The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Summary

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Abstract

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

Introduction

Fractures are among the most common injuries occurring to the musculoskeletal system and are associated with a tremendous socioeconomic burden, including treatment costs that are projected to surpass $25 billion annually in the United States^1,2. Although the majority of fractures heal without incident, healing is associated with substantial downtime and loss of productivity. Approximately 5 - 10% of all fractures result in a delayed healing or non-union, due to age or other underlying chronic health conditions, such as osteoporosis and diabetes mellitus^3,4,5. No FDA-approved pharmacological treatments are currently available to promote efficient bone healing and shorten recovery time.

Fracture healing is a complex and highly dynamic process involving the coordination of multiple cell types. Hence, a comprehensive understanding of the cellular and molecular events associated with bone regeneration is crucial to the identification of therapeutic targets that accelerate this process. As with other human diseases, the establishment of a highly amenable and reproducible animal model is crucial in the study of bone healing. Larger animals, such as sheep and swine, have bone remodeling properties and biomechanics similar to humans, but are expensive, require substantial healing time, and are not readily amenable to genetic manipulation⁶. On the other hand, small animal models, such as rats and mice, offer many advantages, including an ease of handling, low costs of maintenance, short breeding cycles, and a shorter healing time⁷. Furthermore, the mouse genome is fully sequenced, allowing for the rapid manipulation and generation of genetic variants. Thus, the mouse is a powerful model system to study human disease, injury, and repair⁸. In humans, comorbidities like osteoporosis and diabetes mellitus increase the likelihood of a delayed healing. A number of existing mouse models are available to study the effects of comorbidities such as osteoporosis and diabetes mellitus on bone injury and healing. Patients suffering from osteoporosis have a markedly decreased bone formation during the later stages of a fracture healing⁹. Ovariectomized (OVX) mice exhibit rapid bone loss and delayed bone healing similar to that observed in postmenopausal osteoporosis^10,11. Additionally, many mouse models of type I and type II diabetes mimic the low bone mass phenotypes and impaired fracture healing seen in humans¹¹. Moreover, murine fracture models serve as a versatile platform to study the complex biological processes occurring in the callus and explore novel therapeutic strategies that accelerate bone tissue regeneration.

Despite differences in bone structure and metabolism, the overall process of bone fracture healing remains very similar in mice and humans, involving a combination of endochondral and intramembranous ossification followed by bone remodeling. Endochondral ossification involves the recruitment of progenitor cells to less mechanically stable regions surrounding the fracture gap, where they differentiate into chondrocytes that hypertrophy and mineralize the cartilage to produce a soft callus. The second wave of progenitor cells infiltrate the callus and differentiate into mature osteoblasts that secrete new bone matrix^12,13,14,15. During intramembranous ossification, progenitors on the periosteal and endosteal surfaces directly differentiate into matrix secreting osteoblasts and facilitate the bridging of the fracture gap^9,11,12,13. Together, the endochondral and intramembranous ossifications result in the development of a hard callus, which is further remodeled over time to form a strong secondary bone capable of supporting mechanical loads^13,14,15. In healthy humans, the healing process takes approximately 3 months, compared to only 35 days in mice¹⁶.

Fracture healing has commonly been studied using either open or closed surgical models¹⁷. Open surgical approaches, such as the generation of a critically sized defect or complete osteotomy, standardize the injury location and geometry to reduce deviations caused by comminuted fractures. Osteotomies serve as an excellent model to study the underlying mechanism behind a non-union because healing is often delayed compared to closed fractures. Furthermore, a rigid external fixation is required to stabilize the osteotomized bone, meaning the regeneration will primarily depend on the intramembranous ossification. Open surgical approaches use devices such as locking nails, pin-clips, and locking plates to provide axial and rotational stability to the fractured limb; however, such devices are expensive and require significantly more time in surgery^18,19,20,21. On the other hand, closed models are stabilized with a simple intramedullary fixation device, allowing for enough instability to stimulate endochondral healing. As a result, closed fracture models do not readily mimic the conditions of a non-union. Internal fixation techniques, such as intramedullary pins, nails, and compression screws, are advantageous as they are cheap, easy to use, and minimize the time in surgery^21,22,23. In some cases, intramedullary pins are inserted prior to the fracture, but the bending of the intramedullary pin can lead to the angulation or displacement of the fractured femur, contributing to a variable callus size and healing. The fracture location and geometry are more difficult to standardize in closed models, as they are generated using a three-point bending device, wherein a weight is dropped on the diaphysis. However, with the proper technique, this surgical approach offers rapid and consistent results. Moreover, the closed fracture model serves as a clinically relevant tool to study fractures caused by high force impact or mechanical stress²².

This surgical protocol was adapted from previously described methods using an intramedullary pin to stabilize fractured femurs in rats and mice^22,24,25. First, an intramedullary needle of a small diameter is inserted through the intracondylar notch to establish a point of entry, and a guidewire is introduced prior to generating a transverse fracture at the femoral midshaft using a gravity-dependent three-point bending device. Following the successful generation of a closed femoral fracture, an intramedullary rod of a larger diameter is incorporated over the guide wire to stabilize the fractured femur. This method avoids the risk of delayed healing caused by the angulation of the intramedullary pin during the fracture, as the placement of the rod post-fracture allows for the repositioning and optimized stabilization of the injured femur.

Protocol

The following procedure was performed with approval from the Indiana University School of Medicine Institutional Animal Care and Use Committee (IACUC). All survival surgeries were performed under sterile conditions as outlined by the NIH guidelines. Pain and risk of infections were managed with proper analgesics and antibiotics to ensure a successful outcome.

1. Anesthesia and Preparation

1. Weigh the mouse and anesthetize it with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) administered via the intraperitoneal (I.P.) route. Place the mouse in an empty cage and monitor it until it is fully sedated.
2. Ensure that the mouse is sedated using a toe pinch reflex. Apply ophthalmic ointment to its eyes to protect them from drying out.
3. Remove the fur from the right limb. Wipe down the surgical site with an iodine-based scrub and 70% ethanol. Scrub the surgical site starting at the center of the knee and making a circular sweep outward. Repeat this 3x with fresh scrubs, ending with 70% ethanol.
4. Administer a preoperative dose of buprenorphine hydrochloride analgesia (0.03 mg/kg) subcutaneously for the immediate postoperative pain management.
5. Place the mouse on a heating pad covered by a sterile surgical pad.

2. Surgical Approach

NOTE: Prior to the fracture, the weight and the drop height should be empirically determined for the specific strain, age, and sex of the mice prior to the surgery. This surgical procedure is optimized for C57BJ6 male mice at 10 weeks of age.

1. Place the mouse on its back and flex the knee of the operative leg. Using a scalpel blade, make a 1.5 cm incision centered over the knee joint.
2. Laterally displace the patella using forceps to expose the distal end of the femur. Insert a 1.5-in long 25-gauge stainless steel hypodermic needle at the center of the trochlear groove, down the length of the medullary canal in a retrograde manner and through the proximal end of the femur. Take an X-ray to ensure the proper placement of the pin.
   NOTE: The needle should exit the dorsal side of the mouse to create a path for the guidewire.
3. Pass a 4-in long 36-gauge tungsten guidewire through the shaft of the needle, entering through the hub at the distal femur and exiting the bevel on the dorsal side of the mouse.
4. Following the successful placement of the guidewire, carefully remove the 25-gauge needle by gently pulling on the hub while holding the limb and the guidewire in place. Confirm the placement of the guidewire by X-ray.
5. Hold a 391 g weight from a height of 34.6 cm above the impact disc (Figure 1A). Position the femur horizontally across the two supporting points, such that the intertrochanteric and supracondylar regions of the femur rest on the support anvils (Figure 1B) and the lateral side of the limb is facing the loading point (Figure 1C). Drop the weight and carefully remove the mouse from the device immediately following the fracture.
6. Confirm the fracture location by X-ray.
7. Insert the 24-gauge stainless steel hypodermic tubing over the guidewire to stabilize the fractured femur.
   NOTE: This application may require some force as the point of entry was generated using a needle of a smaller diameter. This difference in diameter effectively prevents a potential migration of the 24-gauge rod through the proximal end of the femur. The depth of insertion can be felt manually as the blunt tubing meets the cortical bone of the greater trochanter.
8. Confirm the position of the steel rod and the stabilization of the fractured femur by X-ray before removing the guidewire.
9. Cut the excess tubing at the distal end of the femur using wire cutters. Bury the exposed tubing under the surface of the condyles using forceps to apply a gentle downward force, being careful not to dislocate the knee joint.
10. Reposition the patella using forceps. Close the incision site with a 5-0 absorbable suture.

3. Postoperative Management

1. Following surgery, the mice may be injected with up to 500 µL of sterile saline via the I.P. route to aid them in their postoperative recovery.
2. Monitor the animals on a heated recovery bed until they awaken from surgery. Once ambulatory, return them to their cage.
3. Continue to closely monitor the mice for several days after the surgery to ensure they are healing properly and regain mobility. Administer buprenorphine hydrochloride analgesia (0.03 mg/kg) subcutaneously every 6 h for 3 days following the surgery, and as needed thereafter. Avoid the use of non-steroidal anti-inflammatory drugs (NSAIDs) as they have been shown to impair bone healing after surgery.

Results

The successful implementation of the surgical procedure was monitored with radiographic imaging. Key steps include the insertion of an intramedullary needle, the placement of a guide wire, the induction of a transverse fracture at the femoral midshaft, and the proper stabilization with an intramedullary rod (Figure 2Ai - 2Aiv). The healing progression of the fracture callus was monitored with weekly radiographic images up to ...

Discussion

The goal of this surgical procedure is to generate standardized closed femoral fractures in mice. A key advantage of this model is that the internal fixation takes place after the generation of the fracture, thereby avoiding an angulation of the intramedullary rod. Perhaps the most critical aspect of this protocol is the generation of a standardized transverse fracture at the femoral midshaft, as the fracture geometry is dependent on the applied bending force and the positioning of the hind limb. Improper positioning of ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Oster Minimax Trimmer	Animal World Network	78049-100
POVIDONE-IODINE	Thermo Fisher Scientific	395516
OPHTHALMIC OINTMENT	Thermo Fisher Scientific	NC0490117
Styker T/Pump Warm Water Recirculator	Kent Scientific Corporation	TP-700
1ml Sub-Q Syringe	Thermo Fisher Scientific	309597
ENCORE Sensi-Touch PF	Moore Medical LLC	30347	Latex, powder-free surgical glove
PrecisionGlide 25G Hypodermic Needles	Thermo Fisher Scientific	14-826-49
Ultra-High-Temperature Tungsten Wire,	McMaster-Carr	3775K37	0.005" Diameter, 1/16 lb. Spool, 380' Long
304 stainless steel, 24G thin walled tubing	Microgroup Inc	304h24tw-5ft
#15 Scalpel Blades	Fine Science Tools	10015-00
#10 Scalpel Blades	Fine Science Tools	10010-00
Narrow Pattern Forceps	Fine Science Tools	11002-12	Serrated/Straight/12cm
Iris Forceps	Fine Science Tools	11066-07	1x2 Teeth/Straight/7cm
Dissector Scissors	Fine Science Tools	14081-09	Slim Blades/Angled to Side/Sharp-Sharp/10cm
Fine Scissors	Fine Science Tools	14058-11	ToughCut/Straight/Sharp-Sharp/11.5cm
Olsen-Hegar Needle Holder with Suture Cutter	Fine Science Tools	12002-12	Straight/Serrated/12cm/with Lock
Crile Hemostat	Fine Science Tools	13004-14	Serrated/Straight/14cm
Tungsten Wire Cutter	ACE Surgical Supply Co., Inc.	08-051-90	ACE #150 Wire Cutter, tungsten carbide tips
3-0 VICRYL Suture	Ethicon Suture	J423H	3-0 VICRYL UNDYED 27" FS-2 CUTTING
piXarray 100 Digital Specimen Radiography System	Bioptics, Inc		Cabinet x-ray system
Einhorn 3-Point Bending Device	N/A	N/A	Custom Built