Published online Nov 23, 2022.
https://doi.org/10.5051/jpis.2203420171
Temporal changes of periodontal tissue pathology in a periodontitis animal model
,1,†
Bo Hyun Jung,2,†
Ki-Yeon Yoo,2
Jong-Bin Lee,1
Heung-Sik Um,1
Beom-Seok Chang,1
and Jae-Kwan Lee1
Abstract
Purpose
This study aimed to characterize the early stages of periodontal disease and determine the optimal period for its evaluation in a mouse model. The association between the duration of ligation and its effect on the dentogingival area in mice was evaluated using micro-computed tomography (CT) and histological analysis.
Methods
Ninety mice were allocated to an untreated control group or a ligation group in which periodontitis was induced by a 6-0 silk ligation around the left second maxillary molar. Mice were sacrificed at 1, 2, 3, 4, 5, 8, 11, and 14 days after ligature placement. Alveolar bone destruction was evaluated using micro-CT. Histological analysis was performed to assess the immune-inflammatory processes in the periodontal tissue.
Results
No significant difference in alveolar bone loss was found compared to the control group until day 3 after ligature placement, and a gradual increase in alveolar bone loss was observed from 4 to 8 days following ligature placement. No significant between-group differences were observed after 8 days. The histological analysis demonstrated that the inflammatory response was evident from day 4.
Conclusions
Our findings in a mouse model provide experimental evidence that ligature-induced periodontitis models offer a consistent progression of disease with marginal attachment down-growth, inflammatory infiltration, and alveolar bone loss.
Graphical Abstract
INTRODUCTION
Periodontitis is a chronic inflammatory condition characterized by gingival swelling, alveolar bone loss, and ultimately loss of the tooth. The etiology and pathogenesis of periodontal disease are as follows. Chronic inflammation in periodontal tissue is initiated by bacterial plaque containing various periodontal pathogens [1]. Periodontal disease is also associated with host susceptibility, and host-defense mechanisms appear to be related to periodontium destruction [2]. Risk factors such as smoking and diabetes interact with the host defense mechanisms and bacterial plaque [3]. Plaque bacteria are indispensable, but not sufficient, to elicit periodontitis initiation and development; thus, a susceptible host is essential [4].
Clinical studies on periodontitis face limitations due to the complexity of the disease, which involves interactions between genes, behavior, and dental plaque. Human studies of periodontal diseases may have challenges such as determining the severity of disease, ethical issues, and differences in individuals’ predisposition to periodontitis progression [5]. Experimental animal data can provide models of biological trends before proceeding to human applications. Thus, clinical observations of periodontal disease, along with research in animal models, are significant for the investigation of the pathogenesis of periodontitis and the development of various treatment modalities [6]. Experimental animal models have provided important information regarding the disease pathogenesis outlining clinical, radiographic, and histological features of the disease [7, 8, 9, 10, 11, 12]. Numerous animal species can be used for experimental periodontitis models, and apes, dogs, rats, rabbits, pigs, and mice are the most commonly employed [13]. It is important to select an experimental animal model that has features similar to human anatomy.
Mice have been used for periodontal investigations, primarily focusing on microbiology and immunology [13]. Mouse models have several advantages, such as economic factors, ease of handling, shorter research time, and similarity of the dentogingival area in the molars compared with humans [14]. Moreover, extensive data have been gathered about the immune system, allowing a comparative understanding of the pathogenesis of periodontal disease [9]. Mouse models have been productively employed in the investigation of the pathogenesis of periodontal disease and the development of improved treatments, particularly drugs [7, 10, 15, 16].
Several methods have been employed to induce periodontal diseases in murine models (oral gavage, lipopolysaccharide injections, and calvarial models) [17, 18, 19, 20]. Among the various methods, ligature-induced periodontitis is used in rodents [6, 21, 22]. A braided silk ligature has commonly been tied around the cervical region of the maxillary molar. The major benefit of the ligature-induced periodontitis model in mice is that periodontal disease can be initiated at a preferred time, and connective tissue and alveolar bone loss predictably occur within 15 days [7, 10, 16, 23]. Ligature removal can play a useful role in research on inflammation resolution and the healing response. The mechanism of ligature-induced periodontitis is not due to mechanical trauma but the local accumulation of bacterial plaque and inflammation [7, 24].
A review of the literature demonstrates that the characteristics of ligature-induced periodontal disease in mouse models during the early stages have not yet been elucidated. In this study, we aimed to characterize the early stages of periodontal disease and determine the optimal period for its evaluation in a mouse model. The association between the duration of ligation and its effect on the dentogingival area in mice was evaluated using micro-computed tomography (micro-CT) and histological analysis.
MATERIALS AND METHODS
Experimental animals
Ninety male Institute of Cancer Research mice (aged 4 weeks, weighing 18–20 g) were used. The mice were purchased from Orient Bio Inc. (Seongnam, Korea). The mice were free to access water and food ad libitum and adapted to the laboratory housing conditions for 1 week. The Institutional Animal Care and Use Committee of Gangneung-Wonju National University (protocol number GWNU-2020-39) approved the study design. This study was conducted in accordance with the guidelines established by the international laws and policies (National Institutes of Health [NIH] Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985, revised 1996). The sample size was calculated based on the data from our previous study [25]. When an effect size of 0.45 and a significance level of 0.05 was set using the G*Power program, and the power of 0.8 recommended in scientific research was applied, 10 mice per group were required.
Induction of periodontal disease
To induce periodontal disease, 6-0 black silk (AILEE, Busan, Korea) ligatures were tied around the cervical area of the maxillary left second molar, and the contralateral right second molar that did not receive ligatures served as the control. The ligature was applied and tied gently to prevent unexpected damage to the periodontium. The knot was placed on the buccal side of the second molar to prevent loosening. Microsurgical instruments, such as micro-forceps, needle holders, and micro-scissors, were used under an operating microscope. The ligatures were kept in place in all mice during the experimental period and checked daily. In the following cases, the ligatures were re-tied in the same method: 1) when a ligature was placed in the cervical area but the suture was released, and 2) when a ligature was completely removed.
The 90 mice were randomly allocated into 9 groups as follows:
1. Control group: untreated, n=10
2. Ligation/1 d group: sacrificed 1 day after ligature placement, n=10
3. Ligation/2 d group: sacrificed 2 days after ligature placement, n=10
4. Ligation/3 d group: sacrificed 3 days after ligature placement, n=10
5. Ligation/4 d group: sacrificed 4 days after ligature placement, n=10
6. Ligation/5 d group: sacrificed 5 days after ligature placement, n=10
7. Ligation/8 d group: sacrificed 8 days after ligature placement, n=10
8. Ligation/11 d group: sacrificed 11 days after ligature placement, n=10
9. Ligation/14 d group: sacrificed 14 days after ligature placement, n=10
Tissue processing
Tissue processing for the experimental animals was performed as described elsewhere.[26] At each time point following ligation, animals were euthanized. After cervical dislocation, the thorax was opened, perfusion-washed with 0.9% saline, and perfusion-fixed with phosphate-buffered saline (pH 7.5) containing 4% paraformaldehyde for tissue fixation and extraction. After perfusion fixation, the maxilla was retrieved and post-fixed in the same fixative for 8 hours. Micro-CT images of the fixed tissue were obtained. For histological analysis, maxillae were decalcified using 4% ethylenediaminetetraacetic acid for 4 days. The tissues were immersed for 2 hours in graded ethanol baths at 27°C to dehydrate in succession. Samples were placed twice in fresh pure xylene for an hour each and then embedded in paraffin (Histowax; Leica, Wetzlar, Germany). Histological sections of 5-μm thickness were obtained and mounted on microscopy slides coated with saline.
Micro-CT assessment
Micro-CT scans (SKYSCAN1272, Bruker, Kontich, Belgium) were used to evaluate the alveolar bone loss. The voltage and current of the X-ray tube were 90 kV and 88 µA, respectively, with an exposure time of 500 ms. X-ray projections were obtained at 0.3° intervals with a scanning angular rotation of 180°. The images were digitized at 2,240 × 2,240 pixels. The pixel size was set to 14 μm. All scans were oriented with NRecon software (ver 1.7.04, Bruker, Billerica, MA, USA) and Dataviewer (Bruker) before analysis so that the scan axes and anatomical position were uniformly aligned and both the root apex and the cementoenamel junction (CEJ) appeared in the same slice. For linear measurements of bone loss, the distances from the CEJ to the alveolar bone crest (ABC) were recorded (in mm) along the mesial and distal sides of the ligated tooth.
Histological analysis
Masson trichrome staining was performed to evaluate the changes in the periodontal tissue surrounding the maxillary left second molar. Samples were stained using a trichrome stain kit (Abcam, Cambridge, UK) for descriptive histological analysis. Deparaffinization was performed by washing the samples twice with xylene for 10 minutes. Deparaffinized sections were hydrated by moving them through a gradient of ethanol concentrations. The specimens were then treated with Bouin solution for 60 minutes and chilled for 10 minutes. Treatment with Weigert iron hematoxylin for 5 minutes was followed by washing with running water for 2 minutes. Specimens were stained with Biebrich scarlet-acid fuchsin solution for 15 minutes and rinsed in distilled water (DW).
Finally, the sections were treated with a phosphomolybdic-phosphotungstic acid solution for 10 minutes, moved to aniline blue solution for 5 minutes, and treated with 1% acetic acid solution. The slides were washed with DW, dehydrated with ethanol, and sealed in Canada balsam (Kanto Chemical, Tokyo, Japan). The slides were observed under a microscope (Axio Imager A2, Carl Zeiss, Göttingen, Germany) and images were captured using a digital camera (EOS 100D, Canon, Tokyo, Japan) attached to the microscope.
Statistical analysis
The distances between the CEJ and ABC on micro-CT images were statistically analyzed. Group measurements are expressed as the mean and standard deviation. Differences between the control and ligation groups at the time of sacrifice in each group were analyzed using the Mann-Whitney U test. Differences among the 9 groups were evaluated by 1-way analysis of variance (ANOVA) combined with the Bonferroni correction. All statistical analyses were performed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). The significance level was set at P<0.05.
RESULTS
Experimental animals
All animals remained healthy throughout the experimental period. The body weight did not differ significantly among the groups, and no adverse events occurred.
Micro-CT analysis
Micro-CT scans were analyzed to assess the alveolar bone loss induced by ligatures (Figure 1). To quantify bone loss, the CEJ-to-ABC distance at the mesial and distal sides was measured (Table 1). No statistically significant differences were found between the control and ligation groups until 2 days. However, 3 days after ligature placement, there was an obvious difference in bone height on the mesial (0.161±0.024 mm) and distal sides (0.113±0.015 mm) in the ligation group compared to the control group (P<0.05). From day 3 onwards, there was a significant difference in alveolar bone loss in the ligation groups compared to the control group (Figure 2). ANOVA combined with the Bonferroni correction was used to compare the average value of the mesial and distal sides in the distance between the CEJ and the ABC among the 9 groups (Figure 3). The Ligation/4 d group showed significantly greater alveolar bone loss (0.252±0.035 mm) than the control (0.111±0.006 mm), Ligation/1 d (0.128±0.005 mm), Ligation/2 d (0.131±0.005 mm), and Ligation/3 d (0.137±0.006 mm) groups (P<0.05). The mice in the Ligation/8 d (0.435±0.084 mm) group showed significantly greater alveolar bone loss than the control, Ligation/1 d, Ligation/2 d, Ligation/3 d, Ligation/4 d, and Ligation/5 d (0.291±0.026 mm) groups (P<0.05). The amount of bone resorption in the mice among the ligation groups appeared to have a time-dependent tendency until day 5, while there were no significant differences after day 8.
Figure 1
Representative 3-dimensional images showing the liner measurements performed for each sample. Yellow lines indicate the distances (mm) between the cementoenamel junction and the alveolar bone crest recorded for the M and D locations on teeth.
M: mesial, D: distal.
Table 1
Measurement of the distance between the cementoenamel junction and the alveolar bone crest on the mesial and distal sides of the maxillary second molar at each time point after ligature placement
Figure 2
a)P<0.05, when comparing a ligature group to the respective control group.
Graph representing the average distance between the CEJ and the ABC in the M and D locations on teeth in the groups sacrificed at 1, 2, 3, 4, 5, 8, 11, and 14 days after ligature placement and in the control group. Data are the mean and standard deviation (n=10 mice per group).
CEJ: cementoenamel junction, ABC: alveolar bone crest, M: mesial, D: distal.
Figure 3
a)P<0.05 for comparisons with each group (1-way analysis of variance combined with the Bonferroni correction).
Graph representing the average distance between the CEJ and the ABC in the mesial and distal locations on teeth in the groups sacrificed at 1, 2, 3, 4, 5, 8, 11, and 14 days after ligature placement and in the control group. Data are the mean and standard deviation (n=10 mice per group).
CEJ: cementoenamel junction, ABC: alveolar bone crest, M: mesial, D: distal.
Histological analysis
Sections were obtained from the periodontal tissue of the maxillary second molar, and an analysis was performed in the marginal epithelium and connective tissue between the teeth and alveolar bone. Figures 4 and 5 illustrate representative histological findings at each time point after ligature placement. All mice in the control group showed a normal marginal epithelium and an absence of inflammatory infiltrates (Figure 4A). Gingival crest loss was detected after day 1, and thickening of the epithelium peaked on day 8 in the ligation group (Figure 4B-I). Inflammatory infiltrates and collagen fiber disappearance were evident on days 4 and 5 in the ligation groups (Figure 5F and G). However, the number of collagen fibers increased in the connective tissue, and a fibrous pattern was observed after day 8 (Figure 5H-J).
Figure 4
Representative Masson trichrome-stained sections of gingival tissues harvested 1, 2, 3, 4, 5, 8, 11, and 14 days after ligature placement at low magnification (scaler bar, 100 μm).
Figure 5
Representative Masson trichrome-stained sections of gingival tissues harvested 1, 2, 3, 4, 5, 8, 11, and 14 days after ligature placement at high magnification (scale bar, 50 μm).
DISCUSSION
In this study, we aimed to identify the association between the duration of ligation and the features of periodontitis using an experimental animal model. Our analysis identified an appropriate time for morphometric evaluation of periodontal tissue changes in ligature-induced periodontitis in mice.
In a patient-centered study design, confounding variables such as oral hygiene habits, the presence of other systemic health conditions, and smoking status make data diverse and challenging to analyze. Moreover, human studies often have small sample sizes and do not provide a clear timeline of disease development [27]. To overcome this limitation, studies with animal models must be conducted to explain the pathophysiology of periodontitis. Significant information can be collected by combining human clinical studies with effective animal models [28, 29, 30, 31]. In this study, we selected a mouse model since it has several advantages, such as the similarity of the mouse genome to that of humans (up to 98%), the ability to manipulate mouse strains, and low cost [32, 33, 34]. We used a ligature-induced periodontitis mouse model and investigated the host response through radiographic and histological analyses in the early stages of periodontal disease throughout the 14-day experimental period.
In the present study, we investigated temporal changes in a ligature-induced periodontitis mice model. Based on the micro-CT analysis, we split the mice into 3 groups based on significant differences in alveolar bone loss (Control, Ligation/1 d, Ligation/2 d, and Ligation/3 d groups; Ligation/4 d, and Ligation/5 d groups; Ligation/8 d, Ligation/11 d, and Ligation/14 d groups) (Figure 4). The histological analysis revealed that marginal epithelium loss, periodontal inflammation, tissue breakdown, and gingival epithelium thickening were apparent after day 4 and peaked in the Ligation/8 d group. In the ligature-induced periodontitis model, significant periodontal tissue loss occurred after 4 days and showed a time-dependent relationship for up to 8 days. Previous studies [6, 21, 22] have reported that the onset time of significant alveolar bone loss varies (days 3, 5, and 6). Differences in time courses might occur owing to diversity in ligature models: Marchesan et al. [22] placed the ligature between the first and the second molars using 2 knots, while de Molon et al. [6] tied the ligature around the cervical area of the first and second molars in an “8” shape.
The differences in alveolar bone loss from Ligation/8 d to Ligation/14 d were not statistically significant. In the histological analysis, after 8 days of ligation, gingival fibers increased and showed a fibrous pattern, whereas the occurrence of inflammatory infiltrates and disappearance of gingival fiber were obvious on days 4 and 5. These profile changes in the ligature-induced periodontitis model could be potentially explained by some hypotheses. First, as a consequence of alveolar bone loss, supracrestal attached tissues tend to move toward a more apical position to compensate for the biologic space, causing a reduction in disease severity [35, 36]. De Molon et al. [6] re-placed the ligature on the apical region every 3 days to keep the thread closer to the periodontal tissue and consequently maintain the inflammation. Li and Amar [10] suggested that Porphyromonas gingivalis-soaked ligatures might help aggravate the disease severity. Another report [21] found that at the buccal side of the maxillary second molar in mice, the width of the bone in the apical region is thinner than in the marginal region, accelerating destruction and fenestration in the apical region. Consequently, a false impression may arise that a significant loss of bone has occurred. In that study, a relatively high standard deviation in alveolar bone loss was noted on day 8, reflecting the fact that bone collapse overlying fenestrations did not occur simultaneously in all mice.
Histological analysis revealed that after day 1, the gingival tissue crest disappeared in the ligated animals. Two days after ligature placement, the marginal epithelium appeared to recover; however, the gingival tissue crest was not observed. These results might have been due to trauma during ligature placement. In large animals, the placement of ligatures did not cause mechanical trauma [7, 37]. However, mice have small mouths and teeth; therefore, mechanical trauma caused by ligatures might influence periodontal tissue loss [21]. Nevertheless, disease progression is likely to be affected primarily by bacteria, and a previous report reported that antibiotics significantly reduced bone loss in a ligature-induced periodontitis model [21, 38].
Our findings in this mouse model provide experimental evidence that ligature-induced periodontitis models offer a consistent progression of disease with marginal attachment down-growth, inflammatory infiltration, and alveolar bone loss. The ligature-induced periodontitis model demonstrated no significant differences during the first 3 days. However, from day 4 to day 8, there was progressive inflammation and bone breakdown, and no significant differences were observed after day 8. This study therefore identified the optimal time to investigate host-microbial interactions and inflammation in periodontitis.
Funding:This study was supported by the 2021 Cooperative Research Program (grant number CR2101) of Gangneung-Wonju National University Dental Hospital.
Conflict of Interest:No potential conflict of interest relevant to this article was reported.
Author Contributions:
Conceptualization: Jae-Kwan Lee, Ki-Yeon Yoo.
Data curation: Hyunpil Yoon, Bo Hyun Jung, Ki-Yeon Yoo, Jong-Bin Lee, Jae-Kwan Lee.
Formal analysis: Hyunpil Yoon, Bo Hyun Jung.
Investigation: Hyunpil Yoon, Bo Hyun Jung, Ki-Yeon Yoo, Jong-Bin Lee, Jae-Kwan Lee.
Methodology: Ki-Yeon Yoo, Heung-Sik Um, Beom-Seok Chang, Jae-Kwan Lee.
Software: Hyunpil Yoon, Bo Hyun Jung, Jong-Bin Lee, Jae-Kwan Lee.
Supervision: Ki-Yeon Yoo, Jong-Bin Lee, Heung-Sik Um, Beom-Seok Chang, Jae-Kwan Lee.
Writing - original draft: Hyunpil Yoon, Bo Hyun Jung.
Writing - review & editing: Ki-Yeon Yoo, Jong-Bin Lee, Heung-Sik Um, Beom-Seok Chang, Jae-Kwan Lee.
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