Table 1.

Detailed procedure of the modified method to induce experimental periodontitis.

Step	Treatments (using the left side of the maxillary molars as an example)
1	Exposure of the operating view in the oral cavity	• Hold the curved tweezers with the left hand and point its curved tip upward to prop open the mandible of the mouse and prevent the tongue from falling down to obstruct the view
		• Tie an orthodontic ligature (6–8 cm in length) around the maxillary incisor and tape it to the operating table
2	Pretreatment with C+ files	• Pre-bend 5 mm of the tip of the 8#, 10#, or 15# C+ nickel-titanium root canal files
		• Hold the C+ file with the right hand and carefully use it as a probe to determine the interproximal region between the 1st and 2nd molar and the 2nd and 3rd molar
		• Slowly file into the adjacent space and gently pull back ~2 mm to form a path between the interproximal space. Then separate the filled gingival tissues, for the purpose of the following orthodontic ligature placement
		✧ The reason for the above operation:   1. Because of the flexibility of the orthodontic ligature, it is easily bent and deformed. Thus, it is difficult to insert into the interproximal space because of the blockage of gingival tissues.   2. Being a type of dental root canal file with both stiffness and elasticity, C+ nickel-titanium files are clinically used to locate the narrow orifice of calcified root canals. Because they are sufficiently thin and can pass through the interproximal space, and operators can determine correct placement by touch and feeling feedback, C+ root canal files enable access so that the orthodontic ligature can be successfully placed
3	Insertion of the orthodontic ligature wires	• Prepare the orthodontic ligature wires (0.20 mm in width, 3–5 mm in length).  ✧ If the wire is too long, it could easily pierce the oral mucosa and enter the buccinator, which will adversely affect the ability of mice to eat and could inhibit adequate food intake. Furthermore, a wire that is too long will be easily licked and removed by the tongue, causing it to fall off from the interproximal space. Conversely, if the ligature is too short, it is difficult to clamp and bend it into an appropriate angle for further insertion
		• Slightly bend the orthodontic ligature wire into a “(” shape, in order to form a proper angle for subsequent insertion
		• Hold the needle holder with the right hand, and clamp the ligature with the tip of the needle holder
		• Gently probe the adjacent region prepared by the root canal files above with the orthodontic ligature to determine the correct location, and then gently insert the orthodontic ligature into the interproximal space
4	Completion	• Slightly move the ligature with tweezers to repeatedly check whether the ligature has been properly placed