Research ArticleBIOMATERIALS

Hyperelastic “bone”: A highly versatile, growth factor–free, osteoregenerative, scalable, and surgically friendly biomaterial

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Science Translational Medicine  28 Sep 2016:
Vol. 8, Issue 358, pp. 358ra127
DOI: 10.1126/scitranslmed.aaf7704
  • Fig. 1. Versatility, scalability, and manipulation of 3D-printed HB.

    (A) Easy to synthesize volumes (~100 ml shown) of liquid-based HB inks (inset) can be 3D-printed into a variety of structures: 3D-printed 12 × 12–cm HAPLGA sheet comprising three layers, which can be manipulated in a variety of ways, including rolling, folding, and cutting. Origami methods may be used to create complex folded structures, whereas kirigami methods can produce complex structures from strategic folding and cutting. (B) Full-scale, anatomically correct parts, such as a human mandible, comprising >250 layers, can be designed, 3D-printed from HAPLGA, and washed to rapidly produce a ready-to-implant object. Final image shows 3D-printed mandible next to an adult cadaveric human mandible. (C) Photograph series illustrating that custom-sized HAPLGA sleeves can be snuggly stretched around, cut, and sutured to a soft tissue, such as human cadaveric tendon, facilitating arthroscopic ACL repair and replacement surgery. (D) Independently 3D-printed HAPLGA miniature-scale versions of a human skull, skull cap, mandible, and upper thoracic seamlessly fused together to create highly complex structures by using HB ink applied to points of contact. (E) Black light–illuminated optical photographs of the outside and internal cross sections of HAPLGA fiber with (top) and without (bottom) incorporated recombinant green fluorescent protein (rGFP).

  • Fig. 2. Microstructural characteristics of HB and related 3D-printed systems.

    (A to D) Scanning electron microscopy (SEM) micrographs of representative fibers produced by HA/PCL (1:1 by mass) hot-melt (A), HA/PCL (1:1) room temperature solvent mixture (B), HA/PCL (9:1) room temperature with DCM only (C), and HA/PCL (9:1) with a trisolvent (HAPCL) (D). (E) Schematic representation of proposed HA and elastomer distribution within fibers with single- or graded-solvent mixtures, as a function of time after extrusion. Higher-magnification SEM micrographs of DCM solvent only (F) and HAPCL microstructures (G). Details regarding material compositions and preparations can be found in Table 1.

  • Fig. 3. HB mechanical properties.

    (A) Photograph series showing the compression and recovery of a 1-cm-diameter 3D-printed HAPLGA cylinder over a single compression cycle. (B) Digital representation of average adult human femur and corresponding femoral midshaft section longitudinal and axial views. Axial (C) and longitudinal (D) views of 3D-printed HB femoral midshaft construct using digital file shown in (B). (E) Longitudinal compressive loading profile of HB femoral midshaft (D) and corresponding photographs at indicated percent strain points. Plastic deformation of HB femoral midshaft begins at 2 (10.3% strain) and proceeds to buckle and barrel (3 and 4). Cyclic compression loading profile (10 cycles) of HB femoral midshaft loaded in axial direction (C) in strain domain (F) and time domain (G). (H) Photograph series of a single axial compression cycle displayed in (F) and (G) and the corresponding percent strain. Additional characterization of HB mechanical properties can be found in the Supplementary Materials.

  • Fig. 4. Physical properties of HB.

    (A) Dry and wet densities of HAPCL and natural bone. Asterisk denotes value from literature (53). Upward arrow indicates that the wet density is expected to increase in vivo as water is replaced with proteins and tissues. (B) Time series contact angle profile of water on solid HAPCL surface (top) and small volume of red-colored water being injected into the end of a complex 3D-printed HAPLGA object (bottom). (C to G) Alizarin Red S–stained and Alizarin Red S–unstained (insets) photographs of HAPCL (C), HAPLGA (D), 1:1 HA/PLGA hot-melt (E), and 1:1 HA/PLGA room temperature solvent scaffolds (F) and human cadaveric bone (G). (H) Thermogravimetric profile of as-printed, water-washed, and 70% ethanol–washed HAPLGA scaffolds. Expected evaporation or decomposition temperature ranges for individual components indicated. Note that the PLGA decomposition temperature range and the DBP boiling temperature overlap.

  • Fig. 5. In vitro evaluation of 3D-printed HB scaffolds with MSCs.

    Photograph of 90° offset HAPCL scaffold (A) and the corresponding cross-sectional SEM micrograph highlighting the offset architecture between layers (B). (C and D) (Top) Top-down view, laser-scanning confocal reconstructions of live (green; calcein AM) and dead (red, ethidium homodimer-1) stains. (Bottom) Corresponding cross-sectional SEM micrographs of HAPCL samples 7 days (C) and 28 days (D) after seeding with hMSCs. (E) PicoGreen quantitation of DNA in HAPLGA and HAPCL scaffolds at indicated time points after seeding hMSCs. Values were normalized to average DNA measured on day 1 (n = 3 for all time points; error bars refer to SD). (F) ALP activity normalized to corresponding DNA content from HAPCL scaffolds at indicated time points (n = 3 for all time points; same samples as those used for DNA quantification; error bars refer to SD). (G) Gene expression levels of osteogenic-relevant transcripts in hMSCs grown on HAPCL scaffolds, normalized to sample-specific GAPDH (glyceraldehyde-3-phosphate dehydrogenase) values, followed by normalization to day 0 hMSC values (n = 3; error bars refer to SD). (H) Atomic Ca/P measured in the HA of the scaffold itself (Scaffold HA) and of the nanocrystals within the ECM formed after in vitro culture. Gray box, Ca/P range of natural HA. (E to G) *P < 0.05, over previous time point for the same group. Confidence intervals (P values; two-tailed, equal variance t tests) are as follows: for DNA quantification of HAPCL samples: days 1 and 7, 0.0016; days 7 and 14, 0.013; days 14 and 28, 0.152; for DNA quantification of HAPLGA samples: days 1 and 7, 0.0032; days 7 and 14, 0.043; days 14 and 28, 0.862; for ALP/ng DNA of HAPCL samples: days 1 and 7, 0.034; days 7 and 14, 0.0026; days 14 and 28, 0.0053; and for fold increase in gene expression of HAPCL samples: osteopontin: days 7 and 14, 0.00041; days 14 and 28, 0.00026; collagen I: days 7 and 14, 0.0033; days 14 and 28, 0.017; osteocalcin: days 7 and 14, 0.0049; days 14 and 28, 0.050. Additional in vitro related figures for hMSCs seeded onto 30° advancing angle HB scaffolds can be found in the Supplementary Materials.

  • Fig. 6. Biocompatibility evaluation in vivo with a mouse subcutaneous implant.

    (A) Gross hematoxylin and eosin (H&E) histological image of day 35 HB explant cross section. Blue arrows, HB fiber cross sections. The densely packed HA particles within the HB stain dark purple to black. (B and C) H&E histological image of day 35 explant with HB fiber cross sections (dotted yellow circles), vessels (dashed yellow circles), and capillaries (solid yellow circles). (C) Higher-magnification section of (B). (D) SEM micrograph of day 35 explanted HB scaffold. Several vessels are indicated by dotted yellow circles; soft tissue completely fills the space between the HB fibers. (E) Higher-magnification SEM micrograph of (D) highlighting the structure of HB, new tissue, and the HB scaffold-tissue interface (dashed line). (F) Gross H&E histological image of day 35 hot-melt 1:1 HA/PLGA–explanted scaffold. Because of the solubility of PLGA in common histological solvents, and given that more than half the scaffold volume is composed of PLGA, the 1:1 HA/PLGA scaffold materials and incorporated tissues did not survive histological processing. (G) SEM micrograph of the cross section of day 35 hot-melt 1:1 HA/PLGA–explanted scaffold and tissue. No vessels are visible. (H) Increased magnification SEM micrograph of (G) illustrating the hot-melt HA/PLGA–tissue interface (dashed line). (I) Representative burr cell; these were found throughout the tissue within the hot-melt 1:1 HA/PLGA–explanted scaffolds. Additional related figures can be found in the Supplementary Materials.

  • Fig. 7. Evaluation of HB in vivo for rat spinal fusion.

    (A) Representative radiograph illustrating bilateral placement of HAPLGA (HB) scaffolds across the L4 and L5 vertebral body transverse processes. (B) Photograph of the cross section of rat spinal column containing HB scaffolds 4 weeks after implantation. Placement of HB scaffolds on transverse processes (black arrows) is visible, as is significant tissue incorporation into the scaffolds. (C) Fusion scores of spinal segments with scaffolds explanted after 8 weeks. An established scoring system for the fusion score was used, whereby 0 = no bridging bone, 1 = unilateral bridging bone, and 2 = bilateral bridging bone. ACS, absorbable collagen sponge (historical control); DBM, demineralized bone matrix; HA, hydroxyapatite granules; HB, HAPLGA; HB + BMP, HAPLGA preloaded with 1.5 μg of rhBMP-2 before implantation. (D) Fusion rates were calculated as both percent sides fused (two sides per animal) as well as on a per-animal basis [n = 6 for ACS, DBM, HA, HB + BMP groups; n = 12 (two n = 6 replicates) for HB group]. For the latter, unilateral bridging bone was considered successful fusion (fusion score of ≥1.0). (E) Laboratory microCT–based quantification of new bone volume within and surrounding the HB scaffolds (with and without 1.5 μg of rhBMP-2 per scaffold). n = 6 animals for all groups. (F and G) Representative single-slice synchrotron microCT images of the cross section of HB scaffolds without (F) and with (G) rhBMP-2 added. Green rectangles, region enlarged at right; bright white, native bone; speckled black-white, HB scaffold. Error bars for (C) and (E) refer to SD. Confidence intervals (P values; two-tailed, equal variance t tests) for groups shown in (C) are as follows: HB/HB-BMP, 0.0036; DBM/HB-BMP, 0.0004; HB/DBM, 0.21. Confidence intervals (P values; two-tailed, equal variance t tests) for groups shown in (E) are as follows: per-scaffold HB/HB-BMP, 0.00003; per-animal HB/HB-BMP, 0.0003. *P < 0.05, between indicated groups or when compared with previous time point for the same material group. The double asterisk (**) indicates a value of 0 (no fusion or newbone formation wa observed).

  • Fig. 8. HB as a macaque calvarial defect bone graft.

    (A) Photograph of 40 × 40 × 4–mm 3D-printed HAPLGA (HB) bone graft. The top and bottom layers of the construct are solid, whereas the sides and interior are porous (inset). (B) Photograph of trimmed HB construct from (A) after implantation into ~2 cm across the calvarial defect site. (C) Photograph series of the HB graft and defect site 4 weeks after implantation, and the resulting cranial-HB explant. Note that, during explantation, the extracranial-oriented solid HB surface peeled away from the rest of the construct. (D) Photographs showing (top) intracranial surface of the cranial-HB explant and the corresponding micrograph (bottom) of the surface of the HB graft indicated by purple box. Small vessels emanating from the native tissue on the exterior of the HB graft under the thin surface of the HB graft (arrow). (E) Photographs of gross cross section (top) and the corresponding optical micrographs (bottom) highlighting the cranial graft interface (region a) and the center of graft (region b). (F) Photograph (top) showing the extracranial surface of the cranial-HB explant and the corresponding micrograph (bottom) of the surface of the HB graft indicated by purple box. (G) Mechanical compression results of 4-mm-diameter biopsies taken from the corresponding numbered regions in (E) as well as from the as-printed bone graft in (A) (1) before implantation. The four samples from region 1 were samples from across the width of the 40 × 40 × 4–mm graft. (H) Decalcified H&E histograph of cortical bone from calvarium at site of attachment of the scaffold (region 3), with associated fibrovascular proliferation (yellow line). A small portion of the scaffold survived the decalcification and histological processing (black arrow). (I) New woven bone (blue square) and associated fibrovascular connective tissue along the periosteal surface (region 3) of the cortical bone (yellow line). (J) Evidence of significant soft tissue infiltration and vascularization (arrows) within the interior of the HB scaffold (region 4). (K) Fibrovascular proliferation and associated inflammation composed of macrophages, lymphocytes, and plasma cells within region 4. (L) A false color single slice of synchrotron microCT reconstruction from the rhesus macaque 4-week HB calvarial explant, depicting an area between regions 3 and 4 from (E). The explant is characterized by significant soft tissue (pink) integration, including intimate growth along the edges and into the HB material. Large void spaces between the tissue and the HB within the construct are likely artifacts from the chemical fixation and dehydration procedure after explantation. (M) A possible early-stage new osseous tissue is present (yellow/brown) emerging from or extending into an HB fiber [detail of the box surrounded by dashed white line is in (L)]. Grayscale, contrast-defined reconstructed slices can be found in the Supplementary Materials (fig. S9).

  • Table 1. Summary of formulations, material preparation, 3D printing process, and additional characteristics of the HA-polymer composite systems discussed throughout this work.

    The two materials, HAPCL and HAPLGA, are collectively referred to as HB. 3DP, 3D printing.

    Solid
    composition (%)
    Solvent
    composition (%)*
    3DP preparation3DP
    conditions
    3DP rates
    (mm/s)
    Solidification
    mechanism
    Mechanical
    characteristics
    Trisolvent
    (HAPCL) (9:1)
    HA: 90
    PLGA: 0
    PCL: 10
    DCM: 50
    2-Bu: 33
    DBP: 17
    Ambient wet mixingAmbient0.5–150DCM evaporationElastic
    Trisolvent
    (HAPLGA) (9:1)
    HA: 90
    PLGA: 10
    PCL: 0
    DCM: 50
    2-Bu: 33
    DBP: 17
    Ambient wet mixingAmbient0.5–150DCM evaporationElastic
    Hot-melt (1:1)HA: 50
    PLGA: 50
    PCL: 0
    DCM: 0
    2-Bu: 0
    DBP: 0
    Dry mixing and melting at 200°C200°C0.1–5Temperature-inducedBrittle
    Trisolvent (1:1)HA: 50
    PLGA: 0
    PCL: 50
    DCM: 50
    2-Bu: 33
    DBP: 17
    Ambient wet mixingAmbient0.5–20DCM evaporationPlastic
    Single-solvent (9:1)HA: 90
    PLGA: 0
    PCL: 10
    DCM: 100
    2-Bu: 0
    DBP: 0
    Ambient wet mixingAmbient>50DCM evaporationBrittle

    *Estimation based on initial volume of solvents used for ink synthesis and volume change after sufficient evaporation of DCM.

    Hardware speed limitations of the 3D printing platform used in this work. Higher speeds may be possible with other hardware.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/8/358/358ra127/DC1

      Materials and Methods

      Fig. S1. Additional functionalities and potential applications of 3D-printed HB.

      Fig. S2. Microstructure of 1:1 HA/PLGA hot-melt 3D-printed composite.

      Fig. S3. Additional rheological and mechanical properties of HB.

      Fig. S4. Origin of HB’s mechanical properties and additional mechanical data.

      Fig. S5. Axial compressive loading of HB femoral section by hand.

      Fig. S6. Additional in vitro results: hMSCs seeded onto 30° advancing angle HAPCL scaffolds.

      Fig. S7. Additional mouse subcutaneous implant in vivo results 7 and 35 days after implantation.

      Fig. S8. Additional in vivo SEM micrographs of HB scaffolds explanted 35 days after being subcutaneously implanted into a mouse.

      Fig. S9. Contrast-enhanced grayscale (non–false-colored) version of Fig. 8 (L and M).

      Movie S1. A 32× speed movie of HAPLGA ink being 3D-printed into 14-cm-tall, 6-mm-diameter cylinder composed of hundreds of layers, followed by a 64× speed movie illustrating HAPLGA being 3D-printed into 7.5-cm-tall double helix modeled after the structure of DNA.

      Movie S2. 3D printing and physical manipulation of HB (HAPLGA) sheets.

      Movie S3. HA/PLGA (1:1) hot-melt 3D-printed object being impacted and shattered by a hammer, followed by 3D-printed HAPLGA undergoing a series of hammer impacts and bouncing back.

      Movie S4. Longitudinal compression, axial cyclic, and finger compression of hydrated 3D-printed HB femoral midshafts shown in Fig. 3.

      Movie S5. Synchrotron microCT of 8-week PLF-explanted HAPLGA (HB) scaffolds (black and white speckled object in the movie), without and with 3 μg of rhBMP-2 added, illustrating new bone (dense white) material within and around HB.

    • Supplementary Material for:

      Hyperelastic "bone": A highly versatile, growth factor–free, osteoregenerative, scalable, and surgically friendly biomaterial

      Adam E. Jakus, Alexandra L. Rutz, Sumanas W. Jordan, Abhishek Kannan, Sean M. Mitchell, Chawon Yun, Katie D. Koube, Sung C. Yoo, Herbert E. Whiteley, Claus-Peter Richter, Robert D. Galiano, Wellington K. Hsu, Stuart R. Stock, Erin L. Hsu, Ramille N. Shah*

      *Corresponding author. Email: ramille-shah{at}northwestern.edu

      Published 28 September 2016, Sci. Transl. Med. 8, 358ra127 (2016)
      DOI: 10.1126/scitranslmed.aaf7704

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. Additional functionalities and potential applications of 3D-printed HB.
      • Fig. S2. Microstructure of 1:1 HA/PLGA hot-melt 3D-printed composite.
      • Fig. S3. Additional rheological and mechanical properties of HB.
      • Fig. S4. Origin of HB’s mechanical properties and additional mechanical data.
      • Fig. S5. Axial compressive loading of HB femoral section by hand.
      • Fig. S6. Additional in vitro results: hMSCs seeded onto 30° advancing angle HAPCL scaffolds.
      • Fig. S7. Additional mouse subcutaneous implant in vivo results 7 and 35 days after implantation.
      • Fig. S8. Additional in vivo SEM micrographs of HB scaffolds explanted 35 days after being subcutaneously implanted into a mouse.
      • Fig. S9. Contrast-enhanced grayscale (non–false-colored) version of Fig. 8 (L and M).
      • Legends for movies S1 to S5

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Movie S1 (.mp4 format). A 32x speed movie of HAPLGA ink being 3D-printed into 14-cm-tall, 6-mm-diameter cylinder composed of hundreds of layers, followed by a 64x speed movie illustrating HAPLGA being 3D-printed into 7.5-cm-tall double helix modeled after the structure of DNA.
      • Movie S2 (.mp4 format). 3D printing and physical manipulation of HB (HAPLGA) sheets.
      • Movie S3 (.mp4 format). HA/PLGA (1:1) hot-melt 3D-printed object being impacted and shattered by a hammer, followed by 3D-printed HAPLGA undergoing a series of hammer impacts and bouncing back.
      • Movie S4 (.mp4 format). Longitudinal compression, axial cyclic, and finger compression of hydrated 3D-printed HB femoral midshafts shown in Fig. 3.
      • Movie S5 (.mp4 format). Synchrotron microCT of 8-week PLF-explanted HAPLGA (HB) scaffolds (black and white speckled object in the movie), without and with 3 μg of rhBMP-2 added, illustrating new bone (dense white) material within and around HB.

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