**2. Materials and Methods**

#### *2.1. In Vivo Study*

This study was approved by the Ethics Committee of the Animal Experimentation of the Institutional Animal Care and Use Committee (CRONEX-IACUC 201702003; Cronex, Hwasung, Korea). All experiments were conducted in accordance with the ARRIVE guidelines for reporting in vivo animal experiments [17]. A total of 8 male New Zealand white rabbits (age: 1–2 years; body weight: 2.6–3.0 kg) with no signs of disease were used. The rabbits were anaesthetized via 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 area of the proximal tibia was shaved and washed with betadine, and an antibiotic (Cefazolin, Yuhan Co., Seoul, Korea) was intramuscularly administered. Lidocaine was locally injected into each surgical site. The skin was incised, and the tibiae were exposed after muscle dissection and periosteal elevation. Drills and profuse sterile saline irrigation were used to prepare the implant sites on the flat tibial surface. The drilling was performed with a final diameter of 4.0 mm at the upper cortical bone, in which the implants were installed in cortical bone and medullary space. Only the V-shaped parts of the threads were engaged (Figure 1A). A total of 5 rabbits received acid-etched (SLA) implants only. Each rabbit received 4 SLA implants, 2 on each side of the rabbit tibia. Three rabbits received turned implants only, each receiving 4 turned implants, 2 on each side of the tibia. The cover screw was covered. The muscle and fascia were sutured with absorbable 4–0 Vicryl sutures, and the outer dermis was closed with a nylon suture. The rabbits were separately housed after surgery. All rabbits were sacrificed via an intravenous overdose of potassium chloride after 10 days of bone healing. After 10 days [1,18,19], the tibiae were exposed, all of the inserted implants were removed through unscrewing, and the surrounding bone was surgically removed en bloc with an adjacent bone collar and immediately placed in Karnovsky's solution for cell fixation in falcon tubes, while the specimens for fluorescence immunocytochemistry were preserved in Roswell Park Memorial Institute (RPMI) media and fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in cell culture dish.

**Figure 1.** (**A**) Simplified diagram of, and terminology regarding, the screw-shaped implants used in this study. The inner half, close to the minor diameter of the implant, was defined as the root area. The outer half, close to the major diameter of the implant, was called the crest area. The upper half of the thread was defined as the upper flank (UF), and the lower half was the lower flank (LF). (**B**) Cs-corrected transmission electron microscopy (Cs-STEM) analysis retrieved from focused ion beam specimens of the turned and (**C**) acid-etched (SLA) implants on day 10. There were no cells detected on the turned surface (yellow arrow) beneath the Pt-coated layer (black arrow), whereas, cells were detected on SLA surface (red arrow). (**D**) Confocal laser scanning microscopy (CLSM) of the turned and (**E**) SLA surfaces measured in root, UF, and LF. The turned implant revealed a smooth texture, and no cells were seen after in vivo experiment. The SLA implants displayed cell attachment in the root area, depicted as irregular structure of grey color on top of roughened topography in the 3D mapping of the CLSM.

#### *2.2. Sample Preparation and Implant Surface Modification*

Herein, 26 Ti sandblasted, large-grit, and SLA implants and 18 turned implants were used (Deep Implant System, Inc., Seongnam, Korea). The implants were made of grade 4 commercially pure Ti by computer numerical control (CNC) machining. The implant surface was called 'turned' when the surface had no further modification after CNC machining. The SLA surface was made by sandblasting the implant surface with 250–500 μm alumina particles and by etching the surface with HCl/H2SO4 acid mixture. All of the implants were 4.0 mm in diameter and 5.0 mm in length. A total of 20 SLA implants were used in an in vivo study, and 6 were used in the surface analysis, while 12 turned implants were used in the in vivo analysis, and 6 were used in the surface analysis.

#### *2.3. Surface Characteristics*

Among the 6 SLA implants and 6 turned implants used in the surface analysis, 2 of each type of implant were used for scanning electron microscopy (SEM; Hitachi S-4700, Hitachi, Tokyo, Japan), 2 were used for confocal laser scanning microscopy (CLSM; LSM 800, Carl Zeiss AG, Oberkochen, Germany), and the remaining 2 implants were used for focused ion beam (FIB; Helios 650, FEI, Hillsboro, OR, USA) and Cs-corrected transmission electron microscopy (Cs-STEM; JEM-ARM200F, Cold FEG, FEOL Ltd., Tokyo, Japan), which are capable of producing transmission electron microscopy (TEM) images directly from an undecalcified specimen. SEM was used to observe the topographical features, while CLSM was used to analyze the surface roughness levels. The measured area roughness parameters included the average height deviation value (Sa) and the developed surface area ratio (Sdr). FIB and Cs-STEM were used to observe the undecalcified implant surface directly without any resin embedding.

#### *2.4. Scanning Electron Microscopy (SEM) Analysis*

The retrieved implant specimens and surrounding bony specimens were fixed with Karnovsky's solution and washed in 0.1 M phosphate buffer saline (PBS) 3 times every 15 min. The specimens were dehydrated through a graded 70–100% ethanol series and then treated with hexamethyldisilazane for 15 min. The surrounding bone specimens were cut in half around the round hole in which the implant had been inserted, after degradation with 80% ethanol using rotary discs within an appropriate amount of time. Prior to the SEM analysis, the implant and bone specimens were sputter coated with a thin film of platinum to protect the implant and bony surfaces. All specimens were handled with Ti forceps and surgical gloves in a clean laboratory environment. Each implant and bone sample was attached using adhesive carbon tape, as well as aluminum tape, on the SEM sample stub. The samples were inserted into a Hitachi S-4700 (Hitachi, Tokyo, Japan), which was operated at 20 kV.
