In vitro and in vivo evaluations of glass-ionomer cement containing chlorhexidine for Atraumatic Restorative Treatment

Abstract Objectives: Addition of chlorhexidine has enhanced the antimicrobial effect of glass ionomer cement (GIC) indicated to Atraumatic Restorative Treatment (ART); however, the impact of this mixture on the properties of these materials and on the longevity of restorations must be investigated. The aim of this study was to evaluate the effects of incorporating chlorhexidine (CHX) in the in vitro biological and chemical-mechanical properties of GIC and in vivo clinical/ microbiological follow-up of the ART with GIC containing or not CHX. Material and Methods: For in vitro studies, groups were divided into GIC, GIC with 1.25% CHX, and GIC with 2.5% CHX. Antimicrobial activity of GIC was analyzed using agar diffusion and anti-biofilm assays. Cytotoxic effects, compressive tensile strength, microhardness and fluoride (F) release were also evaluated. A randomized controlled trial was conducted on 36 children that received ART either with GIC or GIC with CHX. Saliva and biofilm were collected for mutans streptococci (MS) counts and the survival rate of restorations was checked after 7 days, 3 months and one year after ART. ANOVA/Tukey or Kruskal-Wallis/ Mann-Whitney tests were performed for in vitro tests and in vivo microbiological analysis. The Kaplan-Meier method and Log rank tests were applied to estimate survival percentages of restorations (p<0.05). Results: Incorporation of 1.25% and 2.5% CHX improved the antimicrobial/anti-biofilm activity of GIC, without affecting F release and mechanical characteristics, but 2.5% CHX was cytotoxic. Survival rate of restorations using GIC with 1.25% CHX was similar to GIC. A significant reduction of MS levels was observed for KM+CHX group in children saliva and biofilm 7 days after treatment. Conclusions: The incorporation of 1.25% CHX increased the in vitro antimicrobial activity, without changing chemical-mechanical properties of GIC and odontoblast-like cell viability. This combination improved the in vivo short-term microbiological effect without affecting clinical performance of ART restorations.


Introduction
Early childhood caries (ECC), mainly in developing countries, is the most prevalent chronic disease in childhood and, consequently, a pending public health problem 6 . Depending on the severity of ECC and the number of dental sources of infection, this disease causes functional, aesthetic and psychosocial disorders that reduce the quality of life of children and their families 6 . The decay process of ECC generally tends repair the longer it remains untreated. An alternative for the treatment of ECC is the Atraumatic Restorative Treatment (ART). ART is a definitive restorative treatment which consists of removing demineralized tooth tissues using minimal intervention to preserve the tooth structure and restoring the dental cavity with glass ionomer cement (GIC) 9 . The correct execution of ART procedures may change the balance of the oral microbiota, reducing cariogenic microorganisms 7 .
This factor is relevant, because children affected by ECC have high counts of cariogenic bacteria in saliva, such as mutans streptococci and lactobacilli, and other species such as Candida albicans 3  One clinical study evaluated the long-term outcome of ART using glass ionomer cement containing CHX 15 . Therefore, the objectives of this study were 1) to evaluate the in vitro concentrations of CHX on biological and physicalchemical properties of a GIC and 2) to investigate in 4 mm diameter) and individually suspended in 24-well plates (Corning Inc., New York, NY, USA) containing 2 mL of BHI broth supplemented with 1% sucrose and 2 μl of inoculum. The plates were incubated in 5% CO 2 at 37°C for 24 h. After this period, GIC samples were washed, immerged in 500μl of 0.9% NaCl solution and sonicated in an ultrasonic cell disruptor at 7 W . This solution was diluted and plated on BHI agar and incubated for 48 h at 37°C. Then, bacterial colonies were counted and expressed in colonies forming units/ mL (CFU/mL). Three independent assays (n=15) were performed for the analysis.

Cytotoxicity assays
These assays were conducted in accordance with Castilho, et al. 5 MDPC-23 odontoblastlike cells were used. The cells were seeded (30,000 cells/cm 2 /well) in sterile 24-well plates and maintained 2 and 95% air at 37°C PA, USA). Ten round-shaped samples of each group (2x4 mm) were prepared in stainless-steel molds, light-cured for 30 s and maintained for 1 h at 37°C in relative humidity. The specimens were then inserted into sterile 24-well plates containing DMEM h. After that, 800 μL of the extract from each well was applied to previously cultured MDPC-23 cells for 24 h. Cell metabolism was analyzed using methyl tetrazolium (MTT) assays. The means were calculated for the groups and transformed into percentages, and metabolism.

Measurement of mechanical properties
Compressive tensile strength and microhardness tests 5 Ten specimens from each group were prepared in cylindrical molds for compressive strength (4x2 mm) and surface microhardness tests (3x6 mm).

Study design
The present study was designed as a randomized controlled clinical trial with parallel groups. One hundred and tirty six three to six-year-old children from four public primary schools of Nova Friburgo (Rio de Janeiro, Brazil) whose parents signed a written consent were examined for dental caries status using the criteria developed by the WHO. Inclusion criteria were (1) good general health; (2) cooperative behavior; (3) at least one cavitated dentin carious lesion (occlusal or occluso-proximal cavities) in primary molars that had an opening wide enough for the smallest ART were children with mixed dentition, teeth with pulpal history of sensitivity and/or spontaneously pain. The dentist gave instructions to caregivers for children not to eat solid food for one hour.            time, but remained measurable after 60 days 11 . It was speculated that it is an interaction between the the precipitation of salts with lower solubility, leaving 11 .
Biocompatibility is a property required for GICs, since these materials are usually applied in deep dentin and could release toxic components, which might indirectly affect the dental pulp 5 However, in contrast to our results, after 9 months the restoration success with GIC containing CHX (60%) was lower than the control group (85%) 14  year of restoration, the experimental group did not to the control group. In a clinical trial study with a chlorhexidine digluconate, it was found that the antibacterial action of the material on residual dentin lasts up to 90 days after the restorative procedure 5 .
In vivo addition of 1% chlorhexidine diacetate to GIC showed comparable results to conventional GIC with regard to microleakage 21 . Differences in the selection of materials, sampling procedures and local of CHX action could explain the controversial results of GIC containing CHX. In this study, we used a conventional high viscosity GIC that may easily release may keep the same product for long time in the matrix, delaying its release. Furthermore, in this study, GIC was exposed to oral environment and it was subject to tooth abrasion that probably accelerated the chlorhexidine release.
The results of this study should be analyzed considering possible methodological limitations. One of them is the dropout rate, approximately 36%, that