Water-soluble fullerene (C60) inhibits the osteoclast differentiation and bone destruction in arthritis

Abstract

Recently, it has been demonstrated that oxygen free radicals have an important role as a signaling messenger in the receptor activator NFkappaB (RANK) signal pathway required for osteoclast differentiation. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against the RANK-induced osteoclastogenesis and osteoclastic bone destruction in arthritis, both in vitro and in vivo. The effects of C60 on the RANK-induced osteoclastogenesis and osteoclastic bone resorption were examined in vitro. Adjuvant-induced arthritic rats were used as an animal model of arthritis. Rats were divided into two subgroups: control and treatment with C60 at 1.0 microM. The left ankle joint was injected intra-articularly with water-soluble C60 (20 microl) in the C60-treated group, while, as a control, the left ankle joint in the control rats received phosphate-buffered saline (20 microl) once weekly for eight weeks. Ankle joint tissues were prepared for histologic analysis. C60 significantly inhibited the responses of osteoclast precursor cells to RANK ligand, including osteoclast differentiation and osteoclastic bone resorption in vitro. In adjuvant-induced arthritic rats, intra-articular treatment with C60 in vivo reduced the number of osteoclasts and alleviated bone resorption and destruction in the joints, while control ankle joints showed progression of joint destruction with time. These findings indicate that C60 downregulates the RANK-induced osteoclast differentiation and is a potential therapeutic agent for inhibition of osteoclastic bone destruction in arthritis.

Keywords: antioxidant; bone resorption arthritis; fullerene; osteoclast.

Figure 1

Inhibitory effects of C60 on the osteoclastogenesis. Osteoclast precursor cells were incubated with osteoclast differentiation factors in the presence or absence of water-soluble C60 (0.1, 1.0, or 10.0 μM) for seven days. Data from four independent experiments were analyzed. A) At the end of incubation period, the osteoclastogenesis was analyzed by the tartrate-resistant acid phosphatase (TRAP) staining. Representative images A) showed that C60 at 10.0 μM inhibited the differentiation of osteoclast precursor cells into TRAP-positive multinucleated giant cells. B) There was a significant difference in the TRAP positivity between the control group and the 10.0 μM C60-treated group (P < 0.01 compared to the control).

Figure 2

Inhibitory effect of C60 on osteoclastic bone resorption in vitro. A) Osteoclast precursor cells were incubated with osteoclast differentiation factors in the presence or absence of C60 (0.1, 1.0, or 10.0 μM). At the end of the incubation period, the ability of osteoclasts to resorb pits was assessed by the total pit areas on ivory dentine slices. Data from four independent experiments were analyzed. As the representative images, C60 (1.0 and 10.0 μM) inhibited the pit formation resorted by the cells. B) There were significant differences in bone resorption between the control and the C60-treated groups (P < 0.01: 1.0 μM C60-treated group, P < 0.01: 10.0 μM C60-treated group).

Figure 3

Intraarticular treatment of C60 inhibits the osteoclastic bone resorption in the arthritis model rats. A) As shown in the representative images of the joint damage at the six-week time point in arthritis model rats, joints that had been treated with phosphate-buffered saline showed severe joint destruction and bony ankylosis. In contrast, inhibitory effects were observed following administration of C60 in the C60-treated group. B) The expression of osteocalstic cells was histologically evaluated in each ankle joint sample (four rats per time point). Although the expression of osteoclastic cells in the joint progressed with time in both control and C60-treated groups, the C60-treated group showed significantly lower expression of osteoclastic cells at four and six weeks compared to the respective control group. C) The degree of bone erosion was histologically scored using the bone erosion score. Bone destruction was graded from 0 to 3, where 0 = normal, 1 = mild loss of cortical bone at few sites, 2 = moderate erosion of cortical and trabecular bone, and 3 = marked destruction of bone at many sites. The C60-treated group showed significantly lower bone erosion at four and six weeks compared to the respective control group.