3.5 Effectiveness of DP scaffold in a GBR model in vivo
A 5-mm-diameter critical-size defect was established in a rat cranium for our in vivo GBR model. No inflammation or infection was observed at the defect sites in the different groups during the experimental period. Micro-CT was applied to evaluate bone regeneration within the defects in each group. Representative 3D reconstructed images are displayed in Figure 5A. For the blank control group, very little bone formation was found in the defect, whereas newly formed bone tissue was evident in defects treated with COL or DP in a centripetal manner with regenerated bone stretching from the margins toward the center. Quantitative analysis of the micro-CT findings is presented in Figure 5B&C. By week 8, the BMD of newly formed tissues in the control group remained as low as 282.2±16.17 mg HA ccm−1. In contrast, the BMD measurements were 529.3±22.67 mg HA ccm−1 and 508.1±14.96 mg HA ccm–1 for the COL and DP groups, respectively, and the differences compared with the control group were statistically significant difference (P=0.0266, n=3). For the ratio of bone volume to total volume (BV/TV), similar trends were observed. Bone regeneration was further evaluated histologically with Masson trichrome staining (Figure 5D). In the control group, the defect areas showed poor repair with mainly blue-stained fibrous connective tissues present. In contrast, in the DP group, the defect was filled with woven bone (blue stained) and cortical-like lamella bone (red stained), and a more mature arrangement of collagen also was observed. Quantitative analysis indicated the new bone percentage in the DP group (49.1%±7.69%) was significantly higher than that in the control group (12.2%±3.94%; P<0.01) and that in the COL group (31.5%±3.45%; P<0.05; Figure 5E). These results indicate that DP promoted significantly more bone regeneration within the rat cranial defect GBR model.
To evaluate the osteoimmunomodulatory properties of DP in the early period of GBR, immunohistochemical staining for CD68 (pan marker of in situ macrophages), iNOS (M1 marker), and CD206 (M2 marker) was performed (Figure 5F). Semi-quantitative analysis of the staining results indicated that a greater percentage of CD206+ M2 macrophages (64.4%±7.64%; P<0.05) was found in the DP group at week 1 compared with the other groups, whereas the number of iNOS+ M1 macrophages in the DP group was relatively low (33.6%±2.52%; P<0.05; Figure 5G). In summary, these results indicate that DP induced polarization of more M2 phenotype macrophages in the early GBR process.