Journal of Dentistry, Oral Disorders & Therapy

This study was aimed to establish the optimal effective concentration of VEGF in bone regeneration in vivo. Four male adult white New Zealand rabbits were equally divided into two experimental groups, A and B, which were also used as control. One circular critical size defect 8-mm was created bilaterally in the mandibular body area of each rabbit. Group A received composite BCP/FS with 250ng/ml VEGF on one side, and the control side received BCP/FS only. Group B received composite BCP/FS with 500ng/ml VEGF on one side, and the control side received only BCP/FS. 4 weeks later, group B showed significant increase in bone formation compared to that of group A, and least bone formation in the control.
Keywords: Critical size bone defects; Composite bone graft; Vascular endothelial growth factor

In human beings, one of the most challenging problems in oral and maxillofacial surgery is the repair of critical-sized bone defects (CSBDs) in the mandible that needs attention [1]. Tai K, et al. [1] demonstrated that treatment of CSBDs using mesenchymal stem cells (MSCs) with suitable biodegradable scaffolds is feasible. However, this approach is not effective for CSBDs due to inadequate vascularisation [2]. In order to encourage the vascularisation process, the cytokines like vascular endothelial growth factor (VEGF) act as strong promoter agent of neovascularisation for effective coupling of angiogenesis and osteogenesis of bone substitutes. The efficacy of VEGF in bone healing process depends on proper dosage, appropriate delivery system that has adequate release kinetics and high biological activity [3]. Thus, this study was aimed to establish the optimal effective concentration of VEGF in vivo. However, the VEGF has some drawbacks. In high dose administration it has negative effects on healing process [3]. Furthermore, its half- life is short when injected into the tissues [4,5]. Considering the drawbacks and challenge from CSBD, we hypothesized that enriched biphasic calcium phosphate (BCP) with VEGF and fibrin sealant (FS) can act as a scaffold with continuous release of low concentration of VEGF into the area of healing to promote neovascularisation.

All animal procedures were conducted under a protocol approved by the Universiti Sains Malaysia Animal Ethics Committee USM (AECUSM) USM/2012/ 1- July- 2012/1- July- 2014), No. of Animal Ethics Approval/USM/ Animal Ethics Approval/2012/ (81) (419). FS was purchased from Baxter Healthcare Corporation, Westlake Village, CA- 91362 USA, US License No. 140. VEGF was purchased from (GIBCO, USA). Macroporous biphasic calcium phosphate (MBCP) ceramic granules were synthesized in Ceramic Laboratory, School of Materials and Mineral Resources, Engineering Campus, Universiti Sains Malaysia (USM). The preparation of FS and VEGF was in accordance with the manufacturer's recommendations. Four male white New Zealand rabbits (Oryctolagus Cuniculus), aged between 5 and 6 months, and with a mean weight 2.63 kg ± 0.35 kg were divided into two experimental groups A and B. The experimental rabbits were also used as their own control.

Rabbits were sedated before surgery with ketamine xylazine I.M injection, ketamine 35 mg/kg and xylazine 5 mg/kg [6]. Anaesthesia was induced and maintained by administration of 1%-3% sevaflourane mixed with oxygen via a face mask. Rabbits were put in lateral position during surgery, and the heart rate, respiratory rate, pulse oximetry, and temperature of the animals were monitored. Before the operation, the area was shaved and prepared with Betadine, and a subcutaneous injection of 0.5% lidocaine with 1:200,000 epinephrine was given in the line of incision (incision line block). One circular critical-sized trephine defect 8-mm ø was created bilaterally in the mandibular body area (the premolar/ molar region) removing only the lateral bony cortex, trabecular bone, and tooth roots (partial thickness).

Group A received composite BCP/FS loaded with 250ng/ml VEGF, while the other side received only composite without VEGF (control). Group B received composite BCP/FS loaded with 500ng/ ml, while the other side received only composite without VEGF (control). The periosteal flap was repositioned and the wound closed in layers using 4-0 coated Vicryl suture. Postoperatively, each animal was observed closely by a veterinary technician and operator until the regained consciousness at which time, it was transferred to the individual cages. Subcutaneous infusion of 5% glucose water was given with oral orange juice for the initial feeding. Thereafter, the animals were fed normally. In each animal the wound site was debrided daily and cleaned with normal saline and povidon iodine for 7 days. Neomycin local antibiotic cream was applied daily for the first week to the incision site. Intramuscular injection of Baytril (Enrofloxacin), 10mg/kg/day once a day for 7 days followed. Tramadol hydrochloric 2 mg/kg once daily was given for pain relief for 7 days. The weight of the rabbits was recorded daily and any change in the dietary habits or activity was monitored closely. The animals were euthanized at 4 weeks after surgery with an intravenous injection of overdose sodium pentobarbitone (100ml/kg) and the tissues from the defect areas were collected. The samples were fixed in 4% paraformaldehyde for 12 h at room temperature. All samples were scanned for bone formation within the defect site using a Scanco system (µCT 80, Scanco Medical Switzerland). After the µCT scanning, all tissue samples were decalcified in 10% formic acid for tissue processing. All sections were cut and stained with haematoxylin and eosin (H&E) for histomorphometric analysis (Carl Zeiss image analysis system MMI 0684).