A Periprosthetic Joint Candida albicans Infection Model in Mouse

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 patients^2,3. 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 tissues^4,5. Besides, C. albicans can form biofilms on the surfaces of biomedical materials such as dentures, catheters, and stents^1,6,7. 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 clearance^6,7. 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 PJI^10,12, 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 strains^14,15,16.

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 antibiotics^11,15. Due to the difficulty in mimicking the surrounding environment of the prosthesis, traditional animal models cannot accurately simulate the actual situation of PJI^17,18.

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 implants^19,20. 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.

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.

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 lesion^22,23. 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

Discussion

The infection caused by the contamination of surgical instruments or the surgical environment during surgery is the major reason for most implant infections^24,25,26,27. 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...

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