Name: A Periprosthetic Joint Candida albicans Infection Model in Mouse (Video) | JoVE Journal
Uploaded: 2024-02-02T12:40:00.000+00:00
Duration: 4 min 37 s
Description: Learn to establish a Candida albicans biofilm-associated periprosthetic joint infection model in mice using titanium-nickel wire implants. This method enables research into PJI prevention and treatment strategies.

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We are grateful for the financial support from the Natural Science Foundation of Shaanxi Province (grant number 2021SF-118) and the National Natural Science Foundation of China (grant numbers 81973409, 82204631).

The animals were purchased from Xi&#39;an Jiaotong University. All animal experiment procedures were approved by the Institutional Animal Ethical Committee of Xi&#39;an Jiaotong University (approval number: SCXK [Shaanxi] 2021-103). The mice were kept for one week with 5 mice per cage. They were allowed free access to food and water. The animals were maintained at room temperature (RT; 24 &#176;C &#177; 1 &#176;C) and light/dark cycle (12 h/12 h) before the study was performed.
1. Buffer and equipment preparation
<ol>
	<li>C. albicans cell culture
	<ol>
		<li>Inoculate a monoclonal colony of C. albicans (SC5314) from a yeast extract peptone dextrose (YPD) plate medium with an inoculating loop into 5 mL of YPD liquid medium (YPD + 50 &#181;g/mL carbenicillin).</li>
		<li>Shake C. albicans cells subsequently at a speed of 220 rpm at 30 &#176;C overnight.</li>
		<li>Centrifuge the suspension at 400 x g for 5 min at RT. Resuspend the C. albicans cells in normal saline and dilute the concentration of cells&#160;to 1 x 106 cells/mL via visually adjusting the turbidity to be the same as a 0.5 McFarland.</li>
	</ol>
	</li>
	<li>Preparation of normal saline
	<ol>
		<li>Weigh 0.9 g of sodium chloride and dissolve in 100 mL of deionized water to prepare 0.9% normal saline.</li>
	</ol>
	</li>
	<li>Surgical instruments preparation
	<ol>
		<li>Autoclave (121 &#176;C, 30 min) the surgical instruments (scissors, forceps, hemostatic forceps, needle holders, suture needles) and titanium-nickel alloy wire (about 0.5 mm in diameter) before use.</li>
	</ol>
	</li>
</ol>
2. Mouse PJI model establishment
<ol>
	<li>Randomly divide 30 C57BL/6 mice (male, 15-20 g) into 3 groups (10 mice/group), namely, control group, blank implant group (titanium-nickel wire implantation without C. albicans infection), and PJI group (titanium-nickel wire implantation with C. albicans infection).</li>
	<li>Anesthetize the mice with 1-4% isoflurane inhalation before removing the hair on the left hindlimb and disinfecting it with iodine. The loss of the righting reflex and no response to the toe stimulation confirms the depth of anesthesia. While&#160;anesthetizing, apply ophthalmic ointment on both eyes to prevent corneal dryness and to replenish heat during surgery and recovery.</li>
	<li>For the mice in the control group, do not provide any treatment. Provide them free access to water and food.</li>
	<li>For the mice in the blank implant group and PJI group, make a 5 mm longitudinal incision on the knee of each left hindlimb with a #10 blade or a sterile razor to expose joints.</li>
	<li>Make a hole of 5 mm in length in the femoral intramedullary canal by inserting a sterile syringe (26 G) needle.</li>
	<li>Insert a smooth nickel-titanium alloy wire (0.5 mm in diameter, 5 mm in length) into the hole before being cut with scissors (Figure 1).</li>
	<li>For the mice in the blank implant group, add 2 &#181;L of YPD medium along the nickel-titanium alloy wire drop by drop before closing the wound layer by layer using a nylon suture (0.15 mm diameter).</li>
	<li>For the mice in the PJI group, inoculate 2 &#181;L of C. albicans cells&#160;(1 &#215; 106 cells/mL) into the joint space of mice along the nickel-titanium alloy wire drop by drop before closing the wound layer by layer using a nylon suture.</li>
	<li>House the mice with free access to water and food for 14 days. Administer meloxicam by subcutaneous injection (4 mg/kg) every 24 h for up to 3 days.</li>
	<li>After 14 days, anesthetize the mice with 3% isoflurane before euthanizing the mice by cervical dislocation.</li>
</ol>
3. PJI model evaluation
<ol>
	<li>Evaluation of infections in major organs
	<ol>
		<li>Collect the kidneys, liver, and spleen from the mice after euthanizing.</li>
		<li>Add 500 &#181;L of sterile normal saline in each organ and grind the tissues on a homogenizer at&#160;4 &#176;C.</li>
		<li>Add 100 &#181;L of the homogenate prepared in step 3.1.2 to a YPD plate before spreading it evenly with a bent rod.</li>
		<li>Place the YPD plates inverted into a 37 &#176;C incubator for 48 h.</li>
		<li>Observe and count the number of colonies visually.</li>
	</ol>
	</li>
	<li>Observation of C. albicans and biofilms on the implants
	<ol>
		<li>Carefully cut the skin over the joint of the mice with scissors before collecting the implant with tweezers.</li>
		<li>Keep the implants immersed in a 2.5% glutaraldehyde solution for fixation at 4 &#176;C for 48 h.</li>
		<li>Rinse the implants with sterile PBS three times before immersing them in 1% osmium acid solution for 3 h.</li>
		<li>Rinse the implants with sterile PBS three times before immersing them in 50%, 70%, 80%, 90%, and 100% ethanol solutions for 15 min for dehydration.</li>
		<li>Keep the implants immersed in tert-butanol for 30 min three times before freeze-drying the implants.</li>
		<li>Fix the implant samples to the sample stage, sputter coat the implant with gold (10 nm coating), and observe it under a scanning electronic microscope (SEM) under a high vacuum and 1.5 kV.</li>
	</ol>
	</li>
	<li>Pathological analysis of the femur tissues
	<ol>
		<li>Collect the femoral tissues with scissors after euthanizing the mice.</li>
		<li>Immerse the femur tissues in 4% paraformaldehyde solution for fixation at 4 &#176;C for 48 h.</li>
		<li>Place the femur tissues in 10% formalin for 1 week.</li>
		<li>Dehydrate the femur tissues by immersing them in 50%, 70%, 80%, 90%, and 100% ethanol solutions for 15 min, respectively.</li>
		<li>Embed the dehydrated femur tissues in paraffin before sectioning the tissues into 4 &#181;m samples using a microtome.</li>
		<li>Stain the femur sections with hematoxylin and eosin by following a standard protocol before pathological analysis21.</li>
	</ol>
	</li>
</ol>

Establishing a Mouse Model for Candida albicans Biofilm-Related PJI

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<li>Stocks, G., Janssen, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infection+in+patients+after+implantation+of+an+orthopedic+device%5BTitle%5D)+AND+%22ASAIO+Journal%22%5BJournal%5D)">Infection in patients after implantation of an orthopedic device.</a> ASAIO Journal. 46 (6), S41-S46 (2000).</li>
<li>Shahi, A., Tan, T. L., Chen, A. F., Maltenfort, M. G., Parvizi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In-hospital+mortality+in+patients+with+periprosthetic+joint+infection%5BTitle%5D)+AND+%22The+Journal+of+Arthroplasty%22%5BJournal%5D)">In-hospital mortality in patients with periprosthetic joint infection.</a> The Journal of Arthroplasty. 32 (3), 948-952 (2017).</li>
<li>Carli, A. V., Ross, F. P., Bhimani, S. J., Nodzo, S. R., Bostrom, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developing+a+clinically+representative+model+of+periprosthetic+joint+infection%5BTitle%5D)+AND+%22The+Journal+of+Bone+and+Joint+Surgery.+American+Volume%22%5BJournal%5D)">Developing a clinically representative model of periprosthetic joint infection.</a> The Journal of Bone and Joint Surgery. American Volume. 98 (19), 1666-1676 (2016).</li>
<li>Stavrakis, A. I., Niska, J. A., Loftin, A. H., Billi, F., Bernthal, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+infection%3A+A+primer+on+animal+models+of+periprosthetic+joint+infection%5BTitle%5D)+AND+%22The+Scientific+World+Journal%22%5BJournal%5D)">Understanding infection: A primer on animal models of periprosthetic joint infection.</a> The Scientific World Journal. 2013, 925906(2013).</li>
<li>Qiao, B., Lv, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrochemical+investigation+of+interaction+of+candida+albicans+with+titanium-nickel+implant+in+human+saliva%5BTitle%5D)+AND+%22International+Journal+of+Electrochemical+Science%22%5BJournal%5D)">Electrochemical investigation of interaction of candida albicans with titanium-nickel implant in human saliva.</a> International Journal of Electrochemical Science. 17 (2), 22028(2022).</li>
<li>Oh, Y. R., Ku, H. M., Kim, D., Shin, S. J., Jung, I. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficacy+of+a+Nickel-titanium+ultrasonic+instrument+for+biofilm+removal+in+a+simulated+complex+root+canal%5BTitle%5D)+AND+%22Materials%22%5BJournal%5D)">Efficacy of a Nickel-titanium ultrasonic instrument for biofilm removal in a simulated complex root canal.</a> Materials. 13 (21), 4914(2020).</li>
<li>Feldman, A. T., Wolfe, D. Tissue Processing and Hematoxylin and Eosin Staining. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFeldman%2C+AT+%22Histopathology%3A+Methods+and+Protocols%22">Histopathology: Methods and Protocols</a>. Christina E, D. ay , Springer, New York. 31-43 (2014).</li>
<li>Sinclair, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Model+development+for+determining+the+efficacy+of+a+combination+coating+for+the+prevention+of+perioperative+device+related+infections%3A+A+pilot+study%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research+-+Part+B+Applied+Biomaterials%22%5BJournal%5D)">Model development for determining the efficacy of a combination coating for the prevention of perioperative device related infections: A pilot study.</a> Journal of Biomedical Materials Research - Part B Applied Biomaterials. 101 (7), 1143-1153 (2013).</li>
<li>Mo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+and+in+vivo+effects+of+the+combination+of+myricetin+and+miconazole+nitrate+incorporated+to+thermosensitive+hydrogels%2C+on+C.+albicans+biofilms%5BTitle%5D)+AND+%22Phytomedicine%22%5BJournal%5D)">In vitro and in vivo effects of the combination of myricetin and miconazole nitrate incorporated to thermosensitive hydrogels, on C. albicans biofilms.</a> Phytomedicine. 71, 153223(2020).</li>
<li>Zahar, A., Sarungi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosis+and+management+of+the+infected+total+knee+replacement%3A+a+practical+surgical+guide%5BTitle%5D)+AND+%22Journal+of+Experimental+Orthopaedics%22%5BJournal%5D)">Diagnosis and management of the infected total knee replacement: a practical surgical guide.</a> Journal of Experimental Orthopaedics. 8 (1), 14(2021).</li>
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<li>Karczewski, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Candida+periprosthetic+joint+infections+-+risk+factors+and+outcome+between+albicans+and+non-albicans+strains%5BTitle%5D)+AND+%22International+Orthopaedics%22%5BJournal%5D)">Candida periprosthetic joint infections - risk factors and outcome between albicans and non-albicans strains.</a> International Orthopaedics. 46 (3), 449-456 (2022).</li>
<li>Cobo, F., Rodriguez-Granger, J., Sampedro, A., Aliaga-Martinez, L., Navarro-Mari, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Candida+prosthetic+joint+infection.+A+review+of+treatment+methods%5BTitle%5D)+AND+%22Journal+of+Bone+and+Joint+Infection%22%5BJournal%5D)">Candida prosthetic joint infection. A review of treatment methods.</a> Journal of Bone and Joint Infection. 2 (2), 114-121 (2017).</li>
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<li>Nishitani, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+the+natural+history+of+biofilm+formation+in+vivo+during+the+establishment+of+chronic+implant-associated+Staphylococcus+aureus+osteomyelitis+in+mice+to+identify+critical+pathogen+and+host+factors%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">Quantifying the natural history of biofilm formation in vivo during the establishment of chronic implant-associated Staphylococcus aureus osteomyelitis in mice to identify critical pathogen and host factors.</a> Journal of Orthopaedic Research. 33 (9), 1311-1319 (2015).</li>
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</ol>

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The infection caused by the contamination of surgical instruments or the surgical environment during surgery is the major reason for most implant infections24,25,26,27. Therefore, a mouse C. albicans biofilm-related PJI model was constructed in this study. Compared to the traditional PJI model in which sterile stainless-steel particles suspended in saline were used as the implant, a ni...

Candida albicans (C. albicans) commensally reside in many parts of the human body1, which is also the most common opportunistic pathogen that causes life-threatening invasive fungal infections, especially in immunocompromised patients2,3. C. albicans can transform between yeast and mycelium states as a polymorphic fungus. The mycelium state exhibits higher virulence, stronger adhesion, and invasion of cells and tissues4,5. Besides, C. albicans can form biofilms on the surfaces of biomedical materials such as dentures, catheters, and stents1,6,7. The dense three-dimensional structure of biofilms restricts the infiltration of antifungal drugs, expresses drug-resistant genes, and down-regulates the metabolism of fungal cells to resist immune system clearance6,7. Therefore, biofilms-related infections&#160;are quite challenging in clinics8.
Staphylococcus aureus, coagulase-negative staphylococcus, and enterobacter are the main pathogens causing PJI9. Although the incidence of fungal PJI is relatively low (about 1%)10, the treatment cost of fungal PJI is higher11, the treatment cycle is longer11, and the treatment success rate is lower10 than bacterial PJI. In recent years, the incidence of fungal PJI has been increasing year-by-year10. Candida PJI accounts for 77%-84% of fungal PJI10,12, and C. albicans is the most common in Candida (54%). Therefore, fungal PJI needs to be studied.
Currently, PJI is treated via revision surgery by (1) removing the infected implant, (2) debridement, (3) antimicrobial treatment, and (4) reimplantation. After thorough debridement, an antibiotic containing bone cement is placed, and the patient is treated with antibiotics systemically for more than 6 weeks to effectively control the infection before a new implant is placed13. However, this method cannot fully eliminate pathogens in tissues, and recurrent infections treated with long-term antimicrobial therapy are highly likely to develop in drug-resistant strains14,15,16.
Establishing animal models of PJI is important for the research and development of new drugs or therapeutics for PJI. In the development of PJI, large dead spaces are formed around the prosthesis, leading to the formation of hematomas, which further block the blood supply of the surrounding tissues and impair the effect of antibiotics11,15. Due to the difficulty in mimicking the surrounding environment of the prosthesis, traditional animal models cannot accurately simulate the actual situation of PJI17,18.
In this paper, a C. albicans biofilm-associated PJI model in mice was constructed by using a clinically widely used titanium-nickel wire to simulate joint implants19,20. This PJI model exhibits the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation. It is expected to be an important model for studying the prevention and treatment of C. albicans biofilm-related PJI.

<table><tbody><tr><td>0.5 Mactutrius turbidibris</td><td>Shanghai Lujing Technology Co., Ltd</td><td>5106063</td><td></td></tr><tr><td>4 &amp;deg;C refrigerator</td><td>Electrolux (China) Electric Co., Ltd</td><td>ESE6539TA</td><td></td></tr><tr><td>Agar</td><td>Beijing Aoboxing Bio-tech Co., Ltd</td><td>01-023</td><td></td></tr><tr><td>Analytical balances</td><td>Shimadzu</td><td>ATX124</td><td></td></tr><tr><td>Autoclaves Sterilizer</td><td>SANYO</td><td>MLS-3750</td><td></td></tr><tr><td>Carbenicillin</td><td>Amresco</td><td>C0885</td><td></td></tr><tr><td>Eclipse Ci Nikon upright optical microscope&amp;nbsp;</td><td>Nikon</td><td>Eclipse Ts2-FL</td><td></td></tr><tr><td>Glucose</td><td>Macklin&amp;nbsp;</td><td>D823520</td><td></td></tr><tr><td>Inoculation ring</td><td>Thermo Scientific</td><td>251586</td><td></td></tr><tr><td>Isoflurane</td><td>RWD</td><td>20210103</td><td></td></tr><tr><td>NaCl</td><td>Xi&#039;an Jingxi Shuanghe Pharmaceutical Co., Ltd</td><td>20180108</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Beyotime Biotechnology</td><td>P0099</td><td></td></tr><tr><td>Peptone</td><td>Beijing Aoboxing Bio-tech Co., Ltd</td><td>01-001</td><td></td></tr><tr><td>RWD R550 multi-channel small animal anesthesia machine&amp;nbsp;</td><td>RWD</td><td>R550</td><td></td></tr><tr><td>SEM</td><td>Hitachi</td><td>TM-1000</td><td></td></tr><tr><td>Temperature incubator</td><td>Shanghai Zhichu Instrument Co., Ltd</td><td>ZQTY-50N</td><td></td></tr><tr><td>Ultrapure water water generator</td><td>Heal Force</td><td>NW20VF</td><td></td></tr><tr><td>Ultrasound machine</td><td>Do-Chrom</td><td>DS10260D</td><td></td></tr><tr><td>Yeast extract</td><td>Thermo Scientific&amp;nbsp;Oxoid</td><td>LP0021B</td><td></td></tr></tbody></table>

a periprosthetic joint candida albicans infection model in mouse

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.

Transferring the samples onto a plate medium and counting colonies after overnight incubation is commonly used to assess the local pathogen load near the lesion22,23. In our study, the microbial culture of liver, kidney, and spleen samples was negative, indicating that the model in this study only led to local infection instead of systemic infection in the mice23.
The SEM images of the implants are shown in <str...

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 ...

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Research

JoVE Journal

Immunology and Infection

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.

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

This video demonstrates the development of an in vivo mouse model to study the biofilm formation of Candida albicans in subcutaneous tissue. The experiment involves implanting Candida-infected devices in the animal's back region for biofilm development.

Candida albicans Biofilm Formation in an In Vivo Mouse Model

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI,  laying a foundation for clinical transformation.

A Catheter-Related Candida albicans Infection Model in Mouse

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

The present protocol describes a mouse model employing subcutaneous dorsal implantation to investigate implant-associated infections, enabling comprehensive investigation of pathophysiological mechanisms and supporting the development of diagnostic criteria with targeted therapeutic strategies.

Animal Model of Implant-Associated Infections in Mice

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A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza A Virus Studies in a Mouse Model of Infection

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Learn to establish a Candida albicans biofilm-associated periprosthetic joint infection model in mice using titanium-nickel wire implants. This method enables research into PJI prevention and treatment strategies.

A Periprosthetic Joint Candida albicans Infection Model in Mouse

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