Design and methodology
In this study, New Zealand white male rabbits (Oryctolagus cuniculus; n = 5) were used. Prior to this study, approval was obtained from the animal ethics committee of the Animal House of the University Malaya Medical Centre (UMMC). All the rabbits were maintained in accordance with the Institutional Animal Care & Use Committee approved protocol.
The rabbits selected for the study were all males, between 28 and 32 weeks of age, and within the weight range of 2.0–2.5 kg at the time of the surgery. All of the rabbits had free access to pellet food and water ad libitum and were housed in the animal experimental block of the UMMC at a constant temperature of 26–28 °C and a humidity of 60 %. The rabbits were caged individually in stainless steel cages measuring 9 cubic meters, employing a fenestrated flooring to allow the feces or droppings to fall into a collection pan.
All the instruments and gauze employed in the surgical procedure were sterilized by autoclaving in the Aesculap 420 laboratory and Eschmann SES 225B autoclave. The instruments were first washed under running water with disinfectant soap and sterilized at a pressure of 220 kPa and a temperature of 134 °C for approximately 15 min. Then they were automatically dried by the autoclaving machine and left for one hour to cool down.
All the surgical procedures were performed in the Animal Theater in the Animal House. Marking the ears of all the rabbits was done with a number assigned to them using a permanent marker for identification. All the cages were also labeled according to their respective numbers. The animals were weighed prior to the surgical procedure, and the amount of anesthesia (Ketamine 20 mg/kg IM) was calculated including the sedative and muscle relaxant, Xylazine (3 mg/kg). Then the antibiotic (Kombitrim 240, 1 ml/10 kg IM) was injected intra-muscularly. The left fore-leg of the rabbit was shaved. All the rabbits gained 0.1–0.3 kg on average throughout the experiment.
After anesthesia had been achieved and the limb shaved, the rabbit then was placed on the operating table in the supine position. The left fore-leg was then cleaned with povidone and properly draped with disposable sterile drapes. The surgery was then performed using the aseptic technique taking adequate precautions all the time.
The center of the ulna bone at the diaphysis was identified and marked on the skin surface. Local anesthesia was administered to the rabbit at the planned incision site. Next, 1.5 cm long longitudinal incisions were made. The intramuscular septum was identified and the mid-shaft of the ulna was exposed. Soft tissues were gently dissected to expose the bone. A longitudinal incision 4 cm in length was made on the anteromedial surface of the left ulna at the cutaneous border, 2 cm proximal and 2 cm distal to the center of the diaphysis which had been marked prior. The fascia was then cut and the extensor muscles were identified and retracted to expose the ulna bone. Care was taken to split the muscular layer by blunt dissection to expose the ulnar bone. The planned site for the osteotomy was marked on the bone, and measured with a small stainless steel ruler. The osteotomy was then performed using an oscillating saw, carefully avoiding damage to the periosteum. Washing was done with some normal saline during the osteotomy. A 5 mm defect was created in each ulna. All the rabbits tolerated the surgery well and resumed their normal activity within a few days. No wound infection was observed. All the rabbits were freely ambulating one day post-surgery.
No neurological insults such as paralysis, convulsion, respiratory distress or signs of pain were observed.
Each rabbit was supplemented (orally) with 5 ml of strontium ranelate solution (2 grams of strontium ranelate mixed in 10 ml of distilled water) in the treatment group using a feeding tube for six weeks.
After necropsy, the radius and ulna were harvested and the soft tissue removed. The bone defect site was observed macroscopically and photographs were also recorded.
X-ray and CT scan
The bone defect healing of all the rabbits was evaluated using the X-ray (DRX-Evolution, 50kVp, 2mAs Carestream, Malaysia) and CT scan. Scanning was done at weeks 1, 3 and 6 for each rabbit. They were scanned weekly for six weeks to determine the progress of defect healing in each group. While under general anesthesia, the rabbits were positioned accurately on the CT scan machine (Siemens Somatom Definition AS 128, Germany) in the right lateral position with the left fore-leg elevated on a sample holder. The long axes of the radius and ulna were aligned orthogonally to the axis of the x-ray beam. All the radius and ulnar bones were scanned using 35.5 μm isotropic resolutions at 80 kVp energy and 60 μA intensity in 150 ms integration time. Images had 0.6 mm slice thickness and resolution of 2048 × 2048 pixels. Images obtained from the scan were saved in DICOM (Digital Imaging and Communication in Medicine) format. The assessment such as calcification, appearance of callus, continuity of bone trabeculae was done by a Senior Orthopedic Surgeon who was blinded using the same score.
Qualitative bone growth evaluation
The radiographs were evaluated objectively and subjectively in collaboration with a consultant orthopedic surgeon. They were evaluated for initial and bridging callus formation. The 3D reconstruction images were obtained for better viewing and bone regeneration evaluation. For this study, we defined union based on the radiographic evaluations. Union was considered to have been achieved in the rabbit ulnar model if visible bridging callus was observed across the fracture on the radiographs, which was grade 4 and above on the Cheung (2000) criteria. Bridging callus needed to be visible in three cortices on two views on the x-ray. We could not do a clinical assessment for the fracture union because the radius was intact and the rabbits were able to weight-bear immediately post -surgery. The pattern and behavior of the bone growth in each group was also evaluated and analyzed from the radiographs enlisting the help of a radiologist.
Serum levels of osteocalcin, ALP, calcium and phosphate at weeks 1, 3 and 6 were obtained via the ear vein and centrifuged at 10,000 x g for 10 min. The plasma was then analyzed for osteocalcin, ALP, calcium and phosphate concentrations using the corresponding kit according to the Standard Operating Procedure in the Tissue Engineering Group Laboratory in the National Orthopedic Center for Excellence and Learning (NORCERAL), University of Malaya. The results were measured with the respective optical densities for each parameter on a microplate reader.
At the culmination of the study after necropsy, the radius and ulnar bones were harvested and the specimens prepared for histopathological slides. Samples for histological evaluation were stored in 10 % neutral buffered formalin for four days and then decalcified for five days in 10 % formic acid. The samples thus processed were embedded in paraffin, sectioned longitudinally and stained with hematoxylin-eosin (Sigma USA). The sections were examined under a light microscope with magnification. The histopathological sections were examined by a single pathologist who was also blinded regarding the content of the current study. The progression of the fracture callus in each specimen was quantified based on the relative percentages of the lamellar bone, woven bone, cartilage, fibrous tissues, endochondral ossification and intramembranous ossification. The qualitative bone growth data were collected using x-ray and CT scan. Statistical analysis was done using the Fisher Exact Test.