Comparison of Osteoconductive Ability of Two Types of Cholesterol-Bearing Pullulan (CHP) Nanogel-Hydrogels Impregnated with BMP-2 and RANKL-Binding Peptide: Bone Histomorphometric Study in a Murine Calvarial Defect Model.

The receptor activator of NF-κB ligand (RANKL)-binding peptide is known to accelerate bone morphogenetic protein (BMP)-2-induced bone formation. Cholesterol-bearing pullulan (CHP)-OA nanogel-crosslinked PEG gel (CHP-OA nanogel-hydrogel) was shown to release the RANKL-binding peptide sustainably; however, an appropriate scaffold for peptide-accelerated bone formation is not determined yet. This study compares the osteoconductivity of CHP-OA hydrogel and another CHP nanogel, CHP-A nanogel-crosslinked PEG gel (CHP-A nanogel-hydrogel), in the bone formation induced by BMP-2 and the peptide. A calvarial defect model was performed in 5-week-old male mice, and scaffolds were placed in the defect. In vivo µCT was performed every week. Radiological and histological analyses after 4 weeks of scaffold placement revealed that the calcified bone area and the bone formation activity at the defect site in the CHP-OA hydrogel were significantly lower than those in the CHP-A hydrogel when the scaffolds were impregnated with both BMP-2 and the RANKL-binding peptide. The amount of induced bone was similar in both CHP-A and CHP-OA hydrogels when impregnated with BMP-2 alone. In conclusion, CHP-A hydrogel could be an appropriate scaffold compared to the CHP-OA hydrogel when the local bone formation was induced by the combination of RANKL-binding peptide and BMP-2, but not by BMP-2 alone.

Effect of tension and compression on dynamic alveolar histomorphometry.

Here, we tested the hypothesis that tensile and compressive stresses generated in the alveolar bone proper regulate site-specific cellular and functional changes in osteoclasts and osteoblasts. Thirty-two 13-week-old male mice were randomly divided into four groups: two experimental groups with vertical loading obliquely from the palatal side to the buccal side of the maxillary molar (0.9 N) 30 min per day for 8 or 15 days and unloaded controls (n = 8). Calcein and alizarin were administered 8 and 2 days before euthanization, respectively, to detect the time of bone formation. Undecalcified sections parallel to the occlusal plane were prepared on the palatal root and the surrounding alveolar bone in the middle of the root length. The alveolar perimeter was divided into 12 equal regions for site analysis, and the bone histomorphometric parameters were obtained for each region. Data from in vivo microfocus computed tomography were used to construct animal-specific finite element models. 2D stress distribution images were overlain on histology images obtained from the same location. Significant differences in the total perimeter between groups and between loading and unloading in each region were statistically analyzed (α = 0.05). Osteoclast counts and the alizarin label ratio were significantly higher in the loaded group than in the unloaded group in regions where the maximum von Mises and principal tensile stresses were the highest along the perimeter. The label ratio of calcein was significantly lower in the 8-day loaded group than in the unloaded group, indicating that the calcein-labeled surface was resorbed by osteoclasts that appeared during the loading period. The effect of loading was mitigated by an increase in the maximum principal compressive stress. We conclude that bone resorption and bone formation are functions of site-specific tension and compression in the alveolar bone proper, confirming our hypothesis. This finding is critical for the advancement of diagnosis and treatment planning in clinical dentistry.

Perforated Hydrogels Consisting of Cholesterol-Bearing Pullulan (CHP) Nanogels: A Newly Designed Scaffold for Bone Regeneration Induced by RANKL-Binding Peptides and BMP-2.

The receptor activator of NF-κB ligand (RANKL)-binding peptide, OP3-4, is known to stimulate bone morphogenetic protein (BMP)-2-induced bone formation, but peptides tend to aggregate and lose their bioactivity. Cholesterol-bearing pullulan (CHP) nanogel scaffold has been shown to prevent aggregation of peptides and to allow their sustained release and activity; however, the appropriate design of CHP nanogels to conduct local bone formation needs to be developed. In the present study, we investigated the osteoconductive capacity of a newly synthesized CHP nanogel, CHPA using OP3-4 and BMP-2. We also clarified the difference between perforated and nonperforated CHPA impregnated with the two signaling molecules. Thirty-six, five-week-old male BALB/c mice were used for the calvarial defect model. The mice were euthanized at 6 weeks postoperatively. A higher cortical bone mineral content and bone formation rate were observed in the perforated scaffold in comparison to the nonperforated scaffold, especially in the OP3-4/BMP-2 combination group. The degradation rate of scaffold material in the perforated OP3-4/BMP-2 combination group was lower than that in the nonperforated group. These data suggest that perforated CHPA nanogel could lead to local bone formation induced by OP3-4 and BMP-2 and clarified the appropriate degradation rate for inducing local bone formation when CHPA nanogels are designed to be perforated.