Name: experimental model of ligature-induced peri-implantitis in mice
Uploaded: 2024-05-17T14:10:00.000+00:00
Duration: 5 min 37 s
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 trained and calibrated operators (Figure 1A).
1. Pre-extraction steps
<ol>
	<li>Perform the procedure in a surgical room complying with all biosafety and protection standards.</li>
	<li>Perform asepsis of all surfaces with triple friction using a 70% isopropyl alcohol solution.</li>
	<li>Anesthetize 3-week-old C57BL/6J male mice with 3% isoflurane. Check for the lack of response to paw withdrawing upon touch to ensure sufficient anesthetic depth is obtained.</li>
	<li>To avoid interfering with procedures in the mouth, use a nosecone to maintain isoflurane anesthesia.</li>
	<li>Have an auxiliary operator to stabilize the animal and maintain the mouth opening (Figure 1B).</li>
	<li>Apply ophthalmic lubricant, to prevent irritation in the eyes before starting the extraction.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66316/66316fig01.jpg" /> 
Figure 1: Operative adaptations: (A) Microscopic magnification. (B) Adapted inhalation anesthesia system and stabilization for mouth opening. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66316/66316fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Dental extraction
<ol>
	<li>For dental extractions, introduce a #5 dental explorer between the first and the second molar to start the elevation and luxation procedures by moving the instrument in the mesio-distal direction until the tooth moves in the socket (Figures 2A).</li>
	<li>Next, introduce the #5 dental explorer in the mesial site of the first molar. Do this by moving the instrument in the mesio-distal direction until the tooth moves in the socket.</li>
	<li>Following elevation, use the tip forceps and the suture tying forceps to remove the 1st molar.</li>
	<li>Next, introduce the dental explorer between the 2nd and 3rd molar to elevate and luxate the 2nd molar (Figure 2B).</li>
	<li>Then, use the tip forceps and/or the suture tying forceps to remove the tooth using the instruments to hold it and take out of the mouth. (Figure 2C).</li>
	<li>After dental extractions, ensure complete hemostasis achievement by using a sterile cotton tipped (Figure 2D) for 1 min (Figure 2E,F).</li>
	<li>Immediately after extraction, provide all animals with pain medication (Carprofen/Rimadyl 5 mg/kg) every 24 h. Administer the medication through subcutaneous injection.</li>
	<li>In addition, replace regular food with a soft diet. Administer the antibiotic (Amoxil 0.25 mg/mL) orally by incorporating the medication into the drinking water. Do this for four weeks after extractions.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66316/66316fig02.jpg" /> 
Figure 2: Initial extraction sequence: (A, B) Maxillary region with 1st and 2nd molar teeth and use of dental explorer for elevation and luxation. (C) Use of the tip forceps and explorer for luxation and tooth removal. (D) Hemostasis. (E, F) Alveolar appearance after extractions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66316/66316fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Implant placement
<ol>
	<li>Using a 15c blade, create a mesio-distal incision through the keratinized tissue in the area corresponding to the previously present teeth. Use the right maxillary molars as a spatial reference (Figure 3A).</li>
	<li>Raise the buccal and palatal full-thickness flaps by using a #5 dental explorer, ensuring complete flap elevation (Figure 3B).</li>
	<li>Perform the osteotomy using a 0.3 mm diameter carbide micro hand drill attached to a Pin Vise (hand drill) and activate it in a clockwise rotation. Create osteotomy sites with approximately 1 mm deep into the healed extraction sockets (Figure 3C).</li>
	<li>Place the custom-designed screw-shaped implants with a smooth machined surface (1.0 mm long and 0.5 mm in diameter) fabricated from 6AL4V titanium rods (Figure 3D), one per animal, by self-tapping in the region of the first and second maxillary left molars using a clockwise screwing motion (Figure 3E-I).</li>
	<li>Immediately after implant placement, administer pain medication (Carprofen/Rimadyl 5mg/kg every 24 h) through subcutaneous injection.</li>
	<li>Allow the implants to heal for four weeks, during which time administer antibiotics and feed as previously described.</li>
	<li>At the end of the healing period, ensure that the mucosal wound is completely closed and has a light pink appearance.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66316/66316fig03.jpg" /> 
Figure 3: Implant placement sequence: (A) Incision using a 15c blade attached to the handle. (B) Full-thickness flaps using #5 dental explorer. (C) Osteotomy using a 0.3 mm carbide micro hand drill attached to a Pin Vise. (D) Titanium dental implant. (E, F) Implant support and implant holder. (G- I) Implant placement using a clockwise screwing motion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66316/66316fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Peri-implantitis induction
<ol>
	<li>Place a silk ligature (6-0) around each fixture immediately apical to the implant head by contouring the surface of the implant and fixing it with a double knot. Keep the ligature for two weeks allowing the peri-implantitis development.</li>
	<li>Check the ligatures every two days to make sure they are still present. If missing, place a new ligature.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66316/66316fig04.jpg" /> 
Figure 4: Ligature-induced peri-implantitis sequence. (A-D) Silk ligature (6.0) placed around the implant head. (E-G) Knot closure. (H) Ligature cut. (I) Final appearance. Clinical images obtained from live animals under sedation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66316/66316fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Sacrifice
<ol>
	<li>After the peri-implantitis induction period (two weeks), sacrifice all animals with a high concentration of inhaled isoflurane, followed by a secondary method of cervical dislocation with a rod pressed into the base of the skull and quickly pulling the hind limbs, causing the separation of the cervical vertebrae from the skull.</li>
	<li>Separate the maxillae using sharp instruments, such as surgical scissors, that will separate the animal&#39;s entire jaw from the rest of the body.</li>
	<li>Photograph the maxillae using an optical microscope, fix it in 10% formalin for 24 h, then store in 70% ethanol.</li>
</ol>
6. Micro-computed tomography (&#181;CT )
<ol>
	<li>Scan the maxillae as previously described18,19 using micro-computed tomography (micro-CT) scanning.</li>
	<li>Turn on the scanner and the computer, then open the scan software, click on the radiation sign, and wait 15 minutes to preheat it.</li>
	<li>Open the equipment door by clicking on the door icon.</li>
	<li>Use a quarter-size rotary table. Place the maxilla on a 15ml conical tube. Fix the tube in the rotary table base. Place the rotary table in the equipment screw.</li>
	<li>Close the equipment door by clicking on the door icon.</li>
	<li>Turn on the X-Ray source.</li>
	<li>Click on the grab image icon and make sure the bone is on the field.</li>
	<li>Adjust a resolution of 10 &#181;m pixel size, X-ray energy of 55 KVp and 181 &#181;A, filter of AI 0.5 mm, rotation step 0.4, average frame 10. These settings will generate an estimated scanning time of 24 minutes.</li>
	<li>Start the acquisition.</li>
	<li>For reconstruction open the reconstruction software. Use beam hardening of 20%. Use ring artifact of 5%. Use a dynamic range of 0-0.13. Deselect label ON. Click on Preview. Choose the adequate view that is seen for the region of interest, and save it as TIFF.</li>
	<li>Orient the images obtained using software for viewing and analyzing data. Make the long axis of the implant head parallel to the sagittal and coronal axes and perpendicular to the axial axis.</li>
	<li>Save the sagittal image as a single image for linear bone analysis.</li>
	<li>Save transaxial image as a dataset for volumetric bone analysis.</li>
	<li>Perform linear bone analysis using the analyzing data software. Measure the distances in millimeters between the implant head and the alveolar bone in the sagittal and coronal planes, including mesial, distal, buccal, and palatal sites.</li>
	<li>Perform volumetric bone analysis using analyzing data software. Track bone loss around implants by drawing the region of interest in all slides corresponding to the bone loss.</li>
</ol>
7. Statistical analysis
<ol>
	<li>Measure linear and volumetric microtomographic bone loss and normalize these values by dividing each value by the average of the control group. Present an average for all groups, as mean &#177; standard error of the mean (SEM).</li>
	<li>Compare significance using a two-way analysis of variance (ANOVA) followed by a Tukey&#39;s test with a 95% confidence interval. (Prism 5). Apply significance levels as follows: p&#8804;0.01**, p&#8804;0.001****.</li>
</ol>

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The authors have nothing to disclose.

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-implantitis26,27,28.
Inevitably, disadvantages such as limited body size and reduced maxillae/teeth dimensions should be considered. In our present protocol, the critical steps refer to the animal size. Despite the ease of obtaining, handling, and maintaining these animals, performing the experimental steps can bring some challenges to the operators, given the small size. To overcome this limitation, two operators are indispensable; where one should open the mouth and stabilize the animal while the other performs the surgeries.
Another important detail is the instrument used for tooth luxation (modified explorer). Considering short dental crowns and the limited space between them, commercially available explorers, even the most delicate ones, are difficult to accommodate between the teeth. Therefore, modifying the tip and obtaining a minimum curvature of 80o was necessary to facilitate handling and positioning. This modification gave the operator greater comfort and operational safety. Another measure in this surgical stage was controlling the force exerted during the luxation to avoid accidents, such as instrument deviation and tissue perforation.
In addition to the same limitations mentioned about small size, the pressure during incision in the palate with a scalpel should also be considered for implant placement. Despite the blade having an adequate size, the millimetric thickness of the tissue to be cut requires extra attention from the operator, who must ensure that no deep or repeated incisions are made. The same goes for the osteotomy, whose vertical pressure accompanied by rotational movements must be gently performed to avoid possible deep perforations in the maxillary sinus. Considering the length of the implants (1.0mm), the use of an optical microscope is a prerequisite. Hence the significance of our method using all the equipment listed.
Although the ligature method has a recurrent and well-established approach in periodontal disease models29,30,31,32, for the peri-implant disease model, some details must be observed, as described in our protocol. Unlike periodontitis induction, in which the dental crowns are visibly exposed, facilitating ligature placement with supragingival visualization, for peri-implantitis induction, implants can be completely or partially covered by keratinized tissue, making it impossible to view. Therefore, a new incision may be necessary, and the ligature placement, in these cases, must be subgingival.
The correct implant site identification and the firm ligature positioning around each fixation were constantly verified every two days, and early replacement in case they had been lost was completed. This ensures that the inflammatory process continues until the proposed model is established33. In all the steps performed, special attention was always given to the constant control of gingival bleeding in mice until hemostasis was achieved. In addition&#160;to a soft diet to minimize local friction, measures like this avoid hemorrhagic conditions for the animals and prevent the failure of the procedures performed18.
Based on our representative results, it is plausible to infer that the present protocol had significant findings since our primary objective was achieved. All clinical, radiographic, and morphological parameters observed here confirmed that the ligature-induced peri-implantitis was successfully established. According to the literature, clinical aspects such as increased soft tissue edema around ligature compared with controls should be expected19. Similarly, the Micro-CT outcomes should reveal increased linear and volumetric bone loss in those mice with peri-implantitis, results also observed in our study and following the current literature34. Finally, our study also includes histomorphological analyses by HE that showed a visibly exacerbated inflammatory infiltrate and bone resorption in those animals with peri-implantitis, like several other studies35.
Although this is a positive finding, critical aspects should be pointed out regarding our protocol. Besides the animal&#39;s size previously mentioned, another considerable limitation is the inability to perform such procedures without optical magnification. Despite expanding the operator&#39;s field of view and confidence, postural discomfort is expected due to the fixed eye contact on the equipment and, at the same time, the detailed surgical performances in a small environment. Despite that, our method has significant merit. It can be applied in different biomedical research areas, such as microsurgeries and bone microarchitecture studies, in addition to precise training for operators&#39; surgical skills improvement.

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><tbody><tr><td>#5 dental explorer</td><td>Hu-Friedy, Chicago, IL</td><td>392-0911&amp;nbsp;</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&amp;mu;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&amp;reg;, 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 &amp;amp; Tip</td><td>D. P. Machining Inc., La Verne, CA</td><td>Unique product&amp;nbsp;</td><td>Implant holder</td></tr><tr><td>Implant support</td><td>D. P. Machining Inc., La Verne, CA</td><td>Unique product&amp;nbsp;</td><td>Implant capture</td></tr><tr><td>Isoflurane&amp;nbsp;</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>&amp;mu;CT scans</td></tr><tr><td>Nrecon Software</td><td>Bruker Corporation, Billerica, MA</td><td>-</td><td>Images reconstruction</td></tr><tr><td>&amp;Oslash; 0.3mm - L 2.5mm Micro Drills&amp;nbsp;</td><td>Sphinx, Hoffman Estates, IL</td><td>ART. 50699&amp;nbsp;</td><td>Osteotomy</td></tr><tr><td>&amp;Oslash; 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 receive ligature (control group).
The success rate of implant osseointegration 
Respecting the four-week healing period and observing the clinical stability, implants in our study had high survival and 100% success rates as none of the placed implants was lost. No other adverse events were observed.
Clinical evaluation 
Using optical microscopy, clinical evaluation was performed through visual inspection and clinical photos immediately after sacrificing the mice (Figure 5). When compared to the control group, inflammation, pocket formation and increased soft tissue edema was observed around the implant in the peri-implantitis group. No evidence of severe clinical phenotype complications was observed.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66316/66316fig05.jpg" /> 
Figure 5: Representative clinical images of (A) non-ligature (NL) and (B) ligature (L) groups 2 weeks after ligature. Increased soft tissue edema was observed around the implant in the peri-implantitis group compared to the other group. 20X magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66316/66316fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Microcomputed tomography analysis (&#181;CT) 
Two weeks after ligature placement, when comparing non-ligature and ligature groups, there were significant differences in bone height observed via by linear analysis (Figure 6A, B and C), and bone loss volume observed via volumetric analysis (Figure 6D). Linear bone loss in the ligature-induced PI group was significantly increased compared to the control group. Likewise, when comparing the volumetric bone loss, the PI group showed more significant circumferential bone loss compared to the control group.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66316/66316fig06.jpg" /> 
Figure 6: Micro-computed tomography analysis. (A) Representative microtomographic sections of control (non-ligature - NL) and (B) peri-implantitis (ligature - L) groups. (C) Graph represents the averaged distance from the implant head to the alveolar bone 2 weeks after ligature. (D) The graph represents the average circumferential volumetric bone loss 2 weeks after ligature. Data represented as average SEM. *p&#60;0.05 (n&#8805;5 for all groups/time points). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66316/66316fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Histological evaluation 
To determine cellular changes, including bone loss around the implant, decalcified samples were sectioned and stained with hematoxylin &#38; eosin (HE) (Figure 7). The decalcification process was carried out by immersing the samples in 15% ethylenediaminetetraacetic acid (EDTA), pH 7.4, for four weeks, changing the solution every three days. With the implants removed, the specimens were embedded in paraffin. So, 5 &#181;m-thick sagittal dimensioned paraffin sections were stained with HE, following standard protocols. As a result, more apical epithelial migration, moderate cellular infiltration, and bone loss in peri-implantitis samples were observed when compared to the control group.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66316/66316fig07.jpg" /> 
Figure 7: Representative sagittal H&#38;E images of non-ligature (NL) (A) and (B) ligature (L) groups. Increased apical epithelial migration and bone loss in the ligature group compared to the control group. 20x magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66316/66316fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
According to the most recent consensus on the classification of peri-implant diseases and conditions, the peri-implantitis diagnosis requires bone loss beyond crestal bone level changes resulting from initial bone remodeling22. Therefore, our study presents well-established and validated peri-implantitis diagnosis parameters. And offer a comprehensive evaluation of the condition under investigation.

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 ...

experimental-model-of-ligature-induced-peri-implantitis-in-mice

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.0K Views.  University of California. Het algemene doel van dit artikel is om het protocol te rapporteren dat wordt toegepast om peri-implantitis bij muizen te induceren door middel van ligatuur en om de effectiviteit ervan te observeren door middel van weefselevaluatie en botverlies rond de implantaten. De volgende procedures werden uitgevoerd in een operatiekamer die voldeed aan alle bioveiligheids- en beschermingsnormen. Alle procedures werden uitgevoerd onder een microscopische vergroting van 10x en uitgevoerd door getrainde ...

Video: Experimental Model of Ligature-Induced Peri-Implantitis in Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Medicine

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, periodontal ligament, cementum, and alveolar bone. In this study, a simple method of PD induction in rats is described. We provide detailed instructions for placement of the ligature model around the first maxillary molars (M1) and a combination of injections of lipopolysaccharide (LPS), derived from Porphyromonas gingivalis at the mesio-palatal side of the M1. The induction of periodontitis was maintained for 14 days, promoting the accumulation of bacteria biofilm and inflammation. To validate the animal model, IL-1&#946;, a key inflammatory mediator, was determined by an immunoassay in the gingival crevicular fluid (GCF), and alveolar bone loss was calculated using cone beam computed tomography (CBCT). This technique was effective in promoting gingiva recession, alveolar bone loss, and an increase in IL-1&#946; levels in the GCF at the end of the experimental procedure after 14 days. This method was effective in inducing PD, thus being able to be used in studies on disease progression mechanisms and future possible treatments.

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

In this study, a rat model of induction of periodontitis is presented via a combination of retentive ligature and repetitive injections of lipopolysaccharide derived from Porphyromonas gingivalis, over 14 days around the first maxillary molars. The ligation and LPS injection techniques were effective in inducing peridontitis, resulting in alveolar bone loss and inflammation.

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, ...

Establishing a Mouse Model for Candida albicans Biofilm-Related PJI

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

Author Spotlight: Advancing Research on Candida albicans Biofilm-Associated Prosthetic Joint Infections 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

Creating a Pulp Inflammation Mouse Model Using a Novel Mouth-Gag 

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

Author Spotlight: Enhancing Dental Pulp Research with Improved Mouse Models

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Experimental Model of Ligature-Induced Peri-Implantitis in Mice

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