The aim of this study was to establish a murine femoral critical-size bone defect model using external fixation technique. Our main finding was that a 3-mm defect is needed to achieve a bony segmental nonunion rate of 100% after a 12-wk observation period.
Other investigators have previously used custom-made femoral external fixators for similar purposes. Cheung et al.
developed an external fixation device composed of two aluminum blocks that were interconnected by two rods [4
]. The femur was stabilized by four nonthreaded stainless steel pins. Closed fractures were created and healed after 14 to 21 d. The postoperative observation period was 60 d. Srouji et al.
described another custom-fabricated external fixation device that uses four 27G needles connected by acrylic dental cement to stabilize the femur [5
]. This fixation device penetrated the skin on the lateral and the medial side of the upper leg. Bone defects of up to 3 mm were created with this device. Two-millimeter defects using this device were reported to be of adequate size to achieve a critical-size defect and larger defects of 2.5 and 3 mm were found to be excessively unstable. The postoperative observation period reported by Srouji et al.
] was 8 wk. The external fixator presented in this paper has several advantages compared to the previously described systems. With a weight of 0.20 g, the external fixator used in the present study was three to five times lighter in comparison to the previously described systems’ weights of 0.6 to 0.8 g [5
] and 1.1 g [4
], respectively. The weight of the implant used in our study is thought to have minimal interference with murine biological movement as it comprises only 0.5% of their body weight. Furthermore, the double-threaded pins we used provide increased stability in comparison to the previously described systems. One end of the pin is threaded and locked into the connecting plastic body and the other end is threaded into the femur in accordance with the principles of a locked plate. The pins are made of TAN (medical grade titanium alloy), which is believed to provide favorable conditions for bone healing due to its osteoconductivity [6
Between the 8th and the 10th postoperative week, we euthanized one animal from the 2-mm group and one from the 3-mm group due to pin loosening. We hypothesize that a chronic pin infection with subsequent osteolysis may have been the cause. Another animal died due to a probable systemically induced abscess on a cubital joint. This finding underscores a disadvantage of external fixation systems when compared to internal fixation systems—the skin-penetrating pins provide an ongoing potential risk of infection [7
]. One may be able to reduce this risk with frequent cleaning and disinfection of the device, as is done for humans.
We found μCT to be the best imaging modality to evaluate the bone morphology of mouse femurs. Artifacts resulting from metal implants currently present a challenging imaging issue. The use of an intramedullary nail or an internal metal-based plate makes it very difficult to obtain high-quality images of the defect area. The described external fixation device allowed us to get an undisturbed scan of the defect area, as the plastic body and the TAN pins do not adversely affect the imaging scan.
Several technical and genetic reasons may explain why different studies have found various critical defect sizes in mice. Beamer et al.
showed that the adult bone density of different inbred strains of mice can be significantly different and that none of the parameters (femur length, density, bone mineral content, volume) was related to the body weight of the mouse [8
]. The fact that the length of a mouse femur increases up until the age of 8 to 12 wk argues strongly for our opinion that mice used for critical-size bone defect models should be at least 12 wk old. Lu et al.
demonstrated a sharp decline in fracture healing ability between juvenile and middle-aged animals, and a more subtle decrease in healing ability was observed between middle-aged and elderly mice [9
Immunodeficient murine animals offer enormous possibilities to investigate xenograft tissues and cells; however, the results of these investigations should be carefully interpreted in relation to the immunocompetent state and the relevance to human bone healing. Generally, mice and rats do not have a Haversian system like that of higher mammals or humans [10
]. Furthermore, adult nude mice have a marked lack of T lymphocytes [11
]. Barbul et al.
found that T lymphocytes play a dual role in wound healing [12
]. They described an early stimulatory role on macrophages, endothelial cells, and fibroblasts, and a later counter-regulatory role, which may be responsible for physiological wound healing. This may impact regenerative capacity and thereby affect bone healing. However, Gan compared the bone healing of immunodeficient nude mice with immunocompetent mice and detected no difference [13
We did not detect statistically significant differences between the three groups with respect to cell numbers stained positively with both bone resorption (TRAP) and bone formation (osteocalcin, osteopontin, and osteonectin) markers. This is not surprising, as no attempts were made to influence fracture healing. The principal mechanism of bone repair is the same at the bone ends irrespective of the defect size. However, these numbers may serve as a baseline for future experiments. It is conceivable that, with modification of the materials used to bridge the defect, the cellular reaction will be different if bone healing can be successfully influenced [14
In addition to the external fixation device used in our study, there are also alternative concepts to create a femoral bone defect in mice. Stabilization of a femoral defect site can be achieved by either intramedullary or extramedullary fixation, each with its own unique set of advantages and disadvantages. The advantages of intramedullary fixation are its ability to minimize soft tissue irritation while providing high axial stability. A disadvantage of this method is that the intramedullary nail causes irritation of the endosteum and the bone marrow. Alternatively, an external fixator [4
] or a plate [15
] can be applied to stabilize the defect site. A lateral approach must be used for this method, as the femur must be exposed from knee to femoral head and at least four pins or screws must be drilled directly into the femoral shaft. The disadvantage of this method is a potentially higher rate of periosteal and soft tissue compromise as compared with intramedullary fixation. The advantages of this method, however, are the ability to protect the endosteum and the bone marrow. Using a femoral bone graft model, Zhang et al.
demonstrated that 70% of newly formed bone mass is attributed to the expansion and differentiation of donor periosteal progenitor cells [16
]. This explains why bone healing is negatively affected when the periosteum is disrupted [17
]. Recent findings that bone marrow [18
], blood vessels [19
], and the soft tissue [20
] around the fracture are sources of osteoprogenitor cells explain why loss of these tissues has a negative impact on bone healing. These findings demonstrate that the plates used in extramedullary fixation can have a detrimental effect on bone healing, as they have a large periosteal contact area. External fixators have the advantage of preserving the soft tissue and the periosteum at the fracture site, thereby avoiding a distorting influence on bone healing.