Experimental Model of Ligature-Induced Peri-Implantitis in Mice

The overall goal of this article is to report the protocol applied to induce peri-implantitis in mice by ligature and to observe its effectiveness through tissue evaluation and bone loss around the implants. The following procedures were performed in a surgical room complying with all biosafety and protection standards. All of the procedures were performed under 10X microscopic magnification and carried out by trained and calibrated operators.

All of the surgeries were done under inhalation anesthesia with isoflurane and oxygen. An assistant operator was needed to stabilize the animal and maintain mouth opening. To prevent irritation in the eyes, ophthalmic lubricant was used before starting the extraction.

For this method, 18 three-week-old C57BL/6J male mice were used and underwent dental extractions, implant placement, and peri-implantitis induction. Dental extraction. For dental extractions, a 5 dental explorer was introduced between the first and second molar to start elevation and luxation.

Next, the dental explorer was introduced into the mesial site of the first molar. Following elevation, the tip forceps and the suture tying forceps were used to remove the first molar. Next, the dental explorer was introduced between the second and third molar to elevate and luxate the second molar.

After dental extractions, complete hemostasis was achieved by using a sterile cotton-tipped applicator for one minute. Immediately after extraction, all animals received pain medication, administered through subcutaneous injection. In addition, for four weeks after extractions, regular food was replaced by a soft diet, and antibiotic was administered orally, incorporating the medication into drinking water.

Implant placement. Using a 15c blade, a mesiodistal incision was made through the keratinized tissue in the area corresponding to the previously present teeth. The right maxillary molars were the spatial reference.

The buccal and palatal full thickness flaps were raised using a 5 dental explorer, ensuring complete flap elevation. The osteotomy was performed using a 0.3 millimeter diameter carbide micro hand drill attached to a pin vise, and activated through clockwise rotation. The osteotomy sites were approximately one millimeter deep into the healed extraction sockets.

After that, titanium implants, one per animal, were self tapped in the region of the first and second maxillary left molars using a clockwise screwing motion. The implants had a four-week healing period during which time the mice were given antibiotics and fed as previously described. Peri-implantitis induction.

Peri-implantitis was induced by placing a silk ligature 6-0 around each fixture, immediately apical to the implant head. Ligatures were maintained for two weeks to develop peri-implantitis. Ligatures were checked every two days to make sure they were still present.

If missing, a new ligature was placed. After this period, all animals were sacrificed. The maxillae were harvested, photographed using an optical microscope, fixed in 10%formalin for 24 hours, then stored in 70%ethanol.

Clinical evaluation was performed by taking pictures immediately after sacrifices using optical microscopy. When compared to the control group, inflammation, pocket formation, and increased soft tissue edema were observed around the implant in the peri-implantitis group. No evidence of severe clinical phenotype complications was observed.

Two weeks after ligature placement, when comparing non-ligature and ligature groups, there were significant differences in bone height observed via linear analysis and bone loss volume observed via volumetric analysis. To determine cellular changes, decalcified samples were sectioned and stained with hematoxylin and eosin. As a result, more apical epithelial migration and bone loss in peri-implantitis samples was observed when compared to the control group.

After watching this video, you should have a good understanding about how to induce peri-implantitis in a mice model, as well as identify the clinical, microtomographic, and histological differences found. Although this method does not represent all the peri-implantitis aspects, it is indispensable for the establishment of a cause and effect relationship, providing information on tissue healing and supporting studies regarding bone loss and peri-implant disease.