## 4 Discussion  
This study compared conventional titanium alloy implants with F-DLC-coated titanium alloy implants to investigate the effects of F-DLC coating on bone formation in vivo. At Week 12, the specimens in Group F were in the process of uncoupling bone formation and resorption. Osteoclasts and bone resorption surface were being reduced, and bone formation was outpacing resorption. Specimens in Group C had not yet reached uncoupling. Lamellar bone, which is formed only in the remodeling phase of bone union, was generated in greater amounts in Group F than in Group C. Immature cells such as fibroblasts and fibrous osteoids were observed in Group C but not in Group F, suggesting that the remodeling phase started earlier in Group F. These findings, which suggest that bone union was more accelerated with F-DLC coated implants than with non-coated titanium alloy implants, are supported by the data showing that bone formation rate/tissue volume increase was more significant in Group F than in Group C.  
DLC coating without fluorine has been reported to promote bone union [22; 23; 24], and the bone union-promoting action of F-DLC in the present study may have been affected by the DLC in the coating as well as by the fluorine portion. [PERSON] et al. have speculated that bone union could be accelerated because the DLC coating is more hydrophilic than titanium coating [26], but that reasoning is inconsistent with the highly hydrophobic properties of the F-DLC coating. The accelerated osteogenesis seen in Group F may instead be attributable to F ions eluted from the F-DLC-coating. Supplemental experiment 2 confirmed elution of F ions from the F-DLC-coating over time. Fluorine reacts with substances such as hydroxyapatite or calcium carbonate to form fluorides such as fluorinated apatite, fluorinated calcium, and sodium fluoride. [PERSON] et al., [PERSON] et al. and [PERSON] et al. have reported that fluoride promotes bone formation by  
Figure 9: Changes over time in the amount of fluorine elution per unit area. The concentration of F ions tended to increase over time. directly stimulating osteoblasts and alkaline phosphatase (ALP) activity [27; 28; 29; 30; 31]. In this regard, [PERSON] et al. and [PERSON] et al. have reported that fluorinated apatite and fluorinated calcium inhibited bone resorption because of the resistance to acidic conditions produced by osteoclasts [32; 33]. [PERSON] et al. have reported that fluorine-containing hydroxyapatite inhibited osteoclast proliferation and promoted apoptosis of osteoclasts [34]. [PERSON] and [PERSON] have described the effects of fluoride on the crystal growth of calcium apatites as dependent on fluoride concentration [35; 36; 37; 38; 39], and [PERSON] et al. have reported that low-level sodium fluoride promoted osteogenesis [40], suggesting that fluoride affected bone formation in a variety of ways. This study showed a decrease in osteoclasts at Week 12, potentially attributable to slow elution of F ions from the implanted F-DLC coated titanium alloy. After reaching a concentration sufficient to affect the osteoclasts, the F ions may have formed fluoride compounds and inhibited bone resorption. Sodium fluoride has been reported by [PERSON] et al., [PERSON] et al. and [PERSON] et al. to demonstrate anti-inflammatory action [40; 41; 42]. During the process of bone repair after a fracture, excessive or prolonged inflammation was associated with extended cell and callus proliferation, leading to excessive callus formation and delaying the remodeling phase. These findings suggested that eluted F ions formed sodium fluoride, and that the resulting anti-inflammatory action shortened the duration of inflammation during early bone union. The shortening of the cell and callus proliferation phases may have accelerated progression to the remodeling phase in Group F.  
Fluorine is a biological trace element and exists in the body at a level of 0.0037%.