Abstract
Infection is a frequent complication of bone-grafted sites. Increasing bacterial resistance to antibiotics is a challenge associated with the prevention or treatment of such infections. Silver nanoparticles (AgNPs) are a potent alternative to antibiotics. We aimed to develop two antibacterial bone regenerative scaffolds with integrated AgNPs.
AgNPs were incorporated into both bovine bone particles (BBX) and light cross-linked gelatin methacryloyl (GelMA) hydrogel. Cubes of bovine bone were treated at temperatures between 100°C to 220°C at 30°C intervals and with pressures ranging from 1.01 to 24.58 Bar, producing five different groups. The samples were characterised topographically using scanning electron microscopy (SEM), and mechanically using atomic force microscopy (AFM) and compression testing. The chemical composition and the organic content were determined using Fourier-transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The BBX was further characterised both pre- and post-chemical treatment (bleaching) and after terminal sterilisation by gamma irradiation. Metabolic activity of osteoblasts and osteoclasts on the constructs was investigated using Prestoblue®. For the GelMA, size and consistency of the constructs were optimised. Interaction and stabilisation of AgNPs in GelMA constructs were characterised using SEM, energy dispersive X-ray spectroscopy, and FTIR. Cell viability of encapsulated human gingival fibroblasts (HGFs) was determined by Prestoblue® assay and live/dead staining with confocal laser scanning microscopy. Transmission electron microscopy was utilised to study retention of AgNPs. AgNP release was measured by inductively-coupled plasma-mass spectrometry. Bacterial viability of encapsulated Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus) was determined using an assay for metabolic activity, and the antibacterial properties of the GelMA-AgNPs constructs were determined using disc diffusion. The safety and regenerative capacity of the optimised AgNPs functionalised BBX and GelMA was tested in a rabbit cranial model.
Increasing the deproteinisation temperature/pressure of the BBX was associated with decreased organic content and compressive strength and increased crystallinity and surface irregularities. Higher osteoblast differentiation and proliferation was obtained from treatments at temperatures below 190°C. Results showed that bleaching and gamma irradiation had limited effect on the surface texture but significantly reduced organic content. A significant dcrease in the collagen content was detected post-bleaching, and in the carbonate content post-gamma irradiation. The presence of AgNPs appeared to enhance proliferation of osteoblasts in vitro compared to AgNP-free controls. Stiff hydrogel constructs showed superior AgNP retention, however high stiffness negatively impacted both handling properties and AgNP diffusion within the constructs. We also found that AgNPs at a concentration of 100 µg/ml inhibited bacteria with minimal adverse effects. Our rabbit model showed that both the optimised BBX at 160°C and 5%wt GelMA hydrogels were biocompatible and had similar regenerative capacity compared to a commercially available product (Bio-Oss®). There were no signs of inflammatory infiltrates, necrosis, or connective tissue encapsulation in the grafted sites.
In conclusion, increasing the processing temperature correlated with significant changes in the characteristics of the BBX, and the deproteinisation temperature can be adjusted to modify the graft properties for various applications. The incorporation of AgNPs in biomaterials ensured stabilisation of nanoparticles, particularly when encapsulated in GelMA hydrogel. A 100 µg/ml dose of AgNPs inhibited bacteria, with minimal adverse effects on the bone cells. Grafts functionalised with AgNPs can provide antibacterial protection and simultaneously act as a scaffold for attachment of bone cells.