Bioactive Natural Protein-Hydroxyapatite Nanocarriers for Optimizing Osteogenic Differentiation of Mesenchymal Stem Cells

Abstract

Improving the controlled release of bioactive growth factors to regulate cell behavior and tissue regeneration remains a need in tissue engineering and regenerative medicine. Inorganic and polymeric nanoparticles have been extensively fabricated as bioactive biomaterials with enhanced biocompatibility and effective carriers of therapeutic agents, however, challenges remain such as the achievement of high loading capacity and sustained release, and the bioactivity preservation of growth factors. Here, a multilayered, silk coated hydroxyapatite (HA) nanocarrier with drug loading-release capacity superior to pure silk or HA nanoparticles was developed. Bone morphogenetic protein-2 (BMP-2) was bound to the silk coatings with a high binding efficiency of 99.6%, significantly higher than that in silk or the HA nanoparticles alone. The release of BMP-2 was sustained in vitro over a period of 21 days without burst release. Compared with BMP-2 loaded silk or HA particles, bone mesenchymal stem cells (BMSCs) showed improved proliferation and osteogenesis when cultured with the BMP-2 loaded composite nanocarriers. Therefore, these silk-HA composite nanoparticles present a useful approach to designing bioactive nanocarrier systems with enhanced functions for bone tissue regeneration needs.

Figure 1

The characterization of different nanoparticles: (A) SEM morphology, (B) Size distribution, (C) FTIR spectra and (D) XRD patterns of silk-HA composite nanoparticles (a), HA nanoparticles (b) and silk nanoparticles (c).

Figure 2

BMP-2 loading and release from the different nanoparticles: (A) Fluorescent microscopy images for FITCBMP-2 (Green) loaded Silk nanoparticles, (B) Fluorescent microscopy images for FITCBMP-2 (Green) loaded HA nanoparticles, (C) Fluorescent microscopy images for FITCBMP-2 (Green) loaded Silk-HA composite nanoparticles, (D) Binding efficiency of BMP-2 on the different particles, and (E, F) BMP-2 release profiles from the different particles. Five replicate samples were used for each condition (n=5). The nanocarriers aggregated to some extent when loaded with B P-2, resulting in the increase of particle sizes.

Figure 3

DNA assay of BMSCs cultured wit different BMP-2 loaded nanoparticles. The samples were as follows: BMP-2, soluble BMP-2; Silk-BMP, BMP-2 loaded silk nanoparticles; HA-BMP, BMP-2 loaded HA nanoparticles; and Silk-HA-BMP, BMP-2 loaded silk-HA composite nanoparticles. Error bars represent mean ± standard deviation with N=5 (*p≤0.05).

Figure 4

Expression of osteogenic markers from BMSCs cultured with different nanoparticles for 1 week: (A) biochemical activity of alkaline phosphatase, (B) calcium content, (C) transcript expression of RUNX2 and (D) transcript expression of osteocalcin. The samples were as follows: BMP-2, soluble BMP-2; Silk, BMP-2 free silk nanoparticles; HA, BMP-2 free HA nanoparticles; Silk-HA, BMP-2 free silk-HA composite nanoparticles; HA-BMP, BMP-2 loaded HA nanoparticles; Silk-HA-BMP, BMP-2 loaded silk-HA composite nanoparticles and Silk-BMP, BMP-2 loaded silk nanoparticles (*p≤0.05, and **P≤0.01,***P≤0.001).

Figure 5

Immunofluorescence staining for collagen I of BMSCs cultured with soluble BMP-2 (a), BMP-2 loaded silk nanoparticles (b), BMP-2 loaded HA nanoparticles (c), and BMP-2 loaded silk-HA composite nanoparticles (d) for 1 week. The blue color (DAPI) represents cell nucleus, while the green color (FITC labeled phalloidin) represents the expression of specific marker protein collagen I.