Orthotopic Modeling Technique

Eight-week-old, female, NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA), with all animal procedures reviewed and approved by the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee. Mice underwent orthotopic tibial implantation as described below and were followed by weekly bioluminescence. They underwent hindlimb amputation when they reached a humane endpoint that included lameness, large tumor burden interfering with the animal’s ability to reach food or water, significant tumor ulceration, guarding behavior, or upon becoming moribund. After hindlimb amputation, mice were followed with weekly bioluminescence imaging until they reached a total body bioluminescent flux of 1 x 10¹⁰ photons or if other endpoints were seen such as persistent poor grooming or lethargy; > 20% body weight loss post-amputation, respiratory difficulty or upon becoming moribund.

LM7.ffluc cells were harvested at confluence and pelleted by centrifugation. 5X collagen neutralization buffer was prepared by mixing 2.5 g minimum essential media (MEM) alpha powder without nucleosides (Thermo Fisher Scientific, Waltham, MA) and 2% wt/vol NaHCO₃ in 45 ml demineralized water, adding 5 ml of 1 M HEPES (Thermo Fisher Scientific, Waltham, MA), and filtering through a 0.22 μm filter. Neutralized high-concentration type I rat-tail collagen was prepared fresh by mixing high-concentration rat tail collagen I (Corning, New York, USA; concentration range 8 – 11 mg/ml) and 5X collagen neutralization buffer in a 5:1 vol/vol ratio on ice. LM7 cells were then resuspended in neutralized high-concentration collagen at 1 x 10⁶ cells per 10 μL collagen, taking care to maintain reagents and pelleted cells on ice during resuspension process and pipetting using wide-bore pipette tips. Once resuspended, cell mixture was pulse-vortexed and pulse-centrifuged for < 5 seconds to disrupt bubbling in mixture. Cells were then plated for individual implants at 10 μL cell mixture per well in a 96-well ultra-low-attachment round-bottom plate (Corning, NY, USA) and allowed to solidify at 37°C in 5% CO₂ for 20 minutes. DMEM supplemented with 10% fetal bovine serum and 1% Glutamax was then added and implants allowed to mature overnight at 37°C in 5% CO₂. The collagen preparation and neutralization protocol described above is as that previously described (21).

Prior to beginning the implantation procedure, mice are anesthetized using inhaled isoflurane at a MAC of 2, depilated, and the right hindlimb prepped from the inguinal area to the paw using 70% alcohol and chlorhexidine solution. Multimodal analgesia was administered both preemptively with meloxicam 5 mg/mL (Boehringer Ingelheim, St. Joseph, MO), subcutaneously at 1 mg/kg and post-operatively with buprenorphine 0.03 mg/mL (Patterson Veterinary, Greeley, CO), subcutaneously at 0.1 mg/kg. The mouse is placed in dorsal recumbency with the right hindfoot gently grasped while flexing the knee. A 5 mm skin incision is made proximal to the patella ( Figure 1A ) and retracted using gentle traction below the patella to expose the proximal anterior tibia ( Figure 1B ). The musculature and soft tissue are gently dissected away from the anterior tibia using a fine hemostat ( Figure 1C ; Fine Science Tools, Foster City, CA, USA). Once cleared of soft tissue, a 2 mm fragment of anterior tibial cortical bone is removed using a 2 mm sharp Rongeur ( Figures 1D, E ; Fine Science Tools). Care is taken to avoid the inferior patellar tendon and to avoid significant entry into the marrow cavity or tibial fracture. An LM7 collagen implant ( Figure 1F ) is then grasped with forceps and placed gently into the cavity left at the osteotomy site ( Figure 1G ). The distal aspect of the skin incision is then gently raised over the implant and the incision allowed to recoil back to its original position proximal to the patella, leaving the implant secured in place by intact skin ( Figure 1H ). Gentle pressure is applied to ensure hemostasis and implant adherence to the bony surface. The skin incision is closed with Vetbond^® surgical glue (3M Animal Care Products, St. Paul, MN). Mice are monitored through until completely recovered from anesthesia ( Figure 1I ), with the entire procedure from incision to closure taking 2 – 5 minutes to perform.

Tibial osteotomy implantation procedure. (A) Incision is made over distal femur. (B) Incision retracted distally to expose anterior tibia. (C) Soft tissue dissected away from anterior tibia. (D) 2 mm tip Rongeur used for osteotomy. (E) Tibial osteotomy. (F) LM7-collagen implant. (G) Implant in place over osteotomy. (H) Final position of implant and incision. (I) Mice demonstrating exploration and weight bearing behavior minutes after anesthesia emergence.

Mice are anesthetized with surgical prep performed from the right hindlimb to the umbilicus. Preemptive analgesia is administered as described above and a 1 mL bolus of sterile saline is administered subcutaneously. The surgical area is draped with the hindlimb extended and secured in place with a sterile adhesive bandage.

A skin incision is made in an elliptical fashion along the inguinal canal from the rostral dorsal iliac spine to the level of the pubic ramus. Gentle blunt dissection with a cotton tipped applicator is used to push the peritoneum and abdominal musculature rostrally to expose the proximal aspect of the femoral neurovascular bundle (artery, vein, and nerve). The bundle is carefully dissected with a fine hemostat and controlled proximally and distally with 5-0 Vicryl^® (Ethicon Inc, Somerville, NJ) braided absorbable suture ties. Axonotmesis is performed on the femoral nerve as the bundle is divided with sharp fine scissors. The musculature overlying the femur is gently divided using sharp dissection and the femur grasped distally using toothed forceps. A heavy scissor is used to divide the femur at the mid-shaft. The distal femur is elevated, allowing visualization of the caudal musculature, which is sharply divided. Axonotmesis is performed on the sciatic nerve as it is visualized caudally using smooth forceps 3 mm proximal to the division site and then ligated. The remaining soft tissue is sharply divided, and the skin incision completed to remove the hindlimb. Care is taken to leave a sufficient posterior muscle flap to cover the proximal femur stump.

After removal of the hindlimb from the field, gentle pressure is used to ensure hemostasis. A single figure of eight suture is used to cover the proximal femur stump with the caudal muscle flap. Skin edges are adhered with an intradermal suture pattern using 5-0 Vicryl^® suture. Vetbond^® surgical glue is applied. The animal is maintained on heat throughout the surgery and during anesthetic recovery with continuous monitoring. The entire procedure from incision to closure lasts 15 – 25 minutes. Amoxicillin 400 mg/50 mls (Sandoz, Princeton, NJ) is added to a 350 ml water bottle at a dosage of 50 mg/kg for one week. Mice are monitored closely at least twice a day, in addition to regular health checks following surgery for 7-10 days by experienced veterinary technologists.