A Periprosthetic Joint Candida albicans Infection Model in Mouse

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.