Is an Impacted Morselized Graft in a Cage an Alternative for Reconstructing Segmental Diaphyseal Defects?

We used 12 Dutch milk goats with 24 femurs (female, Capra Hircus Sana; weight range, 57–64 kg), dividing the 24 femurs into four groups of six femurs (cage reconstruction, cortical graft reconstruction, nonoperated side of the cage group, nonoperated side of the cortical group). We performed a power analysis based on the relevant variable torsional strength. The variation was set at 8.75 Nm, which was based on results of a pilot study and on previous experiments in the femur of goats [15]. The difference in torsional strengths between groups was set at 15 Nm, which is approximately 15% of the normal strength. The relevance of this difference for prediction of fracture risk is difficult because in the test animals, the central nail is still in situ during daily activity, which will contribute substantially to the torsional strength. The expected variation in a group and the difference between groups led to a group size of six goats in this study. All animals were skeletally mature.

The cage was a stainless steel mesh (X-Change system; Stryker Howmedica, Newbury, UK) that was cut to a height of 4.5 cm and folded into a cylinder with a diameter of 22 mm (average diameter of the shaft of a goat femur). This mesh was fixed with three cerclage wires to stabilize the cylinder. We harvested the morselized allograft used to fill the cages from the donor goats’ sternum. The cancellous bone was morselized by hand using a rongeur that produced small (1–3 mm), pure cancellous bone chips. We subsequently processed the bone chips in a Noviomagus bone mill (Spierings Medische Techniek, Nijmegen, The Netherlands) with the smallest rasping blade leading to a particle size of approximately 2 mm. Each batch of cancellous allograft was washed using 2 L of saline with a pulse lavage jet system (Stryker Howmedica). Each cage was filled with five layers of morselized bone graft. The total amount of bone graft used in each reconstruction was approximately 28 g. After each layer, 10 standardized impactions were made with a mass of 1.5 kg that was dropped from a height of 35 cm. The last layer was followed by 40 similar impactions, after which the impacted graft in the cage had a height of 35 mm. At each end of the 45-mm high cages, 5 mm of the cage was left unfilled on both sides, which enabled the host bone to slide into the cage (Fig. 1). During impaction, a central nail was positioned in the cage to reserve space for introduction of the intramedullary nail in the animals.

Under general anesthesia using pentobarbital (doses 0.5 mL/kg, pentobarbital 60 mg/mL), each animal was operated on unilaterally. The goat was positioned on the left side with the right femur fixed. The right leg was shaved and sterilized with Betadine (Mundipharma AG, Basel, Switzerland). Using a lateral approach, the femur was exposed through an 8-cm skin incision. The periosteum was opened with a longitudinal incision and bluntly elevated circumferentially with the overlying soft tissues and left in situ. Subsequently, a 3.5-cm segmental defect was created by two osteotomies performed 7 and 10.5 cm proximal of the lateral joint space line of the knee with an oscillating saw under constant cooling with saline solution. The proximal and distal parts of the femur were marked to prevent rotation malunion. The resected diaphyseal segment of the femur was removed from the operated site in six goats and after 15 minutes placed back in the original position (Fig. 2). In the other six goats, the cortical segment was replaced with a cage filled with morselized impacted bone graft (Fig. 3). The lateral approach was extended with a lateral parapatellar arthrotomy to the knee and the patella was medially subluxated. In the intercondylar femoral notch, the cortex was perforated with a 3-mm drill. With a guide thread, the intramedullary axes were determined. The perforation was enlarged to 11 mm. For fixation, a custom-made unreamed femur nail was used. This 16-cm long, 10-mm thick stainless steel nail was bent preoperatively, meeting the average curvature of the goat femur that was measured on lateral radiographs of 40 different goat femurs. The nail was inserted retrograde using the insertion handle connected to the proximal end of the nail. In the group with the massive cortical graft, the nail was locked in a static way by two proximal and two distal screws, thereby following the most commonly applied technique. In the cage group, the nail was dynamically locked to load the bone graft (Fig. 1). The handle with a custom-made aiming device was connected to the proximal end of the nail which avoided the use of an image intensifier for insertion of the nail and AO locking screws (Synthes, Davos, Switzerland). The soft tissues, including the periosteum, were closed over the defect. The knee cavity was irrigated with saline solution to remove blood clots, and the capsule was sutured. Skin wounds were closed intracutaneously. Antibiotic prophylactics consisted of ampicillin subcutaneously administered for 5 days postoperatively (doses 7.5 mL a day, Albupen 100 mg/mL; Intervet, Boxmeer, The Netherlands).

After the operative procedure, the goats were kept in a hammock for 1 week to prevent postoperative complications and improve wound healing. After the first week, they were allowed unlimited weightbearing. After 2 weeks, the animals were transported from the central animal facility to an outdoor farm. Temgesic (Buprenorfine) at a rate of 0.018 mL/kg was used as an analgesic three times a day for the first 2 days after surgery and thereafter when necessary. To assess new bone formation on histologic sections, each goat received fluorochromes, a subcutaneous injection of calcein green solution (20 mg/kg) at 13 weeks postoperatively for 2 days, and alizarin (30 mg/kg) solution for 2 days before the animals were euthanized.

Radiographs were taken 0, 12, and 26 weeks postoperatively using general anesthesia. The lateral and anteroposterior views of the femur were judged (by BS, MWM) for callus formation, alignment, fixation failure, and disappearance of the host bone-graft junction. The gait of the goats was monitored with the score originally developed by Ypma, in which 0 = not used at all, 1 = supported incidentally, 2 = loaded in a standing position and incidentally while walking, 3 = loaded in a standing position and walking but with a limp, and 4 = normal walking and standing pattern [21].

After 26 weeks, all goats were euthanized using an injection of 20 mL of 200 mg na-pentobarbital/mL into the jugular vein. The operated and contralateral femora were removed and dissected free from all soft tissues. Our institution approved the animal protocol for this investigation, and all investigations were conducted in conformity with ethical principles of research.

After removal of the nail and screw fixation, biomechanical and histologic analyses were performed on the same specimens. The proximal and distal ends of the operated and contralateral femurs were embedded in acrylic cement (AutoPlast; Candulor AG, Wangen, Switzerland) in such a way that a diaphyseal segment, with the former defect located in the center, was free with a margin of 3 cm distal and proximal to the former osteotomy site. The specimens were mounted in a Materials Testing System machine (MTS GMBH, Berlin, Germany), in which the distal femur in cement was loaded in external torsion and the proximal cemented end of the femur was fixated except for axial translation. Throughout the experiment, the specimens were kept moist with Ringer’s lactate (0.9%) at a room temperature of 20° C. All femora were tested for torsion to failure at a rate of 2° per second [15]. The parameter chosen to reflect torsion strength was torque at failure. Torsion strength of the operated femur was expressed as a percentage of torque at failure relative to the contralateral, nonoperated femur of the same animal. Therefore, 100% torsional strength indicated the same strength as the intact case. On the first noticeable crack in the bone, the test was immediately stopped to allow histologic analysis of the whole intact structure.

All specimens were fixed in buffered (0.1 mol/L phosphate buffer, pH 7.4) paraformaldehyde (4%) for at least 1 week. Thereafter, at three levels, thick slices were made for nondecalcified histologic analysis (see Fig. 4 for a schematic representation of the section planes). Of the structural grafts, decalcified histologic analysis was done of adjacent thick slices for quantification of necrosis and bone remodeling. In addition to the cross sections, two 1-cm thick slices were made of the former osteotomy site of the reconstruction with the host bone (Fig. 4). These latter slices were made parallel to the long axis of the bone. All slices were embedded in polymethylmethacrylate. Thin decalcified sections (7 μm) were stained with hematoxylin and eosin. Thicker nondecalcified sections (20–30 μm) were made with a sawing microtome (Leica SP 1600; Leica Microsystems Nederland BV, Rijswijk, The Netherlands) and left unstained for fluorescence microscopy or stained with hematoxylin and eosin.

All sections from each reconstruction were evaluated (LD, PB) by light microscopy, including fluorescence microscopy, for a qualitative assessment of the incorporation process. The quantification of the total bone area inside and outside the cage (only in the cage group) was performed on three slices at equal distance from each other (spaced approximately 875 μm from each other) resulting in one level exactly in the center of the defect and two between the center and osteotomy planes. These cross sections were photographed digitally with an 8-megapixel camera at low magnification and mounted in the computer into a composition of the entire slide. With a digital image analysis system (Soft Imaging GmBH, Münster, Germany), the surface area of the bone inside and outside the cage was measured. In addition, the extent of necrosis in the structural graft was quantified. In each cross section, in four images (one in each quadrant) at a magnification of 10×, the surface area of new bone was measured interactively on digitized images and expressed as a percentage of the surface area of the total bone area. In nonstained thick sections, the distance of bone ingrowth was quantified in four locations in each section based on the penetration of fluorescence labels of calcein green (front at 13 weeks) and alizarin (front at 26 weeks). The torsional strength of the four groups (cage reconstruction, cortical graft reconstruction, nonoperated side of the cage group, and nonoperated side of the cortical group) was compared using an analysis of variance test to show differences between groups followed by a post hoc t-test (Tukey).