Journal of Periodontal Research 17: 202-209, 1982

Histometric analysis of ligature-induced periodontal defects

in beagle dogsLongitudinal evaluation following ligature removal

JAN JANSEN, JOHAN VAN DUK AND TACO PILOT

Department of Periodontology, University of Groningen. The Netherlands

The aim of the present study was to investigate the sequence of events that takes placein iigature-induced periodontal defects following removal of the ligature.

In eight beagle dogs the dentogingival fibres around six lower premolars (jPg, 3P3. 4P4)were cut' to the level of the alveolar bone. Closely adapted copper bands were thencemented around the teeth. After 3 weeks the copper bands were replaced hy cotton flossligatures which were left in place for 11 weeks.

The dogs were sacrificed on different dates so that it was possible to analyse defects3, 7, 15, or 31 weeks after ligature removal. Contralaterai defects one week after ligatureremoval served as controls. Micrographic color slides of the histological sections wereanalyzed using a Ferranti-Cetec digitizer.

Mean values for loss of attachment in the 15 and 31 weeks specimens were greater thanin the control defects, suggesting that apical positioning of the junctional epithelium wasirreversible. However, alveolar hone height tended to increase with experimental time,indicating bone repair. In the 15 and 31 weeks specimens junctional epithelium was oc-casionally found between the root surface and the alveolar hone.

It was concluded that the model does not show all the histological characteristics ofnaturally occurring periodontitis even in those dogs examined 31 weeks after removal ofligatures.

(Accepted for publication September 30, 1981)

Introduction im^^ j . .• ^ j, .. . • , ^L

1975) or dissection of dentogingival fibresArtificial periodontal defects in animals are (Van Dijk 1979). Frequent renewal of liga-widely used in periodontal research. The use tures has also been reported to increase theof piaque retaining ligatures or bands has amount of periodontal breakdown (Lindhebeen shown to induce periodontal break- & Ericsson 1978, Nyman et al. 1980).down effectively. (Kennedy & Poison 1973, When the influence of co-factors or sys-Caton & Zander 1975, Caton & Kowalski temic disorders on the progression of perio-1976, Schroeder & Lindhe 1975, Lindhe & dontal disease is to be tested, a model inEricsson 1978, Schroeder & Lindhe 1980). which the disease promoting ligatures areAdditional procedures obviously enhance left in place during the entire experimentalthe process of degradation of periodontal period has been advocated (Kennedy & Pol-tissaes e.g. surgical removal of alveolar bone son 1973, Lindhe & Svanberg 1974, Erics-(Lindhe & Svanberg 1974, Ericsson et al. son et al. 1975).

A N A L Y S I S O F A R T I F I C I A L P E R I O D O N T A L D E F E C T S 203

When therapeutic or preventive proce-dures are tested, the ligatures or bands areoften removed from the periodontal defectsa certain period of time before the start ofthe actual experiment (Hugoson & Schmidt1978, Caton & Zander 1976, 1979, Van Dijk1979, Caton & Nyman 1980, Nyman et al.1980). The reason to include such a "res-ting period" is that the periodontai tissuesare given time to recover from the presenceof the irritating band or ligature and changefrom an acute defect (Lindhe & Ericsson1978, Schroeder & Lindhe 1980) into amore chronic lesion with the characteristicsof naturally occurring periodontitis. The iossof connective tissue attachment and thealveolar bone loss created in the experi-mental lesion should not be spontaneouslyreversible. The monkey model as describedby Caton and Zander (1975) and Catonand Kowalski (1976) appears to meet tbeserequirements.

Conflicting results have, however, beenreported in the dog model. Reversibility ofartificial periodontal defects upon ligatureremoval was reported by Linghorne andO'Connell (1955), Hugoson and Schmidt(1978), and Jansen et al. (1979), whileLindhe and Ericsson (1978) reported that aresting non-reversibie lesion resulted afterligature removal. Johansen, Nilveus andEgelberg (1978) reported both reversibilityand progression of their furcation defects.

These conflicting results indicate the needfor further research into the dog model.The present longitudinal study in beagledogs was therefore undertaken to investigatethe sequence of events that takes place inligature-induced periodontal defects follow-ing ligature removal, using histometricalanalysis.

Material and Mettrads

Bight beagle dogs 1 to 1.5 years of age wereused. Throughout the experiment the dogs

were fed on a soft diet which favoredplaque accumulation. No oral hygiene pro-cedures were performed, and no attemptwas made to bring the gingivae, which weremildly inflamed at the start of the experi-ment, into a healthy condition.

Periodontal defects were created as de-scribed earlier (Van Dijk 1979, lansen &Pilot 1981). The dentogingivai fibres aroundsix lower premolars (2P9, gPg, 4P4) were cutto the level of the alveolar bone. Closelyadapted copper bands were then cementedaround the teeth to the full depth of thedefects. After three weeks the copper bandswere replaced by cotton floss ligatureswhich were tied around the toothnecks andforced down subgingivaliy as far as pos-sible. These ligatures were left in place foreleven weeks. Upon removal of the liga-tures a notch was made on the mesial anddistal root surfaces to indicate the apicalextent of the soft tissue defect. A smallexcavator was placed in the defect usinglight hand pressure until definite resistancewas felt. The notch was then made in theroot surface with one hucco-lingual stroke.

Pairs of dogs were scheduled for sacrifice3, 7, 15, or 31 weeks after removal of theligature on the right side of the jaw. Contra-lateral defects were created 15 weeks beforethe sacrifice date. One week before sacrificethe ligatures were removed on this side anda notch prepared. These defects on the leftside of the jaw, examined one week afterligature removal, served as controls.

The dogs were sacrificed and preparedfor histological analysis using the methoddescribed by Van Dijk (1979). At the endof the experimental period, the animals wereaaesthesized with sodium pentobarbital andgiven an intravenous injection of heparinto prevent blood clotting. The thorax wasopened, and a canula was placed in the leftatrium. The vascular system was then per-fused with saline followed by a fixative,consisting of 90 g of sodium chloride and

204 J A N S E N , V A N D i J K A N D P I L O T

300 ml of glacial acetic acid in 10 1 of 10 %formalin.

Segments of the jaws involved in theexperiment were removed, sectioned intosmaller blocks, fixed in a 10 % bufferedformalin solution for two weeks, washedwith tap water, and decalcified in a solu-tion of 25 % formic acid and 10 % sodiumcitrate in water. After embedding in paraf-fin, 8 |im thick sections were cut andstained with haemetoxylin and eosin accord-ing to Delafield.

Three sections 40 ^lm apart were selectedfrom the midsagittaJ region of each inter-proximal block. Micrographic color slideswere made and projected with a total mag-nification of 70 on a Ferranti-Cetec digitizertable connected to a Digital P.D.P.-l 1/0.5.computer.

The following points were digitized (Fig.1): Cemento-enamel junction (CBJ), theapical termination of the junctional epi-thelium (ATJE), the apical extension of thenotch and the most coronal extension ofthe alveolar bone opposing the root surfaceat a distance similar to that of the normalwidth of the periodontal ligament.

These points were then projected on atangent along the root surface which par-allels the vertical tooth axis. The distancebetween the projected points were thencomputed. The mean of the three sectionswas taken for further analysis. In this waythe following distances were calculated:CEJ to ATJE (loss of attachment)., alveolarbone to CEJ, bone to ATJE (width of thezone of connective tissue) and notch tobone.

Approximately 25 % of all measurementswere repeated 2 weeks after the first assess-ment Of the 75 repeat scores, 40 were infull agreement, 24 differed 0.1 mm and 11differed by 0.2 mm. This means that 85 %of the registrations were reproducible with-in an accuracy of 0.1 mm.

To be able to relate the experimental

- CEJ.

- AT.J.E.

Fig. 1. Schematic representation ol the points thatwere digitized. ATJE: apicai tBrmination of the junc-tional epEthehum. CEJ: cemento-enamej junction. N:apicai extension ot ttie notch. B: most corohal exten-sion of the aiveolar bone opposing the root surtaceat a distance similar to that ol the normal width ofthe periodontal iigament

recordings to the situation in unaffectedbeagle dogs of the same age, two additionalanimals were sacrificed and the periodon-tium of their lower right premolars wasanalyzed as described above. This situationwill be referred to as baseline.

Seventy-seven out of the 96 possible de-fects were available for analysis, 40 experi-mental and 37 control defects. Nineteenspecimens were eliminated because thesewere either incorrectly sectioned or theCEJ could not be detected.

The number of data at each time pointwas limited. In every evaluation phase only2 dogs with at most 6 defects on each sideof the jaw were available. Furthermore,these defects cannot be cotisidered strictly

A N A L Y S I S O F A R T I F I C I A L P E R I O D O N T A L D E F E C T S 205

independent, mandatory for statistical anal-ysis (Pilot et al. 1980). Therefore, results Resultsare presented as mean values per side of the The measurements of the baseline sectionsjaw for each dog separately, and no effort revealed no loss of attachment. The meanwas made to subject the results to statistical distance from the ATJE to the alveolaranalysis. bone level was 0.64 mm (S.D. = 0.21).

i & A T J E : • • • • • • • - ?

'.•:"!-'•• A T J E

: ' • .

J •.

N ';,

B I .

FIB- 2. a. Control defect, one weeit after ligature removal, b. Experimental deject, 15 weeks after ligatureremoval. ATJE: apical termination of junctionai epithelium. B: most coronal level of aiveoiar bone oppositetile root surface at a distance similar to that of the normal width o( the periodontal ligament. N: notch. Orig-inal magnification x <IO.

206 J A N S E N , V A N D I J K A N D P I L O T

The results of the measurements on thecontrol and experimental sides of dogs num-ber 1-8 are presented in Table 1. Loss ofattachment in control defects was approxi-mately 2 mm, the alveolar bone level wassituated more than 3 mm apical to the CEJ.As a result, the width of the connective tis-sue (distance ATJE-bone) was approxi-mately 1.2 mm. In all but one dog (No. 1)the mean values for loss of attachment atthe experimental sides were greater thanthose of the controls. Mean loss of attach-ment seemed to increase with increasingexperimental periods.

In the 15 and 31 weeks specimens themean values for bone loss (distance "CEJ-bone") were smaller on the experimentalside than on the control side. The notch inthe root surface (made immediately afterremoval of the ligatures from the defects)could be identified in 65 of the 77 defects,(29 control and 36 experimental). Twenty-seven of the 29 notches were situatedcoronal to the alveolar bone level in thecontrol (Fig. 2a). The alveolar bone in theexperimental defects was on average stillsituated apical to the notches in dogs 1 and2 (experimental period 3 weeks), but in theremaining six dogs the alveolar bone wassituated coronal to the notch (Fig. 2b).From the mean values per dog it appearedthat alveolar bone height tended to increasewith experimental time (Table 1, distance"notch-bone"). As a result, the width of con-nective tissue (Table 1, distance "ATJE-bone") became smaller during the experi-mental phase and was eventually less thanhalf that found in the baseline sections. Inthe 15 and 31 weeks specimens junctionalepithelium was occasionally found betweenthe root surface and alveolar bone.

Discussion

Although gingivitis was present in all dogsat the start of the experiment, baseline

- S

o < t

£0 O

ffi OS CL

o o o o o o o o+1 +i +! +1 +1 +1 +1 +1

- r ^ i - ^ T ^ d o O O O

o o o o o o o o-HI +1 -H +1 +1 +1 +1 +1

c r o t^ ^ ^ ^ CDo o o o d e i d o+\ +1 +1 +1 +1 +1 +1 +1g o O <O O CM f^ O

1-; {NI O 1- U) (C pd d d o T ^ d d ' - ^I ! • + + + + + +

+1 -l-l +i +1 +1 +1 +1 +1

o o o o o o o oI I I + I I I I

o o o o o o o o41 +1 +1 +1 +1 +1 +1 +15 a S 2 5 S S R g

c o p > c i N c o < N i c \ i ( ^

d d d d d d d d

d d d d d d d d41 -ft -fi +1 +i +1 +1 +1

+1 +i +i +1 +1 +\ +\ +[

A N A L Y S I S O F A R T I F I O I A L P E R I O D O N T A L D E F E C T S 207

measurements revealed no loss of attach-ment, and the alveolar bone was situatedabout 0.6 mm apicai to the apical termina-tion of the junctional epithelium. Similarfindings have been reported by Lindhe,Hamp and Loe (1975) and Lindhe andEricsson (1978).

Mean loss of attachment on the experi-mental side of the jaw was greater thanon the control side. The difference wasmost pronounced in defects 31 weeks afterligature removal. This result suggests that,after removal of the ligatures, loss of con-nective tissue attachment (apical positioningof the junctional epithelium) is not rever-sible but progressive. Progressive loss ofattachment is a characteristic feature ofnaturally occurring periodontitis in man(Loe et al. 1978) and in dogs (Lindhe etaL 1975, Hamp & Lindberg 1977). Thus,when loss of attachment only is considered,the present dog model seems to meet therequirements for artificial periodontal de-fects.

Mean distance from CEJ — bone in ex-perimental defects 15 and 31 weeks afterligature removal was smaller than that inthe control side. This observation suggestsrepair of bone. The findings that in all butone control defect the alveolar bone wassituated apical to the notch (Table 1, Fig.2a) and that in the experimental defects thebone level became situated coronal to thenotch in the majority of specimens (Table1, Fig. 2b) further substantiate the hypo-thesis that bone repair occurs after liga-ture removal.

Alveolar bone repair can occur uponreduction of periodontal inflammation(Kantor, Poison & Zander 1976, Rosling,Nyman & Lindhe 1976, Rosling et al. 1976,Poison & Heijl 1978). In an earlier pub-lication (Jansen, Pilot & Corba 1981) it wasreported that the histopathological char-acteristi<s of this type of defect changedfrom severe to moderate upon ligature

removal in spite of the absence of oral hy-giene procedures. This change in inflam-matory state may well explain why bonerepair occurred in the present material.Similar findings can he found in the litera-ture. Hugoson and Schmidt (1978) reportedbone repair in experimental bone defects inbeagle dogs after ligature removal. The re-pair was most evident in teeth exposed tomechanical plaque control but could alsobe demonstrated in plaque-infected defects.Johansen et aL (1978) evaluated experi-mental furcation defects in beagle dogs 2and 9 months after ligature removal. In 3out of 5 dogs no change in bone loss wasobserved and in 1 dog bone repair was de-monstrated in the 9 month period after liga-ture removal. In this dog the area of in-filtrated connective tissue was smaller inthe 9 month specimens than in the 2 monthspecimens. Recently, bone repair was de-monstrated in experimental periodontal de-fects in the squirrel monkey after ligatureremoval and the institution of oral hygieneprocedures (Kantor 1980).

Absence of bone repair upon ligature re-moval from experimental periodontal de-fects has, in contrast, been reported byCaton and Zander (1975) and Lindhe andEricsson (1978). Caton and Zander (1975)evaluated elastic induced periodontal de-fects in Rhesus monkeys up to 380 days fol-lowing ligature removal. They concludedthat there was no evidence that either theapical migration of the junctional epithe-lium or the loss of alveolar bone was rever-sible. The overall histopathology of the"long-term elastic-off" specimens was thatof active periodontitis and otily in a minor-ity of cases could a long junctional epithe-lium be seen. It appears that elastic induceddefects in monkeys results in persistance ofseverely inflamed tissues which may pre-vent alveolar bone repair.

Lindhe and Ericsson (1978) evaluated theeffect of ligature removal from experimen-

208 J A N S E N , V A N D I J K A N D P I L O T

tal defects in beagle dogs and concludedthat "in dogs where ligatures were removedbut plaque accumulation continued for an-other 6 months an apparently resting, non-destructive lesion attained." No significantchange in bone level was reported duringthis period. It may well be that the per-sistance of inflammation in a shallowpocket, as was the case in their material,prevented bone repair. Another explanationmay be the size of the defect. In their ma-terial the zone of connective tissue foundbetween the apical termination of thejunctional epithelium and the aiveolar boneat the time of ligature removal was muchsmaller than in the present material. It canbe speculated that a wider zone of connec-tive tissue preserves a space in which osteo-genic cells are still present. These cells may,upoa ligature removal and subsequent re-duction of the inflammation, produce newalveolar bone in the deeper part of thelesion.

The notch in the root surface was madeto indicate the apical extent of the pocketepithelium (bottom of the defect) at thetime of ligature removal. From Table 1 itcan be calculated that the notch was madejust above the alveolar hone level (distancenotch-bone control defects) and about 1mm apical to the apical termination of thejunctional epithelium (distance ATJE-bone,control defects). It can therefore be con-cluded that the notch does not accuratelyindicate the bottom of the pocket but ratherthe alveolar bone level. The severely in-flamed tissues obviously did not act as abarrier to the notch instrument, although itwas inserted with light hand pressure. Thisfinding is of importance for the qualitativeinterpretations of the tissue reactions be-cause it means that tissues coronal to thenotch have not necessarily been exposed tothe oral environment.

The 15 and 31 weeks specimens werecharacterized hy hone repair and a narrow

or sometimes non-existent zone of connec-tive tissue. These are features that are fre-quently found as a result of periodontaltherapy (Lindhe & Ericsson 1978, Caton &Zander 1976, Caton & Nyman 1980, Catonet al. 1980). If a mode! is used to evaluatetherapeutic or preventive procedures, itshould have all histopathological character-istics of naturally occurring periodontitis.The present dog model does not seem tomeet this requirement in fuil.

Acknowledgment

The authors would like to express theirgratitude to the staff of the Central Experi-mental Animal Laboratory of the Univer-sity of Groningen for their skillfull assis-tance during the actual experiment and tothe staff of the Laboratory of Oral Histo-logy of the University of Groningen, HeadDr. H. W. B. Jansen, for the preparation ofthe sections.

References

Caton, J. G. & Zander, H. A. 1975. Primatemodel for testing periodontal treatment pro-cedures I. Histoiogic investigaticm of local-ized periodontal pockets produced by ortho-dontic elastics. Journal of Periodontology 46:7!-77.

Caton, J. G. & Kowalski, C. J. 1976. Primatemodel for testing periodontal treatment pro-cedures. II. Production of contralaterallysimilar lesions. Journal of Periodontology 47:506-510.

Caton, J. G. & Zander, H. A. 1976. Osseousrepair of an infrabony pocket without newattachment of connective tissue. Journal ofClinical Periodontology 3: 54-58.

Caton, J. G. & Zander, H. A. 1979. The at-tachment between tooth and gingival tissuesafter periodic root planing and soft tissuecurettage. Journal of Periodontology 50:462-466.

Catcm, J. G. & Nyman, S. 1980. Histometricevaluation c^ periodontai surgery. I. Themodified Widman flap procedure. Journal ofClinical Periodontology 7: 212-223.

Caton, } . G., Nyman, S. & Zander, H. A.

A N A L Y S I S O F A R T I F I C I A L P E R I O D O N T A L D E F E C T S 209

1980. Histometdc evaluation of periodontalsurgery. II. Connective tissue attachmentlevels after four regenerative procedures.Journal of Clinical Periodontology 7: 224—231.

Van Dijk, L. J. 1979. Surgery and oral hygieneon artificial periodontal defects in beagledogs. University of Groningen, Thesis.

Ericsson, I., Lindhe, 3., Rylander, H. & Oka-moto, H. 1975. Experimental periodonta!breakdown in the dog. Scandinavian Journalof Dental Research 83: 189-192.

Hamp, S. E. & Lindberg, R. 1977. Histopa-thology of spontaneous periodontitis in dogs.Journal of Periodontal Research 12: 46-54.

Hugoson, A. & Schmidt, G. 1978. Influence ofplaque control on the healing of experi-mentally induced bone defects in the dog.Journal of Periodontology 49; 135-141.

Jansen, J., Corba, N. H. C , Pilot, T. & Jan-sen, H. W. B. 1979. Spontaneous regenera-tion of artificially created periodontal defectsin beagle dogs. Journal of Dental Research58: Spec. Issue A 591.

Jansen, J., Pilot, T. & Corba, N. H. C. 1981.Histologic evaluation of probe penetrationduring clinical assessment of periodontal at-tachment levels. Journal of Clinical Perio-dontology 8: 98-106.

Jansen, J. & Pilot, T. 1981. PredictabUity ofprobing depths in ligature-induced perio-dontal defects in beagle dogs. Journal ofPeriodontal Research 16: 372-378.

Johansson, O., Nilveus, R. & Egelberg, J. 1978.Experimental bifurcation defects in dogs.Journal of Feriodontal Research 13: 525-531.

Kantor, M., Poison, A. M. & Zander, H. A.1976. Alveolar bone regeneration after re-moval of infiammatory and traumatic fac-tors. Journal of Periodontology 47: 487-695.

Kantor, M. J980. The behavior of angularbone defects following reduction of inflam-mation. Journal of Periodontology 51: 433-436.

Kennedy, J. E. & Poison, A. M. 1973. Experi-mental marginal periodontitis in squirrelmonkeys. Journal of Periodontology 44: 140-144.

Lindhe, J., Hamp, s! E. & Loe, H. 1975.Plaque induced periodontal disease in beagledogs. Journal of Periodontal Research W:243-255.

Lindhe, J. & Svanberg, G. 1974. Influence oftrauma from occlusion on progression of ex-

perimental periodontitis in the beagle dog.Journal of Clinical Periodontology 1: 3-14.

Lindhe, J. & Ericsson, I. 1978. Effect of liga-ture placement and dentai plaque on perio-dontal tissue breakdown in the dog. Journalof Periodontoiogy 49: 343-350.

Linghorne, W. J. & O'Connell, D. C. 1955.Studies in the reattachment and regenerationof the sufqx>rting structures of the teeth.Journal of Dental Research 34: 164-177.

Loe, H., Anerud, A., Boysen, H. & Smith, M.1978. The natural history of periodontai dis-ease in man. The rate of periodontal de-struction before 40 years of age. Journal ofPeriodontology 49: 607-620.

Nyman, S., Karring, T., Lindhe, J. & Planten,S. 1980. Healing following implantation ofperiodontitis affected roots into gingival con-nective tissue. Journal of Clinical Perio-dontology 7: 394-^1.

Pilot, T., van der Weele, L. T., van Dijk, L. J.& Jansen, J. 1980. Variations in artificialperiodontal defects in beagle dogs. Journalof Dental Research 59: Special Issue B 373.

Poison, A. M. & Heijl, L. C. 1978. Osseousrepair in infrabony periodontal defects. Jour-nal of Clinical Periodontoiogy 5: 13—23.

Rosling, B., Nyman, S. & Lindhe, J. 1976.The effect of systematic plaque control onbone regeneration in infrabony pockets.Journal of Clinical Periodontology 3: 38-53.

Rosling, B., Nyman, S., Lindhe, I. & Jem, B.1976. The healing potential of the perio-dontal tissues following different techniquesof periodontal surgery in plaque-free denti-tions. Journal of Clinical Periodontology 3:233-250.

Schroeder, H. E. & Lindhe, J. 1975. Conver-sion of stable established gingivitis in the doginto destructive periodontitis. Archives ofOral Biology 20: 775-782.

SchroKler, H. E. & Lindhe, J. 1980. Condi-tions and pathological features of rapidlydestructive experimental periodontitis indogs. Journal of Periodontology 51: 6-19.

Address:

Department of PeriodontologyFaculty of DentistryUniversity of GroningenAnt. Deusinglaan I9713 AV GroningenThe Netherlands