In vivo study and histomorphometry
This study was approved by the Animal Research Committee of Seoul National University, Seoul, Korea. All animal experiments were conducted in accordance with the guidelines of the Institute of Laboratory Animal Resources at Seoul National University (approval number: SNU-130619-4) and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for reporting in vivo animal experiments [7]. Five male New Zealand white rabbits (age, 1–2 years; body weight, 2.6–3.0 kg) with no signs of disease were used. All rabbits were anesthetized via an intramuscular injection of tiletamine/zolazepam (15 mg/kg, Zoletil® 50, Virbac Korea Co., Ltd., Seoul, Korea) and xylazine (5 mg/kg, Rompun™, Bayer Korea Ltd., Seoul, Korea). Before surgery, the skin over the proximal tibia was shaved and washed with betadine, after which an antibiotic (Cefazolin, Yuhan Co., Seoul, Korea) was administered intramuscularly. Lidocaine was injected locally into each surgical site. The skin was then incised, and the tibiae were exposed by muscle dissection and periosteal elevation. Drills and profuse sterile saline irrigation were used to prepare implant sites on the flat tibial surface.
In 4 rabbits (the handling of the fifth rabbit is described below), drilling was performed bicortically, with final diameters of 4 mm at the upper cortical bone (to prevent physical involvement, including friction) and 3 mm at the lower cortical bone (to promote stability). If used, the Ti tubes were inserted prior to implant placement; otherwise, implants were installed directly into the tibial bone as shown in Figure 1B. Each of the 4 rabbits received 2 sets of implants: 2 were SLA implants and 2 were modSLA implants. One SLA implant and 1 modSLA implant were inserted within Ti tubes. Thus, in total, 8 SLA and 8 modSLA implants were inserted in the 4 rabbits, and 4 implants of each group were encased in Ti tubes. The implants were inserted into the right and left tibiae of each rabbit (Figure 1C) according to the split-plot design shown in Figure 1D. The muscle and fascia were then sutured with resorbable 4-0 Vicryl sutures, and the outer dermis was closed with a nylon suture.
The rabbits were housed separately after surgery. After a 4-week period of bone healing, they were anesthetized and sacrificed via an intravenous overdose of potassium chloride. The tibiae were exposed, and the implants were surgically removed en bloc with an adjacent bone collar. The implants/bone were immediately fixed in 10% neutral formaldehyde. Histomorphometry specimens were prepared by embedding the implants and bone in light-curing resin (Technovit 7200 VLC, Kultzer, Wehrheim, Germany) [8] and then treating them according to the method described by Donath and Breuner [9]. Thus, undecalcified specimens were ground to a thickness of 50 μm, stained with hematoxylin and eosin (H&E), and subjected to a general histological evaluation under a light microscope (Olympus BX, Olympus, Tokyo, Japan). The image analysis software (Kappa PS30C Imagebase, Kappa Opto-electronics GmbH, Gleichen, Germany) that was associated with the light microscope was used to calculate the bone-to-implant contact (BIC) ratio for each implant. Thus, a stretch of the implant surface measuring 2 mm in length was examined to determine how much of that length was in direct contact with bone. It should be noted that the top of the 2-mm stretch was level with the upper surface of the cortical bone (Figure 1E). The BIC ratios of the left and right sides of each implant were determined. The mean BIC ratio for each implant was then calculated.