Name: experimental model of ligature-induced peri-implantitis in mice
Uploaded: 2024-05-17T14:10:00
Description: Watch this Scientific Journal Video about Experimental Model of Ligature-Induced Peri-Implantitis in Mice at JoVE.com

This work was supported by the NIH/NIDCR DE031431. We would like to thank the Translational Pathology Core Laboratory at UCLA for assistance with preparing the decalcified histological sections.

Procedures involving animal subjects have been approved by the Chancellor&#39;s Animal Research Committee of the University of California, Los Angeles (ARC protocol number 2002-125), and the Animal Research: Reporting In Vivo Experiments (ARRIVE)22. For this method, eighteen 3-week-old C57BL/6J male mice were used and underwent dental extractions, implant placement and peri-implantitis induction. All dental procedures were performed under 10&#215; microscopic magnification and carried out by train...

<ol>
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This protocol presents a descriptive report on surgical procedures for peri-implantitis induction utilizing a ligature model in mice. Working with mice has advantages, such as being cost-effective, the availability of an extensive genetic array given the many backgrounds23 among other aspects24,25. Over the years, several studies have successfully utilized mice in the medical and dental fields, including in peri-implantitis<sup class="xref...

Dental implants are increasingly prevalent as a desirable choice for replacing missing teeth1. The prevalence of dental implants in the US adult population is projected to increase up to 23% by 20262. Based on a market analysis report by Grand View Research (2022), the global market size of dental implants was projected to reach approximately US $4.6 billion in 2022. Furthermore, it is anticipated to exhibit a steady annual growth rate of around 10% until the year 20303. Unfortunately, the use of dental implants can lead to complications, such as peri-implantitis. Peri-implantitis has been defined as a biofilm-induced condition characterized by inflammation in the peri-implant mucosa and subsequent progressive loss of supporting bone4.
A systematic review found that the mean prevalence of peri-implantitis was 19.53% (95% Confidence interval [CI], 12.87 to 26.19%) at the patient level and 12.53% (95% CI 11.67 to 13.39%) at the implant-level5. Peri-implantitis represents a growing public health, due to an increase in implant failure and, consequently, substantial treatment costs6.
Understanding the pathogenesis of peri-implantitis is crucial to developing a systematic approach to prevent its onset and progression and maximize dental implants&#39; longevity in terms of aesthetics and function7,8. In this sense, using murine models in dental research has proven advantageous, given that mice share more than 95% of their genes with humans9,10, the number of available online genetic databases, and the ability to reproduce clinical scenarios11. All the described advantages allow the dissection of genetic mechanisms in different diseases12, accessible accommodation and management, and antibodies widely available as human panels, beyond the genetic modification availability (e.g., knockout and overexpression) for inflammatory tissue assessment and disease mapping13. Although advantageous, there are few publications addressing peri-implantitis in mice. This is due to methodological challenges, among others, including the difficulty in obtaining mini-implants or installing them.
To develop peri-implantitis in mice, many protocols have been described, such as ligature-induced peri-implantitis, bacteria-induced peri-implantitis14, Lipopolysaccharide (LPS)-induced peri-implantitis15, or the combination LPS + ligature-induced peri-implantitis16. Here, we will focus on the ligature model because it is the most widely accepted method to induce periodontitis17,18,19 and, more recently, peri-implantitis20,21. The ligature placed around the implants in a submucosal position stimulates plaque accumulation and, consequently, tissue inflamation. So, the development of this approach is based on the indication of a viable cost-benefit technique for pre-clinical investigations on peri-implant diseases. This study aims to describe an experimental model of ligature-induced peri-implantitis in mice and determine whether there is effectiveness in inducing this disease given the observed bone and tissue changes.
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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 dental explorer</td>
			<td>Hu-Friedy, Chicago, IL</td>
			<td>392-0911&#160;</td>
			<td>Dental luxation</td>
		</tr>
		<tr>
			<td>15c blade and surgical scalpel</td>
			<td>Henry Schein Inc., Melville, NY</td>
			<td>1126186</td>
			<td>Tissue incision</td>
		</tr>
		<tr>
			<td>6-0 silk ligatures</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>NC9201232</td>
			<td>Ligature</td>
		</tr>
		<tr>
			<td>Amoxicillin 50&#956;g/mL</td>
			<td>Zoetis, San Diego, CA</td>
			<td>TS/DRUGS/57/2003</td>
			<td>Oral suspension</td>
		</tr>
		<tr>
			<td>Bacon Soft Diet</td>
			<td>Bio Serve&#174;, Frenchtown, NJ</td>
			<td>14-726-701</td>
			<td>-</td>
		</tr>
		<tr>
			<td>C57BL/6J male mice</td>
			<td>The Jackson Laboratories, Bar Harbor, ME, USA</td>
			<td>000664</td>
			<td>Age: 3-week-old</td>
		</tr>
		<tr>
			<td>CTAn software</td>
			<td>V.1.16 Bruker, Billerica, MA</td>
			<td>-</td>
			<td>Volumetric analysis</td>
		</tr>
		<tr>
			<td>Dolphin software</td>
			<td>Navantis, Toronto, CA</td>
			<td>-</td>
			<td>Linear bone analysis</td>
		</tr>
		<tr>
			<td>Implant carrier &#38; Tip</td>
			<td>D. P. Machining Inc., La Verne, CA</td>
			<td>Unique product&#160;</td>
			<td>Implant holder</td>
		</tr>
		<tr>
			<td>Implant support</td>
			<td>D. P. Machining Inc., La Verne, CA</td>
			<td>Unique product&#160;</td>
			<td>Implant capture</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Vet One, Boise, ID</td>
			<td>NDC13985-528-60</td>
			<td>Inhalational anesthetic</td>
		</tr>
		<tr>
			<td>Micro-CT scan 1172</td>
			<td>SkyScan, Kontich, Belgium</td>
			<td>-</td>
			<td>&#956;CT scans</td>
		</tr>
		<tr>
			<td>Nrecon Software</td>
			<td>Bruker Corporation, Billerica, MA</td>
			<td>-</td>
			<td>Images reconstruction</td>
		</tr>
		<tr>
			<td>&#216; 0.3mm - L 2.5mm Micro Drills&#160;</td>
			<td>Sphinx, Hoffman Estates, IL</td>
			<td>ART. 50699&#160;</td>
			<td>Osteotomy</td>
		</tr>
		<tr>
			<td>&#216; 0.5mm - L 1.0mm Titanium implants</td>
			<td>D. P. Machining Inc., La Verne, CA</td>
			<td>Unique product</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ophthalmic lubricant</td>
			<td>Apexa, Ontario, CA</td>
			<td>NDC13985-600-03</td>
			<td>Artificial tears</td>
		</tr>
		<tr>
			<td>Pin Vise</td>
			<td>General Tools, Secaucus, NJ</td>
			<td>90</td>
			<td>Osteotomy</td>
		</tr>
		<tr>
			<td>Rimadyl 50mg/ml</td>
			<td>Zoetis, San Diego, CA</td>
			<td>4019449</td>
			<td>Anti-inflammatory</td>
		</tr>
		<tr>
			<td>Sterile cotton tipped</td>
			<td>Dynarex, Glendale, AZ</td>
			<td>4304-1</td>
			<td>Hemostasis</td>
		</tr>
		<tr>
			<td>Tip forceps</td>
			<td>Fine Science Tools, Foster City, CA</td>
			<td>11071-10</td>
			<td>Dental Extraction</td>
		</tr>
		<tr>
			<td>Tying forceps</td>
			<td>Fine Science Tools, Foster City, CA</td>
			<td>18025-10</td>
			<td>Ligature placement</td>
		</tr>
	</tbody>
</table>

experimental model of ligature-induced peri-implantitis in mice

Dental implants have a high success and survival rate. However, complications such as peri-implantitis (PI) are highly challenging to treat. PI is characterized by inflammation in the tissues around dental implants with progressive loss of supporting bone. To optimize dental implants' longevity in terms of health and functionality, it is crucial to understand the peri-implantitis pathophysiology. In this regard, using mouse models in research has proven clear benefits in recreating clinical circumstances. This study aimed to describe an experimental model of ligature-induced peri-implantitis in mice and determine whether there is effectiveness in inducing this disease, given the observed bone and tissue changes. The experimental peri-implantitis induction comprehends the following steps: teeth extraction, implant placement, and ligature-inducted PI. A sample of eighteen 3-week-old C57BL/6J male mice was divided into two groups, ligature (N=9) and control non-ligature (N=9). The evaluation of clinical, radiographical, and histological factors was performed. The ligature group showed significantly higher bone loss, increased soft tissue edema, and apical epithelial migration than the non-ligature group. It was concluded that this pre-clinical model can successfully induce peri-implantitis in mice.

For this method, eighteen 3-week-old C57BL/6J male mice were used and underwent dental extractions, implant placement and peri-implantitis induction. There were nine animals per group which was statistically significant, considering linear bone loss achieving 80% power, 15% standard deviation (&#963;) and 95% confidence interval (&#945; =0.05). Mice were fed a soft diet ad libitum during the experiment. Nine mice received a ligature (ligature-induced periimplantitis-experimental group), and nine mice did not rec...

Watch this Scientific Journal Video about Experimental Model of Ligature-Induced Peri-Implantitis in Mice at JoVE.com

Experimental Model of Ligature-Induced Peri-Implantitis in Mice

This is a report on an experimental model of ligature-induced peri-implantitis in mice. We describe all surgical steps, from pre- and post-operative management of the animals, extractions, implant placement, and ligature-induced peri-implantitis.

Dental implants have a high success and survival rate. However, complications such as peri-implantitis (PI) are highly challenging to treat. PI is ...

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.

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