Doxycycline-containing glass ionomer cement for arresting residual caries: an in vitro study and a pilot trial

Abstract In a previous study, we demonstrated that the incorporation of doxycycline hyclate (DOX) into resin-modified glass ionomer cement (RMGIC) inhibited important cariogenic microorganisms, without modifying its biological and mechanical characteristics. In this study, we keep focused on the effect of that experimental material as a potential therapy for arresting residual caries by analyzing other in vitro properties and conducting a pilot clinical trial assessing the in vivo effect of DOX-containing RMGIC on residual mutans streptococci after partial carious removal in primary molars. Specimens of the groups RMGIC (control); RMGIC + 1.5% DOX; RMGIC + 3% DOX; and RMGIC + 4.5% DOX were made to evaluate the effect of DOX incorporation on surface microhardness and fluoride release of RMGIC and against biofilm of Streptococcus mutans. Clinical intervention consisted of partial caries removal comparing RMGIC and RMGIC + 4.5% DOX as lining materials. After 3 months, clinical and microbiologic evaluations were performed. Data were submitted to ANOVA/Tukey or Wilcoxon/Mann-Whitney set as α=0.05. Fluoride release and surface microhardness was not influenced by the incorporation of DOX (p>0.05). There was a significant reduction of S. mutans biofilm over the material surface with the increase of DOX concentration. After clinical trial, the remaining dentin was hard and dry. Additionally, mutans streptococci were completely eliminated after 3 months of treatment with RMGIC + 4.5% DOX. The incorporation of DOX provided better antibiofilm effect, without jeopardizing fluoride release and surface microhardness of RMGIC. This combination also improved the in vivo shortterm microbiological effect of RMGIC after partial caries removal.


Introduction
In developing countries, the prevalence of untreated dental caries is very high. In Brazil, the report from the Brazilian Oral Health Survey (SBBrasil 2010) showed that 48.2% of 5 years-old children have untreated dental caries or almost more than half of the population in this age group had at least one deciduous tooth affected by dental caries 1 . The reasons for the lack of treatment are multiple, such as financial problems, lack of oral health services, pain and fear barriers, and dependency of conventional oral care 2 .
Alternative proposals to the complete caries removal technique have been investigated for permanent and deciduous teeth 3 . Scientific evidences have revealed that incomplete caries removal for teeth with deep cavities preserves a maximum of dental structure and reduces the possibility of pulp exposure [3][4] . Besides being clinically effective in retaining teeth and their vitality for longer, partial caries removal was also considered cost-effective, with significantly reduced long-term costs 5 .
Although encouraging clinical success rates have been reported in several studies [6][7][8][9][10][11] and stated in some systematic reviews and meta-analysis 2,12 , there is still some resistance whether intentionally leaving carious dentin behind is safe, which could cause the failure of the dental treatment. Therefore, by using an effective antibacterial incorporated with lining material it must be possible to solve problems such as the cariogenic microorganisms remained after partial caries removal and consequently avoid caries progression and injuries to the pulp with no costs and less discomfort to the patient [13][14][15][16][17][18] . Even the literature indicates to leave residual carious dentin; the use of an antibacterial material over dentin layer would leave dentists more comfortable.
Doxycycline hyclate (DOX), a derivative of the tetracycline family, has been widely used in the treatment of various infectious diseases in Dentistry, including periodontal and root pathologies 19,20 . Based on study conducted thus far, we support the efficacy of a resin-modified glass-ionomer cement liner containing up to 4.5% DOX against selected cariogenic microorganisms, keeping original biological and mechanical characteristics of the cement 16 . Clearly, DOX exerts biological functions totally independent of its antibacterial properties, since this chemical agent blocks dentin proteolysis 21 . This is an interesting property for a substance, considering that caries progression is not only dependent on the bacterial activity, but is also related to the release of dentin matrix metalloproteinases 22 .
Our research group has focused on therapeutic approaches for caries using agents with specific activities against Streptococcus mutans. Therefore, based on early positive results concerning the mechanical properties of the glass ionomer cement and DOX association 16 developed by the same research group, this study intended to provide more information about physical, antibacterial, and clinical properties of that experimental liner material. Then, we aimed to compare the in vitro microhardness, fluoride release, and antibiofilm properties of a DOX-containing RMGIC, besides to analyze the in vivo efficacy of that material on residual mutans streptococci after partial carious removal in primary molars.

Specimen preparation for in vitro assays
Specimens from three experimental groups (RMGIC-containing 1.5, 3 or 4.5% DOX) and one control group (RMGIC) were prepared for surface microhardness (n=9), fluoride release (n=9), and antimicrobial properties tests (n=9). RMGIC was mixed by agglutination of powder to liquid associated with or without 1.5, 3.0, and 4.5% DOX and then the mixture was placed with Centrix syringe (Centrix Inc., Shelton, USA) into cylindrical moulds (2 mm high x 5 mm diameter) 16 . The specimens were then exposed to a light source (Curing Light XL3000, 3M ESPE), with 410 mW/cm 2 of light intensity for 30 s. Finally, they were stored in distilled water for 24 h at 37ºC before being tested.

Biofilm assays
A new proposal of biofilm assay was used in this

Clinical recordings
Before all dentin collections, the dental cavities were copiously washed and carefully air dried, and the color, consistency, and wetness of the carious dentin were blindly evaluated by a second investigator (CD), based on the following criteria: dentin consistency: 0

Surface Hardness
Fluoride Release (mg F-/mL) Log (UFC/mL) (N)     Maltz, et al. 29 (2002) after the experimental period of 6 months, when all dentin samples exhibited no growth of S. mutans or lactobacilli after partial caries removal using a calcium hydroxide liner. Other studies also had encouraging results, testing the antibiotic mixture (metronidazole, ciprofloxacin, and cefaclor) added to GIC after partial caries removal.
They evidenced more than a 98% mutans streptococci reduction 13 and enhanced clinical/radiographic success after 1, 3, 6, and 12 months of partial caries removal procedures when compared to control without antibiotics. 17 In this study, the clinical effectiveness of DOX