Antibacterial PMMA Composite Cements with Tunable Thermal and Mechanical Properties

Abstract

PMMA-based cements are the most used bone cements in vertebroplasty and total hip arthroplasty. However, they present several drawbacks, including susceptibility to bacterial infection, monomer leakage toxicity, and high polymerization temperature, which can all lead to damage to the surrounding tissues and their failure. In the present study, silver nanowires (AgNWs) have been introduced to bestow antibacterial properties; chitosan (CS) to promote porosity and to reduce the polymerization temperature, without negatively affecting the mechanical performance; and methacryloyl chitosan (CSMCC) to promote cross-linking with methyl methacrylate (MMA) and reduce the quantity of monomer required for polymerization. Novel PMMA cements were formulated containing AgNWs (0 and 1% w/w) and CS or CSMCC at various concentrations (0, 10, 20, and 30% w/w), testing two different ratios of powder and MMA (P/L). Mechanical, thermal, antibacterial, and cytotoxic properties of the resulting composite cements were tested. Cements with concentrations of CS > 10% presented a significantly reduced polymerization temperature. The mechanical performances were affected for concentrations > 20% with a P/L concentration equal to 2:1. Concentrations of AgNWs as low as 1% w/w conferred antimicrobial activity against S. aureus, whereas biofilm formation on the surface of the cements was increased when CS was included in the preparation. The combination of CS and AgNWs allowed a higher concentration of Ag⁺ to be released over time with enhanced antimicrobial activity. Inclusion of AgNWs did not affect cell viability on the scaffolds. In conclusion, a combination of CS and AgNWs may be beneficial for reducing both polymerization temperature and biofilm formation, without significantly affecting mesenchymal stem cell proliferation on the scaffolds. No advantages have been noticed as a result of the reducing P/L ratio or using CSMCC instead of CS.

Figure 1

Synthesis and characterization of CSMCC by FT-IR and ¹H NMR. (A) Schematic representation of the synthesis of methacryloyl chitosan (CSMCC). (B) FT-IR spectra of CS (black), MCC (dark gray), and CSMCC (light gray). (C) ¹H NMR spectrum of CSMCC.

Figure 2

Antibacterial properties of CS and CSMCC. Optical density (OD_600 nm) of S. aureus suspensions in the presence of medium as control (black bars), CS (light gray bars), and CSMCC (dark gray bars) in suspension at different concentrations. Data are reported as a mean ± SD (n = 3). One-way ANOVA returned p < 0.05; results of the Dunnett’s multiple comparison test are reported in the graph (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The t-test performed between CS and CSMCC at different concentrations revealed no statistical difference (p > 0.05).

Figure 3

Morphology of PMMA cements. (A) Representative XCT reconstructed volume of a whole PMMA_2:1 cement and (B) cross section of the same specimen. (C) SEM image of PMMA_2:1 cement surface; the arrow shows a PMMA bead within the solidified cement (see Figure S2 for more images).

Figure 4

Setting properties of cements. Peak polymerization temperatures of PMMA cements containing CS (A) or CSMCC (B), and setting time of PMMA-based cements, containing CS (C) or CSMCC (D), with different ratios of P/L. Data are reported as a mean ± SD (n = 6). One-way ANOVA returned p < 0.05; results of the Dunnett’s multicomparison test, used to compare all of the samples with the PMMA control, and results of the t-test to compare samples with different P/L ratios are reported in the graph (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

Pore volume and pore size of cements. Percentage pore volume (A) of PMMA (black), PMMA after 4 weeks (gray), CS_20% (red), CS_20% after 4 weeks (orange), CSMCC_20% (dark green), and CSMCC_20% after 4 weeks (light green). Data are reported as a mean ± SD (n = 3). T-test was performed between the same cement type before and after soaking (^$p < 0.05). One-way ANOVA returned p < 0.05 (*) when comparing the % pore volume of CS_20% to PMMA before degradation in PBS; results of the Dunnett’s multiple comparison test are reported in the graph. One-way ANOVA returned p < 0.01 when comparing the % pore volume of CS_20% to PMMA after degradation in PBS; results of the Dunnett’s multiple comparison test are reported in the graph (^&&p < 0.01). (B) Pore diameter distribution for PMMA (black), PMMA after 4 weeks (gray), CS_20% (red), CS_20% after 4 weeks (orange), CSMCC_20% (dark green), and CSMCC_20% after 4 weeks (light green).

Figure 6

Compressive strength (MPa) and Young’s modulus (MPa) of composites cements, containing CS (A, C) and CSMCC (B, D). Results are reported as a mean ± SD (n = 6). For compressive strength, one-way ANOVA returned p < 0.05; results of Dunnett’s multicomparison test (used to compare all of the samples with the PMMA_2:1) are reported in the graph (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Unpaired t-test was performed to compare each formulation containing a weight-to-volume ratio of 2:1 with the respective formulation containing a weight-to-volume ratio of 2:0.8 (^$p < 0.05, ^$$p < 0.01). For Young’s modulus, one-way ANOVA returned p < 0.05; results of Dunnett’s multicomparison test, used to compare all samples with the PMMA control, are reported in the graph (*p < 0.05). Unpaired t-test was performed to compare each formulation containing a weight-to-volume ratio of 2:1 with the respective formulation containing a weight-to-volume ratio of 2:0.8 (^$p < 0.05).

Figure 7

Cumulative release of silver ions from bone cements: PMMA_AgNWs_1 (black), PMMA_AgNWs_0.8 (red), PMMA_CS20%_AgNWs_1 (blue), PMMA_CS20%_AgNWs_0.8 (green), PMMA_CSMCC20%_AgNWs_1 (yellow), PMMA_CSMCC20%_AgNWs_0.8 (purple). Data are reported as a mean ± SD (n = 3). One-way ANOVA, at 21 days, returned p > 0.05.

Figure 8

Inhibition of biofilm formation expressed as viable cell count. Absorbance relative to the number of bacterial cells on cement (P/L 2:1) surfaces obtained by an MTT assay after 24 h. (A) PMMA-based cements containing CS. (B) PMMA-based cements containing CSMCC. Data are reported as mean ± SD (n = 4). One-way ANOVA returned p < 0.05; results of the post hoc Tukey’s multicomparison test are reported in the graph (*p < 0.05). Unpaired t-test was performed to compare each formulation with and without AgNWs. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

Figure 9

Cytotoxicity of cements on mesenchymal stem cells. Cytotoxicity was tested against the cement eluates (A, B) and by direct contact of cells with cements (C, D). Toxicity of extracts from cements of P/L ratios 2:1 (A) and 2:0.8 (B) at 24 (left column) and 48 h (right column). Data are reported as mean ± SD (n ≥ 3). One-way ANOVA returned p < 0.05; results of the post hoc Tukey’s multicomparison test are reported in the graph (*p < 0.05). Unpaired t-test was performed to compare each formulation with or without AgNWs. ATP assay for MSCs after 1 day and 3 days of incubation on CS cements (C) and CSMCC cements (D). One-way ANOVA was carried out to compare ATP levels of each cement to PMMA 2:1 at days 1 and 3 and showed a statistical difference (p < 0.05). Results of Dunnett’s multicomparison are reported in the graph; in particular, ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001, and ^$$$$p < 0.0001, at day 1. ^£p < 0.05, ^££p < 0.01, ^£££p < 0.001, and ^££££p < 0.0001, at day 3.The t-test was performed to compare each type of cement at 1 and 3 days (*p < 0.05, **** p < 0.0005).