# Octacalcium Phosphate Bone Substitute (Bontree(r)): From Basic Research to Clinical Case Study
## 3 Results
### Basic Research
#### 3.1.3 In Vivo Study  
On the basis of the results of in vitro testing, Bontree(r) 0.5-granule and Bontree(r) 1.0-granule were selected for further evaluation of new bone formation in vivo. Figure 3A shows low- and high-magnification histological images of transverse cross-sections stained using hematoxylin and eosin; the cortical bone tissue stained light red or pink, and collagen fibers stained pale pink; the black areas represent the implanted Bontree(r) grafts [20]. To examine the size effect of bone grafts, two Bontree(r) grafts (Bontree(r) 0.5-granule and Bontree(r) 1.0-granule) were assessed in this in vivo study. At both 4 and 12 weeks after surgery, bone regeneration was observed in the histological images without any sign of local inflammation or rejection of the bone graft. In the control group (i.e., nongrafted defect), a small amount of new bone tissue was formed only at the edge of the tibial defect, and fibrous connective tissue and bone marrow nearly filled the entire tibial defect after 4 weeks of surgery. In contrast, defects filled with the Bontree(r) 0.5-granule and Bontree(r) 1.0-granule showed a significant amount of newly formed bone tissues that were uniformly distributed over the entire tibial defect and even in the bone marrow cavity. Notably, in the case of the Bontree(r) 1.0-granules, thick and dense new bone tissues were consistently  
Figure 2: (**A**) RANKL activity and (**B**) ALP activity of MG63 cells in Bontree® and MBCP+TM cultures after 7 and 14 days of culture. Statistical significance was set at \({}^{\star}p<0.05\) and \({}^{\star\star}p<0.01\), respectively. RANKL, receptor activator of NF-\(\kappa\)B ligand; ALP, alkaline phosphatase. generated between the graft granules, while relatively thin and less dense new bone tissues were formed with Bontree(r) 0.5-granules. Quantitative analysis of the new bone region (Figure 3B) showed 21.6\(\%\)\(\pm\) 5.3\(\%\) and 26.0\(\%\)\(\pm\) 8.2\(\%\) of new bone volumes after 4 weeks of grafting Bontree(r) 0.5- and 1.0-granules, respectively, which were significantly higher than that of the control (11.2\(\%\)\(\pm\) 2.9\(\%\)) (\(p\) \(<\) 0.01). The histological differences between the samples decreased with increasing implantation time. Twelve weeks after surgery (Figure 3A), all samples exhibited complete sealing  
Figure 3: **(A)** Representative histological images with low- and high-magnifications of the bone graft implantation regions in a rabbit tibia 4 and 12 weeks after surgery. Magnified regions are marked with yellow rectangles in the low magnification images. Black and white bars indicate 2 mm and 1 mm, respectively. Percentages of (**B**) new bone and (**C**) bone graft volumes compared with the tibial defect volume at 4 and 12 weeks after surgery. Statistical significance was set at * \(p\) \(<\) 0.05 and **\(p\) \(<\) 0.01, respectively. of the bone defects; the tibial defects were fully filled with newly formed compact bone, and there was no open gap at the side of the cortical bone defect regions. However, in the control and Bontree(r) 0.5-granule groups, regenerated bone tissues were not fully dense, and they showed macroscopic inner pores within the bone structures. In contrast, tibias grafted with Bontree(r) 1.0-granule showed a fully dense regenerated bone structure at the wound site, and they showed good histomorphological continuity with adjacent old bone tissues. From the quantitative results (Figure 3B), the Bontree(r) 1.0-granule group showed significantly higher new bone volumes (34.5% \(\pm\) 2.5%) than the control (25.8% \(\pm\) 1.5%) and Bontree(r) 0.5-granule (29.2% \(\pm\) 4.8%) groups (\(p\) < 0.05). Regarding bone graft degradation, Bontree(r) 0.5-granule and Bontree(r) 1.0-granule showed apparent degradation over 12 weeks (Figure 3C). Four weeks after surgery, large amounts of bone graft remained inside the tibial defects in both groups, and the retention rates were 5.8% \(\pm\) 3.2% and 21.5% \(\pm\) 8.0% for the Bontree(r) 0.5-granule and Bontree(r) 1.0-granule groups, respectively.