Virus Nanoparticles Mediated Osteogenic Differentiation of Bone Derived Mesenchymal Stem Cells

Abstract

There are few methodologies that allow manipulating a biomaterial surface at nanometer scale, which controllably influence different cellular functions. In this study, virus nanoparticles with different structural features are selected to prepare 2D substrates with defined nanoscale topographies and the cellular responses are investigated. It is demonstrated that the viral nanoparticle based substrates could accelerate and enhance osteogenesis of bone derived mesenchymal stem cells as indicated by the upregulation of osteogenic markers, including bone morphogenetic protein-2, osteocalcin, and osteopontin, at both gene and protein expression levels. Moreover, alkaline phosphatase activity and calcium mineralization, both indicators for a -successful bone formation, are also increased in cells grown on these nanoscale possessed substrates. These discoveries and developments present a new paradigm for nanoscale engineering of a biomaterial surface.

Keywords: biomaterials; mesenchymal stem cells; nanotopography; osteogenesis; virus nanoparticles.

Figure 1

A–I) Molecular models shows surface topography of plant viruses used in this study. A,B) Tobacco mosaic virus (TMV); C) potato virus X (PVX); D,E) turnip yellow mosaic virus (TYMV); F,G) turnip vein clearing virus (TVCV); H,I) cowpea mosaic virus (CPMV). Scale bar indicates 10 nm in (A), (C), (D), (F), and (H) and 5 nm in (B), (E), (G), and (I). The models were generated using Pymol (www.pymol.org) with coordinates obtained from RCSB protein data bank.

Figure 2

Representative AFM images showing the coverage of PDL coated substrate with different virus nanoparticles indicate the viral particles, A) TMV; B) TVCV; C) PVX; D) TYMV; and E) CPMV, are mostly intact and fully cover the coating area. F) Root mean square roughness of different virus nanoparticles coated substrates by AFM analysis. Scale bars indicate 1.25 μm in (A)–(C) and 0.5 μm in (D) and (E). The data are expressed as mean ± s.d. (n = 4) ns indicates nonsignificant and p > 0.05 based on ANOVA.

Figure 3

The expression of osteogenic markers in BMSCs cultured on PDL and different virus nanoparticles coated substrates under osteogenic conditions. Quantitative real‐time PCR analysis (RT‐qPCR) showed upregulation of A) osteocalcin and B) osteopontin in cells grown on TMV, TVCV, PVX, and TYMV (but not on CPMV) coated substrates at 7 d after osteogenic induction. C) Immunohistochemical staining reveals that osteocalcin, a canonical osteogenic marker, is exclusively located in cell aggregates growing on TMV, TVCV, PVX, and TYMV substrates (not for CPMV coated substrate). Color representation: nucleus (blue), osteocalcin (red). Scale bar is 100 μm. The data were expressed as mean ± s.d. (n = 3, *p ≤ 0.05, **p ≤ 0.01 based on ANOVA).

Figure 4

Nearest neighbor analysis of BMSCs cultured on PDL and virus substrates. A–C,G–I) DAPI immunohistochemical staining and D–F,J–L) bright field microscopy images of BMSCs on A,D) PDL, B,E) TMV, C,F) TVCV, G,J) PVX, H,K) TYMV, and I,L) CPMV. M) Schematic diagrams of the nearest neighbor analysis. In this analysis the distribution of cells can range from independent (represented by a theoretical Poisson's distribution), to clustered, or regular. N) Plot of BMSCs spatial distribution on PDL control and virus substrates demonstrated cluster growth pattern which correlated to appearance of cells nodules on TMV, TVCV, PVX, and TYMV virus coated substrates. Scale bar is 200 μm.

Figure 5

Cytochemical analysis of the bone differentiation process of BMSCs on PDL and viruses coated substrates at 4 and 7 d after osteogenic induction. A) Alkaline phosphatase activity of cells cultured on different substrates. The data are expressed as mean ± s.d. (n = 3, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001 based on ANOVA). B) Alizarin red staining of each sample at day 7. Cells on virus substrates are positively stained for calcium deposition, whereas negatively stained is observed on PDL substrates. The data are expressed as mean ± s.d. (n = 3, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 based on ANOVA). C) Absorbance at 548 nm normalized to cell number to indicate a relative amount calcium deposit at day 7 stained by alizarin red solution. The mineralization of cells on TMV substrates doubles that of PDL, while PVX and TYMV substrates increase the mineralization by fourfold. TVCV substrates slightly increase the mineralization of cells compare to PDL control substrates. These evidences suggest an improvement in osteogenesis by virus coated substrates.

Figure 6

Immunochemical staining showing the difference in vinculin size of cells on PDL or virus coated substrates for 24 h. A) Immunofluorescence images of cells on different substrates at 24 h prior to osteoinduction (top panel). Color representation: nucleus (blue), vinculin (green), and phalloidin (red). The bottom panel demonstrates vinculin masking and selection of vinculins for size analysis. The selected vinculin spots are highlighted in blue. Scale bar is 50 μm. B) Average vinculin size of cells on different substrates. The data were expressed as mean ± s.d. (n = 3, * represents p ≤ 0.05 based on ANOVA).