since many years , a great number of studies have been discussing the role of keratinized gingiva ( kg ) in marginal periodontal behavior , either accepting that a minimum of 2.0  mm of kg width is required to maintain marginal periodontal health [ 16 ] or suggesting that kg width is negligible if excellent oral hygiene is performed [ 715 ] .   areas with narrow width of kg seem to be prone to periodontal attachment loss , which could result in recession of the gingival margin in the presence of risk factors .
lang and le   have demonstrated that such areas show clinical signs of inflammation even in the absence of dental plaque , as evidenced by an increase in gingival crevicular fluid ( gcf ) flow rate , which suggests that tissue behavior is mainly associated to pathological more than to physiological processes under these conditions .
an increased gcf flow rate from gingival sulcus is an early sign of clinical inflammation [ 1620 ] . in the absence of dental plaque ,
the increase of gcf flow rate in areas presenting < 2.0  mm of kg width could be influenced by a close proximity of the dentogingival unit to the alveolar mucosa , which is more permeable and mobile to allow primary defense against microorganisms and its products [ 5 , 17 ] .
some factors can explain the increase in gcf flow rate , including ( i ) increase in tissue hydrostatic pressure close to junctional epithelium induced by bacterial plaque accumulation [ 18 , 19 , 21 ] ; ( ii ) mobility of gingival margin and increase in marginal blood flow caused by mechanical stimulus from tooth mobility , frenum pull , toothbrushing , and chewing ; ( iii ) histamine intravenous injection or development of inflammation .
these findings suggest that chemical or mechanical irritation is necessary to the production of gcf [ 21 , 24 ] .  
gcf is considered to develop an important protective role on the defense mechanisms of gingival sulcus through the presence of defensive substance , such as pmn - neutrophils , and through mechanical properties , such as the flushing action , capable of removing carbon particles and bacteria from the gingival sulcus [ 20 , 2428 ] .
taken together , these findings suggest that the absence of gcf in the absence of mechanical stimulation would represent gingival health , while its presence in the absence of mechanical stimulation would represent gingival inflammation .   to the best of our knowledge
, no study has investigated if there are differences in gcf flow rate in areas with a narrow or wide width of keratinized gingiva , aiming at determining if a wider width of keratinized gingiva would allow a better dissipation of gcf .
considering so , the aim of this study was to investigate the role of kg gingiva in the homeostasis of gingival margin by evaluating gcf flow rate in healthy sites before and after a mechanical stimulus .
this study was approved by the committee of ethics in research of school of dentistry at bauru - usp ( no .
the sample was selected according to the following inclusion criteria : presence of a buccal healthy site showing 2.0  mm width of kg at bicuspids or molars and a homologous site presenting < 2.0  mm of kg ; good systemic health ; optimal oral hygiene status .
it was excluded from the study pregnant or lactating women , patients prescribed with restricted drugs or antibiotics in the 6 and 3 months previous to data collection , those taking medicines capable of inducing gingival hyperplasia ( e.g. , calcium channel blockers , cyclosporine , and anticonvulsants ) , smokers , drugs , or alcohol abusers .
a total of 60 patients were initially examined , but only 16 were found in accordance to inclusion and exclusion criteria ( figure 1 ) .
group 1 was composed of 16 strictly healthy premolar and molar buccal sites showing 2  mm of kg width , and group 2 was composed of 16 strictly healthy premolar or molar buccal sites showing < 2  mm of kg width in the same patients , in a split - mouth design . in order to assure the absence of gingival inflammation , all patients were submitted to scaling and root planning and oral hygiene instruction before data collection .
the reliability of these conditions was assessed by the evaluation of plaque index , sulcular bleeding index , and probing depth , measured with a millimeter periodontal probe ( hufriedy , chicago , usa ) .
kg width was measured by a digital caliper ( 727 me , wit , brazil ) as the distance from gingival margin to mucogingival junction at buccal sites of the selected teeth , after staining of the tissues with a schiller solution ( figure 2 ) .
all clinical examinations and procedures were under the responsibility of a single - trained examiner .
clinical measurements were performed 2472 hours before gcf collection to avoid any interference on the gingival sulcus physiology .
the collection of gcf was accomplished by imbibing of periopaper strips ( oralflow , ny , usa ) as proposed by lee and holm - pedersen . after preventing the contact of the tooth by the tongue
the paper strips were closely fitted over the tooth crown and the buccal soft tissue surface , extending up to the alveolar mucosa ( figure 3(a ) ) , allowing the imbibing of the strips by fluid exuding through the bordering edge of alveolar mucosa ( p1a ) and through the gingival margin ( p1b ) .
gcf was also collected at superficial position ( p2 ) , at the entrance of gingival crevice ( figure 3(b ) ) , and deep intracrevicular position ( p3 ) , in which the strip was introduced to the base of gingival crevice ( figure 3(c ) ) , as determined by minimal tactile resistance upon its introduction [ 22 , 25 ] .
paper strips were kept in position for 60 seconds , allowing the imbibing of paper strips in a standardized period , as reported before [ 31 , 32 ] and supported by the results obtained in a pilot study involving 3 patients ( data not shown ) .  
gcf was collected in two different moments : before and after chewing a fresh fibrous food meal ( cooked bovine steak ) for 10 minutes .
collection of gcf before chewing was performed 2472  hs after clinical measurements , in a rested position .
after 24 hours , patients were recalled and eat the cooked fibrous meal during 10 minutes .
the strips were then heat air dried and imbibed in a 2% alcoholic solution of ninhydrine , which provides comparable results to electronic devices   and again heat air dried .
comparisons between groups before and after chewing stimuli were analyzed by unpaired t - test .
intragroup comparison of gcf collection before and after chewing stimuli was analyzed by paired t - test .
a 95% confidence level was established for all statistical analysis (  = 0.05 ) .
periodontal status of test and control sites before collection of gcf is shown at table 1 .
no differences were found between groups in probing depth , plaque index , and bleeding index .
significant differences were observed between groups only in kg width ( p < 0.05 ) .  
the amount of gcf collected from groups 1 and 2 sites before and after chewing stimuli is described in table 2 .
significant differences between groups were found at p1a before mastication stimulus and at p2 and p3 after mastication stimulus .
intragroup analysis by paired t - test showed significant increases in gcf at p1b and p3 in group 1 and at p1a in group 2 .
the results obtained in this study have shown that tissue fluid transudates rather through gingival sulcus than through alveolar mucosa in areas presenting at least a 2  mm kg width , while areas presenting lesser than 2  mm of kg width show transudation of tissue fluid by alveolar mucosa .
these findings suggest that the protective role [ 16 , 21 , 2428 ] exerted by gcf is compromised in areas with a narrow width of kg .  
this study is important because no previous reports have evaluated the role of kg in the homeostatic response of marginal periodontal under strictly healthy and physiological conditions .
miyasato , crigger , and egelberg   also evaluated the homeostatic response of 16 dental students or members of the dentistry faculty showing  appreciable 
( 2  mm ) or  minimal  amount of kg ( < 1  mm ) in plaque - free contralateral or unilateral sites and showed that both areas showed minimal amounts of gcf in a resting state , as observed in the present study . when plaque was allowed to accumulate , a significant increase in gcf flow rate was observed for both groups , with no differences between groups , as gingival inflammation developed , but no mechanical stimulus was performed to induce gcf flow rate .  
gcf flow rate is usually a measure of gingival inflammation , since its exudation increases in the presence of inflammation and either is not present or present in small quantities in healthy situations [ 16 , 1921 , 28 , 29 , 34 ] .
indeed , the volume or resting gf increases as periodontal pockets develop [ 20 , 34 , 35 ] . while in healthy sites gcf represents a transudate of interstitial tissues , in the course of gingivitis and periodontitis , it is transformed into a true inflammatory exudate [ 19 , 20 ] . in this context , the relevance of gcf in the control of a subgingival microbiota compatible with periodontal health should be emphasized [ 17 , 20 , 25 ] .  
no differences between groups were observed at gingival margin ( p1b ) and superficial ( p2 ) and deep ( p3 ) intracrevicular positions before chewing stimuli .
this can be possibly explained by the fact that in clinically healthy conditions the amount of gcf transudation through gingival sulcus is minimal [ 16 , 1921 , 28 , 29 , 34 ] , which might account for differences between the present study and others published in literature [ 1 , 16 ] .
however , a significant difference between groups was found at p1a ( alveolar mucosa edge ) , indicating that , in a resting position , dissipation of tissue fluid occurs mainly by alveolar mucosa , which is more permeable and mobile to allow metabolic interchange .  
this result also seems to indicate that some tissue fluid from alveolar mucosa can dissipate toward the gingival sulcus in narrow kg , suggesting that the greater the distance from gingival sulcus to alveolar mucosa , the lesser the influence of alveolar mucosa on the physiological behavior of gingival sulcus .
according to siegel , the identification of an increased flushing of tissue fluid through the epithelial barrier of the alveolar mucosa is highly suggestive of the permeability of this tissue .  
after mechanical stimuli provided by chewing a fibrous meal , a significant increase in transudation of gcf through gingival sulcus was observed at p1b and p3 positions for sites with 2  mm width of kg , with a trend to increase also at p2 , although not statistically significant .
the same stimulus resulted in increase in transudation of tissue fluid trough alveolar mucosa ( p1a ) , while the amount of gcf collected from gingival sulcus ( p1b , p2 and p3 ) suffered minor not significant variations in sites showing < 2  mm of kg width .
these results are in agreement with other studies [ 1618 , 20 , 22 , 25 ] showing that mechanical stimulus is capable of increasing gcf flow rate even in healthy areas .
these findings also indicate a more pronounced permeability of alveolar mucosa than kg , which allows the crossover of substances from the inner to the outer environment and vice versa . this might be relevant in the control of periodontal homeostatic response through an early recognition of potentially aggressive bacterial antigens .
the increased fluid rate observed at p1a position implies in increased alveolar mucosa transudation influx , probably related to the tissue blood supply necessary to accomplish the metabolic demands for functional tissue mobility . besides , the increase in gcf flow rate at gingival sulcus in group 1 reflects the role of gcf in flushing the gingival sulcus , corroborating the results obtained by previous studies [ 16 , 17 , 20 , 25 , 26 ] .
this protective role was not observed in group 2 , since gcf flow rate remained the same after chewing , suggesting that a wider area of kg is more compatible with marginal periodontium homeostasis , playing a regulatory role on the control of concentration and distribution of gcf flow rate [ 16 , 17 , 22 , 25 , 37 , 38 ] .
these results might explain why areas showing 2  mm of kg width are more compatible with marginal periodontal health than areas < 2  mm wide , which requires a strict maintenance program in order to prevent plaque accumulation [ 18 , 11 , 14 ] .
kennedy et al .   found that gingival health could be maintained in patients under professional plaque control but not in those without professional care in areas presenting an inadequate kg width . in their study , patients previously submitted to free gingival autografts procedures that did not participate in the supportive periodontal treatment for a 5-year period have not shown significant gingival inflammation and/or recession overtime , contrariwise to those portraying inadequate kg .
from these results , it can be assumed , as in the present study , that the existence of an adequate kg width is a natural requirement to allow a reliable homeostatic response of the marginal periodontium when the patient performs personal routine dental plaque control .   in the absence of dental plaque ,
the increase in flow rate of gcf in areas < 2  mm in width could be influenced by a close contact between the dentogingival unit and the alveolar mucosa , which is more permeable and mobile , thus allowing primary defense mechanisms to be activated against bacterial challenge .
additionally , the increase in tissue hydrostatic pressure close to the junctional epithelium may contribute to a more pronounced flow rate of gcf in narrower areas [ 1821 , 39 , 40 ] . since the attached gingiva is a fraction of keratinized gingiva and extends up to the boundaries of the mucogingival junction
, these areas must be capable of neutralizing the tension transmitted by the alveolar mucosa under muscle action in such a way that marginal gingival mobility is prevented .
this study also showed that the mastication of natural fibrous food produces alterations in gcf flow rate which can vary according to the width of kg .
this behavior could be engaged to the dental mobility induced by chewing , that would transmit a stimulus to gingival blood vessel walls through oxytalan fibers [ 4145 ] , giving rise to an increased blood vessel transudation , which in turn would be related to the changes in gcf flow rate [ 18 , 19 ] .
additionally , the mobilization of gingiva can also be attributed to food excursion and/or functional demands of the alveolar mucosa , since both inadequate and adequate kg groups experienced significant variation in gcf flow rate , more pronounced in areas presenting an adequate width of kg .
the main limitation of this study is the small number of subjects included in the study , due to difficulties in finding patients who presented homologous sites with  or < 2  mm of kg width and did not present any of the exclusion criteria .
this might explain why no significant differences were found at p2 position before and after chewing in group 1 , since an increase in gcf was noticed . by the other side , this was a split - mouth design study , which implies that  test  and  control  sites were in the same patients .
all patients were submitted to professional plaque control by scaling and root planning and prophylaxis , and oral hygiene instruction in order to assure that all patients were clinically healthy at the moment of gcf collection .
these results suggest that kg plays a definite role in controlling the physiology of gingival sulcus , allowing the transudation of gcf through the gingival sulcus , resulting in an adequate protective role essential to the maintenance of gingival health and periodontal homeostasis .
the results obtained in this study suggest that a wider area of keratinized gingiva favors physiological behavior of the gingival sulcus by a better dissipation of gcf ; a closer proximity of gingival margin and alveolar mucosa influences the dissipation of tissue fluid through alveolar mucosa , which is more permeable and mobile , impairing primary defense of gingival sulcus by the concentration of gcf .
 
 objective . to shed light on the role of kg , its influence on periodontal behavior was investigated . methods .   
 tissue fluid transudation was assessed in alveolar mucosa ( p1a ) , outer gingival margin ( p1b ) , at entrance of ( p2 ) and within gingival sulcus ( p3 ) , before and after chewing of fibrous food in 16 patients portraying 2  mm  kg at one tooth ( group 1 ) , and < 2  mm at another homologous tooth ( group 2 ) . 
 results . 
 there was a significant increase in gcf after chewing at p1b and p3 in group 1 and at p1a in group 2 ( t - test , p < 0.05 ) . 
 conclusions . 
 the results suggest that kg plays a role in marginal periodontal homeostasis . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions