Introduction
White spot lesions are a common side effect of orthodontic treatment. This laboratory study aimed to explore the suitability of chlorhexidine hexametaphosphate (CHX-HMP) as a coating for orthodontic elastomeric ligatures to provide sustained chlorhexidine (CHX) release.
Methods
Dissolution kinetics of CHX-HMP were firstly explored using spectroscopy and a colorimetric phosphate assay. Elastomeric ligatures were categorized into 3 groups—acetone-conditioned, ethanol-conditioned, and as received—and were then immersed in 5 mM CHX-HMP suspension or 5 mM chlorhexidine digluconate solution and rinsed. CHX release was measured over 8 weeks, and the effects of conditioning and immersion on elastomeric force and extension at rupture and surface topography were investigated.
Results
CHX-HMP exhibited a gradual equilibration that had not reached equilibrium within 8 weeks, releasing soluble CHX and a mixture of polyphosphate and orthophosphate. CHX digluconate–treated ligatures showed no CHX release, whereas CHX-HMP–treated ligatures showed varying degrees of release. As received, CHX-HMP–treated ligatures showed a modest release of CHX up to 7 days. Acetone conditioning did not enhance CHX-HMP uptake or subsequent CHX release and caused a deterioration in mechanical properties. Ethanol conditioning enhanced CHX-HMP uptake (6×) and led to a sustained CHX release over 8 weeks without affecting mechanical properties.
Conclusions
Within the inherent limitations of this in-vitro study, CHX-HMP led to a sustained release of CHX from orthodontic elastomeric ligatures after ethanol conditioning. Conditioned and coated elastomeric ligatures may ultimately find application in the prevention of white spot lesions in orthodontic patients.
Highlights
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Elastomeric ligatures were coated with chlorhexidine hexametaphosphate (CHX-HMP).
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CHX-HMP–coated ligatures provide sustained release of ionic chlorhexidine.
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CHX-HMP uptake is enhanced by ethanol but not acetone conditioning.
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Ligatures release chlorhexidine continually over a period of 8 weeks.
White spot lesions (WSLs) are a common iatrogenic effect of orthodontic treatment- because orthodontic appliances compromise oral hygiene and promote plaque accumulation. Demineralization around orthodontic brackets and bands can be rapid, with WSLs developing after as little as 4 weeks of appliance placement. Although some remineralization usually occurs after orthodontic treatment is concluded, baseline pretreatment levels are never regained. Furthermore, orthodontic appliances can increase the proportions of pathogenic microbes, increasing the likelihood of caries developing. Figures quoted for WSLs in orthodontic patients vary widely, with incidences ranging from 25% to 73%, and these differences are most likely due to the inconsistent definition and reporting of WSLs rather than the necessarily genuine differences in occurrence.
Oral hygiene practices are accepted as the single most important factor in determining whether WSLs will develop, but this preventative method relies heavily on patient compliance. It is known that oral health and hygiene often deteriorate rapidly after a fixed orthodontic appliance is fitted, which is usually explained by the patient experiencing pain and becoming accustomed to their new appliance. Therefore, orthodontic biomaterials such as adhesives, bands, and brackets, which are inherently antimicrobial or anticariogenic, have been the subject of attention in the research community. Orthodontic ligatures are a potentially useful vector for localized antimicrobial delivery to prevent WSLs in orthodontic patients. The ligatures are in close proximity to the enamel surface and are regularly replaced during a course of orthodontic treatment. Sustained, appropriately dosed antimicrobial delivery could therefore reduce dependence on patient compliance. This is not a new concept. Elastomeric ligatures are easy to apply, cheap, and relatively hygienic, making them the most common form of orthodontic ligation. The ligatures used in orthodontics are usually made from latex or polyurethane and are replaced at each orthodontic appointment, owing to their limited resistance to the oral environment.
Chlorhexidine (CHX) is not commonly used as part of orthodontic treatment, although it is used widely in general dentistry. CHX is a cationic bisbiguanide with broad-spectrum antimicrobial activity, which is effective against a wide range of bacteria and yeasts. Being cationic, CHX is attracted to the negatively charged bacterial cell wall and binds to the inner membrane. This increases cell wall permeability, leading to loss of cell components, precipitation of the bacterial cytoplasm, and cell death. CHX is most commonly used as the digluconate salt (CHXdg), which is readily soluble in water, and therefore, convenient to formulate into mouth rinses and other aqueous topical agents.
A novel salt of CHX, CHX-hexametaphosphate (CHX-HMP), has been reported as a material that provides sustained release of the constituent CHX when exposed to an aqueous environment. Owing to the physical and chemical properties of this salt, it can be used as a component of composite materials. Provided the composite has a degree of water permeability, it can provide sustained release of CHX under aqueous conditions. The dose and duration of release are influenced by a number of factors such as doping, local physicochemical conditions (such as flow, temperature, ionic strength, and other ions), and host substrate. For instance, CHX release from glass ionomers doped with CHX-HMP can be sustained for over 2 years, which is substantially longer than in other studies of CHX–modified glass ionomer cements and related materials.
This study aimed to explore whether CHX-HMP might be a useful component of elastomeric ligatures that could provide sustained release of CHX over the usual 4-8-week period between orthodontic appointments and without adversely affecting pertinent mechanical and physical properties of the ligature.
Material and methods
Pure CHX-HMP powder used for characterization was supplied by Pertinax Pharma (Bristol, United Kingdom). Saturated CHX-HMP suspensions were prepared using 10 g/L CHX-HMP in deionized water (DIW). The suspensions were stored in 250 mL lidded glass Schott bottles (n = 3 for each of CHX and phosphate analysis) and incubated at 37°C with constant stirring for the duration of the experiment. The pH of the suspensions was measured at regular intervals. After centrifugation (21,000 g of force, 15 minutes), the resulting supernatants were isolated, diluted as required to cause the expected CHX or phosphate concentration to fall within the range of the calibration standards, and transferred to sealed containers for further use.
For CHX analysis, 1 mL of the supernatant was placed in lidded, semimicro polystyrene cuvettes, which were transparent in the ultraviolet (UV) for spectrophotometry at 255 nm. The reference was the calibration standards of aqueous CHXdg solutions (5-65 μM CHX) with the DIW background signal subtracted.
For phosphate analysis, 10 mL of supernatant was combined with a commercial phosphate analysis reagent mixture (ammonium molybdate, potassium antimonyl tartrate, potassium disulfate, and ascorbic acid; Hanna Instruments, Leighton Buzzard, United Kingdom). Measurements were also conducted using stock solutions of 25 μM sodium dihydrogen orthophosphate (Sigma Aldrich, Gillingham, United Kingdom) and 40 μM sodium HMP (NaHMP; Prayon, Lyon, France), in which the concentration of NaHMP was calculated using the average phosphate chain length given by the manufacturer (16.5). After mixing by agitation for approximately 5 seconds, the mixture was left for 2 minutes before transferring a 1.5 mL aliquot into a lidded, semimicro polystyrene cuvette. The reaction was monitored using absorbance values at 880 nm (λ max of the blue phosphomolybdate complex) with the DIW background signal subtracted at regular intervals over the course of 60 minutes.
For the purpose of functionalizing the ligatures, an aqueous suspension of CHX-HMP was prepared using the method described by Barbour et al, whereby 10 mM solutions of CHXdg and NaHMP (nominal HMP chain length 17; Sigma Aldrich, Gillingham, United Kingdom) were mixed by rapid stirring under atmospheric pressure at room temperature to yield a resultant equivalent CHX concentration, in CHX-HMP solid, of 5 mM. For comparison, a 5 mM aqueous solution of CHXdg was also prepared.
A total of 465 silver-colored polyurethane elastomeric ligatures with occlusal guards (AlastiK; 3M Unitek, Bracknell, United Kingdom) were rinsed in DIW and allowed to air dry for 1 hour before use. To test the effect of solvent conditioning on CHX-HMP release, the ligatures were either used in this as received but in a washed condition (stored in a dry environment) or were immersed in either ethanol or acetone for 60 minutes under agitation. Immediately after conditioning, the ligatures were then immersed in 1 of 3 solutions, namely: DIW, 5 mM CHXdg, or 5 mM CHX-HMP for 10 minutes under agitation. This was followed by a final immersion in DIW for 10 seconds to remove any unbound material and air drying for at least 1 hour before further use.
In this way, a total of 9 experimental groups of ligatures (n = 180) were prepared and were referred to as N, E, or A as prefix (none, ethanol, or acetone as the solvent conditioning) and -DIW, -CHXdg or -CHX-HMP (DIW, CHXdg, or CHX-HMP as the coating treatment) as the suffix. Therefore, as an example, N-DIW refers to no solvent conditioning and only DIW treatment, whereas E-CHX-HMP refers to ethanol conditioning and CHX-HMP treatment.
After functionalization, the ligatures (n = 20) were placed into individual UV-transparent cuvettes containing 2 mL DIW and sealed prior to chemical analysis. The release of CHX as a function of time was measured using UV spectrophotometry as previously described. Cumulative CHX release at the conclusion of the 8-week period was determined.
Physical analysis of ligatures was carried out by examining 2 samples from each group using scanning electron microscopy (Phenom, Eindhoven, The Netherlands). The effect of just the solvents (water, ethanol, and acetone) was also determined after drying for more than 24 hours to avoid transient effects (n = 5). Ligature width and lumen size were measured using light microscopy (magnification ×4) and Cellsens computer software (Olympus, KeyMed [Medical & Industrial Equipment] Ltd, Southend-on-Sea, UK).
To explore the effects of the treatments on the mechanical properties of the ligatures in the 9 groups, 30 specimens of each were tested using a universal testing machine (Zwick/Roell, Herefordshire, UK) with a 500 N load cell and a test rate of 100 mm per minute. Each ligature was mounted using a customised jig comprising two 0.6 mm stainless steel loops fastened to the clamps of the testing machine. Due to the asymmetric nature of the elastomeric ligatures under test, they were mounted with the occlusal guard facing in the same orientation.
Statistical analysis
The data were analyzed with a predetermined significance level of 0.05 using either a 2-way (CHX release and mechanical properties) or one way (physical properties) ANOVA and a Tukey HSD post-hoc test.
Results
The pH of the CHX-HMP suspensions varied between 5.7 and 6.0 over the period of measurement. There was no trend over time.
The aqueous CHX concentration in the CHX-HMP suspensions equilibrated in DIW as a function of incubation time as shown in Figure 1 . There was an initial period (<1 day) during which CHX was released rapidly, reaching a pseudoequilibrium concentration of 200 μM, followed by a period during which [CHX] the concentration changed little. After a lag of approximately 30 days, the CHX concentration increased steadily at approximately 7 μM/d for the remainder of the experiment.
Figure 2 shows an absorbance wavelength of 880 nm, the peak absorbance wavelength for the phosphomolybdate complex as a function of reaction time after mixing stock solutions of sodium dihydrogen orthophosphate and NaHMP with the phosphate assay reagents. The data are expressed as percent of the maximum absorbance reached over the 60-minute period. When only orthophosphate was present there was no change in absorbance over the 60-minute period ( Fig 2 , A ). When only polyphosphate was present, there was a material change of the absorbance at 880 nm by a factor of 2 ( Fig 2 , B ) and therefore in the concentration of the phosphomolybdate complex, over 60 minutes. This was attributed to hydrolysis of the polyphosphate to smaller phosphates, including orthophosphate, under the strongly acidic conditions of the phosphate assay. Therefore, when analyzing unknown phosphate solutions, a stable reading over the 60-minute period after combining the reagents indicates the presence of orthophosphate only, whereas an increasing reading is likely to indicate the presence of polyphosphate(s), possibly in combination with orthophosphate. Not all of the polyphosphate was hydrolyzed to orthophosphate in 60 minutes, and after longer periods, the phosphomolybdate complex was unstable and broke down. Therefore, this assay could not be used under these conditions to ascertain the concentration of polyphosphate in solution, only to infer its presence or absence.
