Table 1.
Primer sequences of osteogenic markers.
Figure 1.
Characterization of CAH/B2 scaffolds.
A. EDX analysis of CAH specimen. B. XRD pattern of CAH specimen.
Figure 2.
Physicochemical properties of CAH/B2 scaffolds.
A. SEM view of CAH/B2 scaffold at a magnification of 100×, B. SEM view of CAH/B2 scaffold at a magnification of 200×. SEM analysis showed that micro-pores were generated in the CAH scaffold, the average pore diameter in the scaffold being 100–110 µm. C. In vitro release behavior of BMP-2 from the CAH scaffold. Col/B2 served as a control. The release velocity of BMP-2 was delayed in the CAH/B2 group compared with release from Col/B2. The cumulative BMP-2 released from the CAH/B2 and the Col/B2 groups almost reached a plateau at 21 and 14 days, respectively. D. SEM view of MSCs cultured on a CAH/B2 scaffold at a magnification of 100×.
Figure 3.
Biocompatibility and effects of the CAH/B2 scaffold on osteoblastic differentiation.
A. MTS assay of MSCs cultured with CAH and CAH/B2 scaffolds. The cells were also cultured in plates as a control. Data represent the mean+SD of n = 5 samples. No statistically significant differences were seen between groups. B. Effects of CAH/B2 on in vitro ALP activity. ALP activity in the CAH/B2 group was higher than in the CAH group. One-way analyses of variance suggest that there are significant differences among three groups. The significant post hoc test results are identified by symbol *. N = 5. P<0.001: CAH vs. CAH/B2. C. The level of calcium deposition in 3-week cultures was evaluated by AR-S. The values indicated are means ± SD, (n = 5) p<0.005 as compared with that deposition in the CAH group. D. qRT-PCR analysis of osteoblast marker genes, showing that MSCs cultured on a CAH/B2 scaffold exhibited increased Col I, OCN and OPN gene expression. Data represent the mean + SD of n = 5 samples. P<0.05.
Figure 4.
CAH/B2 scaffolds enhance bone regeneration in the rat critical-sized calvarial defect model.
A. Image of calvarial bone from the LUNAR PIXImus system, 12 weeks after surgery. The areas of bone regeneration were labeled in different colors. A black area circled with a blue line (*) is a low density area. The dark gray area between inside blue line and inside yellow line (#) is an area of thin bone, the area between two yellow lines(▵) is considered an area of normal bone density, and the density in this area is close to that of normal bone tissue. B. Quantitative analysis of bone density. N = 6, *p<0.05:CAH/B2+M versus four other groups. • p<0.05: CAH+M versus CAH or blank. ♦p<0.05: CAH versus blank. C. Quantitative analysis of bone area in implanted region showed a significantly larger bone area within the CAH/B2+M group when compared with the other four groups. BV, bone area in the implant; TV, total implant area. N = 6, *p<0.05:CAH/B2+M versus four other groups. •p<0.05: CAH+M versus CAH or blank. ♦p<0.05: CAH versus blank.
Figure 5.
Coronal sections through the midlines of defects are shown. (A–E): lower magnification, Bar = 1 mm, yellow arrow: new bone formed; (A′–E′): higher magnification, Bar = 0.25 mm. (A, A′): Blank; (B, B′): CAH; (C, C′): CAH+M; (D, D′): CAH/B2+M. (E, E′)Col/B2+M. Blue arrow: new bone, Yellow arrow: host bone, *:interface between new bone and host bone.