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

Abstract

Growth factors (GFs) are powerful signaling molecules with the potential to drive regenerative strategies, including bone repair and vascularization. However, GFs are typically delivered in soluble format at supraphysiological doses because of rapid clearance and limited therapeutic impact. These high doses have serious side effects and are expensive. Although it is well established that GF interactions with extracellular matrix proteins such as fibronectin control GF presentation and activity, a translation-ready approach to unlocking GF potential has not been realized. We demonstrate a simple, robust, and controlled material-based approach to enhance the activity of GFs during tissue healing. The underlying mechanism is based on spontaneous fibrillar organization of fibronectin driven by adsorption onto the polymer poly(ethyl acrylate). Fibrillar fibronectin on this polymer, but not a globular conformation obtained on control polymers, promotes synergistic presentation of integrin-binding sites and bound bone morphogenetic protein 2 (BMP-2), which enhances mesenchymal stem cell osteogenesis in vitro and drives full regeneration of a nonhealing bone defect in vivo at low GF concentrations. This simple and translatable technology could unlock the full regenerative potential of GF therapies while improving safety and cost-effectiveness.

Keywords: BMP-2; Bone Regeneration; biomaterials; fibronectin; growth factors.

Fig. 1. Fibril-based GF presentation.

(A) FN contains three types of domains that promote integrin binding (III_9–10), GF sequestration (III_12–14), and FN-FN interactions (I_1–5). (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 (FNIII_12–14) 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 FNIII_12–14; 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).