Agreement, correlation, and kinetics of the alveolar bone-loss measurement methodologies in a ligature-induced periodontitis animal model

Abstract Periodontal research involves the use of animal models to better understand the biological processes of periodontal diseases and the potential of new or existing therapies. Currently, ligature-induced periodontitis in rats is the main model used in periodontal research, in this model, alveolar bone loss (ABL) is the main parameter evaluated by radiographic, morphometric, and histological techniques. Interestingly, although these methodologies are widely used, it is not totally clarified neither the kinetics of ABL over the induction time nor the agreement degree (repeatability and reproducibility) of these techniques. Objective: To characterize ABL kinetics at 0, 3, 7, 15, 30, and 60 days after ABL induction by ligature and to evaluate the intra- (repeatability) and inter-examiner (reproducibility) agreement and the correlation among the radiographic, morphometric, and histological methodologies. Material and Methods: 60 male Wistar rats with induced ABL were randomly divided into 6 experimental groups (n = 10 animals/group). After 0, 3, 7, 15, 30, and 60 days, the animals were euthanized and their hemimandibles were removed for ABL determination using radiographic, morphometric and histological techniques. Results: Radiographic and morphometric/linear techniques allowed the detection of statistically significant ABL on the third day, while histological and morphometric/area techniques could only detect ABL after the seventh day (ANOVA/Tukey, p<0.05). After the fifteenth day, except for histological analysis, the ABL was stabilized. Concerning the agreement of the methodologies, Bland Altman's test (intra and inter-examiner evaluations) showed no difference among the measurements (p>0.05). In addition, high correlations (Pearson's test, r2>0.9, p<0.05) were observed. Conclusion: The results indicated that the minimum time for ABL induction could vary from 3 to 7 days, according to the chosen analysis methodology. Agreement and correlation data support the comparison of results between studies with same induction time.


Introduction
In periodontal research, animal models are important for studying the relationship between diseases and external factors, and also for testing potential new therapies. The model selection will depend on the aim of the study, but in general, an adequate model should present biological events similar to that observed in humans, such as the progressive destruction of connective tissue, bone loss, and the formation of periodontal pockets 2 .
The rat is the most widely used rodent to study The radiographic method usually involves analyzing ABL by measuring the linear distance between the cementoenamel junction (CEJ) in the alveolar 8,9,13 . The morphometric method requires measuring the linear distance (mm) or the area (mm 2 ) between the CEJ and the bone crest in the mandibular molars 3,14 . In the histological method, the interproximal bone loss by the linear distance between the CEJ and the bone crest 18 .

Radiographic analysis
The hemimandibles were radiographed using a digital x-ray system (Kavo Dental, Biberach, Baden-Wurttemberg, Germany). The x-ray incidence was lingual, with an exposure time of 0.04 seconds and an object-focus distance of 30 cm. The mesial linear distance (mm) between the CEJ and the alveolar bone software Image J 1:48 ( Figure 1B).

Morphometric analysis
The hemimandibles were boiled for 30 minutes and immersed in hydrogen peroxide solution (3%) to remove the excess tissue. After this procedure, the hemimandibles were stained with methylene blue 1% to demarcate the CEJ. For each specimen, the following were measured: 1) linear distance (mm) between the CEJ and the bone crest of the three mandibular molars, with a total of seven measurements corresponding to the number of roots, and 2) the area (mm 2 ) of bone loss between the CEJ and the bone crest of the three molars. The measurements were made on the lingual surface using the software Image J 1.48 ( Figure 1C/D).
Measurements of the linear distance (mm) between molar, and the second molar were obtained. The measurements were made using the software Image J 1.48 ( Figure 1E).

Data evaluation and statistical analysis
Intra-examiner agreement (repeatability) of the ABL measurement techniques: A trained examiner performed two independent ABL measurements (radiographic, morphometric, and histological techniques) using the G15 group with a t test (one sample) was carried out to determine the intra-examiner agreement.
Inter-examiner agreement (reproducibility) of the ABL measurement techniques: Two trained examiners performed independent ABL measurements (radiographic, morphometric, t test (one sample) was carried out to determine the inter-examiner agreement.
Correlation among the ABL measurement techniques: Using data from G15 group, Pearson's correlation was carried out to determine the correlation among the different methodologies of ABL evaluation.
Kinetics of bone loss observed in the ABL measurement techniques: ANOVA and Tukey's test were used to determine the statistical differences in ABL among the periods of evaluation with the software Graph Pad Prism 6.0e.

ABL Kinetics
Both the radiographic and morphometric/linear 15. After this period, the ABL remained constant until day 60 (Figure 2A and B).
Using morphometric/area analyses, after ABL induction, the minimum time needed to detect bone loss occurred until day 15, and from day 15 to day 60, the ABL remained constant ( Figure 2C).
Using histological analyses, linear analysis showed until day 30. After this time, the bone resorption was uniform ( Figure 2D). Figure 3 shows

Discussion
The use of animal models in periodontal research is becoming increasingly frequent. Between 1966 and 2016, more than 300 articles using experimental periodontitis induced by ligature in rats were published in the PubMed database that aimed to clarify Tukey, p>0.05)  with the control, on the third day after PD induction, when measured by radiographic or morphometric/ linear techniques, while ABL was detected after 7 days using morphometric/area and histological techniques. The rapid bone resorption described for this rat