Research ArticleAPPLIED SCIENCES AND ENGINEERING

3D printing of Haversian bone–mimicking scaffolds for multicellular delivery in bone regeneration

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Science Advances  20 Mar 2020:
Vol. 6, no. 12, eaaz6725
DOI: 10.1126/sciadv.aaz6725
  • Fig. 1 3D printing Haversian bone–mimicking scaffolds integrated with Haversian canals, Volkmann canals, and cancellous bone structure for delivery of osteogenic and angiogenic cells.

    Osteogenic cells were seeded in cancellous bone structure of scaffolds, and angiogenic cells were seeded on Haversian canals. The Haversian bone–mimicking structure–based multicellular delivery system contributed to the formation of new bone and new blood vessels.

  • Fig. 2 3D printing of Haversian bone–mimicking bioceramic scaffolds with cortical bone and cancellous bone structure.

    Cortical bone structure contained Haversian canals and Volkmann canals. (A to E) Optical microscope images exhibited different diameters (D) and numbers (N) of Haversian canal indicated by magenta arrows (A) N = 8, D = 0.8 mm; (B) N = 8, D = 1.2 mm; (C) N = 8, D = 1.6 mm; (D) N = 4, D = 1.6 mm; and (E) N = 2, D = 1.6 mm. Scale bars, 1 mm. (a to e) Micro–computed tomography (CT) images show Volkmann canals (blue arrows) connecting Haversian canals in the interior of scaffolds. Scale bars, 1 mm. (F to J) SEM images presented the microstructure of the scaffolds. Haversian canal on the periphery of scaffolds with different diameters of (F) 0.8 mm, (G) 1.2 mm, and (H) 1.6 mm. Scale bars, 400 μm. (I) Cancellous bone structure in the center of the scaffolds. Scale bar, 400 μm. (J) Microstructure at the surface showed a well-sintered scaffold. Scale bar, 6 μm.

  • Fig. 3 Characterization of Haversian bone–mimicking bioceramic scaffolds.

    (A to C) Micro-CT images of scaffolds with different numbers of Haversian canals, (A) N = 8, (B) N = 4, and (C) N = 2. (D) Compressive strength of scaffolds with different numbers of Haversian canals. (E) Porosity of scaffolds with different numbers of Haversian canals. (F to H) Micro-CT images of scaffolds with different diameters of Haversian canals indicated by blue arrows, (F) D = 1.6 mm, (G) D = 1.2 mm, and (H) D = 0.8 mm. (I) Compressive strength of scaffolds with different diameters of Haversian canals. (J) Porosity of scaffolds with different diameters of Haversian canals. (K to N) Micro-CT images of scaffolds with different numbers of Volkmann canals indicated by red circles for (K) N = 0, (L) N = 1, (M) N = 2, and (N) N = 3. (O) Compressive strength of scaffolds with different numbers of Volkmann canals. (P) Porosity of scaffolds with different numbers of Volkmann canals. n = 6 replicates. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 1 mm.

  • Fig. 4 Haversian bone–mimicking bioceramic scaffolds for the HBMSC-HUVEC coculture system performed better in cell proliferation and angiogenic differentiation than monoculture.

    (A to D) CLSM images of HBMSCs seeded on the cancellous bone structure (A) and HUVECs seeded on the Haversian canal with different diameters, (B) D = 1.6 mm, (C) D = 1.2 mm, and (D) D = 0.8 mm. Scale bars, 100 μm. (E to H) SEM images of (E) HBMSCs seeded on the cancellous bone structure and HUVECs seeded on the Haversian canal with different diameters of (F) 1.6 mm, (G) 1.2 mm, and (H) 0.8 mm. (I and J) The proliferation activity of HBMSC, HUVEC, and cocultured HBMSC-HUVEC seeded on scaffolds with different (I) diameters and (J) numbers of Haversian canals after culturing for 1, 3, 7, and 14 days. n = 6 replicates. (K and L) The osteogenic (K) and angiogenic (L) gene expression of HBMSC, HUVEC, Co-HBMSC (HBMSCs in HBMSC-HUVEC coculture), and Co-HUVEC (HUVECs in HBMSC-HUVEC coculture) for 3 days. n = 3 replicates. *P < 0.05, **P < 0.01, ***P < 0.001, $P < 0.05, $$P < 0.01.

  • Fig. 5 Haversian bone–mimicking bioceramic scaffold–based rBMSC-rSC coculture system performed better in cell proliferation and neurogenic differentiation than monoculture.

    (A to D) The CLSM images of rBMSC in the (A) rBMSC monoculture group and rBMSC-rSC coculture group with the ratio of rBMSC to rSC being (B) 3:7, (C) 5:5, and (D) 7:3 seeded on the cancellous bone of scaffolds. Scale bars, 50 μm. (E to H) The CLSM images of rSCs in (E) the rSC monoculture group and rBMSC-rSC coculture group with the ratio of rBMSCs to rSCs being (F) 3:7, (G) 5:5, and (H) 7:3 seeded on the Haversian canal of scaffolds. Scale bars, 50 μm. (I) The proliferation of rBMSC, rSC, and rBMSC-rSC coculture with different ratio of rBMSCs to rSCs. n = 6 replicates. (J) The neurogenic genes expression of rBMSC, rSC, and rBMSC-rSC coculture with different ratio of rBMSCs to rSCs for 3 days. n = 3 replicates. *P < 0.05, **P < 0.01, ***P < 0.001, $P < 0.05, $$P < 0.01, $$$P < 0.001.

  • Fig. 6 The Haversian bone–mimicking bioceramic scaffold–based RBMSC-RAEC coculture system enhanced the formation of new bone and new blood vessels in rabbit femoral defects.

    (A to E) Digital photographs showed the defects in the (A) blank group and scaffolds implanted into the defect (B) cell-free AKT scaffold group, (C) RAEC monoculture group, (D) RBMSC monoculture group, and (E) RBMSC-RAEC coculture group perfused with Microfil (blue). Scale bars, 3 mm. (F to j) Micro-CT images exhibited (F to J) sagittal view and (f to j) transverse view of the five groups at week 8, respectively (green for newly formed bone and red for scaffolds). Scale bars, 3 mm. (K to O) The sections from Microfil-perfused samples stained with picric acid-acid fuchsin. Scale bars, 2 mm. (k to o) Magnified images of the marked area in (K to O). Scale bars, 500 μm. The red arrows indicated blood vessels (blue). (P) The volume ratio of the newly formed bone to defects (BV/TV) of all groups. n = 4 replicates. (Q) The density of newly formed vessels of all groups. n = 3 replicates. *P < 0.05, **P < 0.01, ***P < 0.001. (Photo credit: Meng Zhang, State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China. Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, P. R. China.)

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/12/eaaz6725/DC1

    Fig. S1. Surface roughness of the Haversian bone–mimicking bioceramic scaffolds.

    Fig. S2. Compressive modulus of the Haversian bone–mimicking bioceramic scaffolds.

    Fig. S3. Flexural strength of the Haversian bone–mimicking bioceramic scaffolds.

    Fig. S4. CLSM images of cocultured HBMSCs and HUVECs stained with DAPI and phalloidin.

    Fig. S5. Cell proliferation of HBMSCs and HUVECs cultured with the extracts from the Haversian bone–mimicking bioceramic scaffolds on days 1, 3, and 7.

    Fig. S6. The osteogenic and angiogenic gene expression of cocultured HBMSC-HUVEC seeded on scaffolds with different diameters and numbers of Haversian canals for 3 days.

    Fig. S7. The CLSM images of rBMSCs and rSCs in monoculture group and rBMSC-rSC coculture group with the ratio of rBMSCs to rSCs being 3:7, 5:5, and 7:3 stained with DAPI (blue) and phalloidin (red).

    Fig. S8. Cell proliferation of rBMSCs and rSCs cultured with the extracts from the Haversian bone–mimicking bioceramic scaffolds on days 1, 3, and 7.

    Fig. S9. The neurogenic gene expression of cocultured rBMSC-rSC seeded on scaffolds with different diameters and numbers of Haversian canals for 3 days.

    Fig. S10. New bone formation evaluated by histological analysis.

    Movie S1. The bottom-up printing process to fabricate Haversian bone–mimicking scaffolds for multicellular delivery.

    Movie S2. The bottom-up printing process to fabricate Haversian bone–mimicking scaffolds with different numbers of Haversian canals for mechanical and porosity tests.

    Movie S3. The bottom-up printing process to fabricate Haversian bone–mimicking scaffolds with different diameters of Haversian canals for mechanical and porosity tests.

    Movie S4. The bottom-up printing process to fabricate Haversian bone–mimicking scaffolds with different numbers of Volkmann canals for mechanical and porosity tests.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Surface roughness of the Haversian bone–mimicking bioceramic scaffolds.
    • Fig. S2. Compressive modulus of the Haversian bone–mimicking bioceramic scaffolds.
    • Fig. S3. Flexural strength of the Haversian bone–mimicking bioceramic scaffolds.
    • Fig. S4. CLSM images of cocultured HBMSCs and HUVECs stained with DAPI and phalloidin.
    • Fig. S5. Cell proliferation of HBMSCs and HUVECs cultured with the extracts from the Haversian bone–mimicking bioceramic scaffolds on days 1, 3, and 7.
    • Fig. S6. The osteogenic and angiogenic gene expression of cocultured HBMSC-HUVEC seeded on scaffolds with different diameters and numbers of Haversian canals for 3 days.
    • Fig. S7. The CLSM images of rBMSCs and rSCs in monoculture group and rBMSC-rSC coculture group with the ratio of rBMSCs to rSCs being 3:7, 5:5, and 7:3 stained with DAPI (blue) and phalloidin (red).
    • Fig. S8. Cell proliferation of rBMSCs and rSCs cultured with the extracts from the Haversian bone–mimicking bioceramic scaffolds on days 1, 3, and 7.
    • Fig. S9. The neurogenic gene expression of cocultured rBMSC-rSC seeded on scaffolds with different diameters and numbers of Haversian canals for 3 days.
    • Fig. S10. New bone formation evaluated by histological analysis.
    • Legends for movies S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). The bottom-up printing process to fabricate Haversian bone–mimicking scaffolds for multicellular delivery.
    • Movie S2 (.avi format). The bottom-up printing process to fabricate Haversian bone–mimicking scaffolds with different numbers of Haversian canals for mechanical and porosity tests.
    • Movie S3 (.avi format). The bottom-up printing process to fabricate Haversian bone–mimicking scaffolds with different diameters of Haversian canals for mechanical and porosity tests.
    • Movie S4 (.avi format). The bottom-up printing process to fabricate Haversian bone–mimicking scaffolds with different numbers of Volkmann canals for mechanical and porosity tests.

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