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 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 i…

Representative 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. <p class="jove_content" fo:keep-toge…

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

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