Development of the local osteoporotic alveolar bone of beagle dogs induced by receptor activator of nuclear factor kappa-B ligand and the effect of injectable β-tricalcium phosphate microsphere with bone morphogenetic protein-2 in the osteoporotic alveolar bone

Abstract

Background: Clinical demands of dental implants have been increased for patients with poor bone quality, in whom it is difficult to get a sufficient osseointegration of dental implant even with extended healing time. Enhanced peri-implant bone regeneration in the osteoporotic alveolar bone by sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2) can increase implant success. In order to achieve such result, a new carrier with optimal release of rhBMP-2 and adequate small size for injectable type is needed. And mandibular alveolar bone of dogs has been regarded as a suitable large animal for bone regeneration research with dental implants and bone substitutes, however, an osteoporotic alveolar bone model of the beagle dog mandible does not exist. Purpose: This study aimed to develop a local osteoporotic alveolar bone model on the beagle dog mandible using a local application of RANKL, to evaluate the release kinetics of rhBMP-2 from β-tricalcium phosphate (β-TCP) microspheres and to analyze the effect of a newly developed injectable β-TCP microsphere with rhBMP-2 in this local osteoporotic alveolar bone model of beagle dogs. Method and material: To evaluate β-TCP microspheres as a BMP-2 carrier, the release kinetics of BMP-2 was assessed in vitro and was compared with that of collagen sponge and other bone graft materials such as 100% hydroxyapatite (HA) granules, biphasic calcium phosphate granules (70% β-TCP + 30% HA), and 100% TCP granules. For in vivo evaluation, the experimental group was administered the injectable bone graft material comprising porous β-TCP microspheres and poloxamer hydrogel with rhBMP-2 (45 μg) and the control group was administered the injectable bone graft material without rhBMP-2

the injectable materials were transplanted in non-osteoporotic extraction sockets of both maxillary first molars in four beagles. After 4 weeks, the maxillary alveolar bone was resected to analyze bone regeneration using micro-CT and histomorphometry. For the development of local osteoporotic alveolar bone, collagen sponges soaked with 0, 20, 40, or 60 µg RANKL were applied into holes created in the mandibular alveolar bone of a beagle dog for two weeks. After the removal of collagen sponges, the bone quality around holes was evaluated with micro-CT to determine the optimal dose of RANKL. After the fabrication of local osteoporosis around five holes in the mandibular alveolar bone of beagle dogs (n = 7) by the application of determined dose of RANKL for two weeks, collagen sponges with RANKL were removed and five dental implants were placed into the holes after the injection of β-TCP microsphere graft materials with four different doses of BMP-2 (0, 5, 15, 45 µg) or without any graft material (control group) in each animal. After four (n = 2) and six weeks (n = 5), animals were sacrificed, and the mandibular alveolar bone was resected to analyze changes of the bone quality at the outside of inter-thread space and peri-implant bone regeneration within the inter-thread space using micro-CT and histomorphometry. Results: Regarding the release kinetics of BMP-2, a significantly more sustained release was observed from porous β-TCP microspheres than from granule bone graft materials (p < 0.05) and much more than that observed from collagen sponge after 12 h (p < 0.001)

the amount of rhBMP-2 released from porous β-TCP microspheres was <21% of the total dose after 26 days. In the maxillary extraction sockets of beagles, the bone volume (BV), BV/tissue volume (BV/TV), and trabecular number were significantly higher in the experimental group than in the control group (p < 0.05). The optimal RANKL dose for the induction of osteoporotic alveolar bone was 40 µg (BV: 41.61% compared to the control). Regarding the bone quality at the outside of the inter-thread space, the BV/TV at 4 weeks was slightly smaller (30.64%) than it immediately after RANKL removal (32.85%). However, the BV/TV and bone mineral density (BMD) were significantly increased at 6 weeks (51.91% and 1.45 mg/cc, respectively) compared to that at 4 weeks (30.64%, 1.36 mg/cc, respectively), which were slight lower to them immediately after the removal of RANKL (32.85%, 1.37 mg/cc, respectively). In the comparison of the bone quality within inter-thread space between the 4 weeks and the 6 weeks, all micro-CT values at 6 weeks were significantly greater than them at 4 weeks. The BV/TV (12.73%) and BMD (1.27 mg/cc), the BA (40.17%) at 4 weeks were increased to 15.10% and 1.31 mg/cc and 53.03%, respectively at 6 weeks (p < 0.001). However, the fold ratio of the increase within the inter-thread space was smaller than it at the outside of the inter-thread space. In the evaluation of peri-implant bone regeneration, the BV/TV and BMD in all five groups were increased at 6 weeks compared to them at 4 weeks. At 6 weeks, the BV/TV in the group with 5 µg BMP-2 (27.22 %) was the highest among the groups and statistically greater compared with that in the Implant only group (23.14%) and the group with 45 µg BMP-2 (22.97%) (p <0.05). The BV/TV in the group with graft material only (26.23%) was statistically higher than that in the Implant only group (23.14%). In the histomorphometric analysis, the bone area (BA) (%) and bone implant contact (BIC) (%) were the highest in the group with 5 µg BMP-2 (43.80 %, 50.34%, respectively) compared to other groups at 4 weeks. The BA (%) and BIC (%) were significantly higher in the group with graft material only (51.97%, 58.10%, respectively) and with 5 µg BMP-2 (58.49%, 66.56%, respectively) compared to the Implant only group (44.05%, 50.05%, respectively) at 6 weeks (p <0.05). While there was no significant difference in the BA between the two groups, the group with 5 µg BMP-2 (66.56%) had a significantly higher BIC than that of the group with graft material only (58.10%) (p <0.05) and with 45 µg BMP-2 (52.44%) (p <0.001). Conclusion: The new injectable bone graft material comprising porous β-TCP microspheres and poloxamer hydrogels possesses the sustained-release property of rhBMP-2, convenient loading property of BMP-2 using the in situ mixing method, and easy handling for transplantation, which can enhance bone regeneration. A new beagle dog model for local osteoporotic alveolar bone of mandible was developed using collagen sponges soaked with 40 µg RANKL, which were applied into holes in the alveolar bone for two weeks. In our RANKL-induced osteoporotic alveolar bone, the initial bone quality at the outside of inter-thread space was slightly decreased at 4 weeks after the removal of RANKL, while it was actively increased at 6 weeks. The bone quality within the inter-thread space at 4 weeks was similar with it immediately after the removal of RANKL, while it was significantly higher at 6 weeks than it at 4 weeks. It seems that the local osteoporotic status in the alveolar bone induced by 40 µg RANKL could be well continued until 4 weeks after the removal of RANKL. And, the new injectable β-TCP microsphere bone grafts could be effectively evaluated with increasing bone formation around the implants, particularly in injectable β-TCP microsphere bone grafts containing 5 µg of BMP-2. However, the bone formation efficacy was not enhanced with increasing BMP-2 concentrations. Our local osteoporotic mandible model of beagle dogs could be useful to evaluate new bone graft materials and implant surfaces to improve bone formation in geriatric patients with osteoporosis.