A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Summary

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Abstract

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

Introduction

Bone healing studies in mice are in great demand because of a broad spectrum of antibodies and genetically-modified animals. These facts allow to study the molecular mechanisms of bone healing¹. In the past few years, different bone healing models for mice have been developed². These models can be divided into open models, in which the bone is osteotomized using an open lateral surgical approach and in closed models, in which the bone is fractured based on the fracture model introduced by Bonnares and Einhorn³. Using this technique, a standardized transverse fracture can be produced by a 3-point bending device and intramedullary implants can be inserted through a small medial parapatellar incision in a minimally invasive technique avoiding a major soft tissue trauma.

The intramedullary screw can be applied for closed fracture stabilization in mice. The screw offers rotational and axial stability. This is achieved by fracture compression through a proximal thread and a distal head⁴. Further advantages of the screw are the simple surgical technique, the low grade of invasivity, the low implant weight and, most notably, a higher stability providing standardized and controlled biomechanical conditions compared to other intramedullary implants⁵. In fact, in the most closed fracture models, the fragments are stabilized only by simple pins, which is associated with a complete lack of rotational and axial stability and a high risk of pin and also fracture dislocation. This can markedly influence the healing process, which may result in delayed healing or non-union formation.

It is well known that the stability of the fracture fixation has a tremendous impact on the healing process^6,7. A high rigid fixation results in intramembranous healing, while a less rigid fixation, which may allow micromovements in the fracture gap, results in endochondral healing. Stabilization of the fracture with the intramedullary screw shows predominantly an endochondral healing with a large amount of callus tissue, particularly after 2 weeks of fracture healing. The possibility to harvest a large amount of callus tissue enables the analysis of multiple parameters by different techniques.

Here, we report on the design and application of the intramedullary screw in mice, as well as on its advantages and disadvantages in experimental studies on normal endochondral bone healing.

Protocol

All procedures were performed according to the National Institutes of Health guidelines for the use of experimental animals and followed institutional guidelines (Landesamt für Verbraucherschutz, Zentralstelle Amtstierärztlicher Dienst, Saarbrücken, Germany).

1. Preparation of Surgical Instruments and Implants

1. Select a scalpel blade (size 15), a small swab, fine forceps, a 27 G needle, a non-resorbable 5-0 suture, scissors and a needle holder from the microsurgical instrument box.
2. Unpack the intramedullary screw, the guide wire (0.3/0.2 mm diameter, 10 cm length), the centering drill bit (0.5 mm diameter) and the hand drill (Figure 1; see Table of Materials).
   NOTE: The intramedullary screw (0.5 mm diameter, 17.2 mm length) is made of medical-grade stainless steel for retrograde implantation into the femur. The screw has a proximal thread (0.5 mm diameter, 4 mm length) with a nose (0.2 mm diameter, 0.4 mm length) at the tip and distal cone-shaped head (0.8 mm diameter, 0.9 mm length) to achieve fracture compression as well as axial and rotational stability.
3. Expose the implants and all surgical instruments to a disinfecting solution (96 % alcohol) for 5 min or sterilize them (steam sterilization, 130 °C, 25 min). After disinfection or sterilization, place the instruments on an operation cloth. Position the operation cloth directly adjacent to the small animal operation table.

2. Animals, Anesthesia, and Analgesia

1. Choose the strain, age, and sex of the mice according to the study question which is addressed.
   NOTE: For this study 12- to 14-week-old male CD-1 mice were used. The appropriate body weight to use the intramedullary screw is between 25-35 g.
2. Anesthetize the mice with an intraperitoneal injection of 15 mg/kg xylazine and 75 mg/kg ketamine. Confirm the anesthetization by toe pinch. Apply eye lubricant to protect the animals' eyes from drying during anesthesia. After induction of anesthesia, place the mouse under a heat radiator to keep the body temperature constant. During the procedure, animals were monitored with repeated toe pinch to ensure an appropriate plane of anesthesia.
3. Apply tramadol-hydrochloride in the drinking water (1.0 mg/mL) for analgesia from day 1 before the surgery until day 3 after the surgery.
   NOTE: Analgesia and infection prevention should be in agreement with the respective guidelines of the country and institution where the experiments are to be performed.

3. Surgical Procedure and Intramedullary Screw Implantation

1. Before surgery, shave the entire right hind leg and apply a depilatory cream. After 5 min, remove the cream and clean the leg with water. Then, apply a disinfecting solution with 96 % alcohol. Betadine or chlorhexidine can be added to the alcohol to ensure complete asepsis.
2. Under aseptic conditions, place the mouse in the supine position on the small animal operation table. Bend the right knee to allow for an anterior approach to the condyles of the knee. Perform a 5-mm medial parapatellar incision at the right knee using the scalpel blade.
3. Mobilize the patellar ligament carefully with the scalpel blade and the swab. Then, shift the patella laterally with the fine forceps to expose the intercondylar notch of the femur.
4. Open the intercondylar notch exactly in the middle of the femur between both condyles. Make sure not to exceed 1.0 mm in depth for the drill hole.
   1. Start manual drilling at a slow speed and a 45 ° offset ventrally to the femur axis using the 0.5 mm centering drill bit and the hand drill (Figure 1C and D, Figure 2). During drilling, continuously decrease the angle to 0 ° offset (parallel with the bone axis of the femur). Stop drilling when a depth of 1.0 mm is reached.
5. After opening the bone at the intercondylar notch, insert the 27 G needle into the intramedullary cavity over the whole length of the femur. Ream the intramedullary cavity of the femur manually through rotary motions of the 27 G needle. Push the needle forward to perforate the cortical bone at the greater trochanter proximally.
6. Remove the 27 G needle and apply the guide wire through the distal part of the femur.
   1. Make a skin incision with a scalpel blade (size 15) proximally over the guide wire and push the guide wire forward until both ends of the guide wire are outside. Make sure to keep the guide wire in place.
7. Create a defined closed fracture by using the guillotine.
   1. Place the mouse in lateral position with the right leg under the guillotine. Make sure that the diaphyseal part of the femur is placed in the middle of the guillotine.
   2. Drop the weight (200 g) from the defined distance of 25.5 cm.
8. Control the fracture configuration and fracture position as well as the position of the guide wire (Figure 3) using the x-ray device (see Table of Materials).
9. Connect the intramedullary screw with the nose at the distal end to the 0.2 mm guide wire and insert it into the femur under continuous pressure, and clockwise rotation.
   1. Shear of the drive shaft when the sufficient torque is achieved.
   2. Remove the guide wire proximally.
10. Reposition the patella and fix the patella tendon to the muscles with one single suture using a 5-0 synthetic, monofilament, nonabsorbable polypropylene suture. Use single sutures of the same material and size to close the wound. Control the reduction of the fragments and the screw position radiologically using the x-ray device (see Table of Materials).
11. Keep the animals under the heat radiator until they recover from anesthesia. Do not leave the animals unattended until they have regained sufficient consciousness to maintain ventral recumbency. Return the animals to single cages in the animal facility. Do not return the animals to the company of other animals during the first 24 h, even if they have fully recovered from anesthesia.
12. Monitor the animals carefully every day. Maintain postoperative analgesia using tramadol-hydrochloride in the drinking water with a dosage of 1.0 mg/mL during the first three days. Continue analgesia if, on day 4 after surgery, the animals still show evidence of pain, as indicated by vocalization, restlessness, lack of mobility, failure to groom, abnormal posture, and lack of normal interest in surroundings. Terminate analgesia when the animals are pain-free.
13. At the end of the experiment euthanize the animal by an overdose of barbiturate.

Results

The operating time from skin incision to wound closure was 20 min. The surgery can be performed without a stereo-microscope. Postoperatively, the animals were monitored daily. Post-operative analgesia was terminated after 2 days because none of the animals showed evidence of pain after this time period. The animals showed also normal weight-bearing within 2 days after surgery. Wound infections were not observed during the entire observation period.

Discussion

Critical steps of the surgical procedure are to find the correct entry point for screw implantation in the middle of the femur condyles at the intercondylar notch as well as the optimal orientation of the needle parallel to the bone axis for reaming of the intramedullary cavity. To avoid an incorrect entry position, the surgeon should prepare the notch until an optimal view is achieved. To control the orientation during reaming, the femur of the mice should be held with the fingers in a stable position. A further critica...

Materials

Name	Company	Catalog Number	Comments
Mouse Screw	RISystem AG	221,100
Guide wire	RISystem AG	521,100
Centering bit	RISystem AG	590,205
Hand drill	RISystem AG	390,130
Cotton-Swab (150 mm, small head)	Fink Walter GmbH	8822428
Suture (5-0 Prolene)	Ethicon	8614H
Forceps	Braun Aesculap AG &CoKG	BD520R
Scissors	Braun Aesculap AG &CoKG	BC100R
Needle holder	Braun Aesculap AG &CoKG	BM024R
27 G needle	Braun Melsungen AG	9186182
Scalpel blade size 15	Braun Aesculap AG &CoKG	16600525
Heat radiator	Sanitas	605.25
Depilatory cream	Asid bonz GmbH	NDXZ10
Eye lubricant	Bayer Vital GmbH	2182442
Xylazine	Bayer Vital GmbH	1320422
Ketamine	Serumwerke Bernburg	7005294
Tramadol	Grünenthal GmbH	2256241
Disinfection solution (SoftaseptN)	Braun Melsungen AG	8505018
CD-1 mice	Charles River	22
X-ray Device	Faxitron MX-20, Faxitron X-ray Corporation	2321A0988
Fracture device small	RISystem AG	891,100