In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Summary

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Abstract

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and µCT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

Introduction

Secondary fracture healing is the most common form of bone healing. It is a complex process mimicking specific aspects of ontogenic endochondral ossification^1,2,3. The early fracture hematoma predominantly consists of immune cells, granulation and fibrous tissue. Low oxygen tension and high biomechanical strains hamper osteoblast differentiation at the fracture gap, but promote the differentiation of progenitor cells into chondrocytes^4,5,6. These cells start to proliferate at the site of injury to form a cartilaginous matrix providing initial stability of the fractured bone. During callus maturation, chondrocytes become hypertrophic, undergo apoptosis, or trans-differentiate into osteoblasts. Neovascularization at the cartilage-to-bone transition zone provides elevated oxygen levels, allowing the formation of bony tissue⁷. After bony bridging of the fracture gap, biomechanical stability is increased and osteoclastic remodeling of the external fracture callus occurs to gain physiological bone contour and structure³. Therefore, the amounts of fibrous, cartilaginous, and bony tissue in the fracture callus provide important information about the bone healing process. Disturbed or delayed healing becomes visible by alterations of callus tissue development both in humans and mice^8,9,10,11. Available in vivo techniques to longitudinally monitor callus tissue development in preclinical fracture healing studies using small animals include digital radiography and µCT imaging^12,13. However, both techniques are only able to discriminate between mineralized and non-mineralized tissue. In contrast, MRI provides excellent soft tissue contrast and might therefore be able to identify soft tissue and cartilage in the fracture callus.

Previous work showed promising results for post mortem MRI in mice with articular fractures¹⁴ and in vivo MRI in mice during intramembranous bone-defect healing¹⁵. However, both studies also stated limited spatial resolution and tissue contrast. We previously demonstrated the feasibility of high-resolution in vivo MRI for longitudinal assessment of soft callus formation during murine endochondral fracture healing¹⁶. Here, we report the protocol for using an MRI-compatible external fixator for femur osteotomy in mice in order to monitor callus tissue development longitudinally during the endochondral fracture healing process. The design of a custom-made mounting device for insertion of the external fixator ensured a standardized position during repeated scans.

Protocol

All animal experiments complied with international regulations for the care and use of laboratory animals and were approved by the regional regulatory authorities (No. 1250, Regierungspräsidium Tübingen, Germany). All mice were maintained in groups of two to five animals per cage on a 14-h light, 10-h dark circadian rhythm with water and food provided ad libitum.

1. Preparation of the Surgical Material and Pre-treatment of the Mice

1. Sterilize all surgical material. Use an autoclaving temperature of 120-135 °C for 20-30 min of sterilization time.
2. Purchase C57BL/6 mice or mice from another strain which are between 19-35 g of body weight. Follow the appropriate animal care and experimental protocols in accordance with national guidelines that is approved by the investigator's Institutional Animal Care and Use Committee. Allow a minimum of 7 days acclimatization period before starting the procedure.
3. Provide analgesia to all mice via the drinking water one day before surgery until the third postoperative day.

2. Surgical Procedure and Application of the External Fixator

1. Place the mouse into a tube preloaded with 5-7% isoflurane and 60 mL/min oxygen. After loss of postural reflexes, remove the mouse from the anesthesia induction tube and maintain the anesthesia via an inhalation mask providing 1-3% isoflurane and 60 mL/min oxygen.
   1. Monitor the breathing pattern and hind paw reflex during anesthesia. Ensure that the breathing rate is around 100 cycles/min and the hind paw reflex is absent.
      ​NOTE: The amount of gas needed is dependent on age, sex, body weight, and strain of the mouse.
2. Prior to surgery, inject the mouse with a single dose of antibiotics subcutaneously (clindamycin, 45 mg/kg). Furthermore, for maintenance of the physiological fluid balance, inject the mouse with a subcutaneous fluid depot of 500 µL saline (0.9% NaCl).
3. To prevent corneal drying, apply eye ointment to the mouse eyes. Place the mouse on a heating plate at 37 °C during the anesthesia and surgical procedure to maintain physiological body temperature.
4. Remove the fur from the right hind limb and scrub the surgical area with an alcohol-based disinfectant. Cover the right hind paw with a small part of a sterile glove to avoid unsterile areas. Disinfect the right hind limb three times. Place a sterile drape over the whole mouse except for the surgical area.
5. Incise the skin approximately 1 cm longitudinally along the anterior side of the right femur with a scalpel. Separate bluntly the m. biceps femoris and the m. vastus lateralis with micro scissor and forceps. Cut the tendon origin side at the femur trochanter with a micro scissor to allow free access to the anterolateral part of the bone. Make sure that the sciatic nerve is preserved.
6. Position the external fixator (axial stiffness of 3 N/mm, Figure 1A) parallel to the femur. Manually drill the boreholes through cortex with a 0.45-mm drill bit and place the ceramic mounting pins into the boreholes. Start with the most proximal pin, followed by the most distal pin, and the two pins in between.
   1. Make sure that there is no tension, compression, or shear stress on the fixator during the mounting procedure, otherwise the achieved osteotomy gap will not be sufficient due to relaxation of the fixator.
7. Humidify the bone with a small amount of sterile NaCl to avoid dehydration during the sawing procedure.
8. Create a 0.4-mm osteotomy through the whole bone between the two inner pins by using a 0.4 mm gigli wire saw.
   NOTE: Optionally, an oscillating micro saw can be used to create the osteotomy. Make sure to avoid any metal chips from the saw at the osteotomy area.
9. Flush the osteotomy gap carefully with 2 mL of sterile NaCl to remove bone chips between the two fractured cortices.
10. Adapt the muscles by using a continuous suture with a resorbable suture (see Table of Materials). Then adapt the skin by using interrupted non-resorbable sutures (see Table of Materials). To avoid wound biting, do not place the suture at the cranial part of the wound.
    NOTE: Do not use skin glue or clips since mice usually remove it from the wound causing further damage to the skin.
11. Clean the surgical area with a disinfectant and place the mouse into its cage. Monitor the mouse and supply sufficient heat (e.g. by infrared light) until it is fully awake. Monitor water, food intake, and body weight after the surgery to make sure the animal is not in pain and distress. Provide analgesia to all mice via the drinking water until the third postoperative day.
    NOTE: Mice may be housed in groups of up to four animals.
12. Monitor the mouse's activity on days 1 to 5 after surgery. During that time course, the mouse should bear weight on the operated limb. Otherwise, the mouse must be excluded from further analysis.

3. MRI Procedure and Image Analysis

1. Prior to the MRI scanning procedure, anaesthetize the mouse according to the protocol in steps 2.1 and 2.3, and keep the respiratory rate around 100 cycles/min. Insert the external fixator at the right hind limb of the mouse carefully into a custom-made mounting device (Figure 1B, C).
   1. Make sure to avoid bending or compression of the fixator during this step since this may interfere with fracture healing.
      Note: The MRI scans can be conducted as early as 3 days after surgery, depending on the animal care and experimental protocol.
2. Place the mouse on a temperature controlled cradle for introduction into the MRI device. Attach the mounting device rigidly to the four-element head coil.
3. Acquire MRI data using a dedicated high-field small-animal MRI system operating at 11.7 T.
   ​NOTE: The MRI data acquisition geometry is aligned with the femur bone, orthogonally to the screws.
   1. Acquire data by applying a proton-density fat-suppressed multi-slice TSE sequence (PD-TSE) using acquisition parameters: echo/repetition time TE = 5.8 ms/TR = 2,500 ms, resolution Δr = 52 × 52 × 350 µm³, field-of-view (FOV) = 20 × 20 mm², and bandwidth Δω= 150 KHz.
   2. NOTE: The total acquisition time for 22 slices is 36 min.
4. Open the acquired data with image analysis software. Enter the voxel size as 0.05 x 0.05 x 0.35 mm³. Segment the different tissues in the fracture callus (bone, cartilage, fibrous tissue/bone marrow) based on their intensity with semi-automatic thresholding as follows.
   1. Click the "Edit New Label Field", click "Add Material", and rename the material to "callus". Distinguish the callus area from the surrounding tissues based on the hypo-intense signal from the periosteum using the "Lasso" tool.
   2. Click "add to material". Click "Add Material" and rename the material to "cartilage". Segment the cartilage by using the "threshold" tool and "Select only current material" from "callus". Click "cartilage" and "add to material". Repeat these steps with "bone" and "bone marrow/fibrous tissue".
5. Generate 3D reconstructions of the fractured femurs based on the tissue segmentation data using image analysis software. Click "Generate Surface", apply "None" for "Smoothing Type" and click "Surface View".
   NOTE: Very small, hyper-intense areas surrounding the ends of the fractured cortices are likely to be artifacts due to the transition from bony to soft tissue. These areas should be excluded from further analysis. Hyper-intense areas in the middle of the fracture callus during the endochondral phase of fracture healing represent cartilaginous tissue. Hypo-intense areas at the fracture callus distal from the osteotomy gap at the endochondral ossification phase and areas with the same intensity throughout the whole fracture callus at later healing stages represent newly formed bony callus tissue. Although these areas have a hypo-intense signal, the signal intensity from mature bone (cortex) is even lower. After thresholding the signal intensity for bony tissue and cartilaginous tissue in the fracture callus, mark the remaining tissue as bone marrow and fibrous tissue. Values for tissue segmentation are: bony tissue (including mature cortex, trabecular bone, and bony callus tissue) is segmented within the range of 1-3.3 (normalized signal intensity to mature cortex), bone marrow/fibrous tissue within the range of 3.4-5.4, and cartilaginous callus tissue within the range of 5.5-6.2.
6. If needed, repeat the MRI scan longitudinally during the fracture healing process. To monitor cartilaginous callus development, scan the mice on days 10, 14, and 21 after surgery.
   NOTE: The time points may depend on the animal care and experimental protocol.

Results

First, the success of the surgical procedure can be confirmed by analysis of the MRI scans (see example in Figure 2). All four pins should be located in the middle of the femoral shaft. The size of the osteotomy gap should be between 0.3-0.5 mm. If the size of the osteotomy gap varies greatly from these values, the mouse should be excluded from further analysis.

Secondly, the evaluation of longitudi...

Discussion

Modifications and Troubleshooting:

The main goal of this study was to describe a protocol for using of an MRI-compatible external fixator for femur osteotomy in the mouse with the ability to monitor callus tissue development longitudinally during the endochondral fracture-healing process. The design of a custom-made mounting device for insertion of the external fixator ensured a standardized position during repeated scans. Semi-automatic tissue segmentation allows the analysis...

Materials

Name	Company	Catalog Number	Comments
Anaesthesia tube	FMI, Seeheim, Germany	ZUA-82-ANA-TUB-Mouse
Anaesthetic machine	FMI, Seeheim, Germany	ZUA-82-GME-MA
Artery forceps	Aesculap, Tuttlingen, Germany	BH104R
Autoclave	Systec, Wettenberg, Germany	DX-150
Autoclaving packaging	Stericlin, Feuchtwangen, Germany	2301-04/06/10/12/16
Avizo software	FEI, Burlington, USA	-	Version 8.0.1
BioSpec 117/16 magnetic resonance imaging system	Bruker Biospin, Ettlingen, Germany	117/16
Bulldog clamp	Aesculap, Tuttlingen, Germany	BH 021R
Carbon steel scalpel no. 11/15	Aesculap, Tuttlingen, Germany	BA211/215
Ceramic mounting pin 0.45 mm	RISystem, Davos, Switzerland	HS691490
Clindamycin (300 mg / 2ml)	Ratiopharm, Ulm, Germany	-
Dressing forceps 115 mm	Aesculap, Tuttlingen, Germany	BD210R
Dressing forceps 130 mm	Aesculap, Tuttlingen, Germany	BD025R
Drill bit coated 0.45 mm	RISystem, Davos, Switzerland	HS820420
Durogrip needle holder 125 mm	Aesculap, Tuttlingen, Germany	BM024R
Foliodrape	Hartmann, Heidenheim, Germany	2513026
Frekaderm	Fresenius, Bad Homburg, Germany	4928211
Gigli saw 0.44 mm	RISystem, Davos, Switzerland	RIS.590.110.25
Hand drill	RISystem, Davos, Switzerland	RIS.390.130-01
Heating plate	FMI, Seeheim, Germany	IOW-3704
Hygonorm gloves	Hygi, Telgte, Germany	2706
Isoflurane	Abbot, London, UK	Forene
Micro forceps 155 mm	Aesculap, Tuttlingen, Germany	BD343R
Micro scissors 120 mm	Aesculap, Tuttlingen, Germany	FD013R
Mouse FixEx L 0.7 mm	RISystem, Davos, Switzerland	RIS.611.300-10
Needle case for drills	Aesculap, Tuttlingen, Germany	BL911R
Needle holder	Aesculap, Tuttlingen, Germany	BB078R
Octenisept	Schülke, Norderstedt, Germany	121403
Osirix software	Pixmeo SARL, Bernex, Switzerland	-	Version 4.0
Oxygen, medical grade	MTI, Ulm, Germany	-
Resolon 5/0	Resorba, Nürnberg, Germany	88143
Saline 0.9%	Braun, Melsungen, Germany	3570350
Scalpel handle 125 mm	Aesculap, Tuttlingen, Germany	BB073R
Scissors 150 mm	Aesculap, Tuttlingen, Germany	BC006R
Sealer for autoclave packaging	Hawo GmbH, Obrigheim, Germany	HM500
Sterican 27 G	Braun, Melsungen, Germany	4657705
Sterile surgical blades no. 11/15	Aesculap, Tuttlingen, Germany	BB511/515
Surgical gloves	Hartmann, Heidenheim, Germany	Peha-micron 9425712
Surgical light	Maquet SA, Ardon, France	Blue line 80
Syringes 5 ml	Braun, Melsungen, Germany	Injekt 4606051V
Tissue forceps 80 mm	Aesculap, Tuttlingen, Germany	OC091R
Tramadol 25 mg/l	Grünenthal, Aachen, Germany	100mg/ml
Vasofix Safety	Braun, Melsungen, Germany	4268113S-01
Vicryl 5-0	Ethicon, Norderstedt, Germany	V30371
Visdisic eye ointment	Bausch & Lomb, Berlin, Germany	3099559