gingival - periodontal diseases include a set of pathologies that affect the protective and insertion tissues of teeth .
a wide range of clinical pictures arises from a complex interrelationship among bacterial aggression , intrinsic host response , and risk factors or disease modifiers .
traditional diagnosis is done by measuring different clinical and radiographic indices : probing depth ( pd ) , attachment level ( al ) , and bleeding on probing ( bop ) and/or spontaneous bleeding , ulceration , exudate , and suppuration ; however , these methods only provide information on past destruction .
given the severity of aggressive periodontitis ( ap ) and its tendency to advance , early detection is a major concern .
variations in the onset , severity , and clinical characteristics can be used to recognize and describe different forms of periodontitis including early - onset periodontitis now called ap , and adult or chronic periodontitis ( cp ) .
both can be sub - classified as laser periodontitis ( lp ) , moderate periodontitis ( mp ) , and severe periodontitis ( sp ) .
aggressive periodontitis usually appears early in life , which means that the etiological agents were able to produce clinically detectable levels of disease within a fairly short time .
this is crucial for understanding these diseases because it implies infection by highly virulent microflora or a high level of susceptibility of the subject to periodontal disease .
its diagnosis requires the exclusion of systemic diseases that can severely deteriorate host defenses and lead to premature loss of teeth .
chronic periodontitis is usually more prevalent in adults but may also be found in children and youths .
the progression of cp is usually slow or moderate , but there may be short periods of rapid destruction
. it may be modified by systemic conditions such as diabetes , smoking , and stress . given the severity of ap and its tendency to advance ,
early detection is a major concern . during the migration process of epithelial cells of the gum from deep layers toward more superficial layers ,
the formation of a layer of keratin which protects the gingival from direct mechanical , chemical , and bacteriological stimuli , thus preserving its integrity , is a phenomenon that translates into cytological changes which can reflect the degree of periodontal health .
oral exfoliative cytology includes the study and interpretation of the features of naturally or artificially exfoliated cells from the oral mucosa .
it consists of observing the morphology of superficial epithelial cells under the microscope after sampling , fixing , and staining .
. , 1990 , suggest that these quantitative techniques based on evaluating parameters such as variations in the size of the nucleus and the cytoplasm and alterations in the nucleus / cytoplasm ratio can increase the diagnostic sensitivity of exfoliative cytology .
the use of exfoliative cytology could be used as a complementary diagnosis method because it is easy to perform and allows repeated sampling without destroying the integrity of the tissue .
cytological study of healthy gum shows exfoliated surface cells and a smaller proportion of intermediate cells from the stratum spinosum .
there are also a few leukocyte - type transitory cells with defense functions , red blood cells , and microbial flora .
the aim of this study was to analyze cytological changes of the periodontal pocket in patients with ap and cp in their clinical sub - stages lp , mp , and sp .
we worked with patients aged 2454 years , of whom 41 had ap ( ages 24.0  9.0 ) ( 21 male and 20 female ) , and 40 had cp ( ages 58.3  7.0 ) ( 13 male and 27 female ) .
the control or clinically healthy group was made up of 40 healthy individuals ( ages 44.0  10.0 ) ( 18 male and 22 female ) .
clinical and dental records were prepared for all subjects , and they signed informed consent for cytological sampling .
an ethical clearance was obtained before the study by the ethical committee of the institution in compliance with the declaration of helsinki of 1975 and the who standards for observational studies .
it included : plaque index ( pi ) , gingival index ( gi ) , pd , al , and bop using a marquis - type periodontal probe ( hu - friedy , nc , usa ) .
patients were classified according to clinical and radiographic criteria into four categories , for both ap and cp : 
 clinically healthy : pd < 3 mm , al < 2 mm , pi < 20% , gi < 1 mm , bop (  ) and bone loss ( )lp : 3  pd < 5 mm , 2  al < 4 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) mp : 5  pd < 7 mm , 4  al < 6 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) sp : pd  7 mm , al  6 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) . 
  clinically healthy : pd < 3 mm , al < 2 mm , pi <
20% , gi < 1 mm , bop (  ) and bone loss (  ) lp : 3  pd < 5 mm , 2  al < 4 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) mp : 5  pd < 7 mm , 4  al < 6 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) sp : pd  7 mm , al  6 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) .
inclusion criteria for patients were : individuals of either sex at least 20 natural teeth , two sites with attachment loss in each quadrant > 3 mm .
exclusion criteria for both the patients and the control group were : smoking , systemic diseases , chirurgic and nonchirurgic periodontal therapy , use of antibiotics , steroidal or nonsteroidal anti - inflammatory agents , all in the 6 months prior to the study .
although smoking , medication , and systemic diseases may affect in different ways the development of the different periodontitis and are frequently associated with chronic disease , these factors were excluded . in this way in order to the cytological founds may be exclusively related to the periodontal diagnose .
samples were collected from the soft wall of the periodontal pocket and gingival crevice of patients and control subjects , respectively , using specific curettes ( hu - friedy , nc , usa ) , after removing supra - gingival bacterial plaque .
the following parameters were observed under a trinocular microscope ( carl zeiss axiostar , gttingen , germany ) : desquamation of surface and intermediate cells , polymorphonuclear neutrophils ( pmn ) , histiocytes , and microbial flora .
wallis test for qualitative variables was used to determine the differences between the ap and cp diagnoses . when there were differences between the groups ,
the anova one - way test was applied , and when the differences were significant , tukey 's test was used .
we worked with patients aged 2454 years , of whom 41 had ap ( ages 24.0  9.0 ) ( 21 male and 20 female ) , and 40 had cp ( ages 58.3  7.0 ) ( 13 male and 27 female ) .
the control or clinically healthy group was made up of 40 healthy individuals ( ages 44.0  10.0 ) ( 18 male and 22 female ) .
clinical and dental records were prepared for all subjects , and they signed informed consent for cytological sampling .
an ethical clearance was obtained before the study by the ethical committee of the institution in compliance with the declaration of helsinki of 1975 and the who standards for observational studies .
it included : plaque index ( pi ) , gingival index ( gi ) , pd , al , and bop using a marquis - type periodontal probe ( hu - friedy , nc , usa ) .
patients were classified according to clinical and radiographic criteria into four categories , for both ap and cp : 
 clinically healthy : pd < 3 mm , al < 2 mm , pi < 20% , gi < 1 mm , bop (  ) and bone loss ( )lp : 3  pd < 5 mm , 2  al < 4 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) mp : 5  pd < 7 mm , 4  al < 6 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) sp : pd  7 mm , al  6 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) . 
  clinically healthy : pd < 3 mm , al < 2 mm , pi <
20% , gi < 1 mm , bop (  ) and bone loss (  ) lp : 3  pd < 5 mm , 2  al < 4 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) mp : 5  pd < 7 mm , 4  al < 6 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) sp : pd  7 mm , al  6 mm , pi  20% , gi  1 mm , bop ( + ) and bone loss ( + ) .
inclusion criteria for patients were : individuals of either sex at least 20 natural teeth , two sites with attachment loss in each quadrant > 3 mm .
exclusion criteria for both the patients and the control group were : smoking , systemic diseases , chirurgic and nonchirurgic periodontal therapy , use of antibiotics , steroidal or nonsteroidal anti - inflammatory agents , all in the 6 months prior to the study .
although smoking , medication , and systemic diseases may affect in different ways the development of the different periodontitis and are frequently associated with chronic disease , these factors were excluded . in this way in order to the cytological founds may be exclusively related to the periodontal diagnose .
samples were collected from the soft wall of the periodontal pocket and gingival crevice of patients and control subjects , respectively , using specific curettes ( hu - friedy , nc , usa ) , after removing supra - gingival bacterial plaque .
the following parameters were observed under a trinocular microscope ( carl zeiss axiostar , gttingen , germany ) : desquamation of surface and intermediate cells , polymorphonuclear neutrophils ( pmn ) , histiocytes , and microbial flora .
the kruskal  wallis test for qualitative variables was used to determine the differences between the ap and cp diagnoses . when there were differences between the groups ,
the anova one - way test was applied , and when the differences were significant , tukey 's test was used .
among the patients selected , those with ap were on average much younger than those with cp , confirming the influence of age on the characteristics of the two types of periodontitis . for the control group , we selected individuals of ages between those of the patients with ap and cp [ table 1 ] .
of all the patients with ap ( n = 41 ) , 25% ( n = 10 ) were diagnosed with lp , 27% ( n = 11 ) with mp and 48% ( n = 20 ) with sp . for
cp ( n = 40 ) , 25% ( n = 10 ) were diagnosed with lp , 25% ( n = 10 ) with mp , and 50% ( n = 20 ) with sp .
characteristics of patients with periodontitis and control subjects regarding the cytological study , figure 1 shows the surface cells and the exfoliated cells of the intermediate layer of patients with ap [ figure 1a ] , pc [ figure 1b ] and a control individual [ figure 1c ] . the first ( surface cells ) show increased cell volume , imprecise edges and blurry appearance of the nucleus , in a patient with periodontitis compared to the control group .
they are rounded or polygonal , some with folded edges , slightly thicker cytoplasm than in surface cells ; the nucleus is central and round .
the cells in the intermediate layer are slightly enlarged in ap [ figure 1a ] , and cp [ figure 1b ] compared to the control group [ figure 1c ] .
surface cells and intermediate cells in patients with aggressive periodontitis ( a ) chronic periodontitis ( b ) and control group ( c ) 400 pap the semi - quantitative difference for surface cells was significant ( p < 0.001 ) when ap was compared to cp and the control group [ figure 2 ] . in turn , within ap , there were significant differences ( p < 0.001 ) among lp , mp , and sp . patients diagnosed with cp showed significant differences ( p > 0.05 ) between mp and the control group .
semi - quantitative analysis of surface cells in patients with aggressive periodontitis , chronic periodontitis , and control group ( c ) the semi - quantitative study of intermediate cells showed statistically significant differences ( p < 0.001 ) between ap and cp , and between ap and the control group [ figure 3 ] .
there was no difference ( p > 0.001 ) in the quantity among groups lp , mp , and sp for patients diagnosed with ap and cp .
semi - quantitative analysis of intermediate cells in patients with aggressive periodontitis and chronic periodontitis and control group ( c ) figures 4a  c show pmns and histiocytes of the soft wall of the periodontal pocket in patients with ap , cp , and control individual , respectively .
the pmns have a polymorphic nucleus and numerous granules in their cytoplasm with differential staining .
more pmns and histiocytes are observed in ap [ figure 4a ] than in cp [ figure 4b ] and control [ figure 4c ] .
neutrophil polimorhphonucleate leukocytes and histiocytes in patients with aggressive periodontitis ( a ) chronic periodontitis ( b ) , and control group ( c ) 400 pap the statistical analysis of pmns showed no significant difference ( p > 0.001 ) between ac and cp , although there were differences ( p < 0.001 ) between stages lp , mp , and sp compared to the control group in ap [ figure 5a ] . in cp patients , mp and
semi - quantitative analysis of neutrophil polimorhphonucleate leukocytes ( a ) histiocytes ( b ) and microbial flora ( c ) in patients with aggressive periodontitis and chronic periodontitis and control group ( c ) statistical analysis showed much higher values for histiocytes in cp ( p < 0.001 ) than in ap [ figure 5b ] .
there were statistically significant differences ( p < 0.001 ) in histiocytes between patients with ap and the control group .
histiocytes in patients with cp , mp , and sp , differed significantly ( p < 0.001 ) from patients with lp , and there were significant differences among all three stages and the control group .
there were statistically significant differences ( p < 0.001 ) [ figure 5c ] among groups lp , mp , and sp in patients diagnosed with ap , as well as between them and the control group ( p < 0.001 ) . in patients with cp , mp , and
sp differed significantly ( p < 0.001 ) from lp and the control group . regarding sex and age of patients , no significant difference ( p >
0.001 ) was found in the number and type of exfoliated cells or microbes ( results not shown ) .
the extent to which the tissues are affected determines the lp , mp , and sp clinical pictures . these pictures , as well as gingivitis , can be studied biochemically , cytologically , and histologically using specific markers .
exfoliative cytology , which is a straightforward noninvasive diagnostic method that can be applied during inflammation , can be considered as a more practical technique to evaluate the oral mucosa .
desquamation of gingival epithelium depends on mitotic activity of basal cells , their enzyme processes , and mechanical irritations of gingival tissue . in recent years
, exfoliative biopsy has been introduced with good results in specification and diagnosis of the lesions buccales .
a few studies have used exfoliative cytology to evaluate changes in the oral mucosa in gingival inflammation and periodontal disease . in periodontal inflammatory processes
lange 1965 suggests an increase in the young cells in the deepest layers of the epithelium such as intermediate and prepyknotic cells , reducing the number of cells containing keratin particles . according to that study
, there is a direct relationship between the degree of inflammation and reduction in keratinization .
endo et al . 2008 , found a reduction of keratinization in superficial inflammations of the gingival .
our study found more superficial and intermediate cells in patients with ap than in patients with pc and the control group .
these results differ from those of other authors , who found fewer of these cells in patients with chronic periodontal disease .
the presence of intermediate cells in the proportion in which they appear in these studies might indicate the onset of periodontal disease in the preclinical , as yet asymptomatic stage .
many authors conclude that exfoliative cytology has a certain value in the diagnosis of precancerous lesions , and particularly in determining their prognosis .
there is currently no cytological study on chronic and aggressive periodontal disease ; nevertheless , our research has shown that the cytological study of the soft wall of the pocket shows tissue damage as an increase in inflammatory cells , cells from deep layers of the epithelium , and disease - related microorganisms .
some studies have shown cell changes in the oral mucosa of patients with systemic diseases such as endocrine disorders and respiratory diseases .
other researchers have found cytomorphometric changes in the cells of the oral mucosa of smokers , similar to those found in diabetics , caused by the chronic inflammation of the oral mucosa .
changes in the oral mucosa similar to those appearing in individuals with type 2 diabetes have also been found in patients with vitamin b12 and folic acid nutritional deficiencies .
histological studies on tissue samples show significant differences in the percentage of collagen fibers and type of cell population such as histiocytes and pmns , in patients with periodontal disease and gingivitis compared to healthy individuals , and also between both groups of patients .
our study showed a high level of histiocytes in mp and sp for cp compared to the control group , as expected , since they are immune cell types that protect the body against infection .
our results showed higher values for pmns and microbial flora in patients with cp and ap than in the group of healthy patients , providing evidence of the cellular immune system defense in these groups of patients .
although the control group was made up of clinically healthy individuals , cytological examination showed the presence of pmns , histiocytes and microbial flora , due to the fact that under normal conditions , the gingival have an inflammatory infiltrate that makes up about 5% of the volume of the connective tissue .
the analysis considering sex and age showed no significant difference in exfoliated cells , coinciding with the results of sguier et al .
2000 . due to the presence of ulceration and mucosal erosion present in stomatitis and gingivitis ,
these cells may vary in size and shape , being mostly increased in their nuclear size , with multiple ovoid nuclei and poorly conserved cytoplasm .
the use of exfoliative cytology for obtaining samples on which to apply sophisticated diagnosis techniques , cytomorphometry , analysis of dna content , and molecular analysis seems to be gaining ground as a reliable method for the diagnosis of oral cancer in its earliest stages .
the main advantages of exfoliative cytology are that the technique is rapid , relatively painless , easy to perform allows repeated sampling of biological material without destroying tissue integrity , which means it can be performed repeatedly in preventive screening programs and during routine dental examinations .
cytological studies of the periodontal pocket of patients with clinically diagnosed ap and cp had greater tissue damage as shown by the increase in cells from the deep epithelium layers , inflammatory cells , and microbial flora compared to the group of healthy subjects . patients with ap had more surface and intermediate epithelium cells from the periodontal pocket than cp patients and the group of healthy subjects . in patients with ap , surface cells increased with the severity of the disease .
the cytological study showed that patients with ap had greater tissue damage , shown by the increase in intermediate and superficial cells of the epithelium of the periodontal pocket compared to the group of healthy subjects and to a lesser extent , to patients with cp . only superficial cells made it possible to differentiate the sub - stages of the disease .
 background : oral exfoliative cytology includes the study and interpretation of the features cells exfoliated from the oral mucosa . the aim of this study was to analyze cytological changes in the periodontal pocket of patients with different clinical stages of aggressive periodontitis ( ap ) and chronic periodontitis ( cp).materials and methods : patients aged 2454 years , of whom 41 were diagnosed with ap , 40 with cp , sub - classified as mild , moderate and severe periodontitis , and 40 healthy individuals who were the control group . 
 samples of the epithelium of the periodontal pocket were taken for the cytological study.results:superficial and intermediate cell values were significantly greater in patients with ap than in patients with cp or the control group . 
 histiocyte number was higher in patients with cp than in those with ap , and differed significantly in both types of periodontitis compared to the control group . 
 there were significant differences in polymorphonuclear neutrophil leukocytes when both types of periodontitis were compared to the control group . 
 microbial flora was statistically higher in patients with cp , and there were differences between patients with periodontitis and the control group.conclusions:the cytological study demonstrated that patients with ap had greater tissue damage , shown by the increase in intermediate and superficial cells of the epithelium of the periodontal pocket compared to the group of healthy subjects and to a lesser extent , to patients with cp . 
 only superficial cells made it possible to differentiate the sub - stages of the disease . 
Introduction
Materials and Methods
Subjects
Dental clinical parameters
Cytological analysis
Statistical analysis
Results
Discussion
Conclusions