A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Abstract

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

Figure 1:. External fixator fabrication.

A: CAD schematic of the assembled external fixator with annotated dimensions for proper fabrication. Each fixator is composed of two aluminum plates held together by two screws. B: Plates are cut from 1.4” x 6” aluminum sheets with “V” grooves cut into bottom sheet. C: Screw holes are drilled into the plates (threaded in the plate with “V” grooves) and all edges and corners are sanded to round and reduce weight. D: Assembled external fixator is tightened with screws (4-40 x 0.25”, 18-8 stainless steel button head cap) once pins are in place in the “V” grooves on the insides of the aluminum plates. The left pin is non-threaded and is most proximal on the femur.

Figure 2:. Schematic of pin placement, fixator placement, and defect creation.

A: The distal pin (1.0 mm threaded k-wire) is placed at the epicondyle metaphyseal region using the jig (blue rectangle) to guide proper pin insertion. The jig is placed onto the anterolateral femoral surface. B: The proximal pin (1.0 mm non-threaded k-wire) is placed using the jig after making a small incision in the gluteal tendon. C: The middle pins (1.0 mm threaded k-wire) are inserted using the jig. D: The jig is removed and the 2 plates are attached to the pins using the 2 screws to secure the plates. The plates are tightened 1 cm above skin level to avoid pressure on the skin. E: A sagittal oscillating saw is used to create a 5 mm defect between the two middle pins.

Figure 3:. Representative high-resolution radiographs show bone healing with rhBMP-2 treatment.

Images for the negative control collagen sponge and the rhBMP-2 soaked sponge groups are shown at 0, 4, and 12 weeks post-operatively. The rhBMP-2 treatment group exhibits significant healing after 4 weeks with callous spanning the defect. The negative control femur ends do not heal with bridging bone and the defect remains non-united.

Figure 4:. Significant new bone formation is seen with rhBMP-2 treatment.

Representative 4x magnified H&E histological images for the negative control collagen sponge and the rhBMP-2 soaked sponge groups both at the cut femur edge and within the defect. New bone formed around the control femur edge, but significant extensions of both new trabecular bone as well as periosteal callous project from the treated femur. No bony material is seen within the control defect, while significant bone formation can be observed throughout the rhBMP-2 treated defect. NB: new bone, F: femur, C: cartilage, H: hemorrhage, PC: periosteal callous. Scale bar: 200 μm.

Figure 5:. Infected femur exhibits hypertrophy and inflammatory cell markers.

H&E histological images of an uninfected femur compared to an infected femur, in full view and at 4x magnification of boxed locations. The uninfected femoral cortex remains organized and delineated, with little sign of inflammation. The infected femur enlarges greatly, as seen in full view, and the cortex is broken up by areas of resorption and necrosis (purple cell clusters indicated by black arrows). F: femur. Scale bar: 200 μm.

Figure 6:. Gaussia luciferase signal detected in defect.

Luminescence of cells transfected with Gaussia luciferase mRNA is imaged with IVIS after external plate removal. Red indicates the highest luminescence intensity at the site of the femoral defect.