Caracterización de Las Lesiones Periodontales Progresivas en Pacientes Con Periodontitts Crónica

8/9/2019 Caracterizacin de Las Lesiones Periodontales Progresivas en Pacientes Con Periodontitts Crnica

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Characterization of progressiveperiodontal lesions in chronicperiodontitis patients: levels ofchemokines, cytokines, matrixmetalloproteinase-13,

periodontal pathogens andinflammatory cells

Silva N, Dutzan N, Hernandez M, Dezerega A, Rivera O, Aguillon JC, Aravena O,Lastres P, Pozo P, Vernal R, Gamonal J. Characterization of progressive periodontallesions in chronic periodontitis patients: levels of chemokines, cytokines, matrixmetalloproteinase-13, periodontal pathogens and inflammatory cells. J ClinPeriodontol 2008; 35: 206214. doi: 10.1111/j.1600-051X.2007.01190.x.

Abstract

Background and aims: Periodontitis is an infection with an episodic nature oftissue support destruction. The aim of this work was to determine the levels ofchemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and

inflammatory cells in periodontal sites characterized by active periodontal connectivetissue destruction.

Material and Method: Fifty-six patients with moderate or advanced severity ofchronic periodontitis were selected. Periodontitis was characterized by at least six sites

with probing depth X5 mm, clinical attachment level X3 mm and radiographic boneloss. Periodontitis progression was determined by the tolerance method. Receptoractivator for nuclear factor k B-ligand (RANK-L), monocyte chemoattractantprotein-1 (MCP-1), tumour necrosis factor-a (TNF-a), IL-1b, MMP-13, Actinobacillusactinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsithia andinflammatory cells levels were determined. Statistical analysis was performed using

the Stata

s

7.0 software. Data were expressed as mean

SD and paired samples t-testand w2 tests were used.

Results: Higher RANK-L, IL-1b and MMP-13 activity levels were observed in activesites (po0.05). The proportion of P. gingivalis, A. actinomycetemcomitans,T. forsythia and the number of CD4

1T were higher in active than in inactive sites

(p40.05).

Conclusion: The detection of periodontopathic bacteria, host matrix

metalloproteinases and cytokines in periodontitis patients with lesions undergoingepisodic attachment loss could partially explain the mechanisms associated with thedestruction of the supporting tissues of the tooth.

Key words: cytokines; inflammatory cells;matrix metalloproteinases; periodontalpathogens; progressive periodontitis

Accepted for publication 25 November 2007

Nora Silva1

, Nicolas Dutzan2

,Marcela Hernandez2, Andrea

Dezerega2, Oriana Rivera2,

Juan Carlos Aguillon3,

Octavio Aravena3, Pedro Lastres4,

Patricia Pozo2, Rolando Vernal2 and

Jorge Gamonal2

1Area de Microbiologia; 2Periodontal Biology

Laboratory, Dentistry School, University of

Chile, Santiago, Chile; 3Programa

Disciplinario de Inmunologa, ICBM, Facultad

de Medicina, University of Chile, Santiago,

Chile; 4Centro de Investigaciones Biologicas

(CIB), Madrid, Espana

J Clin Periodontol 2008; 35: 206214 doi: 10.1111/j.1600-051X.2007.01190.x

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Conflict of interest and source offunding statement

The authors declare that they have noconflict of interests.This study was supported by project grantNo. 1050518 from Scientific and Techno-

logic Investigation Resource (FONDE-

CYT), Santiago, Chile.

Chronic periodontitis is an inflammatorydisease of the supporting tissues of theteeth caused by groups of specificmicroorganisms, resulting in progres-sive destruction of the periodontal liga-ment and alveolar bone with pocketformation, recession, or both (Armitage1999).

Chronic peridontitis is usually relatedto polybacterial infection, including,among others, Porphyromonas gingiva-

lis, Actinobacillus actinomycetemcomi-tans and Tannerella forsithia (Eley &Cox 2003). Destruction of the osseoussupport of the dentition is a hallmark ofchronic periodontitis (Listgarten 1986).The infiltrate present in periodontal dis-eases contains mononuclear cells,mainly transmigrated mononuclear pha-gocytes and lymphocytes. WhereasT lymphocytes predominate in theestablished chronic lesion, the propor-tion of B cells and plasma cellsincreases with disease progression(Saglie et al. 1988, Zappa et al. 1991).

The tissue destruction appears toresult from a complex interactionbetween these bacteria and the hostsimmune and inflammatory responses.Proteolytic activities of these bacteria,including collagenase, may participatein collagen degradation (Sela et al.2003). Activation and over-expressionof host matrix metalloproteinases(MMPs) caused by periodontal patho-gens such as A. actinomycetemcomitansand P. gingivalis, and by inflammatorycytokines have been reported (DeCarloet al. 1997, Okamoto et al. 1997, Ryan

& Golub 2000, Chang et al. 2002,Claesson et al. 2002). Interstitial col-lagenase activity in gingival tissueextracts and gingival crevicular fluid(GCF) from periodontitis sites arepathologically elevated when comparedwith the periodontally healthy gingivaltissue and GCF (Ingman et al. 1994,Golub et al. 1997). Considerable efforthas been made to study the cytokinereleased by different host cells whenexposed to components of periodonto-pathogenic bacteria (Offenbacher 1996).These studies have demonstrated that a

number of pro-inflammatory cytokinesare synthesized in response to period-ontopathogenic bacteria and their pro-ducts, hence inducing and maintainingan inflammatory response in the period-ontium (Page 1991).

Goodson et al. (1982) have presented

evidence indicating that periodontaldisease has dynamic states of exacerba-tion and remission and can be describedin terms of patterns of progression andregression of the disease. In the presentstudy, we investigated the prevalenceand proportions of P. gingivalis,

A. actinomycetemcomitans and T. for-

sithia in active sites, where clear bonetissue destruction occurred, and com-pared these samples with inactive sitesfrom patients undergoing progressionof chronic periodontitis. Bacterial cul-ture was used for the detection and

quantitative evaluation of periodonto-phatic bacteria. The total amount anconcentration of receptor activator fornuclear factor k B-ligand (RANK-L),monocyte chemoattractant protein-1(MCP-1), tumour necrosis factor-a(TNF-a), IL-1b and MMP-13 activitywere measured in samples of GCF. Wealso identified the cell populations pre-sent in lesions undergoing episodicattachment loss from gingival tissuebiopsies.

Material and MethodsPatients

This is a longitudinal clinical study inwhich moderate to severe chronic perio-dontitis patients were followed untilthey developed periodontitis progres-sion. Fifty-six patients were selectedfrom the Center of Diagnostic andTreatment of Northern MetropolitanHealth Services, Santiago, Chile, andconsecutively enrolled between March2005 and January 2006. The criteria forentry were a minimum of 14 natural

teeth, excluding third molars, andincluding at least 10 posterior teeth.Patients with chronic periodontitis hadmoderate to advanced periodontitis (atleast six teeth had sites with probingdepth (PD) X5 mm with attachmentloss X3 mm and extensive bone loss inradiography (430% of sites involved),according to a classification of theseverity periodontal disease based onthe location of the alveolar crest) andhad received no periodontal treatment atthe time of examination. Subjects didnot suffer from systemic illness and had

not received antibiotics or non-steroidanti-inflammatory therapy during the6-month period before the study. Beforethe study, all subjects received supra-gingival prophylaxis to remove grosscalculus for allowing PD. The protocolwas clearly explained to all patients, and

Institutional Reviews Board-approvedinformed consents were signed. Theprotocol stated that, within 2 weeks ofthe detection of disease activity, allpatients would be provided with perio-dontal treatment. Periodontal therapyconsisted of scaling, root planing andoral hygiene instructions.

Clinical measurement and determination

of periodontitis progression

Clinical parameters were evaluated inall teeth, excluding third molars, and

included PD, clinical attachment loss(CAL), dichotomous measurements ofsupragingival plaque accumulation (PI)and bleeding on probing to the base ofthe crevice (BOP). Six sites were exam-ined for each tooth: mesiobuccal, buc-cal, distobuccal, distolingual, lingualand mesiolingual. One calibrated exam-iner monitored the patients and col-lected the clinical reports.

All clinical measurements were takenat baseline, 7 days and 2 months: PD,clinical attachment loss, supragingivalPI and BOP to the base of the crevice.

Disease activity was defined by thetolerance method (Haffajee et al.1983). At the site level, active siteswere considered if they exhibited attach-ment loss X2.0mm during the follow-ing 2-month period. Inactive sites weredefined as those sites with CAL, PD andBOP equivalent to active sites, but with-out attachment loss during the sameperiod. At the patient level, at leasttwo active sites were needed to considerthe patient as undergoing diseaseprogression. Measurements of clinicalparameters were monitored at baseline

and at 2 months samples were collectedfrom one active and one inactivesites simultaneously, on each patientundergoing progression, before theperiodontal therapy was carried out.

Samples of GCF, subgingival plaqueand a gingival biopsy were collectedfrom one active site and one inactivesite from each patient undergoingattachment loss. Eighteen patientsdemonstrating disease activity wereincluded in the present study. Oneinactive site sample, characterizedby no attachment loss, was taken as

Characterization of destructive periodontitis 207

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control from each patient undergoingattachment loss.

Collection of GCF

After isolating the tooth with a cottonroll, supragingival plaque was removed

with curettes (Hu Friedy, Chicago, IL,USA) without touching the marginalgingiva. The crevicular site was thendried gently with an air syringe. GCFwas collected on paper strips (ProFlow,Amityville, NY, USA). The strips wereplaced in the sulcus/pocket until mildresistance was sensed and then left inplace for 30 s. Strips contaminated withsaliva or blood were excluded from thesampled group. After GCF collection,the volume of the sample on the paperstrips was measured using a calibratedPeriotron 8000 (ProFlow). The readings

from the Periotron 8000 were convertedto an actual volume (ml) by reference tothe standard curve. GCF strip sampleswere obtained from one active siteand one inactive site in each patientundergoing periodontitis progression.After GCF collection, strips obtainedfrom the same site were placed inEppendorf vials and kept at 801C.GCF was extracted by centrifugationat 18.000 g for 5 min. at 41C in 100 mlof elution buffer containing 50 mM TrisHCl (pH 7.5), 0.2 M NaCl, 5 mM CaCl2and 0.01% Triton X-100. The elution

procedure was repeated twice, and18 samples from each site were storedat 801C until further analysis.

Quantification of cytokine RANK-L,

MCP-1, IL-1b, TNF-a

Aliquots of each GCF samples wereassayed by enzyme-linked immunosor-bent assay to determine the levels ofRANK-L, MCP-1, IL-1b and TNF-aaccording to the manufacturers recom-mendations (American LaboratoryProducts Company, ALPCO Diagnostic,

Windham, NH, USA). Briefly, 50ml ofstandards and GCF samples were addedin the respective wells in duplicate. Fiftymicroliters of detection antibody wasadded to all wells, except blank, mixedgently, covered with strips with plasticfilm and incubated overnight (1624 h)at 41C. Plates were washed 5 andincubated with 200 ml of conjugate for50 min. at room temperature (18261C).The plates were washed 5 and 200 mlof substrate was added and incubated for30 min. at room temperature (18261C)in the dark. The reaction was stopped by

the addition of 50ml of stop solution,and colour was measured in an auto-mated microplate spectrophotometer(Bio-Tek Instruments Inc., Elx 800,Highland Park, VT, USA).

The amount of RANK-L, MCP-1,IL-1b and TNF-a was determined in

picograms (pg). Cytokine levels in theGCF were calculated with a standardcurve (3.9500 pg) obtained withrecombinant cytokines. Sample cyto-kine concentration values lower thanthe detection limit (below 3.9 pg in ourassay) were considered undetectable.Cytokine concentration (pg/ml) was cal-culated from the volume of GCF esti-mated from the calibration unit reading,according to the following formula:cytokine concentration (pg/ml)5 totalcytokine (pg)/volume GCF (ml).

Subgingival plaque samples

Subgingival plaque samples were col-lected from the active and inactive sitesin each patient and 18 samples fromeach site were collected. After isolatingthe area with cotton rolls and gently airdrying, supragingival deposits werecarefully removed with curettes (HuFriedy). Subgingival microbial sampleswere obtained by inserting two standar-dized N 30 sterile paper points (Johnson& Jonson, Tokio, Japan) into the deepestpart of the periodontal pocket for 20 s.

The samples from each patient wereplaced in a vial containing 1 ml of coldsterilized prereduced transport fluid(RTF) without ethylenediamine-tetraa-cetic acid. Vials with samples weretransported at 41C to the Microbiologi-cal Laboratory of the Faculty ofDentistry, University of Chile, and pro-cessed immediately.

Microbiological procedures

Subgingival plaque samples weredispersed by mixing (Thermolyne maxi

mix II type 37,600, IA, USA) for 45 sfollowed by a 10-fold serial dilution ofthe bacterial suspension in RTF. Ali-quots of 100 ml of the appropriate dilu-tion (10 3, 10 4 and 10 5) wereplated on non-selective Columbia bloodagar (5% defibrinated sheep blood,1 mg/l hemin, and 0.5 mg/l menadione)for total anaerobic cell counts (CFU/ml)and for the detection and quantificationof P. gingivalis and T. forsithia. Theplates were incubated anaerobically at351C for 714 days in a jar containinggas generator envelopes for the produc-

tion of an anaerobic atmosphere (OxoidLimited, Hampshire, UK).

Bacteria were primarily identified bycolony morphology under a stereoscopicmicroscope (Stemi 2000-C, Zeiss, Jena,Germany), pigment production, Gramstaining and rapid enzymatic tests for

oxidase, trypsin-like (BANA) anda-glucosidase detection. In addition,black pigmented colonies of P. gingiva-lis were tested for red-fluorescenceunder UV light (360 nm): negative forP. gingivalis and positive for P. inter-media/nigrescens (18). The anaerobicisolates were further identified bythe BBL-Crystal anaerobe ID System.

A. actinomycetemcomitans was also pri-marily identified by colony morphology(star-like inner structure or ridges) butalso on selective trypticase soy-serumbacitracin-vancomycin (TSBV) medium

(trypticase, 10% horse serum, bacitracinand vancomycin). One hundred micro-litres of undiluted and 10 1 dilutedsamples were inoculated on the agarplates and incubated at 351C for 23days in CO2 candle jars. The percentageof A. actinomycetemcomitans wasobtained using the number of CFU onTSBV as a percentage of the totalanaerobic counts. Gram staining andcatalase production were also used for

A. actinomycetemcomitans identifica-tion. Plates were incubated anaerobi-cally as mentioned above, for 23 days.

Gingival tissue biopsies and preparation

of gingival cells

Gingival tissues biopsies were obtainedfrom periodontitis patients with diseaseactivity. The incisions were made12 mm subgingivally; therefore, speci-mens consisted of the gingival margin,sulcular epithelium and gingival con-nective tissue. Soft issue biopsy speci-mens were washed extensively inphosphate buffer saline (PBS) andimmediately placed in a vial containing

5 ml of cold sterilized transportmedia: RPMI 1640 supplemented with50 UI/ml penicillin, 50mg/ml streptomy-cin and L-glutamine 200 mM (SigmaChemical Co., St Louis, MI, USA).Vials with the samples were transportedat 41C to the Periodontal BiologyLaboratory of the Faculty of Dentistry,University of Chile and processedimmediately. Samples of gingival tis-sues were weighed, minced intoapproximately 1 mm3 pieces and incu-bated in tissue digestion medium ina water bath at 371C for 90min. The

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tissue digestion medium consisted of(RPMI 1640) supplemented with50 UI/ml penicillin, 50mg/ml streptomy-cin and L-glutamine 200 mM, plus200 U/ml of type IV collagenase (GibcoInvitrogen Corporation, Grand Island,NY, USA), in a relation of ml of tissue

digestion medium by 50 mg of gingivaltissue.Cells obtained were washed twice

with PBS and resuspended in cellularculture medium RPMI 1640 supplemen-ted with 50 UI/ml penicillin, 50mg/mlstreptomycin, L-glutamine 200mM and10% foetal bovine serum (Gibco Invi-trogen Corporation). Cell counting wasperformed in a Neubahuer chamberusing phase contrast microscopy (Axio-vert 100, Zeiss Co., Germany). Cellviability was equal to or greater than90% as calculated by Trypan blue dye

exclusion.

Flow cytometry analysis

In order to quantify the immune cellsthat infiltrate the gingival tissues,200,000 total cells in 50ml of PBSwere incubated separately with 10 ml ofphycoerythrin (PE) and fluoresceinisothiocyanate (FITC)-conjugated anti-CD4 (CD41 T cells), anti-CD8 (CD81

T cells) and anti-CD191 (B cells) mono-clonal antibodies (mAb) (Biosciences,San Diego, CA, USA) for 30 min. at 41Cin the dark. Cells were washed once inPBS and resuspended in 300 ml of PBSto be analyzed by flow cytometry

(FACS Calibur Machine, Becton Dick-inson, San Jose, CA, USA). Cells weregated according to their forward andside-scatter characteristics and their spe-cific CD marker. FITC- and PE-conju-gated isotype-matched control mAbwere used to determine the positive

and negative population.

MMP-13 activity measurements

Aliquots of each GCF samples wereassayed by the Fluorokine E activityfluorescent assay (R&D Systems Inc.,Minneapolis, MN, USA) to determinethe basal activity of MMP-13, accordingto the manufacturers recommendations.Activity was expressed as nanograms offluorescent product (ng FP) per site andnanograms of FP per ml of GCF.

Data analysis

Clinical and experimental data werestatistically analysed using softwarestata 7.0 and expressed as subjectmeans standard deviation. w2 testand Students t-test were used to com-pare parameters between the clinicalperiodontitis groups and microbiologi-cal data. In the flow cytometry analysisdifferences were evaluated using the

ANOVA test. The fraction of positive cellswas expressed as a percentage. Cyto-kines, chemokines and MMP-13 activityvalues at each site were recorded and theunpaired Students t-test was used toanalyze differences in levels between

active and inactive sites. Statistical sig-nificance was considered when po0.05.The significance of differences wasassessed using the Tukey test.

Results

The clinical characteristics of the 18patients studied with progressivechronic periodontitis in this study are

grouped in Table 1 (2 months data). Nostatiscal difference existed between bothgroups. Five males and 13 females werestudied, with age range 3556 years(mean age 45.66 6.67). The meanplaque index was 100%, mean BOPwas 100%, mean PD was 4.42 0.46 mm mean attachment level was6.20 0.76 in active sites and meanprobing depth was 4.64 0.62 mm,and mean attachment level was6.42 0.68 in inactive sites (p40.05)(Table 1).

Table 2 describes the microbiologicalfindings in subgingival plaque samplesof active and inactive sites from patientsundergoing progression of chronicperiodontitis. Active sites showed high-er mean percentages for P. gingivalis ofthe total anaerobic than inactive sites,this difference being statiscally signifi-cant (17.9 versus 1.9, po0.05).

One of the main concerns in chronicperiodontitis is the definition of activesites, where tissue destruction, and prob-ably secretion of certain cytokines andMMPs is produced. Active sites areidentified by an increase in attachment

Table 1. Clinical characteristics from baseline measurements of active and inactive sites from periodontitis patients (Mean SD)

Clinical parameters (2 months data) Active sites (n5 18) Inactive sites (n5 18) p value

Mean probing depth (mm) 4.42 0.46 4.64 0.62 40.05

Mean attachment level (mm) 6.20 0.76 6.42 0.68 40.05

% sites with plaque 100 100

% sites with bleeding on probing 100 100Age (years) 45.66 6.67 (3556)

Females (%) 72.22 (13/18)

Table 2. Microbiological finding in subgingival plaque from active and inactive sites

Bacteria Active sites Inactive sites

isolation frequency % of total count isolation frequency % of total count

N % mean SD range N % mean SD range

Actinobacillus actinomycetemcomitans 3 16.6 0.6 05 1 5.5 0.1 01

Porphyromonas gingivalis 6 33.3 17.9n 082 8 44.4 1.9n 011.3

Tannerella forsythensis 3 16.6 0.6 05 1 5.5 0.1 01

n% of total count P. gingivalis, active versus inactive sites po0.05.




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loss during the length of study. Activesites showed a significantly highertotal amount of cytokine RANK-L andIL-1b (125.9 22.2 versus 91.8 10.7; 186.84 20.2 versus 123.78 19.6) (p5 0.0001 and 0.037, respec-tively) than the inactive sites (Table 3).

The mean variations of basal activity ofMMP-13 expressed as nanograms offluorescent product per site and permicrolitre of GCF (FP/site and ng FP/ml GCF, respectively) from active andinactive sites are shown in Table 3.Active sites, identified by an increasein attachment loss during the length of

the study and associated with perio-dontal tissue destruction, containedhigher basal activity levels of MMP-13than those found in inactive sites (1.2 ngversus 0.7 ng FP/site and 0.3 ng FP/mlversus 0.2 n g FP/ ml, respectively;

po0.05).

Levels of CD4

1

, CD8

1

and CD19

1

lymphocyte cells are more abundant inactive sites than in inactive sites,although differences were not so clear(p40.05) (Fig. 1). Figure 1 shows theflow cytometry of a representative sam-ple of periodontitis patients withdestructive periodontitis. Higher levels

of CD41, CD81 and CD191 lympho-cyte cells are associated with active sites(12.81 versus 11.41; 10.26 versus 9.06;5.24 versus 4.41, respectively).

Discussion

This study examined the prevalence andproportions of P. gingivalis, A. actino-mycetemcomitans and T. forsithia, thetotal amount and concentration ofRANK-L, MCP-1, TNF-a, IL-1b andmeasured MMP-13 activity in samplesof GCF, and also identified the cell

CD19+ B cellsCD4+ T cellsCD8+ T cells

INACTIVE SITES

ACTIVE SITES

Fig.1. Immunotypification by the flow cytometry of gingival cells obtained by enzymatic tissue digestion of gingival samples. The fraction ofpositive cells was expressed as a percentage. M15marker of positive cells. M25marker of negative cells. The analysis was made with thered-filled curve; which represents labelled cells with specific antibody. The unfilled curve represents the unlabelled cells used as control.Active sites CD81 T cells 10.26%, CD41 T cells 12.81% and CD191 B cells 5.24%. Inactive sites CD81 T cells 9.06%, CD41 T cells 11.41%and CD191 B cells 4.41%.

Table 3. Cytokine levels of RANK-L, MCP-1, IL-1b, TNF-a and MMP-13 activity in gingival crevicular fluid (GCF) in active and inactive sites(Mean SD)

Active Inactive p value Active Inactive p valuetotal amount (pg) total amount (pg) concentration (pg/ ml) concentration (pg/ ml)

RANK-L 125.95 22.2 91.80 10.7 0.0001 131.17 35.1 108.80 18.9 0.001

MCP-1 101.22 18.2 92.13 22.2 40.05 122.15 18.4 106.12 19.6 40.05

IL-1b 186.84 20.2 123.78 19.6 0.037 205.95 20.4 147.78 24.6 40.05TNF-a 66.18 14.6 47.86 16.8 40.05 73.87 16.6 57.12 18.4 40.05

basal activity (ngFP/site) basal activity (ngFP/site) p value basal activity (ngFP/ml) basal activity (ngFP/ml) p value

MMP-13 1.207 0.429 0.7945 0.205 0.020 0.312 0.091 0.247 0.043 0.01

GCF (ml) 0.99 0.15 0.86 0.15 40.05

MMP-13, matrix mettaloproteinase-13; RANK-L, receptor activator for nuclear factor k B-ligand; MCP-1, monocyte chemoattractant protein-1; TNF-a,

tumor necrosis factor-a.

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populations present in lesions under-going episodic attachment loss and com-pared active sites with inactive sitesfrom patients undergoing progressionof chronic periodontitis. Our resultsdemonstrate that the total amount ofcytokine RANK-L, IL-1b and MMP-13

activity present in GCF of sites under-going progression of periodontitis inlevels is significantly higher than ininactive sites. Active sites showeda higher proportion of P. gingivalis,

A. actinomycetemcomitans, T. forsithiaand CD41 T cells. Our data demonstratethat the detection of periodontophaticbacteria, host matrix metalloproteinases,cytokines and population of infiltratemight correlate with episodic attach-ment loss and could partially explainthe mechanisms associated with thedestruction of the supporting tissues of

the tooth.Three specific pathogens have beenrepeatedly identified as etiologic agentsin the periodontal destruction associatedwith chronic periodontitis: A. actinomy-cetemcomitans, P. gingivalis and T. for-sithia (Zambon 1996). However,evaluation of these three pathogens asrisk factors for identification of attach-ment loss over time has resulted inconflicting evidence. Three studies indi-cated that none of these pathogens wasuseful in predicting periodontal diseaseprogression (Wennstrom et al. 1987,

MacFarlane et al. 1988, Listgartenet al. 1991). In another study, T. forsithiawas predictive of tooth loss among sub-

jects with little or no periodontitis atbaseline (Machtei et al. 1999). In arecent prospective longitudinal study,T. forsithia was identified as a riskmarker for attachment loss in a popula-tion with low prevalence and severity ofchronic periodontitis (Tran et al. 2001).T. forsithia and P. gingivalis were foundto be associated with disease progres-sion in established periodontal patients(Machtei et al. 1997) and were also

found to be associated with alveolarbone loss (Grossi et al. 1995). Perhapsmost relevant to the ability of A. acti-nomycetemcomitans to evade the innatedefences and survive mechanical perio-dontal therapy is its ability to invadegingival tissues (Christersson et al.1998) and, in particular, to invadeepithelial cells (Meyer et al. 1991).T. forsithia possesses several virulencetraits, including the production ofa trypsin-like protease and lipopolysac-charide, but more recently its ability topenetrate host cells or induce apoptosis

has received attention (Arakawa et al.2000). P. gingivalis is one of the bestcharacterized of the opportunistic oralpathogens that inhabit the oral biofilm.P. gingivalis expresses three major viru-lence factors:fimbriae, gingipains andlipopolysaccharides (Genco et al. 1991,

Wingrove et al. 1992, Cutler et al.1995). The three main commonalitiesof A. actinomycetemcomitans, T. for-sithia and P. gingivalis include thefollowing: all are Gram-negative, andtherefore produce lipopolysaccharide,which can modulate the local inflamma-tory response in host cells that expresspattern recognition receptors; all appearcapable of invasion of the mucosalbarrier to infection and possibly of beingsequestered inside epithelial cells; allproduce factors that enable them toevade the antibacterial functions of the

innate immune response either passivelyor actively (Ezzo & Cutler 2003).Our results showed a difference in the

level of the microbial component inactive and inactive sites. However, thedifference in clinical appearance may becaused by factors other than the micro-biological composition of subgingivalplaque. Bacteria are essential, but insuf-ficient to cause disease; a susceptiblehost is also essential and host factors aredeterminative (Page et al. 1997). Perio-dontitis is a family of related diseasesthat differ in etiology, natural history,

disease progression and response totherapy, but with common shared path-ways of tissue destruction (Page & Beck1997). The shared events in the patho-biology are influenced by disease modi-fiers (also known as risk factors andindicators), both genetic and environ-mental or acquired, which may differfrom stage and form of disease toanother (Page & Beck 1997). The clin-ical picture observed is a result of thecomplex interplay among microbialchallenge, shared events and diseasemodifiers. The modifying factors are

major determinants of the differencesobserved in different periodontal condi-tions (Page et al. 1997).

Longitudinal studies on the progres-sion of periodontitis indicate that therate of periodontal tissue destruction islow and that advanced forms of thedisease occur in comparatively few indi-viduals and few tooth sites (Lindhe et al.1989, Hugosan & Laurell 2000). Atten-tion has been focused in the past onthe possibility that periodontal diseasemay not be a continuous process, butmay be characterized by episodes of

activity, followed by periods of relativequiescence (Goodson et al. 1982).Episodic periodontal probing attach-ment loss may be associated with varia-tions in the supracrestal inflammatorycell populations where significantlyhigher numbers of mast cells, mono-

cytes/macrophages and plasma cellsare present in active sites as comparedwith inactive sites (Zappa et al. 1991).However, the contribution of acquiredimmune cells in the progression ofperiodontal disease has long been con-troversial, with its exact role in theprotection versus destruction of thehosts periodontium being unclear(Zappa et al. 1990, Ebersole & Taubman1994). Our results showed higher levelsof CD41 helper T cells in the context ofperiodontal disease progression. Recentstudies show that interactions between

the cell-mediated responses, in particu-lar, CD41 T cells, are critical for thedevelopment of alveolar bone loss(Theill et al. 2002). Other studiesdemonstrated that CD41 mononuclearcells were relatively prominent (consti-tuting approximately 27% of recover-able mononuclear cells) and that theCD81 cells were relatively enrichedcompared with the composition of per-ipheral blood or normal gingival tissues(Taubman et al. 1984, Stoufi et al.1987). Our results confirm that there isa higher degree of inflammation and an

increased number of B cells in theinflammatory infiltrate from biopsies ofperiodontitis patients. CD191 cells arealso abundant in active and inactivesites, whereas the difference was notso evident for T cells. Other authorssuggest that the episodic loss of con-nective tissue in periodontitis patientsis related to the massive presence ofB cells and macrophages in the inflam-matory infiltrate (Zappa et al. 1990,Seymour 1991).

Microorganism-triggered inductionof RANK-L expression on CD41 T cells

and RANK-L-mediated osteoclast acti-vation and bone loss represent the mole-cular explanation for the alveolar bonedestruction observed in periodontalinfections. Moreover, it is known thatthe expression of pro-inflammatorycytokines (i.e., IL-1) can also regulatethe balance of RANK-L/OPG in thebone microenvironment and/or mesen-chymal tissue adjacent to bone(Hofbauer et al. 1999), thus contributingto bone destruction. These cytokinesand inflammatory mediators are capableof acting alone or together to stimulate




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alveolar bone resorption and collagendestruction via tissue-derived MMPs,a major pathway for the breakdown ofbony and soft connective tissue asso-ciated with periodontal disease activity(Offenbacher 1996). However, thepathological mechanisms underlying

the progression of gingivitis to earlyperiodontitis lesions remain unclearand will require further investigation.For example, periodontal pathogenslike A. actinomycetemcomitans, P. gin-givalis or T. forsithia are capable ofstimulating the pro-inflammatory cyto-kines production (i.e., RANK-L, IL-1b)for local tissue destruction. They canthen further activate nave T- and/orB cells for subsequent adaptive immuneresponses (Zambon 1996).

Our findings demonstrate that thetotal amount of RANK-L is significantly

higher in active sites versus inactivesites, considered as a marker of boneresorption. Disease activity is generallyaccepted to be loss of soft or hard tissueattachment to the tooth; a change inprobing attachment level may representa true change in the attachment level ora change in tissue tone or a combinationof the two (Jeffcoat & Reddy 1991). Thepresence of RANK-L in the GCF of siteswith episodic loss of connective tissuesimplies their potential role in themechanisms of tissue destruction asso-ciated with destructive periodontitis.

There are two molecules consideredessential and sufficient to support osteo-clastogenesis: macrophage colony-sti-mulating factor (M-CSF) and RANK-L.The process of bone resorption isinitiated with a resorptive stimulus(Rodan & Martin 2000). These stimula-tors typically affect bone resorptionthrough the activation of M-CSF orRANK-L (Rodan & Martin 2000). Inthe present study, we demonstrated thatthe total amounts of RANK-L present inthe GCF of periodontitis subjects werehigher than those in healthy subjects.

There are few studies in the literaturerelating RANK-L and its potential rolein periodontal tissue. Recently, RANKand RANK-L have been found in dentaltissues and cells in human deciduousteeth (Lossdorfer et al. 2002) andRANK-L has also been associated withalveolar bone tissue destruction duringperiodontal infection using an animalmodel (Rodan & Martin 2000). Further-more, microbial stimulation with

A. actinomycetemcomitans inducedRANK-L expression on the surface ofCD41 cells and in vivo inhibition of

RANK-L function, and using the decoyreceptor OPG diminished alveolar bonedestruction and reduced the periodontalosteoclasts after microbial challenge(Lossdorfer et al. 2002). These resultsindicate that the alveolar bone destruc-tion observed in periodontitis is due, at

least in part, to the action of osteoclastsand is mediated by RANK-L.Our data demonstrate that MMP-13 is

present in the GCF of adult patients withprogressive chronic periodontitis, andsignificantly higher activity levels inactive sites might correlate the presenceof the active form of this enzyme withalveolar bone loss and teeth loss occur-ring during this disease (Manciniet al. 1999). MMP-13 (collagenase-3),a highly expressed collagenolytic MMPin developing bone and cartilage, hasbeen assigned a role in the joint tissue

destruction that is a major feature ofvarious forms of human arthritis (Peltonet al. 1990, Murphy et al. 2002). MMP-13 has critical roles in embryonic devel-opment and remodelling of the skeletonin humans. These roles are reflected inthe collagenase-mediated destruction ofbone and cartilage in several forms ofhuman inflammatory diseases (Mitchellet al. 1996). The molecular weights ofpro-MMP-13 and active MMP-13 arereported to be about 60 and 48kDa,respectively (Neuhold et al. 2001). Ourfindings demonstrate that MMP-13

amounts in periodontitis patients aresimilar, but the level of activation isincreased in active sites compared withinactive ones and basal activity levelsare significantly higher in active sitesthan in inactive sites. During perio-dontitis progression, the main changesbetween active and inactive sites couldoccur at the level of pro-MMP-13 acti-vation, determining significant increasesof enzyme activity in active sites,hence resulting in bone and periodontalligament destruction. MMP-13 is con-sidered as a marker of activity progres-

sion, and this could be a preliminaryapproach, which will help to determinethe function and relevance of this metal-loproteinase during the progression ofperiodontitis.

Acknowledgements

The authors are grateful to Mr. Benja-min Martnez, School of Dentistry,Mayor University, Santiago, Chile, forstatistical assistance, and Miss SabritaChandia, Periodontal Biology Labora-

tory, Dentistry School, University ofChile, for technical assistance.

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Address:

Jorge Gamonal

Facultad de Odontolog a Universidad de Chile

Avenida Olivos 943

Comuna de Independencia

Santiago

Chile

E-mail: [email protected]




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Clinical Relevance

Scientific rationale for the study:

Periodontitis is an infection withan episodic nature of tissue supportdestruction, in which short periodsof destruction are followed bylong periods of remission and

healing.

Principal findings: In summary, wehypothesized that disease progres-sion is due to a combination ofseveral factors, including the pre-sence of periodontopathic bacteria,high levels of pro-inflammatorycytokines, matrix metalloproteinases

and higher levels of CD41

T cells.

Practical implications: The presenceof IL-1b, RANK-L, MMP-13 andhigher levels of CD41 T cellsappears to be a useful tool for knowl-edge on the mechanisms and patho-genesis of active diseases.

214 Silva et al.



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Caracterización de Las Lesiones Periodontales Progresivas en Pacientes Con Periodontitts Crónica

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