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JoVE Journal Immunology and Infection

Immunology and Infection

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Published: February 2, 2024 doi: 10.3791/65263
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1, 1, 2, 1, 1, 1
1School of Pharmacy, Health Science Center, Xi’an Jiaotong University, 2Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University

Summary

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.

Abstract

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&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.

Introduction

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 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.

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Protocol

The animals were purchased from Xi'an Jiaotong University. All animal experiment procedures were approved by the Institutional Animal Ethical Committee of Xi'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 °C ± 1 °C) and light/dark cycle (12 h/12 h) before the study was performed.

1. Buffer and equipment preparation

  1. C. albicans cell culture
    1. 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 µg/mL carbenicillin).
    2. Shake C. albicans cells subsequently at a speed of 220 rpm at 30 °C overnight.
    3. 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 to 1 x 106 cells/mL via visually adjusting the turbidity to be the same as a 0.5 McFarland.
  2. Preparation of normal saline
    1. Weigh 0.9 g of sodium chloride and dissolve in 100 mL of deionized water to prepare 0.9% normal saline.
  3. Surgical instruments preparation
    1. Autoclave (121 °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.

2. Mouse PJI model establishment

  1. 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).
  2. 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 anesthetizing, apply ophthalmic ointment on both eyes to prevent corneal dryness and to replenish heat during surgery and recovery.
  3. For the mice in the control group, do not provide any treatment. Provide them free access to water and food.
  4. 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.
  5. Make a hole of 5 mm in length in the femoral intramedullary canal by inserting a sterile syringe (26 G) needle.
  6. 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).
  7. For the mice in the blank implant group, add 2 µL 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).
  8. For the mice in the PJI group, inoculate 2 µL of C. albicans cells (1 × 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.
  9. 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.
  10. After 14 days, anesthetize the mice with 3% isoflurane before euthanizing the mice by cervical dislocation.

3. PJI model evaluation

  1. Evaluation of infections in major organs
    1. Collect the kidneys, liver, and spleen from the mice after euthanizing.
    2. Add 500 µL of sterile normal saline in each organ and grind the tissues on a homogenizer at 4 °C.
    3. Add 100 µL of the homogenate prepared in step 3.1.2 to a YPD plate before spreading it evenly with a bent rod.
    4. Place the YPD plates inverted into a 37 °C incubator for 48 h.
    5. Observe and count the number of colonies visually.
  2. Observation of C. albicans and biofilms on the implants
    1. Carefully cut the skin over the joint of the mice with scissors before collecting the implant with tweezers.
    2. Keep the implants immersed in a 2.5% glutaraldehyde solution for fixation at 4 °C for 48 h.
    3. Rinse the implants with sterile PBS three times before immersing them in 1% osmium acid solution for 3 h.
    4. 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.
    5. Keep the implants immersed in tert-butanol for 30 min three times before freeze-drying the implants.
    6. 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.
  3. Pathological analysis of the femur tissues
    1. Collect the femoral tissues with scissors after euthanizing the mice.
    2. Immerse the femur tissues in 4% paraformaldehyde solution for fixation at 4 °C for 48 h.
    3. Place the femur tissues in 10% formalin for 1 week.
    4. Dehydrate the femur tissues by immersing them in 50%, 70%, 80%, 90%, and 100% ethanol solutions for 15 min, respectively.
    5. Embed the dehydrated femur tissues in paraffin before sectioning the tissues into 4 µm samples using a microtome.
    6. Stain the femur sections with hematoxylin and eosin by following a standard protocol before pathological analysis21.

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Representative Results

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 Figure 2. No C. albicans adhered or colonized on the surface of the nickel-titanium alloy wire in the blank implant group. However, mature and thick biofilm was observed on the surface of nickel-titanium alloy wire in the PJI group, indicating the successful construction of the C. albicans biofilm-related PJI model in mice 14 days after the surgery23.

The H&E staining of femoral tissues is shown in Figure 3. A clear and complete bone trabecular structure was observed in the control group, while a few bone trabecular tissue defects in the femoral tissues could be seen in the blank implant group (Figure 3, yellow arrows). In the PJI group, the number of bone trabeculae significantly decreased23. These results indicate that the C. albicans biofilm-associated PJI model in mice was successfully established with a significant pathological injury of the femur tissue.

Figure 1
Figure 1: Implantation procedure. The red square in the left panel shows the surgical site where the smooth nickel-titanium alloy wire is inserted. The panel on the right shows a portion of femur (red circle) with the nickel wire. Please click here to view a larger version of this figure.

Figure 2
Figure 2: SEM images of the implant's surface in the blank and PJI groups. Magnifications 1000x (scale bar = 500 µm) and 5000x (scale bar = 100 µm) are shown as representative images. This figure has been modified with permission from Mo et al.23. Please click here to view a larger version of this figure.

Figure 3
Figure 3: H&E staining of femoral tissue. Representative H&E images of the implant, PJI model, and control groups are shown in the figure. The control group shows a clear and complete bone trabecular structure. The blank implant group displayed a few bone trabecular tissue defects in the femoral tissues (yellow arrows). However, the number of bone trabeculae decreased in the PJI group. The magnifications shown are 200x (scale bar = 150 µm) and 400x (scale bar = 75 µm). This figure has been modified with permission from Mo et al.23. Please click here to view a larger version of this figure.

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Discussion

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 nickel-titanium alloy wire, a commonly used implant material, was used in this study to simulate the contact between C. albicans, implant materials, and the bone, which is more similar to the situation in clinics.

The PJI model described in this article can perfectly simulate the physiological environment of PJI in clinics. This model can only be used to study the infection during implantation instead of later blood-borne infection.

C. albicans can be inoculated in two ways. One is directly inoculating the C. albicans at the implant site during surgery28, and the other is culturing the implants with C. albicans for a period of time so that mature biofilms are formed on the surface of the implant before surgical implantation29. The former method was chosen in this study due to its accurate inoculation number of pathogens, which resulted in minimum differences between groups and a more objective evaluation of subsequent treatments. Moreover, the former method is more consistent with the clinical situation.

In this protocol, the insertion of the implant is difficult to perform. The operator has to practice several times to ensure that the implant is inserted into the joint instead of subcutaneously or intramuscularly. Besides, the inoculation number of C. albicans is vital for the repeatability of the PJI model. C. albicans should be thoroughly mixed via vortex to ensure the accuracy of the inoculation number. In addition, the C. albicans should be added along the alloy wire to simulate the route of infection in the clinical situation.

Biofilms could be detected 7 days post bacterial infection, after which biofilms gradually increased and reached a plateau on the 14th day30. Therefore, the success of the established PJI model was inspected on the 14th day. The colonization of C. albicans and the formation of biofilm on the surface of the implant were inspected by SEM. The tissue lesions around the implant caused by local infection were evaluated by pathological analysis after H&E staining. Studies have shown that periprosthetic osteolysis is an important feature due to PJI31. Thus, these indicators are also vital in evaluating therapeutic methods for the prevention and treatment of PJI32.

Microbial culture is commonly used for detecting microbial infection in clinics and laboratories. Therefore, in this study, the microbial culture of the implant, tissues around implants, liver, and other vital organs were performed. For the implant, ultrasonication was applied to remove the C. albicans adhered to the surface of the titanium-nickel alloy wire. Next, the C. albicans were enriched by centrifugation before microbial culture. However, a negative result was found, inconsistent with the SEM result (Figure 2). The SEM result showed that C. albicans adhered to the surface of titanium-nickel alloy wire. Therefore, the result of microbial culture was a false negative, which may be attributed to the tight adhesion of C. albicans to titanium-nickel alloy wire; ultrasonic could not successfully exfoliate the C. albicans from the implant. Similarly, the microbial culture of the tissues around implants and vital organs was also negative. There are two possible reasons: (1) The number of C. albicans inoculated in this study was only 2000 CFU, which may be too small to invade the surrounding tissue and the system during the experimental period; (2) The sensitivity of the method for extracting and separating pathogens from tissues is low. A previously published report suggests microbial culture could easily show false negative results and delayed treatments33. Grocott-Gomori staining can be used to determine the formation of hyphae in the bone and joint32. It may also be helpful to increase the inoculum quantity, prolong the experimental duration or keep the mice in an immunosuppressed state before surgery32. However, it should be noted that long-time infection may lead to deep infection or even systemic infection. Thus, the experimental period should be designed according to the specific purpose.

In summary, this study created a successful mouse model of C. albicans biofilm-associated PJI, which may be of great significance for researching the prevention and treatment of C. albicans biofilm-associated PIJ.

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Disclosures

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.

Acknowledgments

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).

Materials

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

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A Periprosthetic Joint <em>Candida albicans</em> Infection Model in Mouse
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Cite this Article

Yang, C., Zhang, J., Mo, F., Zhang,… More

Yang, C., Zhang, J., Mo, F., Zhang, P., Li, Q., Zhang, J. A Periprosthetic Joint Candida albicans Infection Model in Mouse. J. Vis. Exp. (204), e65263, doi:10.3791/65263 (2024).

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