The mechanical and antibacterial properties of bone cement composites containing silver nanoparticles compounded with various chitosans

doi:10.21203/rs.3.rs-1977919/v1

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The mechanical and antibacterial properties of bone cement composites containing silver nanoparticles compounded with various chitosans

https://doi.org/10.21203/rs.3.rs-1977919/v1

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Objective: To analyse the mechanical and antibacterial properties of bone cement composites containing silver nanoparticles (AgNPs) compounded with chitosan (CS) and carboxymethyl chitosan (CMCS).

Methods: Ten percent wt% AgNPs, CS-AgNPs and CMCS-AgNPs were prepared through an oxidation-reduction reaction under sonic oscillation and mix with polymethyl methacrylate (PMMA). The compressive and bending strengths of these nanoparticles were measured before and after soaking the samples in simulated body fluid (SBF). The Kirby-Bauer (K-B) method and flask oscillation method were used to evaluate the antibacterial effects of the composites.

Results:

The compressive and bending strengths of the AgNP, CS-AgNP, and CMCS-AgNP groups were significantly improved (P < 0.001). After soaking in SBF, the bending strengths of the AgNP, CS-AgNP, and CMCS-AgNP groups were significantly higher than that of the control group (P < 0.001).

The K-B test showed only the 5 wt% and 10 wt% CMCS-AgNP groups exhibited bacteriostatic rings for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). The flask oscillation analyses revealed that the 5 wt% and 10 wt% CMCS-AgNP exhibited good bacteriostatic activity against S. aureus, but only the 10 wt% CMCS-AgNP group for MRSA.

Conclusion: We developed CS/CMCS-AgNP bone cement composites, which show good mechanical properties and antibacterial activity.

Silver nanoparticles

Chitosan

Compressive strength

Bending strength

Antibacterial properties

In recent years, the incidence of periprosthetic joint infections (PJIs) has increased with the extensive application of artificial joint replacements ^[1]. PJIs have strong impacts on the quality of life of patients, creating substantial economic and psychological burdens ^[2]. Currently, for chronic or drug-resistant bacteria-related PJIs, secondary revision surgery is always needed, and an antibiotic-loaded cement spacer is used for joint exclusion.

Antibacterial materials have become a hotspot of research worldwide. Broad-spectrum antibacterial biomaterials, such as silver, silver nanoparticles (AgNPs), chitosan (CS) and carboxymethyl chitosan (CMCS), do not increase bacterial drug resistance and can even inhibit the formation of bacterial biofilms ^[3–6].

Although broad-spectrum antibacterial materials such as AgNPs and CS are currently widely applied in the orthopaedic domain^[7]. Because bone cement plays an important role in arthroplasty and the treatment of PJIs, we developed CS/CMCS-AgNP composites and introduced those composites into bone cement to explore the influence of CS/CMCS-AgNPs on the mechanical and antibacterial properties of bone cement.

1. Main reagents and instruments

Polymethyl methacrylate (PMMA) bone cement (Depuy, USA), CS (Huaxia Ocean Technology, Beijing, China, degree of deacetylation > 95%, viscosity 100-200mpa.s, molecular weight 179.17) and CMCS (McLean Biochemistry, Shanghai, China, degree of substitution ≥ 80%, viscosity 10mPa.s ~ 80mpa.s, molecular weight 220.2) were used in this study. Silver nitrate, acetic acid, sodium borohydride, sodium bicarbonate, polyvinyl pyrrolidine (PVP), absolute ethyl alcohol and acetone were used, and all of these materials were of analytical grade (Putin Conley Technology, Beijing, China). Simulated body fluid (SBF) (Leagene Biotechnology, Beijing, China) was prepared in accordance with the manufacturer’s instructions.

A scanning electron microscope (Hitachi, Japan), ultraviolet (UV)-2550 spectrophotometer (Shimadzu, Japan), X-ray diffractometer (Bruker, Germany), universal material testing machine (SUNS Technology, Shenzhen, China), freeze-drying machine (Labogene, Denmark), and ultrasonic oscillator (Boxun Industry, Shanghai, China) were used in this study.

2. Preparation of AgNPs

AgNPs was synthesized using the wet chemical reduction method^{[8, 9]}. Silver nitrate was dissolved in deionized water to prepare a 0.3 mol/L solution, and after PVP was added, the solution was mixed. Sodium borohydride was dissolved in deionized water to prepare a 0.3 mol/L solution. The silver nitrate-PVP solution was added to the sodium borohydride solution at a velocity of 30 drops per minute under sonic oscillation and magnetic stirring at a constant temperature of 35°C. The mixture was mixed for 1 h before standing to obtain a brown precipitate, which was collected, washed with deionized water, dispersed using ultrasonic oscillation, centrifuged at 1000 rpm for 1 h and then washed with ethanol and acetone twice for precipitation. Finally, the sediment was recentrifuged, refrigerated and dried in vacuum overnight to obtain AgNP powder.

3. Preparation of CS-AgNPs and CMCS-AgNPs

One hundred millilitres of a 3 wt% CS solution was prepared with 2 wt% acetic acid, and 1 ml of 0.3 mol/L silver nitrate solution was added. The mixture was magnetically stirred for 30 min at a constant temperature of 35°C before the addition of 3 ml of 0.3 mol/L sodium borohydride solution. The mixture continued to be ultrasonically oscillated and magnetically stirred for 1 h and was then allowed to stand to obtain a tan colloidal sol, whose pH was adjusted to 7.5 by the addition of sodium bicarbonate solution. Then, the sediment was centrifuged at 1000 rpm for 5 min to obtain a tan jelly-shaped solid, which was washed with ethanol and acetone twice for precipitation. Next, the precipitate was recentrifuged before refrigeration and vacuum drying overnight to obtain CS-AgNP powder.

One hundred millilitres of a 3 wt% CMCS solution was prepared with deionized water, and 1 ml of 0.3 mol/L silver nitrate solution was added. Then, the mixture was magnetically stirred for 30 min at a constant temperature of 35°C before adding 3 ml of 0.3 mol/L sodium borohydride solution. The mixture continued to be ultrasonically oscillated and magnetically stirred for 1 h before standing to obtain a tan colloidal sol, which was precipitated using absolute ethyl alcohol. Then, the sediment was centrifuged at 1000 rpm for 5 min to obtain a tan jelly-shaped solid, which was washed with ethanol and acetone twice for precipitation. Next, the precipitate was recentrifuged before refrigeration and vacuum drying overnight to obtain CMCS-AgNP powder.

4. Characterization of CS-AgNP

4.1 UV absorption analysis

CS and CS-AgNP solutions prepared as mentioned above were stored for one day, and the contents of CS and CS-AgNPs were then qualitatively characterized using a UV-2550 spectrophotometer with a scanning wavelength of 300–800 nm, a high velocity and a sampling interval of 1 nm.

4.2 X-ray diffraction analysis

The CS-AgNP solution prepared as mentioned above was spread on a glass slide and lyophilized in a vacuum freeze-drying machine to form a film. Then, the film was removed and pulverized, and the powder was collected. CS-AgNPs and CS (as the control) were compared using an X-ray diffractometer (Cu Kα, λ = 1.5406 Å, 40 kV, 34 mA) with a 2θ scope of 10–80° and a step of 0.1°/s.

5. Preparation of bone cement samples

To prepare the bone cement samples, 10 wt% AgNP, CS-AgNP, and CMCS-AgNP powders and PMMA polymer powder were blended separately by oscillation. Then, PMMA monomer liquid was added and mixed with the powders together. The mixture was injected into a polytetrafluoroethylene mould and allowed to stand for 30 min at room temperature for solidification before removal. To measure the compressive strength, a cylinder with a diameter of 10 mm and a length of 5 mm was used. The bending strength was measured with a cylinder with a diameter of 10 mm and a length of 25 mm.

6. Characterization of mechanical properties

The differences in mechanical properties among the PMMA, 10 wt% AgNP, 10 wt% CS-AgNP, and 10 wt% CMCS-AgNP PMMA groups were determined using a universal material testing machine before and after soaking. The PMMA group served as the control. Six samples from each group were analysed. To measure the compressive strength, the pressure head applied pressure to the samples at a velocity of 2 mm/min. To measure the bending strength, the diameter of the pressure head was 4 mm, the span was 15 mm, and the pressure head applied pressure to the samples at a velocity of 2 mm/min. After soaking the samples in SBF for seven days, each sample was analysed again. The results are presented as the means ± standard deviations (SDs).

7. Characterization of antibacterial activity

The control groups consisted of the PMMA group and the PMMA containing 4.22 wt% gentamycin sulphate (G-PMMA) group. The experimental groups consisted of the 1, 5, and 10 wt% AgNP, CS-AgNP, and CMCS-AgNP groups and the 10 wt% CS and CMCS groups.

7.1 K-B method

According to the Clinical and Laboratory Standards Institute (CLSI) (2012) ^[10], Staphylococcus aureus and MRSA strains with 0.5 Maxwell turbidity cultured for 18–24 h were inoculated onto M-H culture dishes, and the samples described above were placed on the surface of the culture dish. The samples were distributed evenly, and the distance between the centres of the samples was at least 24 mm. Then, the samples were incubated for 24 h in 35°C incubators, and the sizes of their bacteriostatic rings were observed. The antibacterial efficacy is shown in Table 1. A bacteriostatic ring with a diameter ≥ 14 mm indicated that a sample had good bacteriostatic activity.

Table 1

The diameters of bacteriostatic rings with the *S. aureus* standard strain in the K-B method
Sample	PMMA	G-PMMA	AgNP-PMMA			CS-PMMA			CMCS -PMMA				CS-AgNP-PMMA			CMCS-AgNP-PMMA
Concentration (wt%)			1	5	10	1	5	10	1	5	10	1		5	10	1	5	10
Ring Diameter (mm)	0	30.3	0	0	0	0	0	0	0	0	0	0		0	0	0	17.6	18.5

7.2 Flask oscillation method

According to the GB/T 20944.3–2008 standard ^[11], the antibacterial activity and endurance of samples were tested using the flask oscillation method. The S. aureus and MRSA strains were cultured for 18–24 h, and suspensions with 0.5 Maxwell turbidity were then prepared. Next, each suspension was diluted to 5x10⁵ CFU/ml using TSB solution. Two millimetres of bacterial suspension was added to a 6-well plate. The samples described above were placed in the wells before adding the suspension. The plates were shaken using a horizontal vibrator for culturing at 80 rpm and at a constant temperature of 35°C. Ten microlitres of bacterial suspension was obtained every 0, 2, 4, and 6 h and added to 20 ml of cold normal saline for dilution. One hundred microlitres of the bacterial suspension was then inoculated into a blood agar culture dish, which was then cultured for 24 h in a 35°C incubator.

The colony count on each culture dish was recorded using the plate counting method, and the bacteriostatic rate was calculated. No colony formation was assumed to indicate an antibacterial activity value of 100%, and a value ≥ 90% implied good antibacterial activity.

8. SEM

The samples were bonded to an aluminium sheet by conductive adhesive, fixed and sprayed with Platinum. The morphology of each sample before and after immersion was observed by SEM.

9. Statistical analysis

All data were processed using SPSS 23.0 software (IBM SPSS, US). The results are presented as the means ± SDs. One-way ANOVA was used to evaluate the differences among groups and was followed by Bonferroni's post hoc test. Differences were considered statistically significant at P < 0.01.

1. Verification of the characteristics of CS-AgNPs

1.1. UV absorption analysis

The absorption peak of CS was at approximately 300 nm. The UV absorption peak of the CS-AgNPs was at approximately 430 nm, indicating that a complex with a larger particle diameter than that of AgNPs had formed after chelation of the AgNPs and CS (Fig. 1).

1.2. X-ray diffraction analysis

The CS sample had a peak at 10–30° (Fig. 2). For the CS-AgNP sample, the diffraction peak was observed at approximately 22–28°, indicating the presence of AgNPs (Fig. 2). The diffraction peaks of CS-AgNPs in the same scope were wider and weaker than those of CS, indicating that AgNPs had an influence on the property of CS.

2. Characterization of mechanical properties

2.1 Compressive strength

The compressive strengths of the control, AgNP, CS-AgNP and CMCS-AgNP groups were 120.15 ± 0.37 MPa, 128.79 ± 0.06 MPa, 144.41 ± 0.07 MPa and 151.47 ± 0.03 MPa, respectively (Fig. 3). Compared with that of the control group, the compressive strength of the AgNP, CS-AgNP and CMCS-AgNP groups were all significantly improved (P < 0.001). After being soaked in SBF for seven days, the compressive strengths of the control, AgNP, CS-AgNP and CMCS-AgNP groups were 139.77 ± 0.02 MPa, 136.73 ± 0.04 MPa, 116.66 ± 0.11 MPa, and 117.16 ± 0.04 MPa, respectively (Fig. 3). The compressive strengths of the control and AgNP groups were improved after soaking compared with those before soaking, whereas those of the CS-AgNP and CMCS-AgNP groups were reduced after soaking. The compressive strengths of the CS-AgNP and CMCS-AgNP groups were significantly lower than those of the control and AgNP groups, respectively, after soaking (P < 0.001).

2.2 Bending strength

As shown in Fig. 4, the bending strengths of the control, AgNP, CS-AgNP and CMCS-AgNP groups were 41.18 ± 0.09 MPa, 42.63 ± 0.06 MPa, 54.13 ± 0.12 MPa and 52.63 ± 0.06 MPa, respectively. Compared with that of the control group, the bending strengths of the AgNP, CS-AgNP and CMCS-AgNP groups were significantly improved (P < 0.001). After soaking, the bending strengths of the control, AgNP, CS-AgNP and CMCS-AgNP groups were 49.64 ± 0.08 MPa, 53.8 ± 0.1 MPa, 52.29 ± 0.07 MPa and 55.74 ± 0.07 MPa, respectively. The bending strengths of the control, AgNP and CMCS-AgNP groups were improved after soaking, whereas that of the CS-AgNP group was decreased (P < 0.001). The bending strengths of the AgNP, CS-AgNP, and CMCS-AgNP groups were higher than that of the control group. The differences were significant (P < 0.001).

2.3 SEM observation

As shown in Fig. 5, SEM was used to observe the microstructures of the bone cement samples before soaking. The microstructure of the control group was loose, and a network formed by the crosslinking of macromolecules was observed. Compared with the control group (Fig. 5A), the AgNP (Fig. 5B), CS-AgNP (Fig. 5C) and CMCS-AgNP (Fig. 5D) groups exhibited reduced or no networks, and the surfaces were flatter and more homogeneous. Moreover, in the CS-AgNP and CMCS-AgNP groups, CS and CMCS formed a cotton fibre-shaped layer covering the surface of PMMA, and the PMMA network had disappeared.

Figure 6 shows SEM images of the microstructures of the groups after soaking in SBF for 7 days. The microstructures of the control (Fig. 6A) and AgNP (Fig. 6B) groups showed no obvious changes due to soaking, and networks were still present. The analysis of the CS-AgNP (Fig. 6C) and CMCS-AgNP (Fig. 6D) groups revealed that more pore structures were observed on the surface after soaking. The surface of the CMCS-AgNP group had macropores (> 400 µm), which exposed the porous structure of the PMMA.

The SEM energy spectrum showed that after soaking, the surface of the AgNP group (Fig. 6B) continued to exhibit evenly distributed silver. In contrast, the SEM energy spectra of the the control group ((Fig. 6A), CS-AgNP (Fig. 6C) and CMCS-AgNP (Fig. 6D) groups showed that the surfaces of the two groups had no distribution of silver after soaking, suggesting that the AgNPs on the surfaces of the two groups had been completely released during the soaking process.

3. Characterization of antibacterial activity

3.1 K-B method

As shown in Fig. 7, the use of the K-B method with the S. aureus standard strain revealed that the G-PMMA group (one of the control groups) exhibited an obvious bacteriostatic ring, with a diameter of 30.3 mm. Among the experimental groups, the 5 and 10 wt% CMCS-AgNP groups presented obvious bacteriostatic rings, with diameters of 17.6 mm and 18.5 mm, respectively; the remaining groups had no significant bacteriostatic rings, indicating that these three groups had good resistance to S. aureus (Table 1).

As shown in Fig. 8, the application of K-B method with the MRSA strain revealed that none of the control groups had bacteriostatic rings. In the experimental groups, bacteriostatic rings with diameters of 14 and 15.6 mm were observed around the samples of the 5 wt% CMCS-AgNP group and 10 wt% CMCS-AgNP group, respectively, indicating that these two groups exhibited good resistance to MRSA. The remaining groups had no significant bacteriostatic rings (Table 2).

Table 2

The diameters of bacteriostatic rings with the MRSA strain in the K-B method.
Sample	PMMA	G-PMMA	AgNP-PMMA			CS-PMMA			CMCS -PMMA			CS-AgNP-PMMA			CMCS-AgNP-PMMA
Concentration (wt%)			1	5	10	1	5	10	1	5	10	1	5	10	1	5	10
Ring Diameter (mm)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	14.0	15.6

3.2 Flask oscillation method

As shown in Tables 3, the application of the flask oscillation method with the S. aureus standard strain showed that the G-PMMA group (one of the control groups) had a bacteriostatic rate of 100%. Among the experimental groups, the samples from the 5 and 10 wt% CMCS-AgNP groups showed good bacteriostatic activity, with bacteriostatic rates of 99.5% and 99.7%, whereas the remaining groups had no bacteriostatic activity (P < 0.001).

Table 3

The bacteriostatic rate with the *S. aureus* standard strain in the flask oscillation method.
Sample	PMMA	G-PMMA	AgNP-PMMA			CS-PMMA			CMCS -PMMA			CS-AgNP-PMMA			CMCS-AgNP-PMMA
Concentration (wt%)			1	5	10	1	5	10	1	5	10	1	5	10	1	5	10
Colony count (CFU/ml)	> 4X10⁷	0	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	1.2X10⁵	4X10⁴
Inhibition rate (%)	0	100	0	0	0	0	0	0	0	0	0	0	0	0	0	99.7	99.9

The experiment with the MRSA strain revealed that all of the groups had no significant bacteriostatic activity (P < 0.001), with the exception of the 10 wt% CMCS-AgNP group, which had a bacteriostatic rate of nearly 90% (Fig. 9 and Table 4). Although the CMCS-AgNP group showed good bacteriostatic activity, 4.4X10⁶ CFU/ml bacteria survived in the bacterial suspension, indicating that the bacteriostatic efficacy against MRSA was significantly weaker than that against S. aureus.

Table 4

The bacteriostatic rate with the MRSA strain in the flask oscillation method.
Sample	PMMA	G-PMMA	AgNP-PMMA			CS-PMMA			CMCS -PMMA			CS-AgNP-PMMA			CMCS-AgNP-PMMA
Concentration (wt%)	0	0	1	5	10	1	5	10	1	5	10	1	5	10	1	5	10
Colony count (CFU/ml)	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	> 4X10⁷	4.4X10⁶
Inhibition rate (%)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	89.0

3.3 Observation of bacterial biofilms

After culturing the samples in the bacterial suspension, the MRSA bacteria on the surface of the bone cement samples began to adhere, and the bacterial biofilm formed gradually. As time progressed, the biofilm significantly thickened and became stacked, resembling cotton fibres. SEM images illustrating the changes are shown in Fig. 10.

Among the groups, the control group exhibited the thickest, most solid bacterial biofilm on the surface, as shown in Fig. 10A, and this biofilm was significantly thicker than the biofilms on the surfaces of the other groups. As shown in Fig. 10B, the bacteria on the surface of the G-PMMA group had begun to adhere but did not form a biofilm, and the bacterial biofilms of the AgNP (Fig. 10C) and CS-AgNP (Fig. 10D) groups were denser than the biofilms of the G-PMMA group. Compared with the other groups, the CMCS-AgNP group exhibited the least biofilm formation, and the biofilm obtained with this group presented the lowest thickness among those obtained with all the groups (Fig. 10E). Figure 10F shows the microscopic network structure of bacterial biofilm in the control group, and the latent bacterial colonies could be seen in the network structure.

These results indicated that CMCS-AgNP-PMMA bone cement was more effective than the other types of cement in inhibiting MRSA bacterial biofilm formation.

The incidence of infections after orthopaedic surgery is 1–2% ^[12–14], and some articles have reported that this rate can be as high as 10% ^{[15, 16]}, despite the widespread use of antibiotics. Bacterial resistance is a terrible risk that is gradually becoming prominent. Therefore, novel antimicrobial approaches are required. There are several kinds of novel antimicrobial materials, including organic materials and inorganic metals in the form of salts or nanoparticles and natural compounds. The antimicrobial effect of silver and chitosan has been proven by articles ^[17–19]. Chitosan is a natural organic material and has good bactericidal performance, biodegradability and antivirulence properties. It has been widely applied in the domains of food, textiles, agriculture and medicine ^[5]. Chitosan could be selected as a safe reducing and stabilizing agent for the synthesis of chitosan-based nanoparticles ^[20]. Chitosan-based nanoparticles have gained considerable interest due to their high permeability, high antibacterial activities, nontoxicity to human cells and immunostimulatory properties ^[21]. Carboxymethyl chitosan has a higher solubility at physiological pH (> 6.0) than chitosan ^[6]. It could be applied with antimicrobial, anticancer, antitumour, antioxidant and antifungal biological activities in various areas ^[6].

In research on inorganic antibacterial materials, silver has been of particular interest ^[22]. Silver ions bind to halide ions, such as chloride, and precipitate; therefore, when used directly, their microbicidal activity is shortened. To overcome this issue, AgNPs with small particle sizes and strong reducing properties have shown better antibacterial activity than elemental silver and silver nitrate and have been widely applied in various medical devices ^[9]. Rahimi ^[23] confirmed that a wound dressing with quaternized chitosan and its silver-containing nanocomposites showed potent antimicrobial activity and cytocompatibility. Liu ^[24] reported that nanosilver-coated polyetheretherketones (PEEKs) demonstrated enhanced antibacterial activities and no cytotoxicity. Therefore, we combined the advantages of silver nanoparticles and carboxymethyl chitosan to synthesize CS/CMCS-AgNPs and studied their effect on the mechanical properties of bone cement.

The mechanical properties of bone cement are influenced by many factors, including shape, storage, type, additives such as antibiotics, developing agents, and methods in the preparation process ^[25–27]. It has been reported that the addition of antibiotics to bone cement significantly reduces its mechanical properties ^[28]. He ^[29] found that the addition of more than 4 wt% gentamicin to bone cement reduced its compressive strength. Even the selection of brands of vancomycin and bone cement had a great impact on the release efficacy and mechanical strength of bone cement ^[30]. It has been reported that the addition of developing agents, such as barium sulphate (BaSO4), might agglomerate and be detrimental to the fatigue performance of bone cement in vitro ^[31]. This might lead to loosening of the prosthesis or rupture of the bone cement spacer ^[31]. Makita ^[32] investigated the effects of BaSO₄ concentration on PMMA properties and found that 10–20 wt% BaSO4 had little effect on the compressive strength of PMMA. However, as the percentage approached 30 wt% BaSO4, a steep decrease in the compressive strength was observed ^[32]. The PMMA bone cement used in the present study contained 10 wt% BaSO4, which was unlikely to have affected the mechanical properties and biological activity of the bone cement. For a single additive of chitosan or AgNPs, some studies have revealed the effect on the mechanical properties of PMMA. Slane ^[33] reported that AgNPs at three loading ratios of 0.25, 0.5, and 1.0% wt/wt in acrylic bone cement did not affect the mechanical and material properties compared with those of standard cement. In Shi’s study, mechanical tests indicated that the addition of chitosan nanoparticles allowed the retention of a significant degree of bone cement strength ^[34]. CS-AgNPs and CMCS-AgNPs might be new antimicrobial materials applied in orthopaedic surgery, but few articles have reported the effect of CS-AgNPs on the mechanical properties of PMMA. Therefore, we studied the mechanical properties of PMMA incorporated with CS-AgNPs and CMCS-AgNPs and controlled the additive concentration to no higher than 10 wt%.

The mechanical properties of bone cement are closely related to its composition and structure. Pores in bone cement are mainly formed by spaces between liquid and air (bubbles) during polymerization. Smaller pores result in higher strengths ^[35]. Pore generators, such as carboxymethylcellulose, are responsible for the creation of porosity and determine the final mechanical properties of cements^[36]. Porosity has been reported to be related to the mechanical properties of PMMA ^{[37, 38]}, but there is considerable clinical evidence showing that a reduction in porosity in acrylic cement is not related to the long-term outcomes of total hip arthroplasty ^[39]. Recently, there have been many articles reporting the effect of additives on the mechanical properties of PMMA, such as nanoparticles, microparticles, nanotubes, antibiotics and polymer composites. The porosity of PMMA was studied to load additives and even conduct tissue growth ^[40]. Although some studies demonstrated that silver-loaded nanoparticles or chitosan nanoparticle application did not enhance or preserve the mechanical properties of bone cement ^{[34, 41]}, we found that the compressive and bending strengths of bone cement with AgNP, CS-AgNP and CMCS-AgNP powders all significantly increased in our study. This might be related to the molecular weight of additives. Cierech ^[42] reported that the incorporation of ZnO-NPs to 7.5% in PMMA nanocomposites increased the hardness and hydrophilicity. Bacali ^[43] demonstrated that PMMA resin loaded with graphene-AgNPs presented promising antibacterial activity associated with minimal toxicity to human cells in vitro, as well as improved flexural properties. Kamonkhantikul ^[44] proved that the flexural strength of PMMA incorporated with silanized zinc oxide nanoparticles was significantly greater than that of the nonsilanized group. Most studies have proved that nanoparticle incorporation at a certain weight concentration does not have a detrimental effect on the mechanical properties of bone cement.

The change in the mechanical properties of PMMA is related to the high surface area, fine size, and homogenous distribution of nanoparticles ^[45]. The nanoparticles involved in the polymerization reaction filled the pores well, reducing the number of surface cracks and increasing the homogeneity of the bone cement samples. Our SEM results confirmed that the AgNP, CS-AgNP and CMCS-AgNP groups exhibited reduced or no networks, and the surfaces were flatter and more homogeneous, suggesting infilling of the materials. With the addition of AgNPs, CS-AgNPs or CMCS-AgNPs to the cement, the numbers of internal and surface pores were significantly reduced, possibly increasing the mechanical strength of the bone cement.

CS and CMCS are highly viscous and naturally polar polymer materials. Therefore, when the CS-AgNPs and CMCS-AgNPs were added to the cement, the cement particles were bonded together by these polymeric adhesives. Furthermore, CS and especially CMCS have a strong complexation effect on AgNPs ^[46]. Because CMCS contains more functional groups than CS, such as hydroxyl, amino and carboxymethyl functional groups, CMCS has a stronger ability to adsorb metal particles than CS ^{[47, 48]}. Our X-ray diffraction results showed that the CS underwent a large number of complexation reactions with AgNPs, which might have destroyed their crystallinity and increased their viscosity, thereby improving the mechanical strength of the bone cement samples.

CMCS shows higher water solubility, better biocompatibility, excellent biodegradability, and enhanced antioxidant activity than unmodified CS ^[49]. Consequently, CMCS has been employed as a biomaterial in various biomedical applications and is considered a promising green stabilization agent for the preparation of nanometals ^[50]. In our study, after soaking the samples in SBF for 7 days, the compressive strengths of the PMMA and AgNP groups did not significantly decrease, whereas those of the CS-AgNP and CMCS-AgNP groups decreased by 16.5 and 16.2%, respectively. These decreases were related to the release of CS-AgNPs and CMCS-AgNPs over time. The SEM results were consistent with the mechanical test results. The presence of pores, including large pores (400 µm), was observed on the surfaces of the CS-AgNP and CMCS-AgNP groups after soaking, which explained the decreases in the mechanical strengths of the two groups observed after soaking.

The spectrum analysis showed that the AgNP, CS-AgNP and CMCS-AgNP groups all had silver distributed on their surfaces before soaking. During the soaking of the samples in SBF, the silver on the surfaces of the CS-AgNP and CMCS-AgNP groups gradually disappeared with the slow release of the CS-AgNPs and CMCS-AgNPs. This result confirmed the feasibility of CS and CMCS as antibacterial material delivery carriers. However, the AgNP group contained silver distributed on the surface after soaking. This result was associated with the fact that AgNPs are hydrophobic metals that can only produce Ag + by liquid ionization on the surface of the samples, resulting in a very low release rate.

It has been reported that the axial and transverse compressive strengths of human bone are 131–224 MPa and 106–133 MPa, respectively, whereas the compressive strength of cancellous bone is 30 MPa. According to standards, the average compressive strength requirement of medical-grade PMMA bone cement is ≥ 70 MPa, and the corresponding bending strength requirement is ≥ 50 MPa ^[51]. The mechanical properties of CS-AgNP or CMCS-AgNP groups in this study greatly exceeded these thresholds, indicating that the addition of CS-AgNP or CMCS-AgNP composites to PMMA does not sacrifice the mechanical properties of bone cement.

The results of the in vitro antibacterial tests showed that the 5 wt% CMCS-AgNP, 10 wt% CMCS-AgNP and G groups had good antibacterial activity against S. aureus, whereas the AgNP and CS-AgNP groups exhibited no obvious antibacterial activity. This result is likely due to the following observations. (1) Because of the high viscosity and low porosity of PMMA, the AgNPs are crosslinked inside of the PMMA. (2) The contact sterilization efficiency of AgNPs on the sample surface was very low such that the biological activity of the AgNP group was lower than those of the other two groups. (3) The solubility of CS in a neutral environment is very low ^[52], suggesting that CS cannot quickly release AgNPs into bacterial solution. Therefore, liquid cannot enter the bone cement to elute AgNPs. (4) CMCS shows higher water solubility than CS ^[49], and this finding might explain the antibacterial activity of 5 wt% CMCS-AgNPs and 10 wt% CMCS-AgNPs.

Regarding antibacterial activity against the MRSA strain, the bacteria in the G-PMMA group showed obvious resistance to MRSA, with no bacteriostatic ring formation. Only the samples in the 10 wt% CMCS-AgNP group exhibited good antibacterial properties. This result indicated that CMCS-AgNPs are a broad-spectrum antibacterial material. Because they have many mechanisms to carry out antibacterial activity, they do not increase the risk of developing resistance to bacterial strains ^[53]. Only a few studies have reported bacterial resistance to silver complexes ^[54]. Moreover, we evaluated the inhibition of bacterial biofilm formation. We found that the surfaces of the samples in the CMCS-AgNP group had the least amount of adhered bacteria, indicating that the CMCS-AgNP group had the best resistance among the groups to MRSA biofilm formation. These results were consistent with the results of the in vitro antibacterial tests. Many recent studies have reported the ability of silver bone cement to resist biofilm formation, including MRSA and MRSE biofilms ^{[55, 56]}. However, in this study, the surfaces of the AgNP and CS-AgNP groups were susceptible to the attachment of a large amount of biofilm, indicating that the AgNPs in the bone cement system did not confer obvious resistance to the drug-resistant bacteria. This observation might be related to factors such as the particle size, synthesis method and proportion of AgNPs; the experimental conditions; and the species of bacteria used in this study.

In this study, we developed CS/CMCS-AgNP bone cement composites that good mechanical properties and antibacterial activity.

Ethical Approval

Not applicable

Competing interests

The authors declare no conflicts of interest

Authors' contributions

Zhuoyang Li and Xin Yangfinished the research, wrote and revised the article. Yongping Caoconducted this research and revised the article. Zhichao Meng and Heng Liu helped to finished the research and revised the article. Rui Wang and Hao Wu help to analysed the data and revised the article.

Funding

There was no funding supported.

Availability of data and materials

The data and materials can be accessed by any datasets.

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No competing interests reported.

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The mechanical and antibacterial properties of bone cement composites containing silver nanoparticles compounded with various chitosans

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