Bone regeneration with low dose BMP-2 amplified by biomimetic supramolecular nanofibers within collagen scaffolds

Abstract

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive cytokine that plays a critical role during bone regeneration and repair. In the extracellular environment, sulfated polysaccharides anchored covalently to glycoproteins such as syndecan and also non-covalently to fibronectin fibers have been shown to bind BMP-2 through a heparin-binding domain and regulate its bioactivity. We report here on a synthetic biomimetic strategy that emulates biological BMP-2 signaling through the use of peptide amphiphile nanofibers designed to bind heparin. The supramolecular nanofibers, which integrate the biological role of syndecan and fibronectin, were allowed to form gel networks within the pores of an absorbable collagen scaffold by simply infiltrating dilute solutions of the peptide amphiphile, heparan sulfate, and BMP-2. The hybrid biomaterial enhanced significantly bone regeneration in a rat critical-size femoral defect model using BMP-2 amounts that are one order of magnitude lower than required for healing in this animal model. Using micro-computed tomography, we also showed that the hybrid scaffold was more effective at bridging within the gap relative to a conventional scaffold of the type used clinically based on collagen and BMP-2. Histological evaluation also revealed the presence of more mature bone in the new ossified tissue when the low dose of BMP-2 was delivered using the biomimetic supramolecular system. These results demonstrate how molecularly designed materials that mimic features of the extracellular environment can amplify the regenerative capacity of growth factors.

Figure 1

(A) Chemical structure of heparin-binding peptide amphiphile used. (B) Cryogenic transmission electron micrograph showing the filamentous nature of nanostructures formed by the heparin-binding peptide amphiphile. (C) In vitro analysis of BMP-2 release from nanofiber gels with or without heparan sulfate (n = 3) (ELISA data presented as mean ± standard error of the mean). (D) Scanning electron micrographs of a plain collagen scaffold before infiltration with peptide amphiphile and heparan sulfate solutions. (E and F) Scanning electron micrographs of the surface (E) and interior (F) of hybrid scaffolds. (G) Fluorescence imaging of a cross-section of collagen scaffold containing fibers of heparin-binding peptide amphiphile and FITC-tagged heparan sulfate.

Figure 2

Analysis of the in vivo bone regeneration capacity of heparan sulfate-presenting heparin-binding peptide amphiphile nanofibers. (A) Representative femur reconstructions from micro-computed tomography are shown for the various treatment groups. (B) The number of animals used per condition and the number of animals with a bridged femur after treatment. (C) Quantitative analysis of new bone volume (mm³) within the defect measured by micro-computed tomography. Data are presented as mean ± standard error of the mean; ***P < 0.001. (D) Representative histological longitudinal sections of demineralized femora stained with Goldner's Trichrome. The presence of ossification is characterized by collagen (green) with cytoplasm (red), erythrocytes (orange), and nuclei (dark violet). In the upper box, arrows indicate the proximal (top) and distal (bottom) edges of the defect (scale bars=1 mm). The lower box shows a higher magnification image of the inset indicated above (scale bars, 200 μm).

Figure 3

(A) Schematic representation of extracellular matrix components involved in ligand-receptor interactions during BMP-2 signaling. Syndecan-4 is a transmembrane proteoglycan containing covalently bonded strands of sulfated polysaccharides. These sulfated macromolecules interact non-covalently with fibronectin fibers via their heparin-binding domains and also localize BMP-2 cytokines to facilitate interaction with their cell-surface receptors (27, 51). (B) Schematic representation of our biomimetic strategy to emulate certain aspects of BMP-2 signaling. A supramolecular nanofiber biomimetic of fibronectin displays sulfated polysaccharide strands on its surface, which can localize heparin-binding cytokines such as BMP-2 to facilitate ligand-receptor interactions. The sulfated polysaccharide strands can also bundle with fibronectin fibers through their heparin-binding domains and consequently recruit integrins during signaling (not shown).