several antimicrobial agents have been incorporated in mouthrinses to improve the 
 outcome of mechanical oral hygiene procedures or even to replace mechanical plaque 
 control .
chlorhexidine gluconate ( chx ) is presently the most effective compound having 
 pronounced antimicrobial effects both on gram - positive and gram - negative bacteria as 
 well as on fungi and some viruses .
chx is a positively charged 
 bisbiguanide , which can adsorb to different negatively charged sites including mucous 
 membranes , salivary pellicle on teeth and titanium implant surfaces as well as several 
 components of the biofilm on the tooth surfaces such as bacteria , extracellular 
 polysaccharides and glycoproteins .
however , it has not been recommended for long periods because of 
 the observed side effects .
the reported side effects of cxh are alteration in taste , increase of calculus 
 formation , staining of teeth and mucous membranes and , more rarely , oral mucosa 
 desquamation and parotid swelling . however , the most obvious and important local side effects are the 
 brown staining of the teeth , restorative materials and dorsum of the tongue 
 as well as supragingival calculus formation . non - enzymatic browning ( maillard reactions ) and formation of pigmented metal sulfides 
 are considered the possible mechanisms of tooth discolorations due to chx 
 application . however , clinical and laboratory studies provided 
 strong evidence that staining is caused by interaction or precipitation of dietary 
 chromogens with locally adsorbed cxh . in the presence of food components ,
moreover , when 
 associated with tea and coffee intake , brown staining of tooth and acrylic previously 
 exposed to cxh is more likely to occur . corroborating these findings , addy , et al .
( 1979 ) demonstrated that tea , coffee and red wine were 
 particularly chromogenic .
cxh has proven in many clinical trials to be effective in reducing the formation of 
 dental plaque and preventing gingivitis .
all previous studies 
 reporting chx anti - plaque efficacy and related side effects utilized a previous 
 disruption of present biofilms in all dental surfaces . however , there is a lack of 
 evidence if the presence of biofilm formed on tooth surfaces would increase the side 
 effects of chx .
zanatta , antoniazzi and rsing ( 2007 ) demonstrated that 0.12% chx mouthrinses showed 
 lower anti - plaque and anti - gingivitis effects over previously plaque - covered surfaces 
 compared to plaque - free surfaces .
thus , the working hypothesis of this trial is that the 
 presence of plaque when starting chx rinsing as a sole measurement for plaque control 
 would increase staining and calculus formation
the presence of plaque could be one predisposing factor to 
 staining , since its components would facilitate staining .
the aim of the present study was to compare the effects of 0.12% cxh gluconate on 
 staining and calculus formation in previously plaque - free and plaque covered surfaces , 
 by means of an experimental gingivitis clinical trial .
the study protocol was 
 subjected to and approved by the ethics committee of the lutheran university of brazil , 
 canoas , rs ( cep - ulbra 2006 - 095h ) .
the test panel was recruited from the dental students of the lutheran university of 
 brazil , canoas , rs , brazil . in order to assess the power of the present study , using 
 the power calculation with data from mean calculus formation in the present study at 
 day 25 , taking into consideration the paired design , a mean difference of 0.20 and 
 accepting an -error of 0.05 , revealed a power of 0.85 .
were asked about their medical and dental histories . written and oral explanations 
 detailing the study purpose and design were given for each subject .
subjects that 
 preliminarily met inclusion / exclusion criteria were selected for a dental screening 
 appointment . if the subject met all the inclusion / exclusion criteria , an informed 
 consent was handed out and , upon acceptance , signed by the volunteers . - age between 18 - 35 years ; - male , in order to avoid hormonal influences during the experimental period , 
 especially concerning gingival inflammation ; - no relevant medical conditions that could interfere on the periodontal health ; - willingness to comply . - probing pocket depth > 3 mm and/or clinical attachment loss > 2 mm at any 
 site ; - antibiotic and/or anti - inflammatory therapy within 3 months prior to baseline 
 examination ; - oral mucosal lesions ; - need for antibiotic premedication ; - history of hypersensitivity to chx ; - any condition device that could act as plaque retentive factor ( carious lesions ; 
 inadequate restorations ; dental implants ; orthodontic appliances ; fixed or removable 
 prostheses ) .
the following clinical parameters were assessed in the order listed below from all 
 teeth , excepting the third higher / lower molar . - presence of calculus ( c ) in all teeth , at 6 sites per tooth ( mesiobuccal , 
 mid - buccal , distobuccal , mesiolingual , mid - lingual and distolingual ) was scored as a 
 dichotomous index : 0 - absence of calculus ; 1 - presence of calculus .
- discoloration index proposed by lobene ( 1968 ) and modified by macpherson , et al .
this involves visual stain 
 assessment of the buccal / labial and lingual / palatal aspects of the index teeth .
the 
 modification consisted of dividing each aspect into 4 separate sites ( figure 1 ) , classified as : - gingival ( g ) : 2 mm 
 wide strip running parallel to the gingival margin .
the limit towards the incisal 
 edge given by the end of the interdental papilla ; - body ( b ) : central area of 
 buccal / lingual aspect , between gingival and distal / mesial sites , extending to incisal 
 edge ; - mesial ( m ) : visible area between line angle and adjacent tooth , ending at the 
 interdental papilla and starting at gingival site ; - distal ( d ) : as for mesial ( m ) 
 site .
the stain sites of an anterior tooth : body ( white ) ; gingival ( grey ) ; mesial and 
 distal ( black ) stain was recorded using two separate characteristics , namely intensity and area 
 ( extent ) .
the criteria and codes for intensity were : 0= no stain present with the 
 natural tooth coloration ; 1= slim stain ; 2= clearly visible stain , orange to brown ; 
 3= dark stain , dark brown to black .
the area ( extent ) of the stain was recorded only if an intensity score of 2 or 3 was 
 given .
the area criteria and codes for proximal , gingival and body sites were : 1= up 
 to 1/3 of area affected ; 2= between 1/3 and 2/3 of area affected ; 3= more than 2/3 of 
 area affected . before starting the trial , multiple sessions of training exercises for clinical 
 parameters were performed . after the examiner was trained ,
the intra - examiner 
 reproducibility was assessed . for reproducibility , 4 chronic chx users , who were not 
 included in the study population ,
subjects 
 were evaluated two times with one day intermission . in each session , the examiner 
 scored all teeth , except the third molars .
the results of presence / absence of 
 calculus and stain index were 
 analyzed by kappa - statistics .
the k - coefficient was 0.96 and 0.68 for calculus and 
 stain indexes , respectively .
the outline of the experimental procedures is summarized in figure 2 . to achieve optimal gingival health and to standardize gingival baseline conditions , 
 all subjects participated in a pretrial period during 14 days .
thorough professional 
 scaling and polishing with a rubber cup and prophylactic paste and dental floss was 
 performed . if hygiene was judged unsatisfactory , individual instructions were given 
 on how to improve the performance .
- baseline : on day zero , the presence of calculus and the stain index were recorded 
 in all subjects . after the recordings , a prophylaxis was performed and detailed 
 explanations to discontinue all the plaque control measurements were provided 
 again . - day 4 : each subject had one upper and one lower quadrant randomly assigned by a 
 heads or tails raffle made by the examiner , as " test " ( supragingival plaque - covered 
 sites ) and the other two quadrants as " controls " ( supragingival plaque was 
 professionally removed by the examiner to warrant plaque - free surfaces ) .
after the 
 prophylaxis on control quadrants , the quigley & hein plaque index was recorded in 
 all teeth to ensure that control quadrants were plaque - free .
following , all subjects 
 continued the oral hygiene withdrawal for a 21 days and started to rinse twice daily 
 with 15 ml of a mouthrinse containing 0.12% chx ( noplak , daudt , so paulo , sp , 
 brazil ) . - day 11 and 18 : presence / absence of calculus and stain index was recorded in all 
 teeth .
after the recordings , a professional scaling and 
 prophylaxis was performed to remove stain and calculus and all subjects were free to 
 return the normal habits of mechanical plaque control .
, the examiner was kept unaware of randomization sequence and 
 blinded to which quadrant was test or control . however , because of the nature of the 
 study , it is not possible to assume that the examiner had not become aware of 
 quadrant allocation because plaque accumulation was clinically different between test 
 and control quadrants essentially on days 11 and 18 . whether this problem may have 
 biased
the necessary volume of chx to all the experimental period was 630 ml ( 15 ml- 12/12 
 h ) .
however , to ensure compliance , more than 630 ml of chx solution was given in 
 flasks with 1 l volume , with different volumes in each flask . at the end of the 
 experiment ,
data analysis was performed using commands that take into account clustering of 
 observations within subjects ( stata 9.2 for windows , stata corporation , college 
 station , tx , usa ) .
a robust variance estimator was used to adjust for the clustering 
 of teeth into individuals .
wald tests were used for comparisons , and the p - value was 
 adjusted for multiple comparisons .
the test panel was recruited from the dental students of the lutheran university of 
 brazil , canoas , rs , brazil . in order to assess the power of the present study , using 
 the power calculation with data from mean calculus formation in the present study at 
 day 25 , taking into consideration the paired design , a mean difference of 0.20 and 
 accepting an -error of 0.05 , revealed a power of 0.85 .
were asked about their medical and dental histories . written and oral explanations 
 detailing the study purpose and design were given for each subject .
subjects that 
 preliminarily met inclusion / exclusion criteria were selected for a dental screening 
 appointment .
if the subject met all the inclusion / exclusion criteria , an informed 
 consent was handed out and , upon acceptance , signed by the volunteers .
- age between 18 - 35 years ; - male , in order to avoid hormonal influences during the experimental period , 
 especially concerning gingival inflammation ; - no relevant medical conditions that could interfere on the periodontal health ; - willingness to comply .
- probing pocket depth > 3 mm and/or clinical attachment loss > 2 mm at any 
 site ; - antibiotic and/or anti - inflammatory therapy within 3 months prior to baseline 
 examination ; - oral mucosal lesions ; - need for antibiotic premedication ; - history of hypersensitivity to chx ; - any condition device that could act as plaque retentive factor ( carious lesions ; 
 inadequate restorations ; dental implants ; orthodontic appliances ; fixed or removable 
 prostheses ) .
the following clinical parameters were assessed in the order listed below from all 
 teeth , excepting the third higher / lower molar .
- presence of calculus ( c ) in all teeth , at 6 sites per tooth ( mesiobuccal , 
 mid - buccal , distobuccal , mesiolingual , mid - lingual and distolingual ) was scored as a 
 dichotomous index : 0 - absence of calculus ; 1 - presence of calculus .
- discoloration index proposed by lobene ( 1968 ) and modified by macpherson , et al .
this involves visual stain 
 assessment of the buccal / labial and lingual / palatal aspects of the index teeth .
the 
 modification consisted of dividing each aspect into 4 separate sites ( figure 1 ) , classified as : - gingival ( g ) : 2 mm 
 wide strip running parallel to the gingival margin .
the limit towards the incisal 
 edge given by the end of the interdental papilla ; - body ( b ) : central area of 
 buccal / lingual aspect , between gingival and distal / mesial sites , extending to incisal 
 edge ; - mesial ( m ) : visible area between line angle and adjacent tooth , ending at the 
 interdental papilla and starting at gingival site ; - distal ( d ) : as for mesial ( m ) 
 site .
the stain sites of an anterior tooth : body ( white ) ; gingival ( grey ) ; mesial and 
 distal ( black ) stain was recorded using two separate characteristics , namely intensity and area 
 ( extent ) . the criteria and codes for intensity
were : 0= no stain present with the 
 natural tooth coloration ; 1= slim stain ; 2= clearly visible stain , orange to brown ; 
 3= dark stain , dark brown to black .
the area ( extent ) of the stain was recorded only if an intensity score of 2 or 3 was 
 given .
the area criteria and codes for proximal , gingival and body sites were : 1= up 
 to 1/3 of area affected ; 2= between 1/3 and 2/3 of area affected ; 3= more than 2/3 of 
 area affected .
before starting the trial , multiple sessions of training exercises for clinical 
 parameters were performed . after the examiner was trained , the intra - examiner 
 reproducibility was assessed . for reproducibility , 4 chronic chx users , who were not 
 included in the study population ,
subjects 
 were evaluated two times with one day intermission . in each session , the examiner 
 scored all teeth , except the third molars .
the results of presence / absence of 
 calculus and stain index were 
 analyzed by kappa - statistics .
the k - coefficient was 0.96 and 0.68 for calculus and 
 stain indexes , respectively .
the outline of the experimental procedures is summarized in figure 2 . to achieve optimal gingival health and to standardize gingival baseline conditions , 
 all subjects participated in a pretrial period during 14 days .
thorough professional 
 scaling and polishing with a rubber cup and prophylactic paste and dental floss was 
 performed . if hygiene was judged unsatisfactory , individual instructions were given 
 on how to improve the performance . -
baseline : on day zero , the presence of calculus and the stain index were recorded 
 in all subjects . after the recordings ,
a prophylaxis was performed and detailed 
 explanations to discontinue all the plaque control measurements were provided 
 again . - day 4 : each subject had one upper and one lower quadrant randomly assigned by a 
 heads or tails raffle made by the examiner , as " test " ( supragingival plaque - covered 
 sites ) and the other two quadrants as " controls " ( supragingival plaque was 
 professionally removed by the examiner to warrant plaque - free surfaces ) .
after the 
 prophylaxis on control quadrants , the quigley & hein plaque index was recorded in 
 all teeth to ensure that control quadrants were plaque - free .
following , all subjects 
 continued the oral hygiene withdrawal for a 21 days and started to rinse twice daily 
 with 15 ml of a mouthrinse containing 0.12% chx ( noplak , daudt , so paulo , sp , 
 brazil ) . - day 11 and 18 : presence / absence of calculus and stain index was recorded in all 
 teeth . - day 25 : similar to days 11 and 18 .
after the recordings , a professional scaling and 
 prophylaxis was performed to remove stain and calculus and all subjects were free to 
 return the normal habits of mechanical plaque control .
on days 11 , 18 and 25 , the examiner was kept unaware of randomization sequence and 
 blinded to which quadrant was test or control . however , because of the nature of the 
 study , it is not possible to assume that the examiner had not become aware of 
 quadrant allocation because plaque accumulation was clinically different between test 
 and control quadrants essentially on days 11 and 18 . whether this problem may have 
 biased
the necessary volume of chx to all the experimental period was 630 ml ( 15 ml- 12/12 
 h ) .
however , to ensure compliance , more than 630 ml of chx solution was given in 
 flasks with 1 l volume , with different volumes in each flask .
at the end of the 
 experiment , the remaining solution was turned in to check the individual 
 compliance .
data analysis was performed using commands that take into account clustering of 
 observations within subjects ( stata 9.2 for windows , stata corporation , college 
 station , tx , usa ) .
a robust variance estimator was used to adjust for the clustering 
 of teeth into individuals .
wald tests were used for comparisons , and the p - value was 
 adjusted for multiple comparisons .
checking of compliance revealed that all volunteers adhered to the instructions , with 
 the correct amount of left over rinsing solution given back .
no other side effects , such 
 as taste alterations , allergic reaction , etc . were observed .
gingival inflammation in 
 this test panel evaluated by the gingival index for test was 0.210.02 and 
 0.930.03 at baseline and day 25 , respectively , whereas in control surfaces it 
 was of 0.180.01 and 0.520.03 at the same time points .
the intensity of stain as assessed by discoloration index did not differ among 
 experimental groups at baseline . when test and control surfaces were compared , higher 
 degrees of staining were present in test surfaces at days 11 , 18 and 25 ( figure 3 ) .
mean discoloration intensity index in surfaces initially plaque - free or 
 plaque - covered at different experimental periods * statistically significant differences between initially plaque - free and 
 plaque - covered surfaces ( wald test , p=0.05 ) the stain area ( extent ) was recorded only if an intensity score of 2 or 3 was given . 

both test and control surfaces did n't show any score 2 or 3 at days 1 and 18 . at day 25 , 

the mean extent index was 1.590.13 on control surfaces and 1.620.09 on 
 test initially plaque - covered surfaces .
the frequency of extent scores on day 25 in test surfaces showed was 8.66 % 
 of the surfaces with score 1 , whereas at control surfaces was of 3.03% .
score 2 was 
 detected in 10.22% of test surfaces and 3.88 at control surfaces .
differences were also 
 observed concerning the frequency of score 3 ( 0.71% for test and 0.17% for control 
 surfaces ( figure 4 ) .
frequency of sites with scores 1 , 2 and 3 of stain extent on day 25 * statistically significant differences between initially plaque - free and 
 plaque - covered surfaces ( wald test , p=0.05 ) the presence / absence of calculus was analyzed by a dichotomous index .
the mean 
 percentage of calculus in test and control surfaces is shown in table 1 .
both groups significantly increased calculus from days 11 
 to 18 and 18 to 25 .
when test and control surfaces are compared , statistically 
 significant differences were observed at days18 and 25 ( figure 5 ) .
mean calculus scores on days 0 , 11 , 18 and 25 upper case letters regard to comparison between initially plaque - free surfaces 
 and plaque - covered surfaces in each experimental period .
different letters 
 demonstrate statistically significant differences ( wald test , p=0.05 ) lower case letters regard to within group comparison overtime .
se= standard error frequency of sites with presence of calculus * statistically significant differences between initially plaque - free and 
 plaque - covered surfaces ( wald test , p=0.05 ) comparisons between frequency distribution of sites with calculus did not reveal 
 statistically significant differences among groups at day 11 .
however , a higher 
 frequency of sites with calculus was found in the test group as compared to the control 
 group at days 18 ( 26.19% vs. 4.52% ) and 25 ( 34.4% vs. 12.38% , respectively ) .
the purpose of the present study was to investigate if initial professional prophylaxis 
 before the start of chx plaque control regime would result in different levels of cxh 
 side effects ( stain and calculus formation ) .
initially plaque - covered surfaces ( test 
 group ) showed significantly higher degrees of stain intensity and extent of surface area 
 as well as calculus formation as compared to initially plaque - free surfaces ( control 
 group ) .
these results showed that the initial professional prophylaxis seems to have a 
 greater effect in reducing cxh side effects .
the etiology of extrinsic tooth discolorations due to chx rinsing is not fully 
 understood .
non - enzymatic browning reactions ( maillard reactions ) and formation of 
 pigmented metal ( fe , sn ) sulfides hypotheses is not supported by direct and conclusive 
 in vivo evidence .
however , the main possible mechanism of extrinsic tooth stain is the 
 reaction products of food and beverage , where aldehydes and ketones , natural 
 constituents of various foods , may react with chx forming colored products .
however , a 
 discoloring capacity of chx combined with tea , red wine and coffee has been demonstrated 
 both in vivo and in vitro .
clinical investigations have demonstrated marked variations in the individual 
 discoloration tendency after a period of chx rinsing .
however , the 
 significant increase in stain in previously plaque - free surfaces has been demonstrated 
 by means of staining indexes .
our results are in accordance to these 
 observations . in the present investigation , we found an important effect of the presence 
 of plaque over intensity and extent of tooth staining , since surfaces without 
 supragingival plaque at the beginning of chx rinsing showed significant lower staining 
 as compared to surfaces with established plaque over 21 days ( figures 3 and 4 ) .
thus , 
 initially plaque - covered surfaces were stained earlier than plaque - free surfaces .
( 1997 ) demonstrated in 
 a population with large amounts of plaque and calculus , a significantly higher mean of 
 stain , as assessed by a discoloration index system when compared to a parallel control 
 group ( placebo solution ) . in our study , we used a randomized split - mouth design .
this methodological aspect aimed at reducing 
 inter - individual variability , reducing bias and consequently increasing statistical 
 confidence in the results obtained .
furthermore , a single calibrated clinical examiner 
 ( unaware of group allocation ) assessed all clinical parameters .
the observed results 
 indicate the mechanisms by which staining is higher in plaque - covered surfaces is 
 subject to further investigations
. however , a possible explication could be that plaque 
 presence could increase the chx salivary protein precipitation .
the increase of calculus formation due to chx mouthrinse is a usual finding in early 
 long - term investigations .
control surfaces showed 
 little amounts of calculus formation over 21 days ( about 12% of surfaces ) , while test 
 surfaces presented 34.4% of calculus .
thus , plaque - covered surfaces presented calculus 
 earlier than plaque - free surfaces .
these results may be explained by the fact that 
 supragingival calculus is essentially mineralized plaque .
the process of mineralization and the chx influence on 
 the process are not fully understood , but involves localized supersaturation , 
 nucleation , crystal growth and the transformation of precursor phases such as dicalcium 
 phosphate dehydrate , octacalcium phosphate and amorphous calcium phosphate into more 
 stable , crystalline deposits of hydroxyapatite .
the higher tendency to calculus formation in lingual aspects of 
 lower anterior and buccal aspects of upper posterior tooth surfaces may be due to the 
 location of the submandibular and parotid ducts location . in these areas ,
the abundant 
 supply of urea from the saliva and the high salivary film velocity tend to promote base 
 formation to plaque and calcium phosphate precipitation .
hence , it has been advocated that these locations may be 
 more susceptible to calculus formation because of the low sucrose concentration in 
 saliva with a high saliva film velocity promoting clearance of salivary sugar and acid 
 from plaque , and the higher plaque ph associated to better access to salivary 
 urea . however , these results 
 have to be interpreted with care because our criteria for calculus included both stained 
 and non - stained calculus .
thus , the increased scores could represent the incremental 
 build - up and hardening of stain in the gingival third of the crowns . in the present study
( 2000 ) demonstrated that this stain 
 index was more advantageous because it allowed to assess sites with most visible stain 
 separately and offered higher discriminatory power due to a lower coefficient of 
 variation when compared with conventional counterpart .
the reason to assess calculus in a dichotomous index 
 was to facilitate the assessment in all present teeth due to the most calculus index use 
 only specific teeth and regions .
on the other hand , while these calculus indexes allow 
 for quantification of calculus presence , a dichotomous calculus index shows only 
 calculus presence or absence .
however , it did not compromise our results because we 
 aimed at verifying if calculus formation would be present first over plaque - covered 
 surfaces .
chx was selected for the antimicrobial treatment because it is the best characterized 
 and most effective chemical antiplaque agent known today .
the concentration selected corresponds 
 to that used clinically for plaque control ( 0.12% or 2% )
. moreover , extensive evidence 
 showed similar plaque reduction and gingival inflammation effectiveness when comparing 
 0.2% and 0.12% chx concentrations . 

( 2008 ) recently 
 explored this issue demonstrating that the use of 15 ml of 0.12% chx rinsing did not 
 differ from 10 ml of 0.2% for plaque formation and gingival bleeding in a cross - over 
 double blind 14 days rinsing period study design .
it should be emphasized that for some 
 individuals 15 ml is a high amount and might be uncomfortable .
however , evidences show 
 that 0.12% chx seems to reduce stain side effects , mainly tooth staining
. the results of the present study are intriguing and should be interpreted from a 
 clinical perspective .
the initially plaque - covered and plaque - free surfaces presented tooth stain and calculus 
 formation with different magnitude and timing .
however , significant differences in calculus 
 formation and staining were found and the clinical impact consists in an indication that 
 it is always desirable to diminish such side effects . whenever possible , when starting 
 with chx regimes , plaque should be previously removed in the best achievable way in 
 order to reduce side effects .
 objectivesstudies concerning side effects of chlorhexidine as related to the presence of 
 plaque are scarce . 
 the purpose of this study was to compare the side effects of 
 0.12% chlorhexidine gluconate ( chx ) on previously plaque - free ( control group ) and 
 plaque - covered surfaces ( test group).methodsthis study had a single - blind , randomized , split - mouth , 21 days - experimental 
 gingivitis design , including 20 individuals who abandoned all mechanical plaque 
 control methods during 25 days . 
 after 4 days of plaque accumulation , the 
 individuals had 2 randomized quadrants cleaned , remaining 2 quadrants with 
 plaque - covered dental surfaces . on the fourth day , the individuals started with 
 0.12% chx rinsing lasting for 21 days . 
 stain index intensity and extent as well as 
 calculus formation were evaluated during the experimental period.resultsintergroup comparisons showed statistically higher ( p<0.05 ) stain intensity and 
 extent index as well as calculus formation over the study in test surfaces as 
 compared to control surfaces . thus , 
 26.19% of test surfaces presented calculus , 
 whereas calculus was observed in 4.52% in control surfaces.conclusionthe presence of plaque increased 0.12% chx side effects . 
 these results strengthen 
 the necessity of biofilm disruption prior to the start of chx mouthrinses in order 
 to reduce side effects . 
INTRODUCTION
MATERIAL AND METHODS
Test panel
Inclusion criteria
Exclusion criteria
Clinical Parameters
Assessment of intraexaminer reproducibility
Blindness
Compliance
Statistical analysis
RESULTS
DISCUSSION
CONCLUSIONS