chlorhexidine release. Ultrasound is another method previously used to control the release of antibacterial agents in bone cements, where the synergistic effect of ultrasound and antibiotics enhanced antimicrobial efficiency . More recent studies utilised high frequency ultrasound to deliver antimicrobial chitosan nanoparticles into dentinal tubules, which may open up new approaches for the treatment of root canal infections .
Recently Luo et al. developed a novel chlorhexidine compound, with a unique spherical morphology and the ability to provide controlled release of chlorhexidine. The aim of this study was therefore to investigate the effects of ultrasound from a dental scaler (ultrasound probe); to enhance drug release from novel chlorhexidine spheres in a light cured dental resin. Ultrasound triggered chlorhexidine release may be particularly useful in developing triggered drug delivery from a polymer system incorporating novel chlorhexidine spheres.
2
Materials and methods
2.1
Fabrication of spherical chlorhexidine particles
Chlorhexidine spheres were fabricated and characterized as described previously . Briefly, 15 mg/ml chlorhexidine diacetate (Sigma–Aldrich, Lot: 19H0417) solution and 0.33 M CaCl 2 (Sigma–Aldrich, Lot: SLBF7416V) solution were mixed at 1:1 ratio by volume at room temperature. The mixture was shaken for 1 min, and then centrifuged at 2000 rpm for 1 min (Eppendorf centrifuge 5417C, Germany). The sedimentation was then washed three times with 0.33 M CaCl 2 solution and then centrifuged again. Thereafter, the product was freeze dried (ScanVac Cool Safe Freeze Drying, Denmark) at −107 °C, 0.009 mbar for 1 day, and was used for all the following experiments. Several reproducible batches (3) were produced for this work.
2.2
UV/vis spectroscopy and TGA analysis
The content of chlorhexidine in the spherical particles was determined by UV–vis absorption (Lambda 35, Perkin Elmer, USA). Initially, a series of chlorhexidine diacetate aqueous solutions with standard concentrations of 0.25, 1, 2, 3, 4, 5, 10, 20 ppm were prepared, and the absorption measured. The absorption peak at 254 nm and the reference concentration had a linear relationship, and a standard curve of absorption verses concentration was established (Fig. S1). Residual chlorhexidine in all the supernatants was determined by measuring the UV absorption of the supernatants at 254 nm, and then calculating the concentration using the calibration curve. The proportion of chlorhexidine in the spherical particles was then calculated by subtraction (initial CHX—CHX in all supernatants = CHX in the particles). In addition, the freeze dried chlorhexidine spheres; chlorhexidine diacetate and CaCl 2 powder were analyzed using Thermo-gravimetric analysis (TGA Q50, USA). The TGA was carried out at 10 °C/min in a nitrogen atmosphere, over a temperature range of 50–800 °C.
2.3
Preparation of chlorhexidine UDMA–HEMA resin discs
The resin was prepared by mixing 64% urethane dimethacrylate (UDMA) (Esschem, UK, Lot: 591-22), 36% hydroxyethyl methacrylate (HEMA) (Aldrich, UK), 0.08% of N , N -dimethyl- P -toluidine (Acros Organics, UK) and 0.05% dimethylamino ethyl methacrylate (Aldrich, UK). The mixture was stirred at 800 rpm for 15 min (VWR Stirrer, USA). Finally, camphorquinone (Aldrich, UK) was added at the proportion of 0.1% and the mixture was stirred for another 15 min. The current resin was selected as it was biocompatible when exposed to L929 fibroblasts (ISO 10993) and following a 28 day resin leach out study and DNA assay. Freeze dried spherical chlorhexidine particles were weighed (Salter Ander-180A weighing scale, UK) and incorporated within the resin at 5 wt% (chlorhexidine content), then placed in an Eppendorf tube and mixed for 15 s in a rotomix mixer (120 V/60 Hz, 2850 rotations/min) (ESPE RotoMix, USA). A separate set of samples containing chlorhexidine diacetate powder (5 wt% chlorhexidine content) was also incorporated in the prepared resin in the same way. The resin mixture was then placed into a Teflon mold (10 mm diameter × 2 mm depth) and cured through a Mylar film strip using a curing light (Bluedent LED pen, Bulgaria) (430–490 nm, 600 mW/sq cm) for 30 s on both sides. The discs were next weighed on a microbalance (Salter Ander-180A weighing scale, UK), and the chlorhexidine content in each disc was calculated.
2.4
Labelled chlorhexidine particles and confocal microscopy
For visualization of the chlorhexidine sphere distribution in the resin composites, the spheres were labelled using rhodamine B (Sigma, Lot: 063K3407). Spherical chlorhexidine particles were synthesized as described in Section 2.1 ; however before mixing the chlorhexidine diacetate solution with the CaCl 2 solution, 100 μl of rhodamine B (1 mg/ml) was added. The synthesized particles were then centrifuged, washed and freeze dried as in Section 2.1 . The labelled spherical chlorhexidine particles (5 wt% chlorhexidine content) were mixed with UDMA–HEMA resin and placed into a mold (10 mm length, 10 mm width and 1 mm depth) and cured using a curing light as in Section 2.3 . The rhodamine B (RhB) labelled chlorhexidine particle resin specimen before and after immersion in water (650 h) was then characterized using confocal microscopy (Leica TS confocal scanning system, Germany).
2.5
Chlorhexidine release kinetics
In the release study, all UDMA–HEMA resin discs containing spherical chlorhexidine particles (SCP) or chlorhexidine diacetate powder (CDP) were stored in cuvettes containing 2 ml deionized water at room temperature. To measure the effect of ultrasound on the release kinetics specimens were split into 4 groups (n = 3 per group) for each composite (SCP and CDP). The specimen groups comprised; Gp1:0 s, Gp2:10 s, Gp3:20 s and Gp4:30 s ultrasound exposure. Specimens were treated with a single application of ultrasound at multiple time points. The ultrasound was delivered by contacting the disc specimen surface with an ultrasound probe (27–30 kHz, Piezon Master 400, Electro Medical Systems, Switzerland) at the following time points; 1 h, 3 h, 5 h, 15 h, 25 h, 40 h, 65 h, 95 h, 140 h and 205 h. Ultrasound was applied to the all the samples identically, with the ultrasound probe in contact with the surface of resin discs and moved around the sample during application. Following the last ultrasonic treatment (205 h), release measurements were continued until 650 h (275 h, 350 h, 425 h, 500 h, 575 h and 650 h). Solutions from each time intervals were collected for the UV–vis absorption tests (Lambda 35, Perkin Elmer, USA) and replaced with fresh deionized water. The amount of released chlorhexidine was determined according to the established relationship of absorption and chlorhexidine concentration, and cumulative release curves were plotted. The rhodamine B labelled chlorhexidine particle resin specimen was also immersed in water for 650 h and fresh water added at each time interval.
To further investigate the effect of ultrasound on triggering chlorhexidine release, another set of experiments with the same time intervals and release measurements was conducted on the two composite materials. The first group 1 received no ultrasound treatment. Ultrasound (30 s) was next delivered to the disc specimens at the start of the experiment (0 h, Gp.2); at the start and at 205 h (14.3 h 1/2 ) (Gp. 3) and at the start, 205 h (14.3 h 1/2 ) and 425 h (20.6 h 1/2 ) (Gp. 4). The release profile of each group with different treatment times for each material was compared. The slope of release curves after each of ultrasound treatment was determined by Δy/Δx and compared with that of the control.
2.6
Scanning electron microscopy
Chlorhexidine diacetate powder (C6143, Lot: 19H0417, CDP) and spherical chlorhexidine particle (SCP) powder samples were prepared by placing powder suspensions onto the surface of glass slides, which were stuck onto SEM stubs, and then dried in air. Resin discs containing CPD or SCP from each group before and after the ultrasonication study (Sections 2.3 , 2.5 ), were dipped into liquid nitrogen and broken. Cross-sections were mounted onto SEM stubs. All the samples were gold coated for 45 s at 18 mA, 0.04 mbar using a sputter coater (SC7620, Emitech, UK). All samples were characterized using a scanning electron microscope (FEI Inspect-F, USA), in the secondary electron imaging mode. An accelerating voltage of 10 kV, spot size of 3.5 and working distance of 10 mm was used. Multiple images (10) were collected for each of the samples. The Mean (SD) particle diameter and size range of the chlorhexidine spheres and chlorhexidine diacetate particles was measured using quantitative image analysis (Sigma Scan Pro 5, Systat Software Inc., UK) of the SEM images. Over 100 particles were measured and the data was compared using a t-test (Sigma Stat, version 2.03, SPSS Inc., Chicago, IL, USA), to analyze statistically significant differences between groups (p < 0.05).
2
Materials and methods
2.1
Fabrication of spherical chlorhexidine particles
Chlorhexidine spheres were fabricated and characterized as described previously . Briefly, 15 mg/ml chlorhexidine diacetate (Sigma–Aldrich, Lot: 19H0417) solution and 0.33 M CaCl 2 (Sigma–Aldrich, Lot: SLBF7416V) solution were mixed at 1:1 ratio by volume at room temperature. The mixture was shaken for 1 min, and then centrifuged at 2000 rpm for 1 min (Eppendorf centrifuge 5417C, Germany). The sedimentation was then washed three times with 0.33 M CaCl 2 solution and then centrifuged again. Thereafter, the product was freeze dried (ScanVac Cool Safe Freeze Drying, Denmark) at −107 °C, 0.009 mbar for 1 day, and was used for all the following experiments. Several reproducible batches (3) were produced for this work.
2.2
UV/vis spectroscopy and TGA analysis
The content of chlorhexidine in the spherical particles was determined by UV–vis absorption (Lambda 35, Perkin Elmer, USA). Initially, a series of chlorhexidine diacetate aqueous solutions with standard concentrations of 0.25, 1, 2, 3, 4, 5, 10, 20 ppm were prepared, and the absorption measured. The absorption peak at 254 nm and the reference concentration had a linear relationship, and a standard curve of absorption verses concentration was established (Fig. S1). Residual chlorhexidine in all the supernatants was determined by measuring the UV absorption of the supernatants at 254 nm, and then calculating the concentration using the calibration curve. The proportion of chlorhexidine in the spherical particles was then calculated by subtraction (initial CHX—CHX in all supernatants = CHX in the particles). In addition, the freeze dried chlorhexidine spheres; chlorhexidine diacetate and CaCl 2 powder were analyzed using Thermo-gravimetric analysis (TGA Q50, USA). The TGA was carried out at 10 °C/min in a nitrogen atmosphere, over a temperature range of 50–800 °C.
2.3
Preparation of chlorhexidine UDMA–HEMA resin discs
The resin was prepared by mixing 64% urethane dimethacrylate (UDMA) (Esschem, UK, Lot: 591-22), 36% hydroxyethyl methacrylate (HEMA) (Aldrich, UK), 0.08% of N , N -dimethyl- P -toluidine (Acros Organics, UK) and 0.05% dimethylamino ethyl methacrylate (Aldrich, UK). The mixture was stirred at 800 rpm for 15 min (VWR Stirrer, USA). Finally, camphorquinone (Aldrich, UK) was added at the proportion of 0.1% and the mixture was stirred for another 15 min. The current resin was selected as it was biocompatible when exposed to L929 fibroblasts (ISO 10993) and following a 28 day resin leach out study and DNA assay. Freeze dried spherical chlorhexidine particles were weighed (Salter Ander-180A weighing scale, UK) and incorporated within the resin at 5 wt% (chlorhexidine content), then placed in an Eppendorf tube and mixed for 15 s in a rotomix mixer (120 V/60 Hz, 2850 rotations/min) (ESPE RotoMix, USA). A separate set of samples containing chlorhexidine diacetate powder (5 wt% chlorhexidine content) was also incorporated in the prepared resin in the same way. The resin mixture was then placed into a Teflon mold (10 mm diameter × 2 mm depth) and cured through a Mylar film strip using a curing light (Bluedent LED pen, Bulgaria) (430–490 nm, 600 mW/sq cm) for 30 s on both sides. The discs were next weighed on a microbalance (Salter Ander-180A weighing scale, UK), and the chlorhexidine content in each disc was calculated.
2.4
Labelled chlorhexidine particles and confocal microscopy
For visualization of the chlorhexidine sphere distribution in the resin composites, the spheres were labelled using rhodamine B (Sigma, Lot: 063K3407). Spherical chlorhexidine particles were synthesized as described in Section 2.1 ; however before mixing the chlorhexidine diacetate solution with the CaCl 2 solution, 100 μl of rhodamine B (1 mg/ml) was added. The synthesized particles were then centrifuged, washed and freeze dried as in Section 2.1 . The labelled spherical chlorhexidine particles (5 wt% chlorhexidine content) were mixed with UDMA–HEMA resin and placed into a mold (10 mm length, 10 mm width and 1 mm depth) and cured using a curing light as in Section 2.3 . The rhodamine B (RhB) labelled chlorhexidine particle resin specimen before and after immersion in water (650 h) was then characterized using confocal microscopy (Leica TS confocal scanning system, Germany).
2.5
Chlorhexidine release kinetics
In the release study, all UDMA–HEMA resin discs containing spherical chlorhexidine particles (SCP) or chlorhexidine diacetate powder (CDP) were stored in cuvettes containing 2 ml deionized water at room temperature. To measure the effect of ultrasound on the release kinetics specimens were split into 4 groups (n = 3 per group) for each composite (SCP and CDP). The specimen groups comprised; Gp1:0 s, Gp2:10 s, Gp3:20 s and Gp4:30 s ultrasound exposure. Specimens were treated with a single application of ultrasound at multiple time points. The ultrasound was delivered by contacting the disc specimen surface with an ultrasound probe (27–30 kHz, Piezon Master 400, Electro Medical Systems, Switzerland) at the following time points; 1 h, 3 h, 5 h, 15 h, 25 h, 40 h, 65 h, 95 h, 140 h and 205 h. Ultrasound was applied to the all the samples identically, with the ultrasound probe in contact with the surface of resin discs and moved around the sample during application. Following the last ultrasonic treatment (205 h), release measurements were continued until 650 h (275 h, 350 h, 425 h, 500 h, 575 h and 650 h). Solutions from each time intervals were collected for the UV–vis absorption tests (Lambda 35, Perkin Elmer, USA) and replaced with fresh deionized water. The amount of released chlorhexidine was determined according to the established relationship of absorption and chlorhexidine concentration, and cumulative release curves were plotted. The rhodamine B labelled chlorhexidine particle resin specimen was also immersed in water for 650 h and fresh water added at each time interval.
To further investigate the effect of ultrasound on triggering chlorhexidine release, another set of experiments with the same time intervals and release measurements was conducted on the two composite materials. The first group 1 received no ultrasound treatment. Ultrasound (30 s) was next delivered to the disc specimens at the start of the experiment (0 h, Gp.2); at the start and at 205 h (14.3 h 1/2 ) (Gp. 3) and at the start, 205 h (14.3 h 1/2 ) and 425 h (20.6 h 1/2 ) (Gp. 4). The release profile of each group with different treatment times for each material was compared. The slope of release curves after each of ultrasound treatment was determined by Δy/Δx and compared with that of the control.
2.6
Scanning electron microscopy
Chlorhexidine diacetate powder (C6143, Lot: 19H0417, CDP) and spherical chlorhexidine particle (SCP) powder samples were prepared by placing powder suspensions onto the surface of glass slides, which were stuck onto SEM stubs, and then dried in air. Resin discs containing CPD or SCP from each group before and after the ultrasonication study (Sections 2.3 , 2.5 ), were dipped into liquid nitrogen and broken. Cross-sections were mounted onto SEM stubs. All the samples were gold coated for 45 s at 18 mA, 0.04 mbar using a sputter coater (SC7620, Emitech, UK). All samples were characterized using a scanning electron microscope (FEI Inspect-F, USA), in the secondary electron imaging mode. An accelerating voltage of 10 kV, spot size of 3.5 and working distance of 10 mm was used. Multiple images (10) were collected for each of the samples. The Mean (SD) particle diameter and size range of the chlorhexidine spheres and chlorhexidine diacetate particles was measured using quantitative image analysis (Sigma Scan Pro 5, Systat Software Inc., UK) of the SEM images. Over 100 particles were measured and the data was compared using a t-test (Sigma Stat, version 2.03, SPSS Inc., Chicago, IL, USA), to analyze statistically significant differences between groups (p < 0.05).
3
Results
3.1
Results of the SEM study
The results of the SEM study show a wide particle distribution (6.4–194.7 μm) of angular/tabular chlorhexidine diacetate crystals ( Fig. 1 a, c), with a Mean (SD) diameter of 53.6 (33.7) μm. The spherical chlorhexidine particles ( Fig. 1 b, d) however, produced a narrower particle size range (6.3–21.7 μm) and a Mean (SD) diameter of 17.2 (2.5) μm. The particle size data was compared using a t-test and the chlorhexidine particle group showed a significantly smaller (p < 0.001) Mean particle diameter than the chlorhexidine diacetate group.
