Nanoscale control of silica particle formation via silk-silica fusion proteins for bone regeneration

Abstract

The biomimetic design of silk/silica fusion proteins was carried out, combining the self assembling domains of spider dragline silk (Nephila clavipes) and silaffin derived R5 peptide of Cylindrotheca fusiformis that is responsible for silica mineralization. Genetic engineering was used to generate the protein-based biomaterials incorporating the physical properties of both components. With genetic control over the nanodomain sizes and chemistry, as well as modification of synthetic conditions for silica formation, controlled mineralized silk films with different silica morphologies and distributions were successfully generated; generating 3D porous networks, clustered silica nanoparticles (SNPs), or single SNPs. Silk serves as the organic scaffolding to control the material stability and multiprocessing makes silk/silica biomaterials suitable for different tissue regenerative applications. The influence of these new silk-silica composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on the silk/silica films. The presence of the silica in the silk films influenced osteogenic gene expression, with the upregulation of alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col 1) markers. Evidence for early bone formation as calcium deposits was observed on silk films with silica. These results indicate the potential utility of these new silk/silica systems towards bone regeneration.

Figure 1

SEM images of (A-B) 5%, (C-D) 2.5%, and (E-F) 2.5% (glycerol) wt./vol. silk films with silica nanostructures.

Figure 2

Comparison of FTIR spectra of silk film before and after methanol treatment. Methanol induces β-sheet formation within the silk film as shown as a shift in the FTIR spectrum.

Figure 3

EDAX spectrum of silk/silica film exhibiting strong peaks for elemental silicon and oxygen which confirm that nanostructures observed on the surface of silk film originate from silica.

Figure 4

Optical microscope images of hMSCs on 5% wt/vol silk films mineralized with silica before (A) and after (B) differentiation into osteogenic lineages.

Figure 5

Osteogenic gene expression of cells grown on 5% wt./vol. silk films with silica or silk only (Silk) and tissue culture plastic (TCP) after exposure to osteogenic stimulants for 2 weeks as compared. Each column represents the mean and standard deviation of N = 3 independent cultures. P<0.05.

Figure 6

SEM image and EDAX mapping of 5% wt./vol. silk film with silica after 2 weeks of hMSCs osteogenic culture.

Scheme 1

Method to control silica distribution on silk films.