Research ArticleBONE HEALING

Multifunctional scaffolds for facile implantation, spontaneous fixation, and accelerated long bone regeneration in rodents

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Science Translational Medicine  24 Jul 2019:
Vol. 11, Issue 502, eaau7411
DOI: 10.1126/scitranslmed.aau7411
  • Fig. 1 3D printing and characterization of the physical and thermomechanical properties of HA-PELGA composites.

    (A) Depiction of thermal-responsive shape-memory and hydration-induced stiffening and swelling behaviors of a representative amphiphilic 25% HA-PELGA composite. (B) CAD model of a 3D scaffold with staggered macroporosity (left) and photographs (right) of the temporary shape programming and permanent shape recovery of a 3D-printed 25% HA-PELGA(8/1) scaffold triggered by 50°C water. (C) Stress-based cyclic thermal mechanical test of 25% HA-PELGA(8/1). Rf and Rr were calculated based on the second cycle. Rf, strain fixing ratio; Rr, recovery ratio. (D) Compressive moduli of 25% HA-PELGA scaffolds (n = 5) before (BH) and after hydration (AH). 2/1, 25% HA-PELGA(2/1); 8/1, 25% HA-PELGA(8/1); BH, as-printed/before hydration; AH at 37°C in water for 2 hours. Data are presented as means ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 (one-way ANOVA). (E) Differential scanning calorimetry traces of 25% HA-PELGA scaffolds BH and AH at 37°C in water for 2 hours. (F) Volume swelling ratios of 25% HA-PELGA(8/1) and 25% HA-PELGA(2/1) scaffolds (n = 3) AH at 37°C in water for 2 hours. Data are presented as means ± SEM. n.s., no significant difference, P > 0.05; Student’s t test. RT, room temperature; DI, deionized.

  • Fig. 2 Preparation and facile surgical fitting of 3D macroporous 25% HA-PELGA grafts.

    (A) CAD illustration and photographs of a 3D macroporous HA-PELGA graft fabricated by coprinting a dense HA-PELGA/PVA composite square prism, coring, and subsequent removal of sacrificial PVA material. (B) Photograph of placement of a cylindrical-compressed HA-PELGA graft into a 5-mm rat femoral segmental defect and the graft fixation driven by shape recovery, swelling, and stiffening of the graft upon 37°C saline rinse. (C) Peak forces required to pull HA-PELGA(8/1) grafts, HA-PELGA(2/1) grafts, or collagen sponges (n = 3) from a customized specimen holder simulating a 5-mm rat femoral segmental defect upon hydration as measured by a mechanical test machine (MTS Bionix 370, MTS Systems Corporation). Hydrated collagen sponges were dislodged with negligible force (not detectable, <10 mN). All specimens were prepared in a cylindrical shape (diameter, 3 mm; length, 5 mm) and were hydrated in 37°C DI water for 1 min before the pull-out test. Data are presented as means ± SEM. *P < 0.05.

  • Fig. 3 Graft-guided bone regeneration within 5-mm rat femoral segmental defects in the absence of rhBMP-2/7.

    (A) 3D μCT images and 2D bone mineral density (BMD) color maps (center sagittal slice) of 25% HA-PELGA(8/1)–filled versus 25% HA-PELGA(2/1)–filled defects over time. Global thresholding was applied to include minimum densities of 248.2 mg HA/cm3 (left column) or 518.2 mg HA/cm3 (right two columns) within the defect. HA-PELGA scaffolds were invisible with either thresholding. Red and blue represent higher and lower mineral densities in the color mapping, respectively. (B to C) Longitudinal μCT quantification of bone volume (BV) and BMD (n ≥ 6) within the entire defect [region of interest (ROI)] and the center of scaffold-filled defect (cROI) as indicated by the black and red boxes in (A), respectively. All data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-way ANOVA for longitudinal comparisons within each graft composition over time. Global lower threshold of 518.2 mg HA/cm3 was applied for all quantifications. (D) Trichrome staining of longitudinal sections of 16-week explants showing new bone formation within ROI (boxed), with the cortical bone flanking the defect shown on the left and right sides in the lower-resolution images. Scale bars, 1.2 mm (25× magnification) and 300 μm (100× magnification).

  • Fig. 4 Histology of graft-guided bone regeneration, remodeling, and scaffold degradation within 5-mm rat femoral segmental defects over time.

    (A) Longitudinal sectioning diagram of explanted femurs. (B) Micrographs of hematoxylin and eosin (H&E), alkaline phosphatase (ALP; blue) and tartrate-resistant acid phosphatase (TRAP; red), and toluidine blue (Tol blue)–stained sections from bone treated with 25% HA-PELGA(8/1) versus 25% HA-PELGA(2/1). Boxed regions are shown at higher magnification in bottom rows. CB, cortical bone; HC, healing callus; S, scaffold; NB, new bone; BM, bone marrow. Scale bars, 1.2 mm (25× magnification) and 300 μm (100× magnification).

  • Fig. 5 Accelerated healing of 5-mm rat femoral segmental defects by 25% HA-PELGA(8/1) grafts preabsorbed with 400-ng rhBMP-2/7.

    (A) 3D μCT images and BMD color maps (center sagittal and axial slices) of the ROI showing maturing regenerated bone within the defect over time. Global thresholding was applied to exclude bone densities below 518.2 mg HA/cm3 (HA-PELGA graft invisible at this threshold). (B) Longitudinal μCT quantification of BV and BMD (n ≥ 12) within the ROI over time. Statistical significance over the 4-week data or as indicated by brackets indicated as follows: **P < 0.01, ****P < 0.0001. One-way ANOVA with Tukey’s post hoc test. Data are presented as means ± SEM. The global lower threshold of 518.2 mg HA/cm3 was applied for all quantifications. (C) Histological micrographs of H&E-, ALP/TRAP-, and Tol blue–stained sections of explanted graft-filled femurs over time. Scale bars, 1.2 mm (25× magnification) and 300 μm (100× magnification). Boxed regions shown at higher magnification in bottom rows. HC, healing callus; S, scaffold; NB, new bone; BM, bone marrow. (D) Boxplots of failure torque and stiffness of intact (control, Ctl) versus regenerated femur (8/1 + BMP) 16 weeks after being treated with HA-PELGA(8/1) grafts preloaded with 400-ng rhBMP-2/7 (n = 7). *P < 0.05, n.s.: P > 0.05, Wilcoxon–Mann-Whitney rank sum test.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/502/eaau7411/DC1

    Materials and Methods

    Fig. S1. von Kossa staining of HA-PELGA filaments.

    Fig. S2. Stress-controlled cyclic thermal mechanical tests of 3D-printed macroporous 25% HA-PELGA(2/1) scaffolds.

    Fig. S3. DSC traces of as-prepared 25% HA-PELGA scaffolds during heating.

    Fig. S4. HA-PELGA(8/1) graft preparation without sacrificial PVA.

    Fig. S5. Weight comparison between HA-PELGA grafts prepared with and without sacrificial PVA.

    Fig. S6. Pull-out test of in vitro graft fixation.

    Fig. S7. GPC traces of degradation products of HA-PELGA(8/1) and HA-PELGA(2/1).

    Fig. S8. Cytocompatibility of degradation products of HA-PELGA grafts.

    Fig. S9. Experimental design and μCT quantification of HA-PELGA graft-guided bone regeneration.

    Fig. S10. New bone formation and maturation over time templated by HA-PELGA with or without 400-ng rhBMP-2/7 by polarized light microscopy and trichrome staining.

    Fig. S11. Experimental design, μCT quantification, and torsion test of HA-PELGA and 400-ng rhBMP-2/7 graft-guided bone regeneration.

    Fig. S12. Histological micrographs of H&E-, ALP/TRAP-, and Tol blue–stained femoral sections from HA-PELGA(2/1) and 400-ng rhBMP-2/7–treated group.

    Fig. S13. Representative micrographs of H&E-stained sections of organs.

    Fig. S14. Healing of 5-mm rat femoral segmental defects by collagen sponge preabsorbed with 400-ng rhBMP-2/7.

    Table S1. Literature BMP therapeutics applied to rat femoral segmental defects.

    Data file S1. Individual subject-level data.

    References (7385)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. von Kossa staining of HA-PELGA filaments.
    • Fig. S2. Stress-controlled cyclic thermal mechanical tests of 3D-printed macroporous 25% HA-PELGA(2/1) scaffolds.
    • Fig. S3. DSC traces of as-prepared 25% HA-PELGA scaffolds during heating.
    • Fig. S4. HA-PELGA(8/1) graft preparation without sacrificial PVA.
    • Fig. S5. Weight comparison between HA-PELGA grafts prepared with and without sacrificial PVA.
    • Fig. S6. Pull-out test of in vitro graft fixation.
    • Fig. S7. GPC traces of degradation products of HA-PELGA(8/1) and HA-PELGA(2/1).
    • Fig. S8. Cytocompatibility of degradation products of HA-PELGA grafts.
    • Fig. S9. Experimental design and μCT quantification of HA-PELGA graft-guided bone regeneration.
    • Fig. S10. New bone formation and maturation over time templated by HA-PELGA with or without 400-ng rhBMP-2/7 by polarized light microscopy and trichrome staining.
    • Fig. S11. Experimental design, μCT quantification, and torsion test of HA-PELGA and 400-ng rhBMP-2/7 graft-guided bone regeneration.
    • Fig. S12. Histological micrographs of H&E-, ALP/TRAP-, and Tol blue–stained femoral sections from HA-PELGA(2/1) and 400-ng rhBMP-2/7–treated group.
    • Fig. S13. Representative micrographs of H&E-stained sections of organs.
    • Fig. S14. Healing of 5-mm rat femoral segmental defects by collagen sponge preabsorbed with 400-ng rhBMP-2/7.
    • Table S1. Literature BMP therapeutics applied to rat femoral segmental defects.
    • References (7385)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual subject-level data.

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