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CHARACTERISTICS OF THE ANIMAL MODEL IN PERIODONTAL HEALTH :

1.Clinical Characteristics :
All non-human primates have a deciduous and a permanent dentition. The dental formula of the permanent dentition of baboons (Papio anubis) and cynomolgus monkeys (Macaca fascicularis) is reported to be same as in humans (I 2/2, C1/1, Pm 2/2, and M 3/3), whereas that of the cotton-ear marmosets (Calithrix jacchus), cottontop marmosets (Saguinus Oedipus), squirrel monkeys, and bushbabies are different.

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 to humans with the following exceptions: larger canines, presence of diastemata between anterior teeth primarily at the canine, and an edgeto-edge relationship of the incisors.

This indicates that molar and premolar regions are to be preferred for periodontal investigations in which the presence of diastemata is not desirable. However, the size of the dentition deviates substantially from that of humans. Clinically healthy gingiva has been observed in squirrel and cynomolgus monkeys, but oral hygiene measures, as in humans, must be undertaken in nonhuman primates to establish and maintain clinically healthy gingival.

Toothbrushing, interdental flossing, and application of 2% aqueous chlorhexidine gluconate have been used to prevent supragingival plaque in baboons, rhesus (Macaca mulatta), stump-tailed (Macaca arctoides, cynomolgus, and squirrel monkeys. The frequency of oral hygiene necessary to establish gingival health within 3 to 4 weeks has been reported to vary from daily or three times weekly. The frequency of cleaning may introduce an additional experimental variable due to the effects of food restriction necessary prior to anesthetizing the animals for cleaning.

2.Histology
Clinically healthy periodontal tissue of cotton-top marmosets, baboons, bushbabies, and stump-tailed, squirrel, and howler monkeys has been observed to be histologically quite similar to that of humans. It is characterized by a non-keratinized junctional and oral sulcular epithelium without or with minimal rete ridges, sparse but varying accumulation of inflammatory cells in the connective tissue, and highly organized bundles of collagen fibers in the gingival connective tissue and periodontal ligament.

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.

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-and intra-species variability in the prevalence and severity of plaque accumulation and periodontal disease has been reported. Extensive plaque accumulation was observed in cotton-top marmosets, young adult and middleaged baboons, aged chimpanzess (Pan troglodytes) young adult rhesus, adult cynomolgus, and stump-tailed monkeys, while moderate plaque accumulation was reported in young stumptailed monkeys and slight plaque accumulation in young cynomolgus monkeys.

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 marked gingival telangiectasia in marmosets.

Significant differences have been reported in the prevalence and severity of gingivitis, varying from localized gingivitis in cotton-top marmosets, young chimpanzees, and baboons, slight gingivitis in adults cynomolgus monkeys; moderate gingivitis in adult rhesus monkeys, young stump-tailed, and young adult cynomolgus monkeys; and to generalized gingivitis in young adult and middle-aged baboons, and rhesus and adult cynomolgus monkeys.

The prevalence and severity of periodontitis also vary. Only rarely has periodontitis been reported in young adult baboons, cotton-top marmosets, and young stump-tailed and cynomolgus monkeys. In contrast, the majority of cotton-ear marmosets studied were found to have varying degrees of marginal inflammation; i.e., from mild gingivitis to extensive periodontitis with increased tooth mobility and even tooth loss. Some wild-living howler monkeys also suffer from localized peridontitis, but none of the animals examined exhibited generalized alveolar bone loss. Furthermore, all skulls of wild-living adult mountain gorillas showed alveolar bone loss and some showed tooth loss, indicting that peridontitis is also present in wild living non-human primates.

As in humans where the prevalence and severity of marginal inflammation increase with age, a similar increase with age and duration of captivity has been reported in baboons, bushbabies, chimpanzees, and cynomolgus and squirrel monkeys. A generalized alveolar bone loss was observed in old chimpanzees and a bone loss affecting approximately one-half to three-fourths of the root length was observed in a cotton-ear marmoset held in captivity for several years.

These studies indicate that young adult animals may be appropriate in studies where absence of periodontitis is preferred. However, it should be kept in mind that reports of clinical findings on small samples may be very misleading, since the observed differences in prevalence and severity of plaque accumulation, gingivitis, and periodontitis may be due to different registration criteria, or due to intentional or unintentional animal selection criteria.

2.Histology :
The histological manifestations of spontaneous gingivitis and periodontitis in baboons, bushbabies, chimpanzees, and howler, cynomolgus, and squirrel monkeys, and cottonear and cotton-top marmosets are quite similar to those of humans. Characteristic features are progressive rate ridge formation, ulceration, apical migration of the pocket epithelium with wide intercellular spaces and emigrating PMNs, increased infiltration of inflammatory cells in connective tissue, vascular proliferation, destruction of collagen fibers, and resorption of alveolar bone.

The inflammatory infiltrate of the connective tissue in spontaneously occurring periodontal disease varies significantly between different species. The inflammatory infiltrate in baboons, bushbabies, howler and cynomolgus monkeys, and chimpanzees contains the same inflammatory cells as in that of humans with a predominance of lymphocytes and plasma cells, although one study in cynomolgus monkeys observed a dominance of macrophages. Early lesions contain a small inflammatory infiltrate of predominantly lymphocytes and macrophages, while established lesions are dominated by plasma cells, these observations are also similar to those in humans.

In contrast, cotton-top and cotton-ear marmosets exhibit a predominance of macrophages and almost an absence of lymphocytes and plasma cells. Based on these differences, marmosets appear to be only slightly related to the human condition and therefore may be inappropriate models for studies of periodontal disease pathogenesis which are based on similarities to the human inflammatory and immune responses.

3.Microbiology
In rhesus monkeys with established gingivitis, the total number of bacteria and proportion of anaerobic species are greater than in gingiva health. In cynomolgus monkeys with gingivitis a predominance of Gram-positive cocci and rods, and Gram-negative rods is characteristic. Similar changes are also observed in subgingival plaque from human teeth with gingivitis, but greater numbers of Actinomyces species are commonly found in humans. Actinobacillus actinomycetemcomitans is primarily isolated from humans suffering from juvenile periodontitis, but is also found in > 25% of patients with adult periodontitis. In young cynomolgus monkeys, however, A. actinomycetemcomitans was frequently isolated from subgingival plaque of sties with localized gingivitis and no alveolar bone loss, indicating that the mere presence of these bacteria does not cause overt periodontal disease in this animal species or that the simian strains of A. actinomycetemcomitans were less virulent.

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 of oral hygiene procedures. A simultaneous change to soft diet has been performed to further facilitate plaque accumulation in stump-tailed and cynomolgus monkeys. Therefore, gingivitis can be investigated in non-human primates, but as noted in humans, within a single species there may be a broad time range of gingival responses to plaque accumulation. This may present problems in data interpretation if small numbers of animals are used for experimental gingivitis studies.

b.Histology:
The histological manifestations of experimental gingivitis in rhesus, stump-tailed, and cynomolgus monkeys are similar to gingivitis in humans following the withdrawal of oral hygiene procedures. Although an increase in the size of the inflammatory cell infiltrate in connective tissue predictably follows plaque accumulation, the time frame for histologic changes may vary. The inflammatory cells were dominated by lymphocytes in early lesions in rhesus and stumptailed monkeys, while an increased number of plasma cells was observed in established lesions of rhesus, stump-tailed, and cynomolgus monkeys. Johnson and Hopps found notably numerous macrophages in early lesions in cynomolgus monkeys, but all cells containing phagocytosed material were classified as macrophages regardless of cell morphology, suggesting that the number of macrophages was most likely overestimated.

c.Microbiology:
There appears to be a close similarity between gingival plaque composition in sites from stump-tailed monkeys with experimental gingivitis and from sites in humans with gingivitis. This similarity is also demonstrated in a study using stump-tailed and cynomolgus monkeys placed on soft diet and with minima gingivitis where the subgingival plaque contained 4050% cocci, 20-40% rods, 8-20% fusiforms, 2-6% filaments, 4-7% vibrios, and 2-6% spirochetes.

Inter-species variability exists because supragingival plaque in rhesus monkeys placed on soft diet contained significantly higher numbers of Prevotella and Porphyromonas species, Fusobacterium species, Veillonella species, and Streptococcus mutans than supragingival plaque from cynomolgus monkeys, while significantly higher numbers of Streptococcus mitis and unidentified Streptococci were observed in cynomolgus monkeys. The significance of these differences is presently unknown.

2.Experimental Periodontitis a.Clinical characteristics:


Periodontitis has been induced in non-human primates by a variety of techniques including placement of silk sutures or orthodontic elastics at the cemento-enamel junction (CEJ) and surgical removal of alveolar bone with subsequent placement of mechnical devices, such as wires or metal bands, to accumulate supragingivial plaque and make the lesion more chronic. Frequently, the diet is simultaneously changed to soft in order to increase plaque accumulation. It is necessary to examine sites during active tissue destruction to investigate pathogenesis of periodontitis.

A useful approximation of this process has been through the use of silk-ligated teeth, while placement of orthodontic elastics and surgical removal of alveolar bone predominantly have been used to establish periodontitis for the evaluation of different periodontal therapies. The latter methods will be dealt with only briefly in the end of this section. Placement of silk ligatures at the CEJ in stumptailed, cynomolgus, and squirrel monkeys resulted in increased plaque accumulation, gingival inflammation, pocket formation, and alveolar bone loss of identical depth on contralateral tooth surfaces. When ligatures were applied to several adjacent teeth in squirrel monkeys, a horizontal bone loss occurred, but when applied to a single tooth, angular bone loss was established. Thus, both horizontal and angular bone loss can be induced in non-human primates.

b.Histology:
The histological features of experimentally produced periodontal lesions in rhesus, squirrel, and cynomolgus monkeys are similar to periodontal lesions in humans. The inflammatory infiltrate of cynomolgus monkeys are similar to periodontal lesions in humans. The inflammatory infiltrate of squirre monkeys was different, with a predominance of PMNs and macrophages and few lymphocytes and plasma cells. even though most of these studies in squirrel monkeys are only 14 days studies and/or studies of the connective tissue apically to the pocket epithelium, it must be connective tissue apically to the pocket epithelium, it must be concluded that also squirrel monkeys appear poorly related to the human condition.

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

c.Microbiology:
Ligature-induced periodontitis in the non-human primate has provided a model for studying microbial changes associated with 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 indentify initiation prior to extensive clinical destruction. It should be noted that the acute and rapid nature of induced periodontitis in squirrel monkeys and marmosets produce an extreme 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 microbial interactions. The time frame for microbial changes in nonhuman primates depends on the monkey species and size, previous history of periodontal disease, and the method of disease induction.

d.Hematological and serological characteristics:


Ligature-induced periodontitis for 6 to 8 weeks in cynomolgus monkeys results in changes affecting the experimentally involved teeth and in modulations in the number and proportion of the different peripheral blood lymphocytes and the systemic antibody response to P.gingivalis. The modulations comprised an increasing number of total peripheral lymphocytes and a decreased proportion of helper T-lymphocytes, whereas the proportion of suppressor T-lymphocytes remained constant. Furthermore, serum concentration of antibodies to P. gingivalis increased, which coincided with the increased proportion of this microorganism observed in the subgingival plaque.

NON-HUMAN PRIMATE MODELS FOR TESTING THE EFFICACY AND SAFETY OF PERIODONTAL REGENERATIO PROCEDURES Animal models for testing periodontal regenerative procedures are necessary because controlled quantitatively histological analysis is required to evaluate the quality and extent of the newly formed supporting tissues. Histometric evaluation can determine the amount of new cementum, periodontal ligament, and alveolar bone formed as the result of regenerative periodontal surgery. These studies are not possible in man because of the need to retrieve teeth and their surrounding periodontium in large blocks appropriate for histological analysis. Furthermore, proper evaluation of a new therapy necessarily involves the use of treated and untreated controls which are difficult to obtain in the human. Finally, the testing of potentially harmful new devices and pharmaceuticals may be unethical in man prior to thorough evaluation in higher animals. Animal models used in testing periodontal surgical therapies have primarily involved dogs and monkeys. Monkeys are preferred because the dental anatomy and wound healing physiology closely parallel that of man.

The disease versus the defect :


Periodontitis causes destruction of the tooth supporting tissues due to inflammation induced by bacterial plaque. The disease is treated by mechanical and chemical means which reduce or eliminate the responsible microbial pathogens. Anatomical defects remain, however, such as pockets, bone loss, gingival recession, and root surface alterations. Much of periodontal therapy is directed towards resolution of these defects by resective, reparative, and regenerative surgery.

In order to test the effects of therapy, defects must be produced in laboratory animals which have the same characteristics as those found in humans. These include deep pockets, horizontal and angular bone loss, and root surfaces devoid of Sharpeys fibers which have been exposed to bacterial plaque. Gingival recession may or may not be desirable, depending on the hypothesis being tested.

Choosing the proper animal :


The defects must be created in an animal model which is large enough so that manipulations and devices can be tested. This requires roots which are long and broad enough so that 6 or 7mm of attachment loss can be obtained while the tooth remains stable. Size is also important because the relative distances that cells must travel often determine the outcome of wound healing. If defects are too small, substantial regeneration may occur in sham-operated control sites by the outpouring of cells from the periodontal ligament / alveolar bone.

Design considerations :
Quantitative assessment requires statistical testing of histometric data. A powerful and sensitive way to accomplish this is to produce identical defects on contralateral teeth within the same animal. In this way, the experimental variable can be tested on a tooth with the contralateral tooth serving as the control. Differences between these pairs of teeth are summed for each animal and the mean for the animal calculated.

The grand mean of the differences and the standard deviation of these differences is then calculated from all animals in the study. Appropriate statistical tests are used to determine significant differences. The number of animals in the investigation is determined by estimating the amount and variability of the differences (i.e., regeneration) and performing a power analysis.

Defect production :
There are four types of periodontal defects which have been used for testing the effects of therapy. These include defects produced by naturally occurring periodontitis and three types of experimentally-produced defects: the acute defect model. Naturally occurring periodontitis of sufficient magnitude in large non-human primates occurs late in life and the lesions are asymmetrical. For this reason, these types of defects are seldom used for testing the effects of periodontal surgical procedures.

The acute defect model :


Mucoperiosteal flaps are elevated and the bone, periodontal ligament, and cementum are removed to create the desired defect shape. Flaps are either replaced and sutured, or the experimental variable is inserted prior to flap closure. The time interval between surgery and retrieval of tissue blocks for histological analysis will vary, depending on the hypothesis being tested.

The problem with the acute defect model is that spontaneous regeneration occurs. New bone, cementum, and periodontal ligament which have formed in the 2month healing period. Approximately 50 to 70% spontaneous regeneration can be expected to occur in the acute defect model and this confounds interpretation of attempts to manipulate wound healing variables to produce regeneration. Advantages of the acute wound healing model include cost-effectiveness and reduced experimental time.

Furthermore, if one wants to study periodontal regeneration, this is a predictable model to use. Finally, this model may be used to test the effects of manipulations, devices, and drugs to determine if they have an adverse effect on normal periodontal regeneration wound healing.

The chronic defect model :


Chronic periodontal defects are created in monkeys by placing orthodontic elastics around the circumference of teeth at or slightly apical to the gingival margin. The elastic gradually migrates apically as plaque-induced inflammation destroys the supporting periodontal ligament and bone. Advanced pocketing and bone loss are achieved in 3 to 6 months. The elastics migrate faster on single rooted teeth and those with more tapered roots.

The clinical appearance of chronic periodontal defects produced by orthodontic elastics. Elastic placement is facilitated by using a plastic instrument to wedge teeth apart to allow the elastic to slip apical tot eh contact point. Within a week or two the elastic begins to migrate apically in the presence of severe inflammation. After several months deep pockets are present. If the interproximal space is large, then an angular bony defect will be created. If the interproximal space is narrow or there is an elastic on the adjacent tooth, horizontal bone loss will occur.