Research ArticleBIOMEDICAL ENGINEERING

Material-driven fibronectin assembly for high-efficiency presentation of growth factors

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Science Advances  26 Aug 2016:
Vol. 2, no. 8, e1600188
DOI: 10.1126/sciadv.1600188
  • Fig. 1 Fibril-based GF presentation.

    (A) FN contains three types of domains that promote integrin binding (III9–10), GF sequestration (III12–14), and FN-FN interactions (I1–5). (B) FN adsorption results in an FN nanonetwork spontaneously assembled on the material surface of PEA but not on PMA, allowing us to propose synergistic integrin/GF receptor signaling to direct MSC differentiation in vitro and tissue healing in vivo. (C) FN organized into fibrils on PEA displays higher availability of the GF-binding region (FNIII12–14) than FN adsorbed on PMA in a globular conformation (left). However, similar surface density of BMP-2 on FN-coated PEA and PMA occurs regardless of the organization and conformation of FN on both surfaces (center). Very low release of BMP-2 over 14 days was observed using enzyme-linked immunosorbent assay (ELISA). (D) Atomic force microscopy (AFM) images at different magnifications after the sequential adsorption of FN (3 μg/ml) and BMP-2 (25 ng/ml) on PEA and PMA. No FN/BMP-2 interactions were noted on PMA (only random apposition of both molecules on the surface), whereas on PEA fibrillar FN molecules contained globular aggregates that we proposed to be BMP-2. (E) AFM images of BMP-2 interacting with single FN molecules on PEA (top, phase magnitude; bottom, height magnitude as indicated on the pictures), where a secondary antibody (Ab) bound to a 15-nm gold nanoparticle was used to identify BMP-2 on FN. The Tukey-Kramer method was used with multiple-comparisons posttest analysis of variance (ANOVA). Symbols show statistical significant differences with all the other conditions on PEA (*P 0.001). a.u., arbitrary unit.

  • Fig. 2 Integrin/BMP-2 receptor cosignaling drives MSC osteogenesis.

    (A) Coimmunoprecipitation of integrin β1 and BMPRI occurred on BMP-2 sequestered by FN on PEA, and bands correspond to BMPRIa (60 kD) after precipitation with anti–integrin β1 antibodies. The graphs show quantification of bands relative to the absence of BMP-2. This colocalization can also be seen in individual cells with integrin β1 (stained red) and BMPRIa (stained green). (B) Smad signaling was drastically altered when BMP-2 was presented bound on FNIII12–14; blocking this GF-binding domain of FN (using the monoclonal antibody P5F3 at a molar ratio of 1 with FN to block the GF-binding site) reduces Smad signaling. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Phosphorylation of extracellular signal–related kinase (ERK) 1/2 was significantly enhanced on PEA when BMP-2 was presented at the material interface, sequestered on FN, compared to the presence of the same doses of the soluble factor. (D) In-cell Western assay for Smad, FAK, pERK 1/2, and pRUNX2 with BMP-2 on FN on PEA, soluble BMP-2, and blocking with P5F3 before BMP-2 adsorption. (E) This fulfills the first part of the synergistic signaling hypothesis. (F) Quantitative polymerase chain reaction (qPCR) for osteocalcin (OCN) and osteonectin (ON) after 14 days of culture (PEA and PMA); enhanced expression occurs when BMP-2 was presented bound on FN compared to soluble administration of the GF or when BMP-2 was sequestered on the material surface (PMA) but not bound to FN. (G) Immunofluorescence for osteocalcin and osteonectin confirmed the results obtained at the gene level [red, osteocalcin/osteonectin; blue, 4′,6-diamidino-2-phenylindole (DAPI)]. (H) Alkaline phosphatase (ALP) staining on PEA comparing BMP-2 bound to FN fibrils versus soluble BMP-2. Noggin (50 ng/ml) was used in both conditions as the BMP-2 inhibitor to prevent activity (image quantification included in the graph). Results show means ± SD [n = 3 for all experiments, except for the graph in (H), where 9 images were used]. The Tukey-Kramer method was used with multiple-comparisons posttest ANOVA. Symbols show statistical significant differences with all the other conditions (*P = 0.001, ¥P = 0.01).

  • Fig. 3 Bone regeneration in a critical-size defect with very low doses of BMP-2.

    (A) A cylindric polyimide sleeve was coated with the polymers (either PEA or PMA; the figure shows a picture of the sleeve and the coating is shown with a florescent dye) and implanted in a critical-size defect (2.5 mm) in a murine radius. Faxitron images show the evolution of the defect at different time points after implanting PEA coated with FN and BMP-2. The total amount of BMP-2 was ~15 ng. (B) Three-dimensional (3D) μCT reconstructions for both PEA and PMA polymers after 4 and 8 weeks, with three conditions: polymer only (PEA and PMA), FN coating on the polymer (FN), and FN coating on the polymer followed by BMP-2 adsorption (FN + BMP-2). The positive control is a PEG hydrogel loaded with ~175 ng of BMP-2. (C) μCT measures of bone volume within the defects. (D) Sections of 8-week radial samples stained with Safranin O/Fast Green. Arrow 1 shows the fibroblast-like morphology of cells. Arrows 2 and 3 show the new bone cells coming out of both distal and proximal sides. Arrow 4 shows bone marrow–like cavities found in the new bone. Arrow 5 shows the point at the contact point of new bone, coming out of both distal and proximal sides. Five animals (n = 5) per condition were used. Symbols show statistical significant differences with all the other conditions (*P = 0.001).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/8/e1600188/DC1

    fig. S1. AFM height (left) and phase (right) images of bare PEA and PMA surfaces at different magnifications from 500 nm to 5 μm.

    fig. S2. AFM phase images of BMP-2 and PDGF-BB (100 ng/ml) adsorbed on PEA and PMA, with indication of average size of the GF aggregates on each image.

    fig. S3. Colocalization of integrin β5 and BMPRIa after 1 day of culture on PEA/FN/BMP-2.

    fig. S4. Phosphorylation of FAK on PEA using different conditions for BMP-2 presentation: bound on the surface as a coating versus soluble in the culture medium as well as using P5F3 control.

    fig. S5. Full longitudinal sections of 8-week radial samples stained with Safranin O/Fast Green.

    table S1. Primers for qPCR.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. AFM height (left) and phase (right) images of bare PEA and PMA surfaces at different magnifications from 500 nm to 5 μm.
    • fig. S2. AFM phase images of BMP-2 and PDGF-BB (100 ng/ml) adsorbed on PEA and PMA, with indication of average size of the GF aggregates on each image.
    • fig. S3. Colocalization of integrin β5 and BMPRIa after 1 day of culture on PEA/FN/BMP-2.
    • fig. S4. Phosphorylation of FAK on PEA using different conditions for BMP-2 presentation: bound on the surface as a coating versus soluble in the culture medium as well as using P5F3 control.
    • fig. S5. Full longitudinal sections of 8-week radial samples stained with Safranin O/Fast Green.
    • table S1. Primers for qPCR.

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