Repair of segmental bone defect using Totally Vitalized tissue engineered bone graft by a combined perfusion seeding and culture system

Abstract

Background: The basic strategy to construct tissue engineered bone graft (TEBG) is to combine osteoblastic cells with three dimensional (3D) scaffold. Based on this strategy, we proposed the "Totally Vitalized TEBG" (TV-TEBG) which was characterized by abundant and homogenously distributed cells with enhanced cell proliferation and differentiation and further investigated its biological performance in repairing segmental bone defect.

Methods: In this study, we constructed the TV-TEBG with the combination of customized flow perfusion seeding/culture system and β-tricalcium phosphate (β-TCP) scaffold fabricated by Rapid Prototyping (RP) technique. We systemically compared three kinds of TEBG constructed by perfusion seeding and perfusion culture (PSPC) method, static seeding and perfusion culture (SSPC) method, and static seeding and static culture (SSSC) method for their in vitro performance and bone defect healing efficacy with a rabbit model.

Results: Our study has demonstrated that TEBG constructed by PSPC method exhibited better biological properties with higher daily D-glucose consumption, increased cell proliferation and differentiation, and better cell distribution, indicating the successful construction of TV-TEBG. After implanted into rabbit radius defects for 12 weeks, PSPC group exerted higher X-ray score close to autograft, much greater mechanical property evidenced by the biomechanical testing and significantly higher new bone formation as shown by histological analysis compared with the other two groups, and eventually obtained favorable healing efficacy of the segmental bone defect that was the closest to autograft transplantation.

Conclusion: This study demonstrated the feasibility of TV-TEBG construction with combination of perfusion seeding, perfusion culture and RP technique which exerted excellent biological properties. The application of TV-TEBG may become a preferred candidate for segmental bone defect repair in orthopedic and maxillofacial fields.

Figure 1. The gross view (A) and micro-CT scan (B) of β-TCP scaffold fabricated by Rapid Prototyping technique with designed internal architecture.

Figure 2. Flow circuit schematic diagram in the flow perfusion seeding and culture bioreactor.

The scaffolds were press-fit into the column and the medium was pumped into the scaffolds via a peristaltic pump. The arrows indicated the flow direction of the seeding and culture medium.

Figure 3. Analysis of the D-glucose consumption of the cell/scaffold composites at different time points.

PSPC group exhibited higher D-glucose consumption at all time points followed by SSPC group. ^* p<0.05 vs. SSSC; ^# p<0.05 vs. SSPC.

Figure 4. Assessment of cell viability of the cell/scaffold composites at 8th day of culture.

PSPC group exhibited higher cell viability followed by SSPC group. ^* p<0.05 vs. PSPC; ^# p<0.05 vs. SSPC.

Figure 5. Measurement of ALP activity of cell/scaffold composites at different time points.

PSPC group exhibited higher ALP activity followed by SSPC group.^* p<0.05 vs. PSPC; ^# p<0.05 vs. SSPC.

Figure 6. Histological analysis of the cell/scaffold composites at 8th day of culture.

The cells (blue) and mineralized matrix (red) were visualized by hematoxylin and eosin (H&E) staining. A: PSPC, B: SSPC, C: SSSC. Scale bar: 50 µm (white), 20 µm (blue). Quantitative analysis indicated the higher percentage of cell area/pore area of PSPC group followed by SSPC group. ^* p<0.05 vs. PSPC; ^# p<0.05 vs. SSPC.

Figure 7. The radiographic examination of radius segmental defects repaired with different grafts.

(A) The radiographic images of all the groups at 1 day and 12 weeks. (B) Quantitative evaluation indicated the highest score of PSPC group except for autograft, followed by SSPC group at 12 weeks. ^* p<0.05 vs. PSPC; ^# p<0.05 vs. SSPC.

Figure 8. Compression testing results at 12 weeks post-operation.

PSPC group exhibited higher compression strength followed by SSPC group. ^* p<0.05 vs. PSPC; ^# p<0.05 vs. SSPC.

Figure 9. Fluorochrome labeling of bone regeneration at 12 weeks post-operation.

(A) The fluorescent labeling images of extracted specimens at 12 weeks post-operation. Scale bar: 50 µm (white), 10 µm (blue). (B) Quantitative analysis indicated the faster mineralization apposition rate of PSPC group followed by SSPC group. ^* p<0.05 vs. PSPC; ^# p<0.05 vs. SSPC.

Figure 10. Histological analysis of regenerated bone at 12 weeks post-operation.

(A) The newly formed bone was stained in red color with visible cell nuclei by Van Gieson staining at 12 weeks post-operation. PSPC group exhibited isolated new bone islands (green arrow) in the center of the scaffold. Scale bar: 5 mm (black), 20 µm (green), 10 µm (blue). (B) Histomorphometric analysis showed that the percentage of new bone formation in PSPC group was significantly higher than that of SSPC group, followed by SSSC group. ^* p<0.05 vs. PSPC; ^# p<0.05 vs. SSPC. (C) The percentage of residual material area/total area. There were no statistical differences among the three groups (p>0.05).