Journai of Periodontal Researeh 1983 18: 501-515

A longitudinal study of crevicular fluid inperiodontal disease in beagles

Clinical, histopathological and biochemical observations

SABYASACHI MUKHERJEE, ARUP K. DAS AND MINU K. PATEL

Department of Periodontics, Center for Research in Periodontal Disease and Oral MolecularBiology, and Department of Oral Pathology, College of Dentistry and Research Resources Center,

Biostatistic Facility, University of Illinois at Chicago, Chicago, U.S.A.

A longitudinal study on the composition of crevicular fluid (CF) and serum was carried out inseven beagle dogs during a period from health to 16 weeks of gingivitis and then 8-10 weeks ofligature induced periodontitis (LIPD). The fluids were collected at weekly intervals and the pHand the concentrations of Ca, Mg, Na, and K and the protein profile were determined. Also atweekly intervals, the plaque index, gingival index, calculus index, and pocket depth andattachment loss were recorded for the teeth from which crevicuiar fluid was coiiected.Radiographs were taken on the 0 day and after the dogs were sacrificed. Histopathologicalstudies were carried out following sacrifice of the dogs, and the distances between the cemento-enamel junction and the coronal end of the junctional epithelium and the cemento-enameijunction and the crest of the alveolar bone were measured.

A two-way ANOVA analysis of the two fluids (CF and serum) and of the two diseases(gingivitis and periodontitis) showed significant differences between the two fluids and betweenthe two diseases. Further statistical analysis showed that in serum none of the constituentsstudied differed significantly between gingivitis and LIPD, but a significant rise in theconcentrations of Ca, Mg, and K was observed in CF during LIPD. The pH of CF wassignificantiy iower during LIPD, but no significant change was observed in the concentration ofNa ion. An apparent relationship was observed only between the concentration of K ion in CFand the attachment loss during LIPD.

The serum protein profile remained unchanged throughout the period of this study. Duringgingivitis the CF protein profile was identical to that of serum, but during LIPD a new proteinpeak appeared. This material had a moieeuiar weight of <: 200,000 daltons. Histopathologicaiand radiographic analysis showed bone loss in all but one dog during LIPD. Histopathologicalstudy also showed no significant difference in the distance between CEJ and the coronal end ofthe junctional epithelium between the ligated and the non-ligated sides. This study showed thatin beagle dogs the CF differed significantly from that of serum. Also, LIPD caused significantchanges in both ionic concentrations and protein profile of crevicular fluid.

(Accepted for publication April 21, 1983)

Hancock 1981). Therefore, the basis of cur-Introduction j.gj j therapy is often subjective evaluation of

There is a lack of an objective, accurate, past disease and not current disease activity,non-invasive method of diagnosis of the Sufficient data exist to believe that crevi-severity and activity of chronic inflamma- cular fluid (CF) may be a good source fortory periodontal disease (CIPD) (Hurt 1977, determining the severity and activity of

502 M U K H E R J E E , D A S A N D P A T E L

CIPD (Cimasoni 1974, Golub & Kleinberg1976, Abbott & Caffesse 1977, Hancock1981). The aim of this study was to correlatevarious chemical parameters of crevicularfluid with the progression of chronic in-flammatory periodontal disease. A positivecorrelation between the disease severity andany constituent of CF would be of value inthe diagnosis of CIPD. As short termperiodontitis can be induced in the beagledogs by placing ligatures around the necksof teeth (Ericsson et al. 1975, Schroeder &Lindhe 1975, Lindhe & Ericsson 1978,Schroeder & Lindhe 1980) and the severityand activity of the disease can be evaluatedby both clinical and histopathologicalmeans, this study was conducted in beagledogs. The protein proftle, pH, and the con-centrations of Ca, Mg, Na, and K in crevi-cular fluid and serum were studied.

Method and Materials

Seven two-year-old male beagle dogs wereused. The dogs, upon their arrival, werequarantined for two weeks and properlyvaccinated. They were then thoroughlyexamined to determine their periodontalhealth. All of them showed heavy depositsof plaque and calculus, gingival inflamma-tion, and pocket formation (1-4 mm). Onedog, # 15, had furcation involvementaround the right upper 2nd premolar. Fol-lowing oral examination their teeth werescaled and polished, daily brushing wasinstituted, and they were fed a hard diet(Egelberg 1965). After eight weeks of dailybrushing all teeth, gingivae, and sulcusdepths were examined. Most teeth showed asulcus depth of 0 mm, while some showed adepth of 1-2 mm. There was no gingivalinflammation around any tooth. One biopsyspecimen was taken from the upper molarregion of each dog, and the gingival healthwas further confirmed by histologic means.An absence of inflammatory cells in the

connective tissue of the gingiva was taken asan evidence of health. Daily brushing wasthen withdrawn and the dogs were given asoft diet (Purina Dog Chow softened withwater). At weekly intervals, blood sampleswere drawn, crevicular fluid samples werecollected from upper right premolar teeth( P \ P^, P^, P*), and clinical indices of theseteeth were recorded. After 16 weeks ofchronic gingivitis, periodontitis was inducedby placing silk ligatures (3-0 silk, Ethicon,USA) subgingivally around the right upperpremolar teeth ( P \ P^ P^ P*) of each dog.A mesial and distal groove was preparedsubgingivally around each tooth to keep theligature from moving either coronaiiy orapically. Weekly collections of blood sam-ples and crevicular fiuid samples and re-cording of clinical indices were continued.The fluid was also collected from the upperleft premolars and the clinical indices ofthese teeth were measured occasionally afterthe ligation of upper right premolars. Eightto ten weeks after ligation, the dogs weresacrificed and the upper jaws were separatedand processed for histologic evaluation. Fig.1 shows the experimental design. For clin-ical assessment of gingivitis and periodontit-is the following indices were used:

Plaque Index (Silness & Loe 1964),Calculus Index (Greene & Vermillion

1960), andGingival Index (Loe & Silness 1963).Attachment loss was measured from an

amalgam restoration as reference point onthe crown of each upper premolar tooth

)ay 0 Llgatlofi1 GINGrvmSde wka)—.J PERIODCWTmS (e-10 wk»)

Dally brushing

(8 wka)I i U

Fig

1 =

Day 0

. 1. Experimentai design.

Llpper Right Premoiars: Ciinicai indices and CF andbiood sampies obtained we8i<iy from day 0.Upper Left Premoiars: Ciinicai indices and CFsampies obtained occasionaliy from day 0.i-iistopathoiogy of aii upper premoiar teeth.

C R E V I C U L A R F L U I D IN P E R I O D O N T A L D I S E A S E 503

(Lindhe, Hamp & Loe 1973) to the bottomof the sulcus.

Pocket depth was measured at six pointsaround each tooth (distobuccal, buccal,mesiobuccal, distolingual, lingual, andmesiolingual).

One probe (Williams, Hu-Friedy Immun-ity, USA) was used throughout the experi-mental period for both attachment loss andpocket depth measurements.

Alveolar bone loss was determined by X-ray (Rosenberg, personal communication).After i.v. sedation the dog was positioned insuch a way that the head was kept at a 45°angle. Each quadrant was separated fromthe others as much as anatomically possibleand extraoral lateral radiographs weretaken. Kodak Readypak M2 (Industrex)films were used and processed manuallyaccording to the instructions of the manu-facturer. The X-rays were placed on a view-box and the teeth to be evaluated weremeasured from the CEJ to the apex of theroots and from the coronal crests of thealveolar bone to the apex of the roots. Themesial and distal sides of each upperpremolar tooth were measured, and for eachmultirooted tooth similar measurementswere recorded in relationship with the furca-tion points. The X-rays were taken on the 0day and following sacrifice of the dogs. Thepositions of the alveolar bone in relation toCEJ on these two occasions were comparedto determine the amount of bone loss forindividual tooth.

Kodachrome color pictures of the gin-givae of the dogs were taken on arrival, day0, on ligature placement, and the day ofsacrifice.

At weekly intervals, during the morning,the serum and crevicular fiuid samples werecollected as follows. Venous blood (around4 ml) was first collected and kept standingfor at least one hour. The serum was thenseparated from the clot by centrifugation at1450 xg for 15 minutes. Following the col-

lection of venous blood the dogs were anes-thetized by an intravenous injection of Sur-ital (Thiamylal sodium; Park Davis, N.J.).An estimated dose of lcc/kg was given untileffect and the state of anesthesia was main-tained by titration of same solution.

Crevicular fluid was collected after re-cording the plaque index but before takingany other indices. The teeth were isolatedwith gauze sponges and dried before collec-tion of fiuid. The fiuid was obtained sepa-rately from upper right and left premolarteeth. Volumetric micropipette - 1 , 3, or 5iu\ - (Drummond Scientific Co., U.S.A.) wasplaced extracrevicularly near the margin ofthe gingiva. No gingival massaging wasdone and fiuids visibly contaminated withblood were discarded. All sarnples werefrozen immediately after collection.

Analysis of erevicular fiuid and serumsampiesProtein profile. Immediately following thecollection of crevicular fluid and serumsamples the elution pattern of protein inthese fiuids was deterrnined by hquidchromatography on a Sephacryl S 200 gelcolumn (16.5 x 1.5 cm; bed volume 29 ml).Fifteen fi\ of sample fiuid containing 900-1000 fig of serum protein or 675-1000 /Jg ofCF protein was mixed with 100 1 of eiutingbuffer (0.5 M Tris HCl with 0.1 M NaCl and0.02% NaN3; pH 8.0). The mixture wascentrifuged in a Beckman Microfuge at12,000 rpm for 2 min. About 80 ^1 of thesupernatant was then carefully pipettedfrorn the centrifuge tube and applied on tothe column. The elution was monitoredcontinuously by a Pharmacia Dual Unit UVmonitor (Model 200) at 280 and 254 n.m.The eluted fiuid was collected in fractions of3.0 ml by a Gilson fraction collector at afiow rate of 12 ml/hour.

Ca, Mg, Na, and K eontents. The estim-ation of Mg and Na was carried out at

504 MUKHERJEE, DAS AND PATEL

wavelengths of 285.2 nm and 589.0 nm,respectively. The concentrations of Ca, Mg,Na, and K in serum and crevicular fiuidwere determined by Atomic AbsorptionSpectroscopy using an injection techniqueand an air acetylene fiame (Mukherjee1983). The sample (1-5 fil) was diluted 200-300 times in the diluent. For the determin-ation of Na concentration, 50-100 /A of thediluted sample was further diluted 1; 10 withthe diluent. Perkin Elmer multielement hol-low cathode lamps - Ca-Mg and Na-K -were used.

Determination of pH. The pH of serumand crevicular fiuid was determined by amicro-electrode (MI410; MicroelectrodesInc., NH, U.S.A.) and an Orion pH meter(Model 807A). Only those crevicular fiuidsamples (15-20 fxl) that were to be analyzedfor protein profile were used for the deter-mination. Twenty jA of serum samples werepipetted into a micro-centrifuge tube andthe pH determined.

HistopathologyThe animals were sacrificed with a lethaldose of pentobarbital sodium. The jawswere then removed, separated into right andleft halves, and fixed in 10% neutral buf-fered formalin. Following fixation, the jawswere decalcified in 10% formic acid for fiveweeks. Then eight blocks of tissues weretaken from premolars of each side of upperjaw. Four blocks consisted of bucco-hngualsections that were cut through the amalgampoints and corresponding roots of four pre-molar teeth. The other four blocks consistedof mesio-distal sections passing between thetwo adjacent teeth, i.e. between 1st and 2nd,2nd and 3rd, 3rd and 4th premolar teeth,and 4th premolar and 1st molar teeth. Theblocks were then processed routinely andvacuum embedded in paraffin. Multiple sixfi thick sections were taken from each blockand stained with hematoxylin and eosin,Masson's trichrome, and Van Geison's

method. The slides were projected by meansof Leitz Micro Promer shde projector, andthe images were drawn at x 70 magnifica-tion. The following measurements weretaken on each magnified drawing:

1. The distance from the cemento-enameijunction to the most coronal cells of thejunctional epithelium.

2. The distance from the cemento-enameijunction to the crest of the alveolar bone.

The numbers were divided by 70 to obtainthe actual distances.

Statistieai anaiysisThe data on concentration of various ions inCF were summarized to give the number ofobservations with its corresponding meansand standard deviations for each weekperiod. The data on concentrations of vari-ous ions in CF and serum during gingivitisand during ligature induced periodontitis(LIPD) were analyzed by two-way ANOVA(Sokal & Rohlf 1981). The responses werethe values of the elements and the twofactors of interest were fiuid and disease.After removing the main effects and interac-tion from total variability, one is left withthe inherent variability which is used tomake the necessary comparisons betweenthe specified means. The / values were calcu-lated at specified levels of significance. Allthese and other relevant calculations werecarried out using the BMDP (1975) series.

Results

Piaque Index (PI I), Gingivitis Index (GI),Calculus Index (CI)The mean weekly PI I, GI, and CI ofexperimental teeth (right upper premolars)of all dogs are shown in Fig. 2. It shows thatall the indices increased during gingivitis.After ligation there was an immediate in-crease in PI I and GI and a decrease in CI.

C R E V I C U L A R F L U I D IN P E R I O D O N T A L D I S E A S E 505

INDEX50-

a 2 4 6 8 10 15 16 1 1 3 5 7 9 1 0 WEEKSUgatuffl

Fig. 2. Mean G.I. (•), Pi.I. {•), C.I. (A), and AttachmentLoss (D) index of all rigiit upper premoiar teeth of aildogs, each weei<, during gingivitis and iigatureinduced periodontitis (LiPD).

After the initial increase in PI I, there waslittle further increase during the remainingperiod of ligation. In contrast, the GI de-creased during 2nd, 3rd, and 4th week afterligation and then increased during 5th and6th week and fiuctuated during the remain-ing period of ligation. The CI showed asteady rise during most of the ligationperiod except during 8 to 10 weeks. Statisti-

Table 1

Correlat ion between the t ime (week), tooth, GI,PII, and Cl of all dogs

index Week

GIPiiCi

»**

0.3871***0.7504***0.5004***

P< 0.001.P<0.01.

Tooth

-0.13560.11680.3825

GI

0.5497***0.2630**

Pii

0.5934***

cal analysis showed a significant correlation(p< 0.001) between time and all the clinicalindices (Table 1). The table also shows asignificant correlation between (1) plaqueindex and gingivitis index, (2) plaque indexand calculus index at p< 0.001, and (3)gingivitis index and calculus index atp<0.01.

Foeket depth and attachment lossDuring gingivitis the pocket depth remainedunaltered around most teeth. However, inthose areas where no sulcus was presentduring health a depth of 1 mm was noticedat the end of 16 weeks of gingivitis. Anincrease in depth of 2 mm was noticedaround two teeth - buccal of P^ of dogs#12 and #15. In these same areas at-tachment loss was also noted. Otherwise noattachment loss was observed in the gin-givitis period.

During ligature induced periodontitis thepocket depth increased around P* in alldogs except #16. Some increase in pocketdepth was also noticed around other teeth.Nevertheless, 43% of the total number ofteeth showed no change in pocket depthduring this period. The mean weekly attach-ment loss index of right premolar teeth of alldogs showed an increase during 1st and 2ndweek, a sharp decrease during the 3rd week.

Table 2Amount of bone loss (in mm) as determined by X-ray method

Tooth

Dog#

121314151i|1718

P'M

2.001.02.0

Absent00

D

1.001.03.0

1.00

M

2.01.00.51.01.01.02.0

P'D

3.002.01.01.01.00

F

1.003.02.51.02.01.5

M

2.002.01.001.01.0

D

2.5

__0

-

F

2.5000000

Tofai

16.01.09.5

10.53.06.04.5

Mean

20.141.361.50.50.860.64

Rank

1732645

= Mesiai. D = Distai. F = Furca.

506 MUKHERJEE, DAS AND PATEL

Table 3

Mean distance (in mm) from CEJ to pocket base and CEJ to alveolar crest of all teeth groupedaccording to side and according to non-ligated and ligated teeth

Side

BuccaiLingualDistaiMesiai

N

26232223

CEJ to PocketNon-Ligated

Mean

0.080.060.030.08

S.D.

0.880.540.550.16

N

25252221

BaseLigated

Mean

0.200.210.170.27

S.D.

0.090.200.120.07

N

29272527

CE.i to AiveoiarNon-Ligated

Mean

1.090.951.000.87

S.D.

0.100.140.230.07

N

25242122

CrestLigated

Mean

1.481.421.431.30

S.D.

0.240.290.490.39

and then a gradual rise during the 4th and5th weeks (Fig. 2).

Radiographic evaluationTable 2 shows the bone loss which occurredbetween day 0 and the day of sacrifice asdetermined by X-ray. It can be seen thatbone loss occurred following ligation in alldogs. The highest loss occurred in dog # 12(16 mm, total) and lowest for dog #13. Thetooth P* was excluded from this analysis,because it was difficult to read on most X-rays.

Histopathologieai observationsOf the 224 possible sections (112 sectionseach for ligated and non-ligated teeth) 95sections from non-ligated teeth and 92 sec-tions from the ligated teeth were available

for the study. The mean distances from theCEJ to the coronal end of the junctionalepithelium and from the CEJ to the crest ofthe alveolar bone on buccai, lingual, mesial,and distal aspects of ligated and non-ligatedteeth are shown in Table 3. It shows that allthe values were higher for ligated than thenon-ligated teeth. Also, for both ligated andnon-ligated teeth the mean measurementsare similar on all surfaces of teeth. Table 4shows the mean distances from the CEJ tothe coronal end of the junctional epitheliumof the hgated and the non-ligated teeth foreach dog. Only identical sections weremeasured and compared by a paired / test.The results show that e;ccept for dog # 13there was a slight increase in the distance forthe ligated group of teeth for all dogs.However, statistically these differences were

Table 4

Histopathological determination of the distance from CEJ to the coronal end of junctional epitheliumon ligated and non-ligated sides. Only same type and equal number of sections for both sides were

considered

Dog #

12131415161718

N

912121111159

Non-Ligated

Mean

0.0800.080.040.010.030.03

SD

0.2500.190.080.030.110.08

Ligated

Mean

0.2800.250.430.020.150.44

SD

0.2900.380.490.050.340.57

Paired

t

-1.49_

-1.142-2.57-0.38-1.33-2.13

•f Test

P

0.16-

0.17 .0.020.710.190.05

Aii measurements in mm.

C R E V I C U L A R F L U I D IN P E R I O D O N T A L D I S E A S E 507

Table 5

Histopathological determination of the distance from the CEJ to the alveolar crest on ligated and

non-ligated sides. Only same type and equal number of sections were considered

Dog #

1213

141516

1718

N

912121111

159

Non-Ligated

Mean

0.931.040.940.991.121.110.90

SD

0.290.290.390.430.28

0.280.22

Ligated

Mean

1.651.131.461.581.27

1.621.41

SD

0.780.41

0.550.490.64

0.560.53

Paired

t

- 2 . 6 0- 0 . 6 4- 2 . 6 9- 2 . 9 9- 0 . 7 0- 3 . 1 9- 2 . 6 4

't' Test

P

0.0190.5320.0130.0080.491

0.0030.018

All measurements in ntim.

significant for dogs # 15 and 18 only. Table5 shows the measurements of the samesections from the CEJ to the crest of thealveolar bone. This shows that the distancewas greater for the ligated sides than thenon-ligated sides for all dogs. Statistically,the paired differences were significant for ailbut dogs #13 and #16.

Analysis of serum and crevicular fluid duringgingivitisThe mean Ca, Mg, K, and Na concentra-tions in crevicular fluid during each week ofgingivitis were calculated. Data obtainedfrom dogs # 12 and 15 were excluded in thisanalysis, because these dogs showed attach-ment loss during the gingivitis period (see

above). A week to week variation in con-centration of all elements in CF was clearlyevident. When the data were plotted (Figs. 3& 4), the results indicated a rise in Mg, K,and Na level during the experimentalperiod. Table 6 shows the concentration ofeach element in crevicular fluid at the high-est and lowest gingival index in the samedog. A paired t test between the GI valuesand between each element of CF showed asignificant difference between the GI values(t = 3.733, p<0.05), but not between anyelement in crevicular fiuid (Ca: t = 0.439,p<0.70; Mg: t = 0.999, p<0.40; Na: t= 1.076, p<0.40; K: t= 1.850, p<0.20).

The mean weekly attachment loss indexof the same dogs also showed variations

1.0-

\2D-

rao-ao-6D-

4D

2D-

GINGIVAL INDEX

0 1

Fig. 3. Meanin CF duringtive teeth is

e 10 WEEKS2 3 4 5 6

weekiy Ca (•) and Mg ( D ) concentrat ionsgingiv i t is . Mean weekiy Gi of the respec-

shown in the box.

mEq/L

1500-

130.0^

110.0-9.0-

TO

SiO-

30-

" ' " 1 GINGIVAL INOEX

r ^• *\ /"/

I 2 3 4 5 6 7 8 9 10 WEEKS

Fig. 4. Mean weekiy K ( A ) and Na ( A ) concentrat ions inCF dur ing gingiv i t is . Mean weekly Gi of the respect iveteeth is shown in the box.

508 M U K H E R J E E , D A S A N D P A T E L

Table 6

The concentrations of Ca, Mg, Na, and K in crevicular fluid according to the highest and lowestgingival index of each dog during the longitudinal study

Dog #

14161718

MeanS.D.

G.i.

1.501.081.191.13

1.230.19

Week

9979

High G.I.

Ca

10.0411.609.749.08

10.121.07

Mg

2.45

1.462.22

2.040.52

Na

95.50210.80

125.00

143.7759.90

K

4.6511.495.486.18

6.953.09

G.I.

0.940.920.880.88

0.910.03

Week

5866

Low

Ca

10.069.34

10.269.63

9.820.42

G.i.

Mg

2.20

2.392.90

2.500.36

Na

104.0136.3

111.0

117.116.99

K

4.457.233.855.54

5.271.48

Table 7Two way ANOVA of concentration of each element between fluids (serum and crevicular fluid) and

between diseases (gingivitis and periodontitis)

FiuidDiseaseinter-action

Error/ResiduaiMean Square

df

11

1

19

pH

f

10.98010.035

4.389

P

0.0040.005

0.050

0.5579

df

11

1

97

Ca

f

6.0510.609

6.376

P

0.0160.437

0.013

2.70461

df

11

1

83

Mg

f

25.3423.422

5.172

P

0.0000.068

0.026

0.33405

df

11

1

84

Na

f

0.1720.134

0.719

P

0.6790.716

0.399

1043.73

df

11

1

80

K

f

32.56210.497

5.441

P

0.0000.002

0.022

4.96422

(Fig. 5). However, an apparent relationshipcan be observed only between the meanweekly attachment loss index and meanweekly concentration of the K in crevicularfluid (Fig. 5).

The data were further analyzed to testwhether there were any significant differ-ences between the various ions of CF andserum and between the concentrations ofeach element in each fluid (CF and serum)during gingivitis and periodontitis. Table 7shows the results of a two-way ANOVA testfor each element between the two fluids(serum and CF) and between the two dis-eases (gingivitis and periodontitis). Therewas a signiflcant difference between thefiuids (CF and serum) for pH and theconcentrations of Ca, Mg, and K but not for

Na. A significant difference between thediseases (gingivitis and LIPD) for pH andthe concentrations of Mg and K are also

mm"'5-|ATTACHMENT INDEX

Fig. S. Mean weekiy Ca (o) and Mg (n) concentrationsin CF during LiPD. Mean weekiy attachment loss indexof the respective teeth is shown in the box.

C R E V I C U L A R F L U I D I N P E R I O D O N T A L D I S E A S E 509

Table 8

Mean pH and concentration of Ca, Mg, Na, and K in serum during gingivitis and inducedperiodontitis

PHCaMqNaK

Gingivitis

# of samples Mean

620

171711

7.859.661.90

141.274.13

S.D.

0.050.950.31

22.610.66

Periodontitis

# of samples

527272627

Mean

7.749.091.85

132.774.60

S.D.

0.210.980.24

25.250.59

Statistical

t

-1.721.170.280.84

-0.59

significance

df

945424136

P

0.100.300.800.850.50

Concentrations of Ca and Mg are in mg%.Concentrations of Na and K are in Mq/L.

shown. The interaction between fluid andthe disease was significant for all elementsexcept for the concentration of Na. Theresults were then statistically analyzed by apaired / test to determine whether the dif-ferences in the concentration of each ele-ment in each fluid during gingivitis andperiodontitis were statistically signiftcant.The results for such analysis for serumsamples are shown in Table 8. The pH andthe concentrations of Ca, Mg, Na, and K inserum did not differ significantly betweenthe two diseases. The results of a similar ttest for CF are shown in Table 9. Theconcentrations of Ca, Mg, and K weresignificantly higher during periodontitisthan during gingivitis. The pH was signifi-cantly lower during periodontitis than dur-

ing gingivitis, but there was no significantdifference in the concentration of Na in CFcollected during gingivitis and periodontitis.

Protein profileSerum. Whenever CF sarnples were avail-able for chromatography the correspondingserum samples were also analyzed. Seven-teen serum samples were analyzed duringgingivitis and ten during periodontitis. Thechromatograms were identical for all dogsand remained unchanged during the experi-mental period (Fig. 7). Three definite peakswere observed at 280 nm and 2-3 peaks wereobserved at 254 nm. Peak A had a molecularweight of > 67,000 daltons and was prob-ably composed mainly of serum albumin.

CF. Twenty-one CF samples were ana-

Table 9

Mean pH and concentrations of Ca, Mg, Na, and K in crevicular fiuid during gingivitis and inducedperiodontitis

Gingivitis Periodontitis Statistical significance

Mean S.D. Mean S.D. df

PHCaMgNaK

626181918

8.389.642.25

132.425.89

0.232.010.50

34.792.00

628252628

7.8610.732.77

135.808.80

0.352.100.92

40.883.42

-3.81-2.43-2.91-0.35-4.32

1052414344

0.010.020.010.700.001

Concentrations of Ca and Mg are in mg%.Concentrations of Na and K are in n-iEq/L.

51G MUKHERJEE, DAS AND PATEL

Fig. 6. Mean weekly K (A) and Na (*) concentrations inCF during LIPD. Mean weekly attachment loss index ofrespective teeth is shown in the box.

lyzed during gingivitis. All CF samples pro-duced identical chromatograms and did notchatige during gingivitis (Fig. 8). It can beseen that this chromatogram is similar tothat of serum (Fig. 7). The major peak (A)had a molecular weight of > 67,000 daltonsand probably consisted mainly of serutnalbumin.

Seventeen CF samples were chromatog-raphed during the period of LIPD. Fig. 9shows a chromatogram during this period.An additional peak (AJ appeared in this

9 19 29 34ml

Fig. 8. Chromatogram of CF sample during gingivitis(week 8). Dog # 15, 16 /il CF; 0.5 M Tris-HCl Buffer with0.1 M NaCl and 0.02% NaNji pH 8.0. 280 nm--254nm.

chromatogram which was not observed inatiy CF sample collected during gingivitis.The peak A^ appeared one week after lig-ation and was observed in all samples collec-ted during this period. The only exceptionwas the sample from dog # 13 which did not

AO

34 ml •

Fig. 7. Chromatogram of serum sample. Dog #15, 15/li serum; 0.5 M Tris-HCl. Buffer with 0.1 M NaCl and0.02% NaNj, pH 8.0. 280 nm -- 254 nm.

Fig. 9. Chromatogram of CF sample during LIPD (week10). Dog #15. 15 ii\ CF; 280 nm - - 254 nm.

C R E V I C U L A R F L U I D I N P E R I O D O N T A L D I S E A S E 511

show the presence of peak Aj. The rnole-cular weight of the peak was < 200,000daltons, and the peak appeared in the voidvolume. Also, on the day of sacrifice, CFsamples from both right (ligated) and left(non-ligated) sides were available from threedogs (#12, 15, 18). In each occasion thepeak A] was observed in the chromatogramof CF sample collected from the right side,but this peak was absent in the chromato-gram of CF collected from the left side. Toinvestigate whether peak A^ appeared dueto contamination with the supra-gingivalplaque of the right side, CF was first collec-ted and then the supragingival area wasscaled and rinsed with distilled water. FluidWas collected again. The chromatograms ofthe samples were identical, and both showedthe presence of peak A^.

DiscussionDuring the course of this study, it wasobserved that the flow rate of crevicularfiuid varied from dog to dog. This variationwas observed both before and after theliga tion of teeth. Dog # 16 had little or nocrevicular fiuid for most of the occasions,and we could collect fiuid from this dog onlyfive times during the 26 weeks, but the fiuidfrom two dogs, #12 and 18, was readilyavailable. Generally, we observed that dogswith severe periodontitis provided moreerevicular fiuid than those that sufferedfrom mild periodontitis. The reasons forsuch variations are difficult to explain atpresent. Rate of fiow of CF correlates withthe severity of gingivitis (Brill 1960, Egel-berg 1964, Loe & Holm-Pedersen 1965,Biswas, Duperon & Chebib 1977, Borden,Golub & Kleinberg 1977), but its correl-ation with periodontitis is controversial(Valazza et al. 1972, Tanner et al. 1980). Thestudy also showed that the severity ofperiodontitis in LIPD in beagles varied

greatly between the dogs. Schroeder andLindhe (1980) reported that the severity ofthe disease depended to a large extent on theplacetnent of the ligature and the degree ofulceration of the sulcular epithelium. Theligatures that were placed supragingivally orcaused less ulceration of the sulcularepitheliutn induced less bone destruction. Inthis study all ligatures were placed subgin-givally. Therefore, the variations in theseverity of the disease probably dependedon factors such as subgingival ulceration,immune responses, and pathogenicity of thesubgingival plaque. It should be noted thatthe dog # 13 which showed minimum boneloss developed a swelling on the throat andwas given a course of antibiotics (Strepto-penicillin; 400,000 U Procaine Penicillin Gand 0.5 g Dihydrostreptomycin; subcuta-neously for seven days) ten days prior toligation. This could have destroyed thepathogenic fiora resulting in mild infiatnm-ation and minitnum bone loss.

Our clinical results during gingivitis con-firmed the earlier findings that followingwithdrawal of oral hygiene the PlI, GI, andCI increased with time (Saxe et al. 1967,Lindhe et al. 1973, Schroeder & Lindhe1975). The cause for decrease in CI immedi-ately following ligation was probably due toinstrumentation during ligation of teeth.The decrease in CI seen again during 8 to 10weeks following ligation was probably dueto removal of calculus for evaluation ofattachment loss. Following ligation we ob-served variations in the GI and attachmentloss. The rise in GI one week after hgationfollowed by decrease during the next threeweeks could be due to initial inflammationcaused by trauma from ligation and sub-sequent healing. The variation in attach-ment loss is tnore difficult to explain. It ispossible that attachment loss is not a uni-directional process. Also the infiammationof the gingiva which varied from week toweek could infiuence the attachment loss

512 l U K H E R J E E , D A S A N D P A T E L

because the periodontal probe could easilypass the junctional epithelium when thegingivae were severely infiamed (Listgarten1980). However, it should be noted that theextent of variation in attachment loss ob-served in this study was 0.75-0.25 mm andwas mostly within the approximation of thereading of the periodontal probe (0.5 mm).Further, the presence of ligature causeddifficulty in correct reading of the base ofthe pocket. Schroeder and Lindhe (1980)showed that in LIPD in beagles the boneloss preceded the apical migration of junc-tional epithelium. Our study confirms thisfinding (Tables 4 & 5). The bone loss wasevident both radiologically and histopath-ologically, and the severity of bone loss ascalculated by both methods correlated wellwith each other.

Our finding of a significantly higher con-centration of Ca and K in crevicular fluidcompared to serum agrees with studies ofKaslick et al. (1970) and Bang et al. (1973)in humans. However, in contrast to thesestudies we found a significant inerease in Caconcentration in CF during periodontitis.Bang et al. (1973) found no correlationbetween concentration of Ca in CF andclinical parameters such as gingival in-fiammation, pocket depths, and bone loss inhumans. We noticed no apparent correl-ation of Ca concentration in CF with theattachment loss. Our study showed that ofthe ions studied the concentrations of K inCF correlated best with the severity of thedisease and the attachment loss. This is inagreement with the findings of Bang et al.(1973) who found a similar relationship withthe severity of the disease as measured bypocket depths and bone loss in humans.

Kasliek et al. (1970, 1970a, 1970b) foundthe Na concentration in CF to be signifi-cantly higher in moderately infiamed gin-givae than clinically healthy gingivae. Theyconcluded that the concentration of Na inCF was "least affected by variables other

than degree of infiammation and, therefore,could serve as the best ionic indicator ofseverity of gingivitis." But Bang et al. (1973)did not find any correlation between theseverity of gingival infiammation and theconcentration of Na in CF. However, theyfound a slightly negative correlation be-tween the concentration of this ion in CFand the severity of bone loss. In this studywe found no apparent correlation betweenthe attachment loss and the concentrationof Na ion. Our finding of an increasedconcentration of Mg in crevicular fiuid com-pared to serum was opposite to those foundby Kaslick et al. (1973) in humans.

Little is known about the pH of thecrevicular fluid of beagle dogs. Our studyshowed that the pH of dog crevicular fiuidwas more alkaline than serum, and the pHbecame significantly less alkaline as thedisease progressed from gingivitis toperiodontitis. Kleinberg and Hall (1969)observed a decrease in pH of human gin-gival crevice in areas with increased pocketdepth. However, it should be noted that themean pH of dog serum (7.74-7.85) observedin this study is higher than published values(7.30-7.40) (Kirk 1977). The higher pH ofserum observed in this study could be due toloss of CO, following collecfion of bloodand during measurement of pH in a verysmall sample volume (15-20 ^1). BecauseCF samples were analyzed in an identicalmanner it was possible that loss of CO2occurred also in these samples. Thus theactual pH of CF could have been lower thanthose observed in this study. However,because we analyzed all samples (serum andCF) in an identical manner the differencesobserved between the serum and the CFsamples and between the CF samples col-lected during gingivitis and periodontitisshould be considered valid.

The character of erevicular fiuid proteinhas been studied by various workers (seeNovaes Jr., Ruben & Kramer 1979 for a

C R E V I C U L A R F L U I D IN P E R I O D O N T A L D I S E A S E 513

review). Most investigators found that theCF collected during periodontal disease wassimilar to that of serum (Brill & BronnestamI960, Mann & Stoffer 1964, Carraro etal. 1964, Sueda, Cimasoni & Held 1966,Schenkein & Genco 1977) or plasma(Brandtzaeg 1965, Tollefsen & Saltvedt1980). Our study shows that the proteinprofile of serum and CF collected duringgingivitis are identical and supports theseprevious findings. However, it was foundthat the profile changed during periodonti-tis. A distinct high tnolecular peak appearedin CF collected during this period. Thispeak might consist of increased amounts ofimmunoglobulins. However, most studies inhumans have shown the immunoglobulincontents of CF to be similar to that of serum(Holmberg & Killander 1971, Tollefsen &Saltvedt 1980) or less than that of serum(Shillitoe & Lehner 1972, Schenkein &Genco 1977). Other possibilities include (1)breakdown product of gingiva and alveolarbone and (2) bacterial metabolites.

This study shows that distinct changesoccurred in the ionic composition and pro-tein profile of crevicular fiuid due to LIPDin beagle dogs. Further investigations intothese areas are urgently needed as fruitfulinformation may be obtained regarding thediagnosis and pathogenesis of periodontitis.

Acknowledgement

The authors thank Dr. Henry M. Rosen-berg, Head of the Department of Radi-ology, College of Dentistry, University ofIllinois at Chicago, for his guidance intaking and evaluating X-rays, and Mr. Ri-chard J. Graczyk, radiographer, of the samedepartment, for taking many X-rays. Theauthors are also grateful to Dr. John M.Crawford, Department of Periodontics,University of Illinois at Chicago, for hiscriticism and correction of the manuscript.Supported in part by Campus Research

Board (University of Illinois at Chicago)grant and a contract from Naval DentalResearch Institute. ONR #0128-80-C-0029.

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