Titanium bone screw with machined surface 1.2 × 4.5 mm (screw head 1.5 mm, fixture 3 mm) was purchased from ACS Surgical Supply (Brockton, MA) that was used as implant by Freire and coworkers [15].

Six Sprague-Dawley, male, 400–450 g in which the body weight is based on reported article [15]. To achieve a stable implantation, the thickness of alveolar bone is crucial. Therefore, body weight is crucial for success of implantation. Animals were maintained and experiments were performed according to a protocol that was approved by the Rutgers Institutional Animal Care and Use Committee (IACUC).

The time course is illustrated at Fig. 1. Rats were randomly located into 3 groups. Group 1 is for the baseline of 7 weeks after implantation, group 2 is for ligature induced peri-implantitis, and group 3 receives sham ligature to be the control of peri-implantitis 2 weeks after the procedure of ligature (Fig. 1a).

Anesthesia is achieved by intraperitoneal injection of ketamine HCl/xylazine solution in dose of 80 mg/kg/ketamine and 5 mg/kg/xylazine. During the anesthesia, breath and blood circulation were intensively monitored by observing chest and stomach movement, and color of the tip of nose and tongue. Animals were not sent back to the animal facility until the animals were completely awakened.

Implantation is performed under anesthesia. When well anesthetized, the rat is laid in supine position. Jaws are kept open by pulling jaws up with silk suture loops tightened on upper and lower incisors. After cleaning the oral cavity with 0.12% chlorhexidine, a 0.5 mm × 0.5 mm niche in the alveolar ridge is made transmurally with a ball-shaped carbide dental bur with a diameter of 0.5 mm in a low speed to avoid overheating. Then, the implant is placed manually with an equipped screwdriver till no movement can be done. Each rat receives implantation of 1.2 mm × 4.5 mm titanium implants on the maxillary alveolar ridge in each side of the natural diastema (total of two implants per rat). Peri-implantitis is induced with ligature of silk suture at cervical part of implant that will lead to local inflammation and alveolar bone loss to mimic the clinical peri-implantitis. This procedure is modified from procedure to induce periodontitis [16, 17].