Clarifying the cellular mechanisms of periodontitis with an improved animal model

Researchers from Tokyo Medical and Dental University (TMDU) have developed a technique that allows them to analyze in detail the development of periodontitis over time.

Periodontal disease, represented by periodontitis, is the leading cause of tooth loss and affects almost one in five adults worldwide. In most cases, this condition occurs as a result of an inflammatory response to a bacterial infection of the tissues around the teeth.

As the condition worsens, the gums begin to recede, exposing the roots of the teeth and bone. Notably, the incidence of periodontitis increases with age, and as populations around the world increase in life expectancy, it is important to have a solid understanding of its underlying causes and progression.

In a study published in Nature Communications, TMDU researchers found a way to achieve this goal by improving a widely used animal model for studying periodontitis.

Direct study of periodontitis in humans is difficult. As a result, scientists often turn to animal models for preclinical research. For example, the "mice ligation-induced periodontitis model" has, since its introduction in 2012, allowed researchers to study the cellular mechanisms underlying this condition.

Simply put, in this model, periodontal disease is artificially induced by placing silk sutures on the molars of mice, which causes plaque accumulation. Although this method is convenient and effective, it does not cover the complete picture of periodontitis.

Schematic illustration of inflammatory gene expression profiles during periodontitis and the role of the IL-33/ST2 axis in controlling acute inflammation. Source: Tokyo Medical and Dental University.

"Although periodontal tissue is composed of gingiva, periodontal ligament, alveolar bone and cementum, analysis is usually performed exclusively on gingival samples due to technical and quantitative limitations," notes lead study author Anhao Liu. "This sampling strategy limits the conclusions that can be drawn from these studies, so methods are needed that can analyze all tissue components simultaneously."

To address this limitation, the research team developed a modified model of ligature-induced periodontitis. Instead of the classic single ligature, they used a triple ligature on the upper left molar of male mice. This strategy expanded the area of bone loss without significant bone destruction around the second molar, increasing the number of different types of periodontal tissue.

"We isolated three major tissue types and assessed RNA yield between the two models. The results showed that the triple ligation model effectively increased yield, achieving four times the amount of normal periradicular tissue and supporting high-resolution analysis of different tissue types," explains Sr. By Dr. Mikihito Hayashi.

After confirming the effectiveness of their modified model, the researchers began studying the effects of periodontitis on gene expression among different tissue types over time, focusing on genes associated with inflammation and osteoclast differentiation.

One of their main findings was that Il1rl1 gene expression was significantly higher in periradicular tissue five days after ligation. This gene encodes the ST2 protein in receptor and decoy isoforms, which binds to a cytokine called IL-33, which is involved in inflammatory and immunoregulatory processes.

To gain further insight into the role of this gene, the team induced periodontitis in genetically modified mice that lacked the Il1rl1 or Il33 genes. These mice demonstrated accelerated inflammatory bone destruction, highlighting the protective role of the IL-33/ST2 pathway. Further analysis of cells containing the ST2 protein in its receptor form, mST2, showed that the majority were derived from macrophages.

"Macrophages are generally classified into two main types, pro-inflammatory and anti-inflammatory, depending on their activation. We found that mST2-expressing cells are unique in that they simultaneously express some markers of both types of macrophages," comments senior author Dr. Takanori Iwata. "These cells were present in the periradicular tissue before the onset of inflammation, so we called them 'resident periodontal macrophages.'"

Together, the results of this study demonstrate the power of the modified animal model to study periodontitis at a more detailed scale, down to the biomolecular level.

"We propose the possibility of a novel molecular pathway, IL-33/ST2, regulating inflammation and bone destruction in periodontal disease, along with specific macrophages in the periradicular tissue, which is deeply involved in periodontal disease. This will hopefully lead to the development of new treatment strategies and prevention methods," concludes senior author Dr. Tomoki Nakashima.