Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

Summary

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

Abstract

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

Introduction

Research on fracture healing is necessary to address a large clinical and economic problem. Each year over 12 million fractures are treated in the United States¹, costing $80 billion per year². The likelihood of a male or female suffering a fracture in their lifetime is 25% and 44%, respectively³. Problems associated with fracture healing are expected to increase with increased comorbidities as the population ages. To study and address this problem, robust models of fracture generation and stabilization are required. Rodent models are ideally suited for this purpose. They provide clinical relevance and can be modified to address specific conditions (i.e., multiple injuries, open, closed, ischemic, and infected fractures). In addition to replicating clinical scenarios, animal fracture models are important for understanding bone biology and developing therapeutics and devices. However, attempts to study differences between interventions may be complicated by the variability introduced by inconsistent fracture generation. Thus, generating reproducible and consistently closed fractures in animal models is essential to the field of musculoskeletal research.

Despite properly controlling for potential subject heterogeneity by ensuring the appropriate genetic background, sex, age, and environmental conditions, the production of clinically-relevant consistent bone injuries is a significant variable affecting reproducibility that must be controlled. Statistical comparisons using inconsistent fractures are plagued with experimental noise and a high variability⁴; in addition, fracture variability can result in unnecessary animal death because of the need to increase the sample size or the necessity to euthanize animals with comminuted or malpositioned fractures. The purpose of the method described herein is to optimize the fracture generation parameters that are specific to sample type and yield a consistent fracture location and pattern.

Current models of fracture generation fall into two broad categories, each with their own strengths and weaknesses. Open-fracture (osteotomy) models undergo surgery to expose the bone, after which a fracture is induced by cutting the bone or weakening it and then manually breaking it^5,6,7,8. The benefits of this method are the direct visualization of the fracture site and a more consistent fracture location and pattern. However, the physiological and clinical relevance of the approach and mechanism of injury is limited. Additionally, open methods of fracture generation require a surgical approach and closure with prolonged periods during which the rodents are exposed to an increased risk of contamination.

Closed techniques address many of the open technique's limitations. Closed techniques produce fractures using an externally applied blunt force trauma which induces injury to the bone and surrounding tissues, more similar to those seen in human clinical injuries. The most common method was described by Bonnarens and Einhorn in 1984⁹. They described a weighted guillotine being used to impart blunt trauma to break the bone without causing any external skin wounds. This method has been widely adopted to study the effect of genetics^10,11, pharmacologic therapy^12,13,14,15, mechanics^16,17, and physiology^18,19,20 on bone healing in mice and rats. While the benefit of closed methods is physiologically relevant fractures, experimental reproducibility and rigor are limited by fracture heterogeneity. The inconsistent fracture generation results in a limited between-group differentiation, lost specimens, and an increase in animals needed to achieve statistical significance.

Controlling the variability in fracture generation and stabilization is essential to produce meaningful results. In order to properly study the biology of fracture repair, a simple yet robust fracture model is needed. The model should be translatable to rodent species, bone types (femur or tibiae, for example), and across variable mouse genetic backgrounds and induced mutations. Furthermore, the ideal procedure should be technically simple and produce consistent results. To address fracture heterogeneity, the method described herein is the construction of a well-controlled fracture device that can then be used to optimize parameters and generate consistently closed fractures regardless of age, sex, or genotype.

Protocol

This protocol was developed to ensure that animals are not used needlessly and are spared all unnecessary pain and distress; it adheres to all applicable federal, state, local, and institutional laws and guidelines governing animal research. The protocol was developed under the guidance of a university-wide Laboratory Animal Medicine Program directed by veterinarians specialized in laboratory animal medicine. The protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).

1. Fracture Tower Construction

Note: All parts are listed in the materials section (Table of Materials). Detailed technical drawings are provided for the machined and 3D-printed parts in Supplementary Figures 1-12. The subassembly technical drawings include fastener details for all mounted parts (Supplementary Figures 1, 2, 7, and 9).

1. Support subassembly
   NOTE: For a technical drawing of the support subassembly, see Supplementary Figure 1.
   1. Attach the Beam Support--Jaw Section at the midpoint of the Beam Support--Horizontal Section.
   2. Attach the Beam Support--Vertical 1 to the top surface of the Beam Support--Jaw Section, 2 in from the Beam Support--Horizontal Section.
   3. Attach the Beam Support--Vertical 2 to the top surface of the Beam Support--Horizontal Section at the midpoint (7 in from the end).
   4. Attach the Beam, Support--Plate Mount to the end of both Beam Support--Vertical 1 and Beam Support--Vertical 2. The end of the plate support should be flush with the back of Beam Support--Vertical 2.
2. Ram subassembly
   Note: For a technical drawing of the ram subassembly, see Supplementary Figure 2.
   1. Machine the Block Stop and the Block Guide (Supplementary Figure 3); the Rod Ram (Supplementary Figure 4); the Screw Alignment (Supplementary Figure 5); and the Plate Mounting (Supplementary Figure 6).
   2. Attach the Plate Mounting to the Beam Support--Plate Mount of the support subassembly.
   3. In the following order, slide the first Linear Sleeve Bearing; the Block Guide; the second Linear Sleeve Bearing; and the Block Stop on the Rod Ram. Attach the guides and the blocks to the Plate Mounting.
   4. Attach three ⅜-in nuts to the threaded portion of the Rod Ram. One should be flush with the end of the rod to engage with the electromagnet. The other 2 will be used to adjust the fracture depth.
   5. Align the grove in the Rod Ram to face forward and insert the Alignment Screw into the threaded hole of the Block Guide.
3. Magnet subassembly
   Note: For a technical drawing of the magnet subassembly, see Supplementary Figure 7.
   1. Solder the Electromagnet leads to the wire (polarity is not a factor for the electromagnet operation). Allow enough length to reach the floor, where the fracture device will be positioned. Use zip ties or another form of attachment to stress relieve the wire.
   2. Strip the Power Supply's end and connect it to the Foot Pedal. Finally, connect the wire to the Foot Pedal in an "off" (normally open) configuration. Test the circuit to ensure the Electromagnet is on when the Foot Switch is not pressed. This will hold the ram up before the fracture.
   3. Print the Mount Magnet (Supplementary Figures 8A and 8B) using an additive manufacturing device, or machine the part from aluminum.
   4. Attach the Electromagnet to the Mount Magnet.
   5. Attach 2 Corner Brackets to the Beam Support--Magnet.
   6. In the following order, thread the Rod Magnet through the top Corner Bracket and add one ¼-in nut; the Mount Magnet; two ¼-in nuts; and the bottom Corner Bracket. Secure the assembly with two ¼-in nuts on each end.
4. Complete assembly
   Note: For a technical drawing of the complete assembly, see Supplementary Figure 9.
   1. Attach the Magnet Subassembly to the top surface of the Beam, Support--Plate Mount.
   2. Adjust the alignment of the Beam Support--Magnet so the magnet engages with the Rod, Ram.
      NOTE: If the rod does not release when the foot pedal is pressed, reduce the contact area between the electromagnet and the rod by moving the Beam Support--Magnet.
   3. Machine the Brackets Leg Jaw (Supplementary Figure 10).
   4. Attach the two Brackets Leg Jaw to the Beam Support--Jaw Section. When dropped, the tip of the ram should be at an equal distance from each jaw.
   5. Place the Platform Fracture (Supplementary Figures 11A and 11B) above the jaws.
   6. Print the Jig Positioning Fracture (Supplementary Figures 12A and 12B) and the Jig Pin Gauge (Supplementary Figures 13A and 13B) using an additive manufacturing device, or machine the parts from aluminum.
      NOTE: The dimensions of the Jigs will be calculated in the optimization steps detailed in step 2.
   7. Attach the Jig Positioning Fracture to the Platform Fracture.
   8. Confirm that the depth of the impact can be adjusted using the two stop nuts on the Rod Ram.
   9. Confirm that the speed of the impact can be adjusted by moving the Mount Magnet up and down.
   10. Confirm that the width of the fracture can be adjusted by moving the Brackets Leg Jaw closer or further away from the Rod Ram.

2. Fracture Optimization

1. Fracture location
   1. Obtain radiographs of the limb (femur or tibia) to be fractured in a representative sample of 5 euthanized animals.
      NOTE: The sample should be matched to the specimens, which will be used in the experimental protocol based on age, genotype, and sex. Even if the final protocol calls for only one fractured limb, both sample limbs will be used.
   2. Position the limb tangential to the x-ray beam to acquire true-lateral and anterior/posterior views to the bone. Place an object of known dimension at the imaging plane to provide a scale for analysis.
   3. Note: If imaging femurs, ensure the limb is in full extension, where the femur is in the same axial plane as the tibia.
   4. Mark the desired location of the fracture on the radiograph of the limb to be fractured (Figure 1A - dashed line). Measure from the calcaneal-tibial joint to the level of the marked fracture (Figure 1A). Calculate the mean fracture length (FL) for all trial specimens. Measure from the intercondylar notch for femur fractures.
2. Fracture-positioning jig
   1. Measure the distance from the outside surface of one support anvil to the center of the guillotine impact (CGI) (Figure 2). Subtract the CGI from the FL, described in step 2.1.4, to calculate the fracture-positioning jig depth (JD). Machine or 3D-print a U-shaped channel with a height and a width equal to the anvil, and a depth equal to the JD (Figure 3A). A sample technical drawing and CAD file are included in Supplementary Figures 12A and 12B.
      NOTE: When the limb is placed in the jig, the dorsum of the foot should lie against the surface furthest from the guillotine impact. Modify the U-shaped channel if additional clearance is required for the limb.
   2. Position the specimen in the fracture apparatus in the prone position for femur fractures or in the supine position for tibia fractures (Figure 4). Press the dorsum of the foot against the end of the fracture-positioning jig. Manually depress the guillotine until the limb fractures. Obtain a radiograph of the fractured limb to confirm the jig size and fracture location (Figure 2B).
   3. Increase JD if the fracture location is too distal on the bone, or decrease JD if the fracture location is too proximal on the bone.
3. Stabilization of the pin parameters
   1. Pin length: Using the radiographs obtained in step 2.1, measure the limb length (LL) from the tibial plateau to the level of the posterior malleolus for tibia fractures, or the intercondylar notch to the greater trochanter for femur fractures. Multiply the bone length by 0.9 to calculate the pin length (PL) (Figures 1A and 3B).
   2. Pin Width: Using the radiographs obtained in step 2.1, measure the minimum medullary diameter (MD) in the fractured limb (Figure 1A). Select a needle with a gauge approximately equivalent to the medullary diameter and a length more than 1.5 x PL.
      NOTE: An approximate pin size for a 14-week-old C57BL/6J mouse is 22 G, 1½ in and 27 G, 1¼ in for femur and tibia, respectively.
4. Pin cutting gauge
   1. 2.4.1. Machine or 3D-print a gauge with a length equal to PL minus the needle length (CGL) (Figure 3B; Supplementary Figures 13A and 13B). One end should have an overhang to rest against the hub of the needle and the other should indicate where the pin should be cut. A sample technical drawing and CAD file are included in Supplementary Figures 13A and 13B.
5. Intramedullary pin fracture stabilization
   1. Using the non-fractured trial specimens from step 2.1, remove hair with an electric clipper or depilatory cream from mid-tibia to mid-femur, exposing the knee joint.
   2. Tibia pinning: Insert the needle percutaneously lateral to the patellar ligament. Retract the patellar ligament medially and align the tip of the needle to the axis of the tibia. Using a reaming motion, gently breach the tibial plateau and guide the needle down the medullary cavity.
   3. Femur pinning: Insert the needle percutaneously lateral to the patellar ligament. Retract the patellar ligament medially and align the tip of the needle to the axis of the femur in the intercondylar notch. Using a reaming motion, gently breach the articular surface of the intercondylar notch and guide the needle down the medullary cavity.
   4. Using the gauge manufactured in step 2.4, ream until the exposed needle is equal to the gauge length. Retract the needle to provide enough room (~3 mm) to cut the needle at the level indicated by the gauge.
      NOTE: Be sure to hold the proximal (plastic) end of the needle while cutting, so it does not become a hazardous projectile.
   5. Crimp 0.3 mm of the distal end of the pin using a pin cutter and then cut the pin at the level of the gauge. Sink the pin to the articular surface using a rod with a diameter 1.5x larger than the diameter of the needle.
      NOTE: Crimping prevents rotation of the needle and migration by increasing the needle-bone contact.
   6. Obtain radiographs to confirm the needle extends the length of the medullary canal of the limb and does not protrude from the proximal or distal end (Figure 1C).
6. Impact depth
   1. Using the radiographs obtained in step 2.1, measure the diameter of the cortex at the level of the desired fracture (Figure 1A). Calculate the mean cortical diameter (CD) for all trial specimens.
   2. Position a pinned trial specimen from step 2.5 in the fracture device with the fracture-positioning jig manufactured in step 2.2. Rest the impact ram on the uninjured limb.
      NOTE: Do not allow the ram to drop; the bone should remain intact during this optimization step.
   3. Apply enough downward force on the ram to compress soft tissue, but not fracture the bone. Adjust the impact depth (ID) to 0.75 x CD (Figure 2).
      NOTE: The ideal impact depth is 0.5 x CD when fracturing a bone without any soft tissue. Using 0.75 accounts for the additional soft-tissue compression.
7. Anvil width
   1. Set the anvil width (AW) to 0.4 cm for the mouse tibia or femur (Figure 2).
      NOTE: A wider width is recommended for larger specimens such as rats.
8. Ram weight
   1. A minimum weight of 250 g is recommended for murine specimens.
      NOTE: Additional weight can be threaded on to the ram for larger specimens (Figure 2).
9. Impact velocity
   1. Set the drop height (DH) to 2 cm (Figure 2). Position the ram in its starting position by connecting it to the activated electromagnet.
   2. Position a trial limb in the fracture apparatus. Press the dorsum of the foot against the fracture-positioning jig manufactured in step 2.2. Briefly depress the footswitch to release the ram and then reset it to its starting position.
   3. Radiograph the impacted trial limb. Analyze the limb for any evidence of a fracture (Figure 1D).
      NOTE: This can be subtle when using low velocities with a controlled impact depth.
   4. If no fracture is generated, repeat steps 2.9.1 - 2.9.3 and increase the drop height by 2 cm.
   5. If a fracture is generated, record the drop height, and multiply it by 1.1. This is the new DH.
   6. Using the DH from step 2.9.5, fracture the next trial limb.
   7. If no fracture is generated, repeat steps 2.9.1 - 2.9.6 and increase the drop height by 2 cm.
   8. If a fracture is generated, repeat steps 2.9.6 - 2.9.7 until all test samples are used. Record the final DH and all parameters (FL, CGI, JD, PL, MD, PS, CGL, CD, ID, AW, and RW) from the optimization. Record the trial specimens' age, sex, genotype, and weight.

3. Closed-stabilized Fracture Generation

1. Set-up
   1. Sterilize all equipment and instruments via autoclave, hot bead immersion, or their equivalent.
   2. Place a heating element on the surgical table and set it to the optimal temperature. Cover the element with a surgical drape. Prepare 3 x 3 in² of surgical drape with a 0.75-in circle cut out in the middle.
   3. Confirm the adjustment of the fracture tower before each trial (Figure 2). Set the ID, AW, RW, and DH to the values derived from the optimization protocol specific to the sex, age, and genotype for the specimen to be studied.
   4. Weigh and record the weight of the animal.
2. Surgery
   1. Adequately sedate the mouse using inhalant anesthetics (isoflurane: 4 - 5% for induction; 1 - 2% for maintenance) or another established laboratory anesthesia protocol. The respiratory rate should be 55 - 100 breaths/min. The animal should not be responsive to a hind-limb toe pinch.
   2. Administer the first dose of the postoperative analgesia buprenorphine (0.1 mg/kg subcutaneously).
   3. Apply ocular lubrication to prevent corneal drying.
   4. Remove the animal's hair with an electric clipper from mid-tibia to mid-femur, exposing the knee joint. Clean the site of excess hair using non-reactive tape. Prepare the pinning site with a wet swab moistened with 70% EtOH. Repeat as necessary to remove all hair from the incision area.
   5. Prepare and clean the pinning area with alternative swabs of povidone-iodine and 70% EtOH. Use two alternative swab sequences to ensure sterility.
   6. A drape is then placed around the surgical site after the skin has been appropriately disinfected.
   7. Pin the limb to be fractured using the protocol described in step 2.5. Acquire radiographs to confirm the pin extends the length of the medullary canal but does not protrude from the proximal or distal end.
   8. Turn on the electromagnet and connect the impact ram to place it in the starting position.
   9. Position the specimen in the fracture apparatus by placing it in a prone position for femur fractures or in a supine position for tibia fractures. The pinned limb should be placed across the anvils and in the fracture-positioning jig with the dorsum of the foot pressed against the outside of the jig.
   10. While pressing the foot with one hand and ensuring only the limb is in the impact ram target area, briefly depress the footswitch to release the ram. Replace the ram in the starting position.
   11. Acquire radiographs and confirm the fracture location and type.
3. Postoperative management
   1. Monitor the animal every 15 min during its recovery from anesthesia until the animal is conscious, can maintain sternal recumbency, and is ambulatory. Confirm the animal is able to ambulate over a 72-h period.
   2. House the animal individually until it has completely recovered.
   3. Maintain analgesia over a 48-h period with buprenorphine (0.1 mg/kg subcutaneously) administered every 12 h.
   4. Monitor and record the health status of the animal daily for 7 - 10 d or until euthanasia.
4. Post-fracture analysis
   1. Measure FL, PL, CD, MD, and the fracture pattern. Record the measurements in a master data file.

Results

The guillotine previously used in our laboratory was developed in 2004 and was based on models published by Einhorn²¹. The design did not permit adjustments to adequately account for any differences in bone morphology and did not permit a reproducible positioning of the limb. Furthermore, the previous apparatus required two people to operate it. Therefore, we designed, engineered, and built a new fracture apparatus. The main design goal was the possibility to the h...

Discussion

This fracture optimization and generation protocol provides researchers with an efficient method to derive at fracture parameters and perform a minimally invasive procedure, which produces precise, repeatable, transverse fractures. Additionally, this protocol establishes a common set of fracture generation parameters, which promotes method consistency amongst researchers. These parameters will enable the creation of a common fracture database to establish fracture standards based on a variety of parameters (e.g....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Support Subassembly			Supplementary Figure 1
Beam, Support--Jaw Section	80/20	1003 x 9.00	w/ #7042 at A, C, in Left End
Beam, Support--Horizontal Section	80/20	1002 x 14.00
Beam, Support--Vertical 1	80/20	1050 x 10.50	w/ #7042 at A in Left End and at A in Right End
Beam, Support--Vertical 2	80/20	1010 x 10.50	w/ #7042 at D, B in Left End and at A in Right End
Beam, Support--Plate Mount	80/20	1030 x 8.00	w/ #7036 at Left End
Beam, Support--Magnet	80/20	1010 x 13.50	w/ #7042 at A, C, in Right End
Anchors (3)	80/20	3392
Double Anchor (3)	80/20	3091
Bolt Assembly (6)	80/20	3386	1/4-20 x 3/8"
Button Head Socket Cap Screw (6)	80/20	3604	1/4-20 x 3/4"
Ram Subassembly			Supplementary Figure 2
Block, Stop	Custom		Supplementary Figure 3
Block, Guide	Custom		Supplementary Figure 3
Rod, Ram	Custom		Supplementary Figure 4
Alignment Screw	Custom		Supplementary Figure 5
Plate, Mounting	Custom		Supplementary Figure 6
Linear Sleeve Bearing (2)	McMaster-Carr	8649T2
Hex Nut (3)	McMaster-Carr	92673A125	3/8-16 UNC
Socket Cap Screw (8)	McMaster-Carr	92196A108	4/40 x 3/8"
Socket Cap Screw (6)	McMaster-Carr	92196A032	4/40 x 1 1/8"
Socket Cap Screw (1)	McMaster-Carr	92196A267	10/32 3/8"
Magnet Subassembly			Supplementary Figure 7
Mount, Magnet	Custom		Supplementary Figure 8
Power Supply	McMaster-Carr	70235K23
Foot Switch	McMaster-Carr	7376k2
Electromagnet	McMaster-Carr	5698k111
Wire - 10 feet	McMaster-Carr	9936k12
Rod, Magnet	McMaster-Carr	95412A566	1/4" Threaded Rod x 7"
Corner Bracket (6)	80/20	4108
Socket Cap Screw (1)	McMaster-Carr	92196A705	10/32 1 1/4"
Hex Nut (4)	McMaster-Carr	92673A113	1/4-20 UNC
Complete Assembly			Supplementary Figure 9
Bracket, Leg Jaw (2)	Custom		Supplementary Figure 10
Platform, Fracture	Custom		Supplementary Figure 11
Jig, Positioning-Fracture	Custom		Supplementary Figure 12
Other
Pin Cutter	Medical Supplies and Equipment	150S
Needles	Sigma	Z192430, Z192376	23g x 1.5" - mouse femur, 27g x 1.25" - mouse tibia