## 2 Materials and Methods  
This study was designed to investigate the effects of F-DLC coated titanium alloy on in vivo osteogenesis and was conducted with the approval of the Animal Research Ethics Review Board at the authors' institution (Approval No. 2017-65). Twelve beagle dogs (females, 10 months of age, body weight 10 kg) were used. This experiment focused on local bone response rather than systemic response. The experiment used both femurs of each dog to minimize the number of animals used, out of consideration for animal welfare. Test specimens were of Ti-6 Al-4V metal alloy, consisting of 90% titanium, 6% aluminum, and 4% vanadium by mass fraction. The alloy is widely used in bone anchoring implants because of its high bio-affinity. The coating method was as reported by [PERSON] [21]. In this study, the coating was applied in three layers sequentially, using a radiofrequency inductively coupled plasma chemical vapor deposition (CVD) technique. The first layer of silicon carbide (SiC) coating was applied over the metal alloy and was followed by a second layer of DLC coating (a-C:H structure), and a third layer of F-DLC coating (23 at.%F+a-C:H:F structure). Coating thickness was 0.1 \(\upmu\)m for SiC, 0.1 \(\upmu\)m for DLC, and 0.5 \(\upmu\)m for F-DLC (Figure 1). The test pieces were shaped into solid columns 5 mm in diameter and 20 mm in length. Parallel slits 0.5 mm wide and 2 mm deep were made in each column along the short axis  
Figure 1: Coating thickness and structure. in 12 locations at 1 mm intervals. The test pieces were divided into the group with F-DLC coating (Group F, Figure 2a) and the control group with no coating (Group C, Figure 2b). For surgery, the dogs were placed under general anesthesia with vecuronium (0.3 mg/kg), midazolam (0.1 mg/kg), and propofol (5 mg/kg/h). The antibiotic cefazolin sodium (50 mg/kg) was administered preoperatively. Both hind legs were shaved, the hind legs and area around the tail were disinfected with isodine, and the skin was anesthesized with 0.5% lidocaine hydrochloride. A 10 cm skin incision was made in the anterior lateral margin of the femur, from the greater trochanter towards the patella level. The initial cut was made between the tensor fascia lata muscle and the quadriceps femoris muscle. The diaphysis was exposed by placing forward traction on the quadriceps femoris muscle. A \(\phi\)5.0 mm drill was used to create holes at 3 sites along the long axis in the diaphragm region of the femur (the center of the diaphysis, 2 cm proximal to the center, and 2 cm distal to the center). The diameter of each hole was then enlarged using a \(\phi\)5.1 mm rearner. During this process, the drill and reamer were cooled continuously to minimize irritation to the bone. After the holes were completed, they were washed with saline solution to remove residual tissue. Three implants from Group F or Group C were inserted into the three holes in each femur, one implant per hole, from the outside inward. In total, 6 implants were placed in each animal. At 4, 8, or 12 weeks after implantation, the dogs were placed under general anesthesia in the same manner as described for implantation. Animals were then sacrificed by intravenous injection of KCL, and femoral specimens were collected (Figure 3). At each time point (4, 8, and 12 weeks), 8 femur specimens were collected. In preparation for double staining of the bones, tetracycline had been injected subcutaneously for yellow fluorescence labeling 6 days and 2 days prior to implantation, and calcein had been injected subcutaneously for green fluorescence labeling 6 days and 2 days prior to the collection of bone specimens. Under fluorescence microscopy, it was thus possible to differentiate between bone calcified before and after implantation. Immediately after collection of the femur specimens, each bone was fixed with 70% ethanol and cut into 3 pieces, so that each piece contained one implant. Each piece was polished and stained with Villanueva Osteochrome Bone Stain to create cross-sectional femoral specimens.