Animals
Twenty-four (24) male Nigerian local rabbits with body weight 2.5±0.5kg were acquired for the purpose of this study and were allowed to acclimatize for two weeks. They were randomly divided into three groups; ABG, NC and CDBM group. Ethical approval was sought from the Faculty Animal Research Ethics Committee of the Faculty of Veterinary Medicine, Usmanu Danfodiyo University, Sokoto with the reference number UDUS/FAREC/2019/AUP-R0-5.
Preparation of CDBM
Long bones of goat were collected from the slaughter-house for the preparation of DBM. All soft tissues were removed and the bones were cut into 1cm pieces with a saw. The pieces were decalcified in 0.6 mol/L HCl (BIC Chemicals, Maharashtra, India) at 4°C for 8 days as described by Monazzah et al. [24]. After the eight days, the DBM were washed with distilled water, packed and stored at 4°C till use.
Critical bone defect model and bone graft implantation
Anaesthesia was achieved using an intramuscular injection of ketamine (Ketalar®, International Limited, Lagos, Nigeria) (100 mg/kg, IM) and xylazine hudrochloride (XYL-M2®, VMD, Arendonk, Belgium) (50 mg/kg, IM). The left forelimbs were clipped and prepared aseptically for surgery using chlorhexidine (Purit®, Saro Lifecare Ltd., Lagos, Nigeria), methylated spirit (La Onyz®, Samstella Nigeria Limited, Abule Oba, Nigeria) and povidone iodine (Wosan®, Jawa International Limited, Lagos, Nigeria). The limbs were draped with sterile draping materials.
A skin incision was made over the ulnar bone, and the bone was exposed by retracting the extensor muscles. A non-union bone model, an osteoperiosteal segmental defect of approximately 1 cm was created on the ulna bone according to standard procedure previously described [12, 24, 25].
In ABG, the defected areas were filled with same ostectomized bone to serve as autologous graft, the defects were left unfilled in NC and the defect was filled with same length of caprine DBM in CDBM. The grafts were secured in place with the surrounding muscles and sutured using simple continuous suture pattern with chromic catgut (Agary Pharmceuticals, Jiangsu, China) size 3/0. The skin was sutured using interrupted horizontal suture pattern with nylon (Agary Pharmceuticals, Jiangsu, China) (size 2/0).
Post operative analgesia and antibiosis were achieved using diclofenac sodium (Yanzhou Xier Kangtai Pharmaceutical, Yanzhou, China) at 3mg/kg intramuscularly for three days and Penicillin-Streptomicin (Hebei Hope Harmony Pharmaceutical Co. Ltd., China) at 25mg/kg intramuscularly for five days.
Radiological Evaluations
For radiological evaluation, radiographs of the defected areas were taken immediately post-surgery to ascertain the defect and correct filling of the defected area. Subsequently, the radiograph was taken at 14th, 28th, 42nd and 56th postoperative days. Radiological criteria evaluated were bone formation, bone union, and remodelling. The radiological findings were scored with modification according to radiological scoring earlier reported [17, 26, 27]. All radiographs were taken under light sedation (xylazine, 25 mg/kg) to prevent animal movement during exposure. The radiographs were evaluated by two independent radiologists that were blinded with the experimental design.
Histopathological Evaluations
On the 56th postoperative day, the experimental animals were euthanized by means of cervical dislocation after anaesthesia for histopathological evaluations [28]. The operated limbs were harvested and the bones were dissected free of soft tissue. The defect sites and the graft were ostectomized and fixed in 10% buffered formalin. The formalin-fixed bone samples were transferred into 0.6mol/L HCl for eight days at 4oC for decalcification [17]. Standard histologic processing and Haematoxylin and Eosin staining technique was carried out as stated by Bigham-Sadegh and Oryan [12]. The slides were examined under a light microscope and photomicrographs were taken. All histopathological sections were evaluated and scored using Emery histopathological formation criteria as reported by Bigham et al. [29]. The histological evaluation was conducted by two independent histopathologists that were blinded to the experimental design.
Statistical Analysis
All retrieved data from radiological scoring were analyzed using a two-way ANOVA repeated measures mixed model approach with rank transformation of the scores prior to analysis to stabilize the variance. The histopathological data were analyzed using Kruskal-Wallis, non-parametric ANOVA and significance level was determined when P value is < 0.05, pair-wise group comparisons was performed using Mann-Whitney U Test (InVivoStat version 4.0.2).