Animal Models2

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CHARACTERISTICS OF THE ANIMAL MODEL IN

PERIODONTAL HEALTH :

1.Clinical Characteristics : All non-human primates have adeciduous and a permanent dentition.The dental formula of the permanent

dentition of baboons (Papio anubis) andcynomolgus monkeys (Macacafascicularis) is reported to be same as inhumans (I 2/2, C1/1, Pm 2/2, and M 3/3),

whereas that of the cotton-earmarmosets (Calithrix jacchus), cotton-top marmosets (Saguinus Oedipus),squirrel monkeys, and bushbabies aredifferent.


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Macroscopically, tooth morphology,

occlusion, and gingiva of baboons,howler monkeys (Allouta caraya),cynomolgus and squirrel monkeys,and cotton-top and cotton-ear

marmosets are quite similar tohumans with the followingexceptions: larger canines, presenceof diastemata between anterior teethprimarily at the canine, and an edge-to-edge relationship of the incisors.


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This indicates that molar and premolarregions are to be preferred for periodontalinvestigations in which the presence ofdiastemata is not desirable.

However, the size of the dentition deviatessubstantially from that of humans.

Clinically healthy gingiva has beenobserved in squirrel and cynomolgusmonkeys, but oral hygiene measures, as inhumans, must be undertaken in non-human primates to establish and maintainclinically healthy gingival.


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Toothbrushing, interdental flossing, andapplication of 2% aqueous chlorhexidinegluconate have been used to prevent

supragingival plaque in baboons, rhesus(Macaca mulatta), stump-tailed (Macacaarctoides, cynomolgus, and squirrelmonkeys.

The frequency of oral hygiene necessary toestablish gingival health within 3 to 4 weekshas been reported to vary from daily or threetimes weekly.

The frequency of cleaning may introduce anadditional experimental variable due to theeffects of food restriction necessary prior to

anesthetizing the animals for cleaning.


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2.Histology

Clinically healthy periodontal tissue ofcotton-top marmosets, baboons,bushbabies, and stump-tailed, squirrel,and howler monkeys has been observed tobe histologically quite similar to that of

humans. It is characterized by a non-keratinized

junctional and oral sulcular epitheliumwithout or with minimal rete ridges, sparse

but varying accumulation of inflammatorycells in the connective tissue, and highlyorganized bundles of collagen fibers in thegingival connective tissue and periodontalligament.


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3. Microbiology :

Subgingival plaque from teeth with no or

minimal gingivitis seems to contain few

bacteria, and those present appear to be

predominantly cocci and straight rods,while motile rods, spirochetes, and curved

bacteria are either absent or sparse in

humans, and stump-tailed monkeys and

squirrel monkeys.


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CHARACTERISTICS OF THE ANIMAL MODEL IN

THE TRANSITION FROM HEALTH TO

GINGIVITIS TO PERIODONTITIS IN

SPONTANEOUS PERIODONTAL DISEASE :1. Clinical Characteristics :

Among animals kept in captivity great inter-andintra-species variability in the prevalence and

severity of plaque accumulation and periodontaldisease has been reported.

Extensive plaque accumulation was observed incotton-top marmosets, young adult and middle-

aged baboons, aged chimpanzess (Pantroglodytes) young adult rhesus, adultcynomolgus, and stump-tailed monkeys, whilemoderate plaque accumulation was reported inyoung stumptailed monkeys and slight plaqueaccumulation in young cynomolgus monkeys.


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The clinical manifestations of

spontaneous gingivitis and

periodontitis in chimpanzees, baboons,

and cotton-top and cotton-ear

marmosets are quite similar to that of

humans, with the exception of markedgingival telangiectasia in marmosets.


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Significant differences have been reportedin the prevalence and severity of gingivitis,

varying from localized gingivitis in cotton-top

marmosets, young chimpanzees, andbaboons,

slight gingivitis in adults cynomolgusmonkeys;

moderate gingivitis in adult rhesusmonkeys, young stump-tailed, and young

adult cynomolgus monkeys; and to generalized gingivitis in young adult and

middle-aged baboons, and rhesus andadult cynomolgus monkeys.


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The prevalence and severity of periodontitis alsovary.

Only rarely has periodontitis been reported inyoung adult baboons, cotton-top marmosets, andyoung stump-tailed and cynomolgus monkeys.

In contrast, the majority of cotton-ear marmosetsstudied were found to have varying degrees ofmarginal inflammation; i.e., from mild gingivitis to

extensive periodontitis with increased toothmobility and even tooth loss.

Some wild-living howler monkeys also suffer fromlocalized peridontitis, but none of the animalsexamined exhibited generalized alveolar bone loss.

Furthermore, all skulls of wild-living adultmountain gorillas showed alveolar bone loss andsome showed tooth loss, indicting that peridontitisis also present in wild living non-human primates.


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As in humans where the prevalenceand severity of marginal inflammationincrease with age, a similar increasewith age and duration of captivity hasbeen reported in baboons,bushbabies, chimpanzees, andcynomolgus and squirrel monkeys.

A generalized alveolar bone loss wasobserved in old chimpanzees and abone loss affecting approximatelyone-half to three-fourths of the rootlength was observed in a cotton-earmarmoset held in captivity for severalyears.


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These studies indicate that young adultanimals may be appropriate in studieswhere absence of periodontitis ispreferred.

However, it should be kept in mind that

reports of clinical findings on smallsamples may be very misleading, sincethe observed differences in prevalenceand severity of plaque accumulation,gingivitis, and periodontitis may be dueto different registration criteria, or dueto intentional or unintentional animal

selection criteria.


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2.Histology :

The histological manifestations of spontaneousgingivitis and periodontitis in baboons,bushbabies, chimpanzees, and howler,cynomolgus, and squirrel monkeys, and cotton-ear and cotton-top marmosets are quite similar to

those of humans. Characteristic features are progressive rate ridge

formation, ulceration, apical migration of thepocket epithelium with wide intercellular spacesand emigrating PMNs, increased infiltration of

inflammatory cells in connective tissue, vascularproliferation, destruction of collagen fibers, andresorption of alveolar bone.


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The inflammatory infiltrate of the connective tissuein spontaneously occurring periodontal disease

varies significantly between different species. The inflammatory infiltrate in baboons, bushbabies,

howler and cynomolgus monkeys, andchimpanzees contains the same inflammatory cellsas in that of humans with a predominance of

lymphocytes and plasma cells, although one studyin cynomolgus monkeys observed a dominance ofmacrophages.

Early lesions contain a small inflammatory infiltrateof predominantly lymphocytes and macrophages,while established lesions are dominated by plasmacells, these observations are also similar to thosein humans.


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In contrast, cotton-top and cotton-earmarmosets exhibit a predominance of

macrophages and almost an absenceof lymphocytes and plasma cells.

Based on these differences,

marmosets appear to be only slightlyrelated to the human condition andtherefore may be inappropriatemodels for studies of periodontal

disease pathogenesis which are basedon similarities to the humaninflammatory and immune responses.


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3.Microbiology

In rhesus monkeys with established gingivitis, the total

number of bacteria and proportion of anaerobic species aregreater than in gingiva health.

In cynomolgus monkeys with gingivitis a predominance ofGram-positive cocci and rods, and Gram-negative rods ischaracteristic. Similar changes are also observed insubgingival plaque from human teeth with gingivitis, but

greater numbers of Actinomyces species are commonlyfound in humans.

Actinobacillus actinomycetemcomitans is primarily isolatedfrom humans suffering from juvenile periodontitis, but is alsofound in > 25% of patients with adult periodontitis.

In young cynomolgus monkeys, however, A.

actinomycetemcomitans was frequently isolated fromsubgingival plaque of sties with localized gingivitis and noalveolar bone loss, indicating that the mere presence ofthese bacteria does not cause overt periodontal disease inthis animal species or that the simian strains of A.actinomycetemcomitans were less virulent.


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CHARACTERISTICS OF THE MODEL IN

EXPERIMENTAL GINGIVITIS AND

PERIODONTITIS :

1.Experimental Gingivitis :a.Clinical characteristics:

As in humans, increased plaque accumulation and gingivitis

can be induced rapidly in rhesus monkeys by cessation oforal hygiene procedures.

A simultaneous change to soft diet has been performed tofurther facilitate plaque accumulation in stump-tailed andcynomolgus monkeys.

Therefore, gingivitis can be investigated in non-human

primates, but as noted in humans, within a single speciesthere may be a broad time range of gingival responses toplaque accumulation.

This may present problems in data interpretation if smallnumbers of animals are used for experimental gingivitisstudies.


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b.Histology:

The histological manifestations of experimentalgingivitis in rhesus, stump-tailed, and cynomolgus

monkeys are similar to gingivitis in humansfollowing the withdrawal of oral hygiene procedures.

Although an increase in the size of the inflammatorycell infiltrate in connective tissue predictably followsplaque accumulation, the time frame for histologic

changes may vary. The inflammatory cells were dominated by

lymphocytes in early lesions in rhesus and stump-tailed monkeys, while an increased number ofplasma cells was observed in established lesions of

rhesus, stump-tailed, and cynomolgus monkeys. Johnson and Hopps found notably numerous

macrophages in early lesions in cynomolgusmonkeys, but all cells containing phagocytosedmaterial were classified as macrophages regardlessof cell morphology, suggesting that the number ofmacrophages was most likely overestimated.


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c.Microbiology:

There appears to be a close similaritybetween gingival plaque compositionin sites from stump-tailed monkeyswith experimental gingivitis and from

sites in humans with gingivitis. This similarity is also demonstrated

in a study using stump-tailed andcynomolgus monkeys placed on soft

diet and with minima gingivitis wherethe subgingival plaque contained 40-50% cocci, 20-40% rods, 8-20%fusiforms, 2-6% filaments, 4-7%

vibrios, and 2-6% spirochetes.


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Inter-species variability exists becausesupragingival plaque in rhesus monkeysplaced on soft diet contained significantly

higher numbers of Prevotella andPorphyromonas species, Fusobacteriumspecies, Veillonella species, andStreptococcus mutans than supragingivalplaque from cynomolgus monkeys, while

significantly higher numbers ofStreptococcus mitis and unidentifiedStreptococci were observed in cynomolgusmonkeys. The significance of these

differences is presently unknown.


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2.Experimental Periodontitis

a.Clinical characteristics: Periodontitis has been induced in non-human

primates by a variety of techniques includingplacement of silk sutures or orthodonticelastics at the cemento-enamel junction (CEJ)and surgical removal of alveolar bone withsubsequent placement of mechnical devices,such as wires or metal bands, to accumulatesupragingivial plaque and make the lesion

more chronic. Frequently, the diet is simultaneously

changed to soft in order to increase plaqueaccumulation. It is necessary to examine sitesduring active tissue destruction to investigate

pathogenesis of periodontitis.


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A useful approximation of this process has beenthrough the use of silk-ligated teeth, while placementof orthodontic elastics and surgical removal ofalveolar bone predominantly have been used toestablish periodontitis for the evaluation of differentperiodontal therapies. The latter methods will bedealt with only briefly in the end of this section.

Placement of silk ligatures at the CEJ in stump-tailed, cynomolgus, and squirrel monkeys resulted in

increased plaque accumulation, gingivalinflammation, pocket formation, and alveolar boneloss of identical depth on contralateral toothsurfaces. When ligatures were applied to severaladjacent teeth in squirrel monkeys, a horizontal bone

loss occurred, but when applied to a single tooth,angular bone loss was established. Thus, bothhorizontal and angular bone loss can be induced innon-human primates.


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b.Histology:

The histological features of experimentally producedperiodontal lesions in rhesus, squirrel, andcynomolgus monkeys are similar to periodontallesions in humans. The inflammatory infiltrate ofcynomolgus monkeys are similar to periodontal

lesions in humans. The inflammatory infiltrate of squirre monkeys was

different, with a predominance of PMNs andmacrophages and few lymphocytes and plasmacells. even though most of these studies in squirrel

monkeys are only 14 days studies and/or studies ofthe connective tissue apically to the pocketepithelium, it must be connective tissue apically tothe pocket epithelium, it must be concluded thatalso squirrel monkeys appear poorly related to thehuman condition.


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long-term studies of ligature-induced periodontitis in squirrelmonkeys have demonstrated progressive loss of attachmentand alveolar bone occurring primarily during the first 10weeks making non-reversible lesions.

c.Microbiology: Ligature-induced periodontitis in the non-human primate has

provided a model for studying microbial changes associatedwith the initiation and progression of periodontitis.

Monitoring this transition period is not possible in humans,due to low incidence of initiation and our inability to indentifyinitiation prior to extensive clinical destruction.

It should be noted that the acute and rapid nature of inducedperiodontitis in squirrel monkeys and marmosets produce anextreme time compression of microbial changes associated

with disease initiation and microbial changes. Macaque species have a more extended period of disease

induction that allows observation of more complex microbialinteractions. The time frame for microbial changes in non-human primates depends on the monkey species and size,previous history of periodontal disease, and the method ofdisease induction.


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d.Hematological and serological

characteristics:

Ligature-induced periodontitis for 6 to 8 weeks incynomolgus monkeys results in changes affectingthe experimentally involved teeth and inmodulations in the number and proportion of thedifferent peripheral blood lymphocytes and the

systemic antibody response to P.gingivalis. The modulations comprised an increasing number

of total peripheral lymphocytes and a decreasedproportion of helper T-lymphocytes, whereas theproportion of suppressor T-lymphocytes remained

constant. Furthermore, serum concentration of antibodies to

P. gingivalis increased, which coincided with theincreased proportion of this microorganismobserved in the subgingival plaque.


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NON-HUMAN PRIMATE MODELS FOR TESTING THE

EFFICACY AND SAFETY OF PERIODONTAL REGENERATIOPROCEDURES

Animal models for testing periodontal regenerative procedures

are necessary because controlled quantitatively histologicalanalysis is required to evaluate the quality and extent of thenewly formed supporting tissues.

Histometric evaluation can determine the amount of newcementum, periodontal ligament, and alveolar bone formed asthe result of regenerative periodontal surgery.

These studies are not possible in man because of the need toretrieve teeth and their surrounding periodontium in largeblocks appropriate for histological analysis. Furthermore,proper evaluation of a new therapy necessarily involves theuse of treated and untreated controls which are difficult toobtain in the human.

Finally, the testing of potentially harmful new devices andpharmaceuticals may be unethical in man prior to thoroughevaluation in higher animals.

Animal models used in testing periodontal surgical therapieshave primarily involved dogs and monkeys. Monkeys arepreferred because the dental anatomy and wound healingphysiology closely parallel that of man.


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The disease versus the defect :

Periodontitis causes destruction of the toothsupporting tissues due to inflammationinduced by bacterial plaque.

The disease is treated by mechanical and

chemical means which reduce or eliminatethe responsible microbial pathogens.

Anatomical defects remain, however, such aspockets, bone loss, gingival recession, and

root surface alterations. Much of periodontal therapy is directed

towards resolution of these defects byresective, reparative, and regenerative

surgery.


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In order to test the effects of therapy,defects must be produced in laboratory

animals which have the samecharacteristics as those found in humans.

These include deep pockets, horizontaland angular bone loss, and root surfaces

devoid of Sharpeys fibers which havebeen exposed to bacterial plaque.

Gingival recession may or may not bedesirable, depending on the hypothesis

being tested.


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Choosing the proper animal :

The defects must be created in an animal

model which is large enough so thatmanipulations and devices can be tested.

This requires roots which are long andbroad enough so that 6 or 7mm of

attachment loss can be obtained while thetooth remains stable.

Size is also important because the relativedistances that cells must travel oftendetermine the outcome of wound healing.

If defects are too small, substantialregeneration may occur in sham-operatedcontrol sites by the outpouring of cellsfrom the periodontal ligament / alveolar

bone.


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Design considerations : Quantitative assessment requires

statistical testing of histometric data. A powerful and sensitive way to

accomplish this is to produce identicaldefects on contralateral teeth withinthe same animal.

In this way, the experimental variablecan be tested on a tooth with the

contralateral tooth serving as thecontrol.

Differences between these pairs ofteeth are summed for each animal and

the mean for the animal calculated.


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The grand mean of the differencesand the standard deviation of these

differences is then calculated from allanimals in the study.

Appropriate statistical tests are used

to determine significant differences. The number of animals in the

investigation is determined byestimating the amount and variabilityof the differences (i.e., regeneration)and performing a power analysis.


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Defect production :

There are four types of periodontal defectswhich have been used for testing the effectsof therapy.

These include defects produced by naturallyoccurring periodontitis and three types ofexperimentally-produced defects: the acutedefect model.

Naturally occurring periodontitis of sufficientmagnitude in large non-human primates

occurs late in life and the lesions areasymmetrical.

For this reason, these types of defects areseldom used for testing the effects of

periodontal surgical procedures.


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The acute defect model :

Mucoperiosteal flaps are elevated and thebone, periodontal ligament, and cementumare removed to create the desired defectshape.

Flaps are either replaced and sutured, or theexperimental variable is inserted prior to flapclosure.

The time interval between surgery andretrieval of tissue blocks for histologicalanalysis will vary, depending on thehypothesis being tested.


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The problem with the acute defect modelis that spontaneous regeneration occurs.

New bone, cementum, and periodontalligament which have formed in the 2-month healing period.

Approximately 50 to 70% spontaneous

regeneration can be expected to occur inthe acute defect model and this confoundsinterpretation of attempts to manipulatewound healing variables to produceregeneration.

Advantages of the acute wound healingmodel include cost-effectiveness andreduced experimental time.


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Furthermore, if one wants to studyperiodontal regeneration, this is apredictable model to use.

Finally, this model may be used totest the effects of manipulations,devices, and drugs to determine ifthey have an adverse effect on normalperiodontal regeneration woundhealing.


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The chronic defect model :

Chronic periodontal defects are created inmonkeys by placing orthodontic elasticsaround the circumference of teeth at orslightly apical to the gingival margin.

The elastic gradually migrates apically asplaque-induced inflammation destroys thesupporting periodontal ligament and bone.

Advanced pocketing and bone loss areachieved in 3 to 6 months.

The elastics migrate faster on single rootedteeth and those with more tapered roots.


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The clinical appearance of chronicperiodontal defects produced by orthodonticelastics.

Elastic placement is facilitated by using aplastic instrument to wedge teeth apart toallow the elastic to slip apical tot eh contactpoint. Within a week or two the elastic begins

to migrate apically in the presence of severeinflammation.

After several months deep pockets arepresent. If the interproximal space is large,then an angular bony defect will be created.

If the interproximal space is narrow or thereis an elastic on the adjacent tooth, horizontalbone loss will occur.


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Animal Models2

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