the influence of xylitol on the control of risk factors and prevention of dental caries 
 has already been assessed in several studies .
the outcomes of this strategy seem to depend on the detection of 
 minimal salivary levels of that polyol along time .
however , the rapid clearance of xylitol from the oral cavity can 
 explain the short - term increase of salivary xylitol concentrations after the use of 
 vehicles such as candies , chewing gums , dentifrices and solutions .
high frequencies of intake of xylitol have been employed in most of the studies so that 
 its anticariogenic effects could be observed in clinical trials . however , these protocols could cause discomfort to the 
 patient and strongly rely on compliance , which turns difficult the use of xylitol in daily clinical 
 practice .
thus , the need of vehicles that could allow sustained xylitol release along 
 time has been recognized .
fluoride , chlorhexidine , copper and iron have been shown to be successfully delivered when incorporated into 
 dental varnishes because their adherence to tooth surface leads to longer maintenance of 
 these different agents in the oral cavity .
however , to our knowledge , no attempt was 
 made to include xylitol into a varnish formulation so far .
hence , the present in 
 vitro study aimed at analyzing the xylitol concentrations in artificial 
 saliva along time after application of varnishes containing 10% and 20% xylitol .
enamel specimens were obtained from lower bovine incisors . after visual inspection , 
 stained and/or cracked teeth were excluded . besides , soft tissues were removed from 
 the coronal and root surfaces with the aid of a periodontal curette 
 ( duflex , sswhite , rio de janeiro , rj , brazil ) .
fifteen enamel 
 specimens ( 8x4 mm ) were obtained after two double sections of the widest portion of 
 the dental crowns , as described by magalhes , et al.19 ( 2008 ) . 

subsequently , the specimens were numbered and randomly allocated to 3 different 
 groups ( n=5/group ) , according to the type of varnish that would be applied : ( 1 ) 10% 
 xylitol ( fgm / dentscare , joinville , sc , brazil ) ; ( 2 ) 20% xylitol ( fgm / dentscare ) and 
 ( 3 ) no xylitol ( control ; fgm / dentscare ) .
three different varnishes ( control , containing 10% and 20% xylitol ) were especially 
 manufactured by fgm / dentscare for the present research .
xylitol concentrations were 
 determined by the maximum incorporation of that polyol into the varnish that would 
 not lead to precipitation .
varnishes used contain colophonium , synthetic resin , 
 thickening polymer , essence and ethanol in their composition ( informed by 
 manufacturer ) .
xylitol was supplied by danisco ( xylitab 300 , danisco 
 brasil ltda , cotia , sp , brazil ) .
enamel specimens were weighed before and after varnish application to standardize the 
 amount used . after a pilot study ,
4 mg of the respective varnish were applied on a 
 dry bovine enamel specimen with a microbrush .
it was the maximum amount that could be 
 applied considering the area of the specimens .
after 10 min , each specimen was 
 inserted into a microcentrifuge tube containing 500 l of artificial saliva ( 1.5 
 mmol / l ca(no3)2.h2o ; 0.9 mmol / l 
 na2hpo4.2h2o ; 150 mmol / l kcl ; 0.1
mol / l 
 h2nc(ch2oh)3 ( tris ) ; 0.05 g / ml naf , ph 
 7.0 ) . sample size and times of immersion of the specimens in artificial saliva were chosen 
 based on results of a pilot study ( data not shown ) .
after 1 , 8 , 12 , 16 , 24 , 48 and 72 
 h from the first immersion , each specimen was removed , washed with deionized water 
 and placed into another microcentrifuge tube containing 500 l of fresh artificial 
 saliva at room temperature .
the analysis of xylitol concentrations in artificial saliva was performed by a 
 colorimetric method , using a spectrophotometer ( ultrospec 2000 uv / visible 
 spectrophotometer , pharmacia biotech , cambridge , usa ) and an enzymatic kit 
 d - sorbitol / xylitol ( boehringer mannheim , r - biopharm , darmstadt , germany ) .
a standard 
 curve was obtained in triplicate using five different amounts of xylitol ( 0.5 , 2.0 , 
 5.0 , 8.0 and 10 g ) .
a mathematical equation 
 determined by the manufacturer of the enzymatic kit was used to convert the values of 
 absorbance into xylitol concentration ( mg / l ) .
all samples were analyzed in duplicate 
 and the mean repeatability of the readings was 98.6% .
graph pad instat version 3.0 for windows and graph pad prism version 4.0 for windows 
 ( graph pad software inc .
, san diego , usa ) were used . since data presented a normal 
 distribution ( kolmogorov - smirnov test ) and homogeneity ( bartlett test ) ,
they were 
 analyzed by two - way repeated - measures anova followed by bonferroni 's post 
 hoc test for individual comparisons .
enamel specimens were obtained from lower bovine incisors . after visual inspection , 
 stained and/or cracked teeth were excluded . besides , soft tissues were removed from 
 the coronal and root surfaces with the aid of a periodontal curette 
 ( duflex , sswhite , rio de janeiro , rj , brazil ) .
fifteen enamel 
 specimens ( 8x4 mm ) were obtained after two double sections of the widest portion of 
 the dental crowns , as described by magalhes , et al.19 ( 2008 ) . 

subsequently , the specimens were numbered and randomly allocated to 3 different 
 groups ( n=5/group ) , according to the type of varnish that would be applied : ( 1 ) 10% 
 xylitol ( fgm / dentscare , joinville , sc , brazil ) ; ( 2 ) 20% xylitol ( fgm / dentscare ) and 
 ( 3 ) no xylitol ( control ; fgm / dentscare ) .
three different varnishes ( control , containing 10% and 20% xylitol ) were especially 
 manufactured by fgm / dentscare for the present research .
xylitol concentrations were 
 determined by the maximum incorporation of that polyol into the varnish that would 
 not lead to precipitation .
varnishes used contain colophonium , synthetic resin , 
 thickening polymer , essence and ethanol in their composition ( informed by 
 manufacturer ) .
xylitol was supplied by danisco ( xylitab 300 , danisco 
 brasil ltda , cotia , sp , brazil ) .
enamel specimens were weighed before and after varnish application to standardize the 
 amount used . after a pilot study ,
4 mg of the respective varnish were applied on a 
 dry bovine enamel specimen with a microbrush .
it was the maximum amount that could be 
 applied considering the area of the specimens . after 10 min , each specimen was 
 inserted into a microcentrifuge tube containing 500 l of artificial saliva ( 1.5 
 mmol / l ca(no3)2.h2o ; 0.9 mmol / l 
 na2hpo4.2h2o ; 150 mmol / l kcl ; 0.1 mol / l 
 h2nc(ch2oh)3 ( tris ) ; 0.05 g / ml naf , ph 
 7.0 ) .
sample size and times of immersion of the specimens in artificial saliva were chosen 
 based on results of a pilot study ( data not shown ) .
after 1 , 8 , 12 , 16 , 24 , 48 and 72 
 h from the first immersion , each specimen was removed , washed with deionized water 
 and placed into another microcentrifuge tube containing 500 l of fresh artificial 
 saliva at room temperature .
the analysis of xylitol concentrations in artificial saliva was performed by a 
 colorimetric method , using a spectrophotometer ( ultrospec 2000 uv / visible 
 spectrophotometer , pharmacia biotech , cambridge , usa ) and an enzymatic kit 
 d - sorbitol / xylitol ( boehringer mannheim , r - biopharm , darmstadt , germany ) .
a standard 
 curve was obtained in triplicate using five different amounts of xylitol ( 0.5 , 2.0 , 
 5.0 , 8.0 and 10 g ) .
a mathematical equation 
 determined by the manufacturer of the enzymatic kit was used to convert the values of 
 absorbance into xylitol concentration ( mg / l ) .
all samples were analyzed in duplicate 
 and the mean repeatability of the readings was 98.6% .
graph pad instat version 3.0 for windows and graph pad prism version 4.0 for windows 
 ( graph pad software inc . , san diego , usa ) were used . since data presented a normal 
 distribution ( kolmogorov - smirnov test ) and homogeneity ( bartlett test ) , they were 
 analyzed by two - way repeated - measures anova followed by bonferroni 's post 
 hoc test for individual comparisons .
regardless of the time of immersion , xylitol could not be detected in artificial saliva 
 samples of the control group .
consequently , data obtained only after application of 
 xylitol - containing varnishes are presented .
mean xylitol concentrations in artificial saliva ranged between 63.42.7 mg / l ( 1 h ) and 
 32.70.9 mg /
l ( 72 h ) after application of 10% xylitol varnish and between 169.01.5 mg / l 
 ( 1 h ) and 12.70.4 mg / l ( 72 h ) after application of 20% xylitol varnish ( figure 1 ) .
significant differences were detected for 
 the variables varnishes ( f=33 , p=0.0004 ) and times of immersion 
 in saliva ( f=2,466 , p<0.0001 ) , as well as a significant interaction 
 between these criteria ( f=1,486 , p<0.0001 ) .
mean accumulated xylitol concentrations ( mg / l ) in artificial saliva over time 
 after application of 10% and 20% xylitol varnishes .
distinct lowercase letters indicate significant differences among time points for 
 each varnish ( two - way rm anova , p<0.05 ) in overview , a reduction of xylitol concentration was observed along time for both 
 varnishes , except for 48 h after immersion in artificial saliva .
nevertheless , a greater 
 reduction could be noticed after application of 20% xylitol varnish than after 
 application of 10% xylitol varnish ( figure 1 ) . in 
 the short - term ( up to 8 h ) ,
xylitol concentrations in saliva were significantly higher 
 after application of 20% xylitol varnish in comparison to its counterpart ( p<0.001 ) . 

this relationship was inverted after 12 h from application of the xylitol varnish during 
 the remaining time ( p<0.001 ) .
when the use of xylitol is aimed to prevent dental caries , important parameters such as 
 frequency of administration , dose - response , and minimal salivary concentration must be 
 regarded . however , such clinical conditions have not been established yet .
the amount of 5 g of xylitol , divided in at least 3 
 times per day , is supported for a considerable number of studies as the 
 minimum clinical effective dose to achieve prevention of dental caries . on the other 
 hand ,
it has been recently 
 reported that 0.01% xylitol was able to inhibit in vitro growth of 
 mutans streptococci , but the extent to which this information can be transferred to the 
 clinical situation demands further investigation .
randomized clinical trials normally use daily xylitol amounts that vary between 2 and 15 
 g , divided in 3 to 5 times per day , delivered mainly by chewing 
 gums
it is desirable to 
 develop new vehicles able to promote a sustained release of xylitol in saliva along 
 time .
hence , xylitol - containing varnishes were formulated and tested in the present 
 study .
dental varnishes can be used in lower frequencies and mastication is not required 
 for their effectiveness which improves patient 's compliance .
additionally , the varnishes 
 were manufactured with a fruit flavor to make them more suitable for babies and young 
 children , compared to chewing gums or solutions .
it also has been recognized that the frequency of use of xylitol seems to be more 
 important in the prevention of dental caries than the amount of that polyol .
xylitol is rapidly 
 removed from the oral cavity after application of the vehicles commonly available .
( 2006 ) observed 
 that salivary xylitol levels can be significantly increased up to 16 min by mastication 
 of chewing gums containing 1.32 g of xylitol or by mouth rinsing with 10% xylitol 
 solution .
tablets , candies and dentifrices were able to enhance salivary levels of that 
 polyol up to 8 min after be used .
this pioneer study evaluated the potential of a newly developed varnish to release 
 xylitol in a medium that resembles the composition of natural saliva along time . 

therefore , a positive control group with xylitol delivered via another vehicle , such as 
 chewing gum , was not included .
bovine enamel specimens were immersed in artificial 
 saliva and they were kept under gentle agitation to simulate the movements that occur in 
 the mouth .
conditions as salivary flow and temperature of the oral cavity were not 
 simulated . in this study
, the amount of varnish ( 4 mg ) applied on each specimen might contain 40 
 and 80 mg of xylitol in 10% and 20% varnishes , respectively , which is very low when 
 compared to that is recommended for prevention of dental caries .
however , the volume of 
 varnish usually applied in the whole mouth is around 0.2 ml ( ~200 mg ) , nearly 50 times 
 larger than that used in the present research .
consequently , it could be clinically 
 expected that around 2 and 4 g of xylitol would be found in the oral cavity after 
 applying respectively 10% and 20% xylitol varnishes .
higher concentrations of 20% 
 xylitol could not be incorporated into the varnishes without precipitation .
regarding safety considerations , high doses of xylitol ingested by adults and children 
 can cause adverse gastrointestinal effects .
adults can tolerate the intake of up to 200 g of xylitol per 
 day , while children can ingest up to 45 g per day .
then , even 
 if the xylitol varnishes would be applied on all dental surfaces , the amount of xylitol 
 that could be ingested is completely safe .
we believe that more homogeneous varnishes might provide more sustained xylitol release 
 along time and , perhaps , this hypothesis could explain the present results . 

significantly higher xylitol concentrations were released in saliva after application of 
 the 20% xylitol varnish than of the 10% xylitol varnish on a short - term basis ( up to 8 
 h ) .
thereafter , this trend was inverted from 12 h until the end of the experimental 
 period ( 72 h ) .
therefore , the 10% xylitol varnish was able to produce sustained degrees 
 of xylitol release along time .
however , both varnishes were not removed from the dental 
 surfaces throughout the time . this situation is not observed under clinical conditions , 
 when dental varnishes are usually removed from tooth surfaces after 6 to 12 h from 
 application .
taking this into account , a better clinical performance of the 20% xylitol 
 varnish might be expected in relation to its 10% counterpart . before any speculative 
 conclusion , the performance of these varnishes needs to be tested in 
 vivo regarding xylitol release and their caries - preventive effect .
considering the limitations of this pioneer in vitro study , it can be 
 concluded that sustained xylitol releases can be obtained in artificial saliva after 
 application of 10% xylitol varnish , although 20% xylitol varnish released larger amounts 
 of that polyol in the short - term .
these varnishes seem to be viable alternatives to 
 increase salivary xylitol levels and , therefore , they should be clinically tested to 
 confirm their effectiveness .
 objective the present study analyzed xylitol concentrations in artificial saliva over time 
 after application of varnishes containing 10% and 20% xylitol . 
 material and methods fifteen bovine enamel specimens ( 8x4 mm ) were randomly allocated to 3 groups 
 ( n=5/group ) , according to the type of varnish used : 10% xylitol , 20% xylitol and 
 no xylitol ( control ) . after varnish application ( 4 mg ) 
 , specimens were immersed in 
 vials containing 500 l of artificial saliva . 
 saliva samples were collected in 
 different times ( 1 , 8 , 12 , 16 , 24 , 48 and 72 h ) and xylitol concentrations were 
 analyzed . 
 data were assessed by two - way repeated - measures anova ( p<0.05 ) . 
 results colorimetric analysis was not able to detect xylitol in saliva samples of the 
 control group . 
 salivary xylitol concentrations were significantly higher up to 8 h 
 after application of the 20% xylitol varnish . 
 thereafter , the 10% xylitol varnish 
 released larger amounts of that polyol in artificial saliva . 
 conclusions despite the results in short - term , sustained xylitol releases could be obtained 
 when the 10% xylitol varnish was used . 
 these varnishes seem to be viable 
 alternatives to increase salivary xylitol levels , and therefore , should be 
 clinically tested to confirm their effectiveness . 
INTRODUCTION
MATERIAL AND METHODS
Preparation of bovine enamel specimens
Varnish application
Times of immersion in artificial saliva
Analysis of xylitol in artificial saliva
Statistical analysis
RESULTS
DISCUSSION
CONCLUSIONS