A variety of methods have been proposed by different studies to induce experimental animal models of periodontitis (23). It is generally believed that periodontitis animal models should represent the obvious processes of plaque attachment, gingival inflammation, attachment loss and alveolar bone loss observed in human disease (24). For these reasons, we choose to induce animal model by alveolar bone removal surgery in association with ligature placement. We successfully reduced the obstacles of fixing rat’s body position and exposing the surgical region in the narrow oral cavity by designing a customized dental surgery bed (Fig. 1A 1–3). Overall, the application of our process can effectively improve the efficiency of periodontitis modeling in rats (the average successful rate is 82.6%, data not shown).
Periodontitis is a chronic inflammatory response that results from the interaction between the host immune system and oral pathogens(12). That’s why previous experimental studies generally used models that resulted from plaque accumulation which gradually induce periodontitis. However, the induction period of such approach takes longer than 5–7 months to develop primary clinical gingivitis in dogs(15). In our study, the maximum peak periodontitis was obtained as early as the 9th day (Fig. 3B-E). Animals such as monkeys, miniature pigs and beagle dogs are seldom the first choice for periodontium regeneration research because they are expensive to culture and require a high standard for experimental equipment (25). The breeding and housing costs of rodent animals are relatively low, making it possible to carry out studies with sufficient mass for statistical analysis (26). Rat modeling was therefore faster, easier and more cost-effective. Nowadays, gene knockout rats have been widely cultured in recent years especially for the study of the specific roles of genes in regulating pathological process, inflammation responses and tissue regeneration of periodontitis. A large number of studies have used genetically-engineered rats to study the underlying mechanism of systemic inflammation and its effect on periodontal healing. Rats can be ideal animals for the study of periodontal diseases, which are suitable not only for the study of teeth, but also for the dynamic interaction of soft-hard tissue related to oral inflammation (26).
Meanwhile, previous study found that ligature alone did not induce stable and lasting periodontal bone loss in rats, as the regression of inflammation and the healing of alveolar bone were too observed in the ligature group of our study from Day 12 to Day 18 (Fig. 3A 8–9). The decrease of CEJ-ABC was also rather random in individual rat, making it difficult to achieve standardized measurement for comparable data analysis. Therefore, we also optimized the surgical bone removal protocol in rats according to pre-described anatomical landmarks (Fig. 1A 4–6). The 3D micro-CT reconstructed images of the mandibular first and second molars showed similar triangular area of bone defect after operation, which proved that the operation location was reliably repeatable (Fig. 3A 13&19). The method proposed in this study not only produces a standardized morphological defect area, but also ensures reliable data for repetitive comparison research according to any given scheme. For an instance, any amount of regenerative osseous tissue provoked by certain regenerative periodontal treatment (manifesting as a blurring margin and the shallowing of the triangle surgical defect, Fig. 1D) might be easily identified, measured and quantitated. Compared to the mainstream ligation method, the model we induced manifest a significantly faster progression, longer duration and a more standardized bone absorption area of experimental periodontitis during the same period. Therefore, we believed that the present established a model of periodontitis in rats by alveolar bone defect in association with silk ligature confirmed its superiority to previous methods, proving its essentiality to be a suitable experimental model for regenerative periodontal treatment evaluation.
By far, no satisfying model similar to the pathologic process of human periodontitis has been proposed (23). In present study, we obtained various methods to evaluate the model outcomes of periodontitis at different time points, including the two-dimensional CT panel of labial-lingual section, the micro-CT reconstructed three-dimensional model and HE stained histopathological sections, all reporting obvious time-pattern changes and specificity. The present rat models we established, induced by acute alveolar bone defect and chronic silk ligature, is the first to successfully mimic the pathological changes in periodontal tissue and stages divisions in human periodontitis (Table 2).
Table 2
Differences of stages of gingivitis and periodontitis between humans and rats
Stage
|
Humans
|
Rats
|
Time (days)
|
Clinical Findings
|
Underlying microscopical features
|
Time (days)
|
Clinical Findings
|
Underlying microscopical features
|
Ⅰ.Initial lesion
|
2–4
|
• Gingival fluid flow
|
• Vascular dilation
• Infiltration by PMNs
• Perivascular collagen loss
|
0–3
|
• NOT clinically evident
• subclinical gingivitis
|
• gingival vasculature
• PMNs exudation
• fibrin deposition
|
Ⅱ.Early lesion
|
6–8
|
• Erythema Bleeding on probing
|
• Vascular proliferation
• Lymphocytes infiltration
• Increased collagen loss around infiltrate
|
3–6
|
• erythema of the gingival margin
• “marginal gingivitis”
|
• PMNs into pocket area
• collagen destruction
• proliferated capillaries and capillary loops
|
Ⅲ.Established lesion
|
14–21
|
• Changes in color, size, texture, and so on
|
• Vascular proliferation and blood stasis
• Plasma cells infiltration
• Continued loss of collagen
|
6–9
|
• bluish tinge may become superimposed on the reddened gingiva (anoxemia)
• gingival edema
|
• plasma cells increase
• widened intracellular spaces with PMNs
• congested vessels
• RBCs extravasate
|
Ⅳ. Advanced lesion
|
>28
|
• consistent bleeding (gingival index = 2)
• more attachment loss
|
• fibrosis of the gingiva
• widespread tissue damage
• plasma cells and neutrophils dominating epithelium
|
9–12
|
• probing bleeding
• pocket form
• attachment loss
|
• slight alveolar bone loss
|
Ⅴ.periodontitis
|
For years
|
• plaque and calculus, gingival swelling, redness
• Deep PDD
• BOP(+)
• Attachment and bone loss (angular/vertical or horizontal)
• Increased tooth mobility
|
• Degeneration and inflammatory exudate
• edema
• inflamed engorged connective tissue, expanding rete pegs
|
15–18 onwards
|
• oral inflammatory changes (erythema, edema, hemorrhage) intensify
• horizontal and vertical (or angular) bone loss
• Increased tooth mobility
|
• alveolar bone is lost via osteoclastic activity
|
At day 3, experimental region between the mandibular first and second molars of rats showed clear-cut triangular bone defect area (Fig. S1). Mild hyperemia of blood vessels was observed in the gingival tissues with low clinical indexes of BI and TM (Fig. 2C&D), indicating that ligation hadn’t led to obvious plaque accumulation yet. This stage can be regarded as the initial lesions of human periodontitis, showing similarity in clinical manifestations and pathological progress (27). In 6–9 days, the activity of periodontitis increased due to plaque accumulation, initiating inflammatory cell infiltration. As more of the gingiva becomes affected, bleeding may be spontaneous as the clinical manifestations of marginal gingivitis (28) (Fig. S1). Rough surface of alveolar bone was captured both by micro-CT and histopathologic sections, corresponding to the early lesions of human periodontitis (Fig. 3A 19). With the progress of the experiment, the destruction of periodontal tissue exacerbated (Fig. 4A and Fig. S1) in the Day 9 to 12 (Fig. 4B 13). The connection between the junctional epithelium and the dentin surface was greatly loose with polymorphonuclear leukocytes (PMNs) infiltration. The connective tissue showed significant RBC extravasation and collagen fibers disappearance (Fig. 4B 14). In our study, bone loss peaked at 9 days postoperatively (Fig. 3B). Significant horizontal and vertical bone resorption was formed causing the height of alveolar crest decreased, and the loss of buccal bone reached more than 1/2 (Fig. 3A 20&23). By far, the expression of periodontal tissue is close to the clinical manifestations of the established stage in human periodontitis. From 15 to 18 days, the acute gingival inflammation was slightly alleviated (Fig. S1). This may be a result of the conversion of the innate responses of the rat immune system to adaptive responses to produce protection of periodontal tissue, but at this point the reduced alveolar bone height is not significantly recovered and chronic periodontal destruction will persist (Fig. 3A 21). Periodontal loss is considered to be irreversible, meaning that lost bone cannot be regained without advanced regenerative surgeries(29). Therefore, the optimal experimental period of our model is from 9 to 12 days.