Study materials
Deproteinized bovine bone substitute material (BBSM; Bio-Oss; Geistlich Pharma AG, Wolhusen, Switzerland; granularity 1-2 mm), as well as resorbable, non-cross-linked collagen membranes (Bio-Gide; Geistlich Pharma AG, Wolhusen, Switzerland) were used. BBSM is deproteinized bovine cancellous bone with a structure similar to human bone and osteoconductive characteristics. In brief, it consists of a natural, non-antigenic, porous bone mineral matrix and it is produced by removal of all organic components from bovine bone [3, 30]. The porcine-derived type I and III collagen membrane has a bilayer structure, consisting of a compact outer layer and a porous inner layer of collagen fiber bundles [10]. Both materials, alone and in combination by means of GBR-procedures, are frequently used in preclinical as well as clinical regenerative maxillofacial surgery [3, 18, 31-33].
Experimental animal model
The study was planned prospectively in accordance to the ARRIVE guidelines [34] and the EU Directive 2010/63/EU for animal experiments. The animal experiments were approved by the Research Ethics Committee for Laboratory Animals at the University of Medicine and Pharmacy “Nicolae Testemitanu”, Chisinau, Moldova. Fourty, 9 months old, 4–5 kg, female New Zealand white rabbits were used for the in vivo experiments. All animals were treated in accordance with both policies and principles of laboratory animal care and with the European Union guidelines. The rabbits were housed in individual cages in an animal room maintained at 22°C and 55% relative humidity with ventilation 18–20 times/h and a 12-h light–dark cycle. They were allowed free access to diet and water. The treatment consisted of two surgical approaches under general anesthesia (intramuscular injections of a combination of a dose of 35 mg/kg body weight ketamine and a dose of 5 mg/kg body weight xylazine) each. Prior to any surgical intervention, local anesthetic was applied (4% articaine with 1:200.000 epinephrine (Ultracaine DS, Sanofi, Frankfurt am Main, Germany)) followed by disinfection using chlorhexidine (Chlorhexamed FORTE 0.2%, GlaxoSmithKline Consumer Healthcare, Bühl, Germany). At the first surgical step, a full thickness critical size perforating bone defect removing both cortical plates and the trabecular bone [35] (1 x 1 cm) was created at the right side of the mandible in all animals after incision of the skin and elevation of the periosteum. The wounds were closed with absorbable sutures (Vicryl 4–0 (Ethicon GmbH, Norderstedt, Germany)). Eight weeks after formation of the defect, the regeneration procedure was carried out. In brief, using the same surgical approach as in the first step, the bone was carefully skimmed with a straight fissure carbide bur under copious irrigation with sterile 0.9% physiological saline to remove remaining soft tissue and to lay open fresh bone tissue (figure 1). In no defect, osseos healing was seen and each defect was augmented using BBSM. In a randomized approach using a computerized list, the animals either received a collagen membrane to cover the BBSM-containing defect (group +; n=20) or none such membrane (group -; n=20). The membranes were put under the periosteum and no further fixation was conducted. The mucoperiosteal flaps, muscles, subcutaneous tissue and skin were advanced, repositioned anatomically and fixed via interrupted and mattress sutures with Vicryl 4–0. Postoperatively, Ibuprofen (2-10 mg/kg body weight orally) was used for analgesia.
Measurements
Bone turnover marker
The outcomes of bone regeneration were measured by activities of total acid phosphatase out of the peripheral blood (TAcPh; figure 2), bone acid phosphatase out of the ground bone from the augmented site (BAcPh), total alkaline phosphatase (TAlPh) as well as bone alkaline phosphatase (BAlPh). The peripheral blood (TAcPh; (TAlPh) as well as the bone (BAcPh; BAlPh) samples were collected immediately after 24 and 72 hours, 7 days, 2 weeks and 3 weeks after surgery with n=4 samples per group. Blood samples were obtained from anesthetized animals before sacrifice. Animals were sacrificed with an intravenous overdose of pentobarbital (100 mg/kg body weight). Half of the augmented sites were removed en bloc, ground into particles and processed for enzyme analysis, using standard kits and following the manufacturer’s protocol (Acid Phosphatase Assay Kit CS0740, Sigma-Aldrich, Taufkirchen, Germany; Alkaline Phosphatase Detection Kit AFP, Sigma-Aldrich, Taufkirchen, Germany). The serum was separated by centrifugation at 3000g for 10 min at 37 °C. Aliquots were stored at 80°C in appropriate cuvettes.
Histology
Using the other half of the samples, histological analysis on formation of new bone was carried out. For this purpose, following fixation in 4% buffered formaldehyde and dehydration, the specimens were embedded in a 1:1 combination of glycol-methacrylate and ultraviolet light-activated polymethyl-methacrylate (Technovit 7200® VLC; Heraeus Kulzer, Hanau, Germany) for 5 days. After penetration of the whole specimen by Technovit 7200® VLC, the slides were carefully photopolymerized and processed applying the sawing and grinding technique [36] by using the microgrinding system EMS (Exact, Norderstedt, Germany) to a thickness of 10 – 20 μm and stained with toluidine blue. Finally, five representative cut and ground sections from the core for each defect were digitized using a color scanner with a resolution of 2400 dpi. Additionally, an empty histology slide with millimeter scale was scanned for calibration. The pictures were digitally edited with imaging software (Adobe Photoshop CS, Adobe Systems Software Ireland Ltd., Dublin, Ireland) in order to amplify the contrast between remaining bone graft material and soft tissue by color-coding. The slides were evaluated histomorphometrically using the computer software Analysis® (Soft-Imaging-Systems, Münster, Germany). For each slide, the amount of new-formed bone within the augmented matrix (%) was analyzed as described before [16]. For each defect, mean values were created out of 3 slides and used for further calculations. All measurements were therefore performed in triplicates. Examiners were blinded to the kind of augmentation.
Statistics
The study was carried out as a pilot study as there was no prior analysis on enzymatic activities after guided bone regeneration procedures in the literature. The case number of n=4 per time point and group is comparable to other animal studies reporting GBR-procedures [3, 37, 38]. Comparisons were conducted between the membrane groups and between the different time periods for each group. A non-parametric Kruskal–Wallis test was used to identify statistical differences between the experimental groups or the time points. Whenever a statistical difference was found, the Mann–Whitney test was applied. Analyses were made using SPSS Version 24 software (SPSS, Inc., Chicago, USA) and the significance level was set at p<0.05. The data are presented as the mean ± standard deviation. P-values <0.05 were described as ‘‘statistically significant’’, although no adjustment for multiple tests has been applied and the p-values are reported descriptively only.