RANKL inhibition through osteoprotegerin blocks bone loss in experimental periodontitis

Abstract

Background: Prevention of alveolar bone destruction is a clinical challenge in periodontal disease treatment. The receptor activator of nuclear factor-kappa B ligand (RANKL) inhibitor osteoprotegerin (OPG) inhibits osteoclastogenesis and suppresses bone resorption.

Methods: To study the effects of RANKL inhibition on alveolar bone loss, an experimental ligature-induced model of periodontitis was used. A total of 32 rats were administered human OPG-Fc fusion protein (10 mg/kg) or vehicle by subcutaneous delivery twice weekly for 6 weeks. Negative or positive controls received no treatment or disease through vehicle delivery, respectively. Biopsies were harvested after 3 and 6 weeks, and mandibulae were evaluated by microcomputed tomography (microCT) and histology. Serum levels of human OPG-Fc and tartrate-resistant acid phosphatase-5b (TRAP-5b) were measured throughout the study by enzyme-linked immunosorbent assay (ELISA). Statistical analyses included analysis of variance (ANOVA) and Tukey tests.

Results: Human OPG-Fc was detected in the sera of OPG-Fc-treated animals by 3 days and throughout the study. Serum TRAP-5b was sharply decreased by OPG-Fc treatment soon after OPG-Fc delivery and remained low for the observation period. Significant preservation of alveolar bone volume was observed among OPG-Fc-treated animals compared to the controls at weeks 3 and 6 (P <0.05). Descriptive histology revealed that OPG-Fc significantly suppressed osteoclast surface area at the alveolar crest.

Conclusion: Systemic delivery of OPG-Fc inhibits alveolar bone resorption in experimental periodontitis, suggesting that RANKL inhibition may represent an important therapeutic strategy for the prevention of progressive alveolar bone loss.

Figure 1

In vivo rhOPG-Fc pharmacokinetics after a single subcutaneous administration. A) The maximum level of serum rhOPG was seen at 3 days after administration. Serum rhOPG decreased within 14 days. At 21 days after injection, the delivered rhOPG was eliminated completely. Bars represent SDs. *P <0.01 compared to baseline. B) Human OPG immunostaining in the proximal tibial metaphysis. Young male rats received a single infusion IV with human OPG-Fc at 5 mg/kg. The delivered human OPG circulated primarily in the blood rather than integrating into the bone matrix. The highest serum level of administered human OPG appeared 12 hours after IV infusion, and the serum OPG was eliminated over time. N = 6 animals per group. Bars = 25 μm. RBCs = red blood cells; h = hours; d = days.

Figure 2

A) Serum rhOPG levels after twice weekly subcutaneous administration. Higher serum rhOPG levels were seen in OPG delivery groups, whereas rhOPG levels remained almost undetectable in vehicle-treated animals. At 6 weeks, the serum rhOPG levels between the OPG + disease and the OPG alone groups were statistically significantly different (^†P <0.05). *P <0.05 compared to the vehicle group. Bars represent SDs. B) Serum TRAP-5b levels after repeated subcutaneous administration. Serum TRAP-5b levels in rhOPG-injected animals decreased sharply at 10 days and remained very low until 6 weeks, whereas the serum TRAP-5b levels in non-rhOPG-injected animals gradually declined. Except at baseline, the TRAP-5b levels in non-rhOPG-treated groups were significantly higher than those in rhOPG-injected groups. *P <0.05 compared to non-rhOPG-treated groups. Bars represent SDs. N = 4 animals per group. Veh = vehicle.

Figure 3

OPG-Fc blocks alveolar bone loss in experimental periodontitis. Left panels show the mesial view of μCT 3-D images of mandibular first molars of each group at 3 weeks. The vehicle + disease group shows the greatest degree of bone loss. Right panels show the histology of the alveolar bone at the mesial side of mandibular first molars of each group at 3 weeks (TRAP immunostaining; magnification, ×200). TRAP-positive osteoclasts are stained brown. Higher numbers of positively stained osteoclasts were noted on the surface of alveolar bone in the vehicle + disease groups. Few osteoclasts were seen at the alveolar bone crest in the OPG + disease group. Furthermore, no positively stained cells were found in the OPG alone group. Blue arrows demarcate the CEJ. Yellow arrows indicate the alveolar bone crest. Black arrows indicate positive TRAP-stained osteoclast-like cells. Veh = vehicle; B = bone crest; D = dentin. N = 4 animals per group.

Figure 4

OPG-Fc blocks alveolar bone loss in experimental periodontitis as measured by linear and volumetric μCT. A) The linear measurement of the distance from the CEJ to alveolar bone crest at the mesial roots of mandibular first molars in each group. The greatest difference was seen in the vehicle + disease group at both 3 and 6 weeks, whereas there was no significant difference among other groups. B) The fractions of bone volume versus the total volume surrounded by the superficial surfaces of five roots of first mandibular molar. At 3 and 6 weeks, the vehicle + disease group showed the smallest BVF, whereas the OPG alone group displayed the greatest BVF. No significant difference was found between the no treatment and OPG + disease groups. C) BMD for the regions surrounded by the superficial surfaces of all roots of first mandibular molars of each group. At 3 and 6 weeks, BMD in the vehicle + disease group was the lowest, whereas the OPG alone group displayed the highest BMD. No significant difference was found between no treatment and OPG + disease groups. N = 4 animals per group.*P <0.05; ^†P <0.01; bars represent SDs. Veh = vehicle.

Figure 5

OPG administration inhibits osteoclast formation at the alveolar bone crest in experimental periodontitis. The osteoclast surface coverage at the coronal 0.5-mm region of interest in alveolar bone at the mesial of the mandibular first molars was evaluated histomorphometrically at 3 and 6 weeks using TRAP immunostaining. At 3 weeks, osteoclast coverage area was the greatest in the vehicle + disease group, whereas there were no significant differences noted among the other three groups. At 6 weeks, the vehicle + disease group had the greatest osteoclast coverage at the alveolar crest compared to OPG administration groups, with no significant difference with the no treatment group. *P <0.05. N = 4 animals per group. Veh = vehicle.