Figure 1.
Experimental design and surgical procedure.
(A) The experimental design of the whole study. (B) A 16 mm-long segmental defect was created on the radius of each rabbit and implanted with four types of β-TCP scaffolds with different architectures.
Figure 2.
Characterization of β-TCP scaffolds.
Micro-CT 3D image (upper row) and gross view (lower row) showed the general constructions of the four types of scaffolds. Two porous scaffolds (Group A and B) with porosity of around 40% and pore size of 500 µm, the tubular scaffolds (Group C) with inner diameter of 2 mm and the solid scaffolds (Group D) were manufactured and used in this study.
Figure 3.
X-ray and gross examinations of the radius with different treatment at 12 weeks post-surgery.
X-ray examination (upper row) showed evident ingrowth of new bone tissue into the porous (Group A and B) and tubular (Group C) scaffolds. Tubular scaffolds showed better graft/bone integration than porous ones, which was comparable to the implantation of autologous bone graft (Group E). The solid scaffolds (Group D) showed the poorest osteointegration as evidenced by clear boundary line (red arrow) between the scaffold and bone tissue. Non-union healing of the bone defect in Group F (no treatment) validated that the segmental defect used in this study was the critical sized bone defect. Gross view of the radius (lower row) showed treatment with porous or tubular scaffolds and autologous bone graft exerted better bony union than solid scaffolds.
Figure 4.
Fluorochrome labeling of bone regeneration at 12 weeks post-surgery.
(A) The fluorescent labeling images indicated that more new bone growth into the porous (Group A–B) and tubular scaffolds (Group C), while very limited bone tissue formed around the solid scaffolds (Group D). (B) Quantitative analysis showed there were no significant differences in the bone mineralization apposition rate (p>0.05), indicating similar bone growth rate in these four kinds of scaffolds. Scale bar: 50 µm (white), 10 µm (blue).
Figure 5.
Micro-CT analysis of new bone formation and biodegradation rate of scaffolds at 12 weeks post-surgery.
(A) Micro-CT images showed more bone ingrowth within the tubular scaffolds (Group C) than porous ones (Group A and B), especially in the center of grafts (red arrow). Little new bone generated in solid scaffolds (Group D). (B) The ratio of bone volume/total volume (%BV/TV) in Group C was higher than that of Group A, B and D. (C) Micro CT images indicated that porous scaffolds experienced faster degradation rate with ruptured overall structure, while tubular scaffolds maintained intact for its overall structure. (D) Quantitative analysis confirmed lower biodegradation rate in tubular scaffolds (Group C) than porous scaffolds (Group A and B) (*p<0.05 vs. Group D, +p<0.05 vs. Group A and B).
Figure 6.
Compression testing of the scaffolds and radius defect.
The results of compression testing showed that tubular scaffolds (Group C) demonstrated improved mechanical properties with higher compression strength in vivo than porous ones (Group A and B), indicating better bone formation and integration with the tubular scaffolds; the native solid scaffolds (Group D) showed the highest compression strength, but the lowest in vivo data after implantation (*p<0.05 vs. Group D, +p<0.05 vs. Group A and B).
Figure 7.
Histological analysis of new bone formation at 12 weeks post-surgery.
Van Gieson staining showed more bone formed in tubular scaffolds (Group C) than porous (Group A and B) and solid (Group D) ones, especially in the center of grafts (blue arrow). The center region of porous scaffold was occupied by soft tissue with poor osteogenesis (red arrow). The tissue stained in red color was the newly formed bone with visible cell nuclei. The tissue stained in yellow/green/blue color was fibrous tissue. Scale bar: 1 mm (white), 100 µm (blue).
Figure 8.
Histomorphology analysis of new bone formation.
Quantitative analysis showed the percentage of new bone formation in Group C was significantly higher than that of Group A, B and D at 12 weeks post-surgery (*p<0.05 vs. Group D, +p<0.05 vs. Group A and B).
Figure 9.
ECT analysis of vascularization at 2, 4, 8 and 12 weeks post-surgery.
(A) ECT images of rabbits showed that despite of no difference among the four groups at 2 weeks post-surgery, higher counts were observed in tubular scaffolds (Group C) than other groups (Group A, B and D) since week 4 post-surgery, suggesting enhanced vascularization and metabolic activities. (B) Quantitative measurement of the uptake ratio (T/NT) further confirmed better vascularization and higher metabolic activities of tubular scaffolds than others since week 4 post-surgery (+p<0.05 vs. Group A, B and D).