Department of Orthopaedic Surgery, University of California, San Francisco
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Yu, Y. y., Bahney, C., Hu, D., Marcucio, R. S., Miclau, III, T. Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects. J. Vis. Exp. (62), e3552, doi:10.3791/3552 (2012).
Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.
All Procedures were approved by the UCSF Institutional Animal Care and Use Committee and conform to national guidelines.
1. Preparation of Fixators Prior to Surgery
2. Anesthesia, Fracture Creation, and Fixator Application
3. Distraction Osteogenesis (see also: 12,13)
To modify this procedure to accommodate distraction osteogenesis is straightforward. The rings are held in position by threaded rods, and by turning the nuts holding the rods in place the rings can be moved apart.
4. Creation of a Critical Sized Defect
To create a critical sized defect the external fixators are applied as previously described with the following modifications.
5. Representative Results
When properly applied, the external fixators provide more rigid stability of the closed tibial fracture with excellent reduction (Figs. 1, 2). However, in some cases inadequate reduction (an obvious and large gap between bone ends or multiple fractures occur (Fig. 3), and these mice are excluded from analyses. Fractures stabilized using this method heal primarily via intramembranous ossification (Fig. 4). In contrast, if the fracture is not stabilized a large cartilage callus is formed in the fracture gap (Fig. 5), and this is replaced by bone through the process of intramembranous ossification.
Figure 1. Radiographs illustrating an external fixation device used to stabilize tibial fracture. Radiograph taken after fracture showing a well-aligned bone segments (arrowhead).
Figure 2. Image of a mouse after the fixator has been applied.
Figure 3. Radiograph taken after fracture shows misaligned and fragmented bone segments (arrowhead).
Figure 4. Stabilized fracture heals via intramembranous ossification. Trichrome staining of stabilized fracture shows some new bone (b) at fracture site. Scale bar = 500 μm.
Figure 5. Non-stabilized fracture heals via endochondral ossification. Trichrome staining of non-stabilized fracture shows cartilage (c) and bone (b) at fracture site. Scale bar = 500 μm.
Bones heal by two different modalities depending on mechanical stability (reviewed in: 14). When left unstable a large cartilage template forms in the fracture gap that is replaced by bone to bridge the two ends of broken bone. Proximally and distally to the break, bone forms directly by intramembranous ossification within the periosteum and endosteum. In contrast, in stable fractures healing occurs exclusively via intramembranous ossification 3. However, the specific mechanisms that regulate the switch between these two processes are unknown. There is evidence that stem cell fate in response to the mechanical environment is controlled genetically and can be altered. In mice that lack Mmp9 cartilage forms in the fracture site of stable fractures suggesting that Mmp9 is involved in stem cell fate decisions during fracture repair 5. Cartilage can also be induced to form in stable fractures if the Bone morphogenetic protein (BMP) signaling pathway is activated during fracture repair 8,9. In order to draw these conclusions providing rigid stabilization during the early stages of healing is important, because these stem cell fate decisions are made during the first few days after injury11.
Our method of external fixation, when applied correctly can be used to stabilize bone to assess intramembranous ossification during fracture healing. Our method creates a fracture along with other limb injuries that occur in a high velocity impact. In contrast, drill hole models that are used to assess intramembranous ossification may not reflect trauma in patients. Further, the method described here is closed, so complications associated with an open injury do not confound interpretation of the outcomes. Another common method for stabilization uses placement of an intramedullary pin prior to fracture (e.g.4). This technique is widely used, and placement of the pin is simple and efficient. While this approach produces lateral stabilization it does not provide for rotational stability. The resulting injury heals primarily via endochondral ossification which can be achieved without stabilization at all 3. Plated fixation has also been used to achieve fracture stabilization in mice 15. Here an open fracture is created and specially designed internal fixators are attached to the bone after an osteotomy is made. Similar to our approach the internal fixation method provides stability and healing occurs via intramembranous ossification. We do not observe significant complications, pin-tract infections, or other co-morbidities associated with this procedure, and the animals are able to move around their cages easily. The major drawbacks are in the length of time it takes to become proficient at this method, the time it takes to complete the procedure, and the requirement for teams of two individuals to perform the procedure. In combination with genetic or physiologic manipulations to the mice, comparing stabilized to non-stabilized fracture healing provides considerable insight into the mechanisms that regulate stem cell fate during fracture healing 5, and has allowed an investigation of distraction osteogenesis14 while also providing us with a model of non-union13.
We have nothing to disclose.
This work was funded by R01-AR053645 from NIAMS.
|0.25mm insect pin||Fine Science Tools||26000-25||Blacked Anodized Steel, 0.25mm rod diameter, 4cm length|
|Stainless Steel Hex Nut||Small Parts, Inc.||#2-56||1/8" length, 56 threads per inch|
|Stainless Steel Hex Nut||Small Parts, Inc.||#0-80||1/8" length, 80 threads per inch|
|Stainless Steel Machine Screw||Small Parts, Inc.||#0-80||1/8" length, 80 threads per inch|
|Stainless Steel Machine Cut Threaded Rod||Small Parts, Inc.||#0-80||6" length, 80 threads per inch|
|18-8 Stainless Steel Head Machine Screw||McMaster-Carr||2-56 Threads, 3/6" length|
|External Fixation Device||Machine shop||Custom-designed|