Guidelines regarding the care of animal research subjects were strictly followed the ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital, Korea (IACUC No. BA1205-104/034-01). Researches support that canines show more promise as animal models for the testing of bone implant materials because their bone structures had more similarities to the humans. Beagle was selected as animal models.
Animal preparation and surgical procedure
Four beagle dogs (5-6 months old, weighing 8-10 kg in good health) were used in the study. Beagles were fed with commercial diet (Dog Chow GoldPet, #35520, Cargill Agri Purina, Inc., Pyungtaek, Korea) and housed in individual cages. The animals were prepared with a subcutaneous injection of atropine 0.005 mg/kg (Daihan Pharm. Co., Ansan, Korea) under supine position and anesthetized with an intramuscular injection of zoletil (Zoletil50, Virbac S.A., Carros, France) 5.0 mg/kg and xylazine (Rompun, Bayer Korea, Ansan, Korea) 0.2 mg/kg after 15 min. After endotracheal intubation of a 6.5-sized tube, general anesthesia was maintained with enflurane 2.2% (JW Pharmaceutical, Hwasung, Korea) and oxygen level at 3.0 l/min. Animals were injected intramuscularly with cefazolin 30 mg/kg (Chongkundang Pharm, Cheonan, Korea) before surgical procedures.
The surgical field was scrubbed with povidone-iodine solution. Local anesthesia for hemostasis using 2% lidocaine with 1:100,000 epinephrine (Yuhan Co, Ltd., Seoul, Korea) was injected on both submandibular areas. Skin incisions were made on submandibular area, and subperiosteal dissection was raised to expose lateral surface of mandibular body area. A standardized 4 round bone defects of 8.0 mm size simulating cystic lesion with bony window using large trephine bur on both mandible [18]. Each depth of defect was made until the lateral cortex was completely removed. Cold saline irrigation was performed to prevent heating by the burr (Fig. 1a)
Each bony defects was filled with OSTEON II (HA:β-TCP = 3:7, Genoss Co., Suwon, Korea) The experimental defects were covered Osteoguide membrane, and the others were closed without membrane coverage (Fig. 1b and c). All surgical sites underwent primary closure using polyglactin 4-0 (Vicryl, Ethicon, Menlo Park, CA). Post-operatively, 1.0 ml of dexamethasone-21-isonicotinate (Voren, Boehringer Ingelheim Korea Ltd., Seoul, Korea) was injected for once, and clemizole penicillin G and sodium penicillin G (Antipen-SM, WooGene B&G Ltd., Seoul, Korea) 1.0 ml/10.0 kg were injected for three times on every third day. Two beagles were sacrificed each time on 4 and 8 weeks later through formalin perfusion (Fig. 1d).
Histologic analysis
Block sections (8 × 8 × 5 mm) including grafted sites were harvested. The sections were fixed with 10% buffered neutral formalin (Sigma Aldrich Co. LLC., St. Louis, USA) for 2 weeks and decalcified in formic acid (Shadon TBD-1, Thermo Fisher Scientific Inc., Kalamazoo, USA) following water rinse. The specimens were decalcified in tissue processor (Shadon Citadel 2000, Thermo Fisher Scientific Inc., Kalamazoo, USA) and embedded in paraffin with embedding center (Shadon Histocentre 3, Thermo Fisher Scientific Inc., Kalamazoo, USA). Serial sections of 3.0 μm in thickness were cut using Microtome (Shadon Finesse 325, Thermo Fisher Scientific Inc., Kalamazoo, USA). And each specimen was stained with hematoxylin and eosin (H&E).
And dehydration of the specimens was done in ethanol. Specimens were embedded with ethanol mixed resin solution (Technovit 7200 VLC, Heraeus Kulzer, Hanau, Germany). Blocks were fixed in light polymerization unit (EXAKT 520, EXAKT Technologies Inc., Oklahoma City, USA) and sections of 300 μm thickness were cut using EXAKT cutting system (EXAKT 300 CP, EXAKT Technologies Inc., Oklahoma City, USA) and grinded in 35 μm thickness. Each specimen was stained with H&E and Goldner-Trichrome stains.
To obtain histomorphometric measurements, image was acquired from a light microscope (BX51, Olympus Co., Tokyo, Japan) connected with computer, CCD camera (SPOT Insight 2Mp scientific CCD digital camera system, Diagnostic Instruments Inc., Sterling Heights, USA), and adaptor (U-CMA3, Olympus Co., Tokyo, Japan). SPOT Software V4.0 (Diagnostic Instruments Inc., USA) was used for the image analysis. Tissue sample images were taken at low magnifications for histologic examination (× 12.5) and histomorphometric measurement (× 40). New bone formation was measured using Pro Plus® (Media Cybernetics Inc., Warrendale, USA) analysis software.
For histometric evaluation in this experiment, because OSTEON II was distributed irregularly in the entire treatment area, it is impossible to accurately evaluate the area of the treatment site, which is a conventional measurement method. Newly formed bony volume (BV) was evaluated within 12 mm2. The boundary between the defect and the normal bone is first set, and the area of the newly formed bone is calculated as the percentage of the total area of the defect. Remaining grafting volume (RG) was evaluated as same as BV (Fig. 2).
In order to compare the differences in the degree of osteogenesis according to the presence or absence of membrane in the control and experimental groups, the Mann-Whitney test statistical analysis was performed at the significant probability of 95% using statistic program SPSS, version 25.0 (SPSS Inc., Chicago, IL, USA).