Sustained Release from Injectable Composite Gels Loaded with Silver Nanowires Designed to Combat Bacterial Resistance in Bone Regeneration Applications

Abstract

One-dimensional nanostructures, such as silver nanowires (AgNWs), have attracted considerable attention owing to their outstanding electrical, thermal and antimicrobial properties. However, their application in the prevention of infections linked to bone tissue regeneration intervention has not yet been explored. Here we report on the development of an innovative scaffold prepared from chitosan, composite hydroxyapatite and AgNWs (CS-HACS-AgNWs) having both bioactive and antibacterial properties. In vitro results highlighted the antibacterial potential of AgNWs against both gram-positive and gram-negative bacteria. The CS-HACS-AgNWs composite scaffold demonstrated suitable Ca/P deposition, improved gel strength, reduced gelation time, and sustained Ag⁺ release within therapeutic concentrations. Antibacterial studies showed that the composite formulation was capable of inhibiting bacterial growth in suspension, and able to completely prevent biofilm formation on the scaffold in the presence of resistant strains. The hydrogels were also shown to be biocompatible, allowing cell proliferation. In summary, the developed CS-HACS-AgNWs composite hydrogels demonstrated significant potential as a scaffold material to be employed in bone regenerative medicine, as they present enhanced mechanical strength combined with the ability to allow calcium salts deposition, while efficiently decreasing the risk of infections. The results presented justify further investigations into the potential clinical applications of these materials.

Keywords: antimicrobial activity; bone regeneration; chitosan hydrogels; controlled release; silver nanowires.

Figure 1

Characterization of silver nanowires-AgNWs: (A) Typical color change of the reaction mixture at different temperatures; (B) the corresponding Ultra Violet spectra; (C) Scanning Electron Microscopy image of AgNWs suspended in a solution of chitosan in lactic acid (scale bar 4 µm); (D) Transmission Electron Microscopy image of AgNWs (scale bar 100 nm); (E) X Ray Diffraction pattern of the synthesized AgNWs (red bars: Ag, PDF No. 04-0783; blue bars: AgCl, PDF No. 31-1238).

Figure 2

Physical characterization of the hydrogels: Chitosan (CS, black), chitosan gel with chitosan hydroxyapatite composite (CS-HACS, light grey) and chitosan gel with chitosan hydroxyapatite composite and silver nanowires (CS-HACS-AgNWs, dark grey). (A) Gelation time calculated by the inverted tube method. Data are reported as mean ± SD (n = 9). One-way Analysis of Variance (ANOVA) returned p < 0.0001. Post-hoc Tukey’s comparison test results are shown in the graph: **** indicates p < 0.0001 as compared to the control (CS); (B) Gel strength calculated by texture analysis before (empty bars) and after (filled bars) gelation. Data are represented as mean ± SD (n = 3). The t-test performed on all samples before and after gelation showed significantly different strength values for all gels (^$$ indicates p < 0.01 and ^$$$$ p < 0.0001). One-way ANOVA to compare the different formulations returned p < 0.01 before and p < 0.0001 after gelation. Post-hoc Tukey’s test results showed that before gelation only CS is different from all other gels (^# p < 0.05), while the individual results for after gelation are reported on the graph (** p < 0.01; **** p < 0.0001).

Figure 3

Further physical characterization, enzymatic degradation and bioactivity determination of hydrogels: CS (black), CS-HACS (light grey) and CS-HACS-AgNWs (dark grey). (A) Equilibrium swelling (Q%), data are reported as mean ± SD (n = 6). One-way ANOVA returned p < 0.05, results of the post-hoc Tukey’s multi-comparison test are reported in the graph (* p < 0.05); (B) Gels porosity determined by gravimetric method, data are reported as mean ± SD (n = 3). One-way ANOVA returned p < 0.05, results of the post-hoc Tukey’s multi-comparison test are reported in the graph, (* p < 0.05 and ** p < 0.01); (C) Gels degradation in the presence of lysozyme (full bars), the empty bars represent the relative control gels treated in PBS; data are reported as mean ± SD (n = 3). One-way ANOVA returned p < 0.05, results of the post-hoc Tukey’s multi-comparison test are reported in the graph (^# p < 0.05). A t-test was performed against the PBS incubated samples for each gel at each time point; * p < 0.05, ** p < 0.01 and *** p < 0.01; (D) Percentage surface calcification of hydrogels incubated at 37°C in SBF for 7, 14 and 21 days as calculated by image analysis via ImageJ and BoneJ from SEM images. Controls were only incubated for few hours in deionized water. Results are reported as mean ± SD (n = 3). Samples labeled with the symbol ^# resulted to be significantly different from their control sample (p < 0.05). One-way ANOVA returned p < 0.05 only when testing CS-HACS at different time points, the results of the post-hoc Tukey’s multi-comparison test are reported in the graph (* p < 0.05, ** p < 0.01).

Figure 4

Energy-dispersive X-rays spectroscopy (EDS) coupled SEM images (1000× magnification) of CS, CS-HACS and CS-HACS-AgNWs hydrogels incubated in SBF for 7, 14 and 21 days. Control samples were incubated in water for 5 h. Images show an overlay of SEM pictures with EDS data: Ca (green), P (red) and co-localisation of Ca/P (yellow).

Figure 5

Cumulative release profile of silver cations from AgNWs (triangle) and CS-HACS-AgNWs (square).

Figure 6

Growth curves of different bacteria in suspension in the presence of 0 (grey), 12.5 (green), 25 (blue), 50 (red) and 100 (black) µg/mL of AgNWs.

Figure 7

Growth of S. aureus, E. coli, MRSA and S. saprophyticus in suspensions containing CS-HACS (light grey), CS-HACS-AgNWs (dark grey) and LB only (black). Results are reported as mean ± SD. One-way ANOVA was performed between different samples, when significant a Tukey’s post-hoc multicomparison test was performed, results are reported on the graph. * represents the statistical difference between scaffolds and bacterial suspensions (* p < 0.05, ** p < 0.01; *** p < 0.001 and **** p < 0.0001) while ^# represents the statistical difference between CS-HACS and CS-HACS-AgNWs scaffolds (^# p < 0.05, ^## p < 0.01; ^### p < 0.001 and ^#### p < 0.0001).

Figure 8

(A) ATP produced by MC3T3-E1 cells grown in the presence of the following hydrogels: CS (black), CS-HACS (light grey) and CS-HACS-AgNWs (dark grey). Cultures grown on tissue-culture plates were taken as control (white). Data are reported as mean ± SD (n = 3). One-way ANOVA returned p < 0.0001 for 7 and 14 days; (B) ALP expression by MC3T3-E1 cells grown in the presence of the same hydrogels. Data reported as mean ± SD (n = 3). One-way ANOVA returned p < 0.0001 for 7 days and p = 0.0005 for 14 days. The results of the post-hoc Tukey multi comparison test for (A,B) are reported in the graph: ****, p < 0.0001; ***, p < 0.001; **, p < 0.01 compared to the control; ^#, p < 0.05, ^## p < 0.001, ^### p < 0.001.