Injectable nanosilica-chitosan microparticles for bone regeneration applications

Abstract

This study was aimed at assessing the effects of silica nanopowder incorporation into chitosan-tripolyphosphate microparticles with the ultimate goal of improving their osteogenic properties. The microparticles were prepared by simple coacervation technique and silica nanopowder was added at 0% (C), 2.5% (S1), 5% (S2) and 10% (S3) (w/w) to chitosan. We observed that this simple incorporation of silica nanopowder improved the growth and proliferation of osteoblasts along the surface of the microparticles. In addition, the composite microparticles also showed the increased expression of alkaline phosphatase and osteoblast specific genes. We observed a significant increase ( p < 0.05) in the expression of alkaline phosphatase by the cells growing on all sample groups compared to the control (C) groups at day 14. The morphological characterization of these microparticles through scanning electron microscopy showed that these microparticles were well suited to be used as the injectable scaffolds with perfectly spherical shape and size. The incorporation of silica nanopowder altered the nano-roughness of the microparticles as observed through atomic force microscopy scans with roughness values going down from C to S3. The results in this study, taken together, show the potential of chitosan-tripolyphosphate-silica nanopowder microparticles for improved bone regeneration applications.

Keywords: Chitosan; differentiation; nano-roughness; nanosilica; osteoblasts; proliferation.

Figure 1.

Representative SEM images for microparticle showing the spherical morphology at low magnification (a) (100×) and the surface at high magnification (b) (2000×). The bar graph on c shows the quantitative analysis of elemental Si along the surface of microparticles. Highest wt. % Si was observed in the S3 groups containing 10% (w/w) nSiO₂. EDS spectrum (d) shows the elements present at the surface of the microparticles.

Figure 2.

Comparison of Fourier transform infrared (FTIR) spectrum of pure CS and sample microparticle group. S1 spectrum is shown in the figure as a representative spectrum for sample group as the spectrum looked similar irrespective of nSiO₂ content.

Figure 3.

AFM height images showing 3D representation of the surface scanned over 1 μm region along the surface of C (a), S1(b), S2 (c) and S3 (d). The image is in the scale of 125 nm. The bar graph e shows the average roughness (Rq) for different group of microparticles. The avergae Rq decreases with increase in nSiO₂ content in the mocroparticles. * represents siginificant difference in Rq between and C and S3 group.

Figure 4.

WST-1 assay results showing the O.D. values for cells cultured with media extracted from different groups.

Figure 5.

Cytation 5 images of microparticles with cells proliferating along their surface on day 10 imaged after staining with calcein AM. The cell proliferation was higher on S2 (c) and S3 (d) compared to C (a) and S1 (b) (scale: 1000 μm).

Figure 6.

SEM images showing the morphology of cells attached to the surface of microparticles at day 5. Cells had a flattened morphology on all groups with more elongated structure on sample groups (scale: 30 μm).

Figure 7.

Amount of DNA obtained from the cells attached and proliferated along the surface of the microparticles on day 7 and 14. * represents the significant difference from the control group on same day and # represents significant difference for same group on different days (p < 0.05).

Figure 8.

ALP activity of the microparticles normalized with the total protein amount at different days of culture. Apparently, the sample groups containing nSiO₂ had significantly higher ALP activity during the whole study period.

Figure 9.

Expression profile for osteoblast specific genes over 14 days. The expression of different genes by the cells growing on the sample groups (S1, S2 and S3) was higher than that expressed by the cells growing on the control group (C). * represents the significant difference from the control group on same day and # represents significant difference for same group on different days.