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 ossification1,2,3. 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 chondrocytes4,5,6. 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 mice8,9,10,11. Available in vivo techniques to longitudinally monitor callus tissue development in preclinical fracture healing studies using small animals include digital radiography and µCT imaging12,13. 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
Representative Results
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
We thank Sevil Essig, Stefanie Schroth, Verena Fischer, Katja Prystaz, Yvonne Hägele, and Anne Subgang for excellent technical support. We also thank the German Research Foundation (CRC1149, INST40/499-1) and the AO Trauma Foundation Germany for funding this study.
Materials
| 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 |
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