Antibiofilm properties of silver nanoparticles incorporated into polymethyl methacrylate used for dental applications

Abstract

BACKGROUND:

Acrylic resins used in dental and biomedical applications do not have antimicrobial properties, their surface is susceptible to colonization of microorganisms.

OBJECTIVE:

The aim of this study was to evaluate the antibiofilm properties of silver nanoparticles (AgNPs) deposited in a polymethyl methacrylate (PMMA) surface against a Staphylococcus aureus biofilm.

METHODS:

The PMMA was impregnated with AgNPs by using the in-situ polymerization method. To determine the solubility of the incorporated silver (Ag⁺) atomic absorption spectrophotometry was used (AAS) at 24 h, 48 h, 7 days, and 30 days. Thirty specimens of PMMA with AgNPs and without NP (control group) were assembled in the CDC Biofilm Bioreactor system with a cell suspension of S. aureus. The specimens were removed at 6, 12, 24, 48, and 72 h to determine the viability profile and quantify the Arbitrary Fluorescence Units (AFU).

RESULTS:

The AgNPs showed an irregular and quasispherical shape with an average size of 25 nm. AAS analysis demonstrated a low solubility of Ag⁺. The formation of the S. aureus biofilm increased as the evaluation periods continued up to 72 h. The experimental group showed poor growth, and a decrease in the intensity of the fluorescence demonstrated a statistically significant inhibition of the formation of the biofilm (P < 0.05) in relation to the control group at 6, 12, 24, 48, and 72 h.

CONCLUSION:

AgNPs incorporated into PMMA decreased the growth and maturation of S. aureus biofilm.

Keywords

Polymethyl methacrylate silver nanoparticles bioreactor system biofilm

1. Introduction

The polyacrylates are a family of polymers derived from unsaturated carboxylic acids that have a wide range of medical applications. The most frequently used of these is polymethyl methacrylate (PMMA), an alloplastic material used in the manufacture of maxillofacial prostheses, intraocular lenses, and cranial and orthopedic surgical devices, as well as in dentistry for orthodontics appliances, provisional fixed prostheses, and denture prostheses [1–3].

Silver nanoparticles (AgNPs) have also been applied in several uses in dentistry [4], including to avoid or at least to decrease the microbial colonization over dental materials, and to increase oral health levels, including dental prostheses [5], restorative dentistry [6], and endodontic treatments [7,8] and in implant dentistry [9]. AgNPs have been shown to be effective antimicrobial agents, through the slow release of Ag⁺ cation and the nanomechanical mechanism of action of AgNPs that promotes the destroy bacterial membranes, the antimicrobial effect is related to the generation of reactive oxygen species and the induction of oxidative stress, however, AgNPs are presented as clusters of atoms with diameters in the range of 1 to 100 nm, thereby their size allows their entry into both bacteria and cells [10–14]. Therefore, they have been incorporated into the polymer surfaces of dental biomaterials and appliances [15], important because of the increase of opportunistic bacteria in the oral cavity of patients with removable orthodontic appliances and denture prostheses [16].

Biofilms are complex structured aggregates of bacteria capable of surviving hostile environmental conditions and exhibit resistance to different chemotherapeutic agents and the host’s immunity [17]. Since infections caused by bacterial biofilm are difficult to treat, testing novel biofilm inhibitors is required [18]. The effect of antimicrobial agents on the formation, viability, and distribution of a biofilm should be tested in a manner similar to that of the clinical application and should be used for the development and official registration of commercially available disinfectants against biofilm bacteria [19,20].

PMMA specimens with AgNPs incorporated have been reported to show significantly less Candida albicans adherence, without cytotoxicity or genotoxicity effects and without changes in the mechanical properties of PMMA [21,22]. The biofilm is considered an ecosystem which hosts microbial pathogens, Staphylococcus aureus is one of the main organisms responsible for the growth of biofilms that cause chronic infections and that present highly virulent characteristics [23,24].

Different in vitro models have been implemented over the last decade to evaluate biofilm formation and antimicrobial activity in biomaterials, including the microtiter plate-based assays, the Calgary device, substratum suspending reactors (CDC Biofilm Reactor), and the flow cell system [25]. A biofilm disinfection assay method must include all the biological and chemical components of conventional suspension or dried-surface tests. In addition, it requires factors that mechanically stimulate biofilm growth such as a biofilm reactor for growing a reproducible biofilm with the key attributes of the naturally occurring biofilm where the antimicrobial alternative will be applied [25,26]. Another key factor is the microscopic technique for assessing biofilm growth. Confocal laser scanning microscopy (CLSM) offers advantages over other methods of biofilm growth assessment and is a semiquantitative method commonly used with vital staining techniques to determine the viability profile, architecture, and spatial distribution in microbial biofilms [26].

The physicochemical characterization techniques are tools to determine the size, diameter, and shape of the NP, as well as dispersion and agglomeration, and to determine whether the experimental conditions are appropriate for NP synthesis. The particle size is visualized and verified through transmission electron microscopy (TEM), and dynamic light scattering (DLS) is used to determine the size distribution [27]. The Ag⁺ as an antibacterial agent has antibacterial activity of ample spectrum, and its effect has been demonstrated at low concentrations; however, it is necessary to evaluate whether the Ag⁺ ion release is controlled in the applied environment [28,29]. The cytotoxicity effect of AgNPs is associated with the mechanisms of entry at the cellular level, concentration, exposure time, cell affinity and cell cycle phase, the main target for their toxicity is the alteration of the cell wall and the bacterial cell membrane [11,12].

The aim of this study was to evaluate whether incorporating AgNPs on the surface of a PMMA (Orthocryl^®) substrate using an in-situ polymerization method develops an effect on the inhibition of viable cells in S. aureus biofilm by a dynamic model with a CDC biofilm reactor with fluid dynamics and CLSM to assess and describe the spatial distribution and viability of S. aureus biofilm formation.

2. Material and methods

2.1. Synthesis of AgNPs

Silver nanoparticles were synthesized using Gallic acid in an aqueous chemical reduction of silver nitrate (AgNO₃, 99.99%; Sigma-Aldrich, St Louis, MO, USA) according to Martínez-Castañon et al. [30]. The physicochemical characterization and the antibacterial effect (E. coli and S. aureus) of the colloidal dispersion has been previously reported [31]. The AgNPs were then dried with a microcellulose filter (StarLab Scientific^®) for the preparation of specimens. The redispersion of the silver dust was evaluated by TEM with an acceleration voltage of 100 kV and DLS (Z. sizer), and the AgNPs were characterized by UV–Vis spectroscopy using a S2000-UV–Vis spectrometer (Ocean Optics Inc).

2.2. Preparation of PMMA/AgNP specimens

Thirty Ø12 × 4-mm specimens were fabricated in silicone molds from a PMMA resin that met ISO 1567/1999 Standard of the American Dental Association [32] (Self-polymerizing Acrylic Resin; red - 161-620-00; Orthocryl^®, Dentaurum, PA, USA). The coating was prepared by in-situ polymerization using 200 μg/mL of AgNPs mixed with methylmethacrylate (MMA) monomer. This solution (16 μL) was deposited on the slab surface of PMMA with a micropipette. The average weight of the specimens was 298 mg and the total silver content on the surface of each specimen of PMMA/AgNP was 3.1 μg, equivalent to 0.001% wt. Once the polymerization process had been completed after 20 min under room temperature conditions, the specimens were washed with running water for 30 min to remove excess monomer. The specimens were then placed on the mounted bases of the bioreactor specimens, and the reactor was assembled, filled, wrapped, and sterilized by autoclaving at 121 °C for 20 min.

2.3. Characterization of AgNPs and silver release quantification

The AgNPs were characterized through TEM (JEOL JEM-1230) at an accelerating voltage of 100 kV, and DLS analysis was performed in a Malvern Zetasizer Nano ZS. An Atomic Absorption Spectrophotometer (AAS; AA-7000 Shimatzu, Japan) was used to measure the solubility of silver coupled in the PMMA according to ANSI/ADA specification No. 139 [33]. A total of 12 PMMA/AgNP specimens, 3 for each group, were immersed in 10 mL of deionized water and were incubated at 37 ± 1 °C for 24 h, 48 h, 7 d, and 30 d. Measurements were made in triplicate for each group. The limits of quantification and the analytical method were determinate according to PMMA/AgNP characteristics.

2.4. Strains and culture reactor conditions

The S. aureus strain American Type Culture Collection (ATCC) 25923 was used in this study. It was seeded on sheep blood agar plates and incubated at 37 °C for 24 to 48 h. From this fresh pure culture, one bacterial colony was removed and transferred into 5 mL of YPD broth (Yeast extract, Peptone, Dextrose; Becton Dickinson, Mexico City, Mexico). The use of YPD has been previously standardized by our team, providing excellent growth of the biofilm of bacterial strains of S. aureus [34]. This culture was incubated overnight at 37 °C with constant 80-RPM agitation. After this, each tube was centrifuged in order to continue a series of washes with phosphate buffered saline (PBS) solution. A cell suspension of the reference strain at an approximate cell density of 1–1.5 × 10⁸ cells/mL for bacteria was prepared from this initial suspension, with a final density of 1–1.5 × 10⁴ cells/mL in the reactor flask with the YPD broth. The density was verified by serial dilutions of the suspension and the subsequent culture on plates of Trypticase Soy Agar (TSA; Becton Dickinson), and the number of colony forming units (CFUs) was counted; the logarithm of CFU per milliliter (log CFU/mL) was calculated by the same examiner.

The suspension in YPD broth was agitated at 80 RPM for 10 h, and the culture was again removed from the CDC biofilm reactor (model CBR 90–1, BioSurface Technologies Corp) by washing with 750 mL of PBS to remove the nonadherent cells and to leave only the cells of the primary biofilm on the surface of the specimens. Later the reactor was activated to a flask containing 750 mL of YPD, and the reactor was completely assembled. A flow rate of 1.8 mL/min was activated, and, after 15 to 20 min, agitation was activated at 80 RPM, taking care to maintain an incubation temperature of 36 °C. The culture medium was refreshed every 24 h to 72 h, and the specimens were removed at 6, 12, 24, 48, and 72 h.

2.5. S. aureus biofilm evaluation

The viability and the qualitative/semiquantitative evaluations of the formation of S. aureus biofilm cells adhering to the specimen surfaces were conducted by incubation with the Live/Dead Bacterial Viability Kit (Invitrogen, CA, USA) fluorescent stain for 15 to 30 min in the dark at 30 °C before examination by CLSM (Leica, DMI 4000B, Wetzlar, Germany). The stained biofilm specimens were examined with an argon ion laser with 480/500 nm excitation for the SYTO 9 stain and 520/650-nm emission for propidium iodide stain of wavelengths. For analysis with the LAS AF Lite program, all metabolically active cells were visualized by green fluorescence, and those with lower metabolic activity by diffuse red fluorescence. The tests were carried out in triplicate. A total of 10 images (×40) were quantified for each specimen for all measurements of arbitrary fluorescence units (AFU). Ten+ values were collected (20 × 20 μm) for each image, and a three-dimensional (3D) reconstruction was performed of the entire thickness of the biofilm formed for the entire time period.

2.6. Statistical analysis

Descriptive statistics of the Live/Dead bacterial relationship were performed in both groups and at their different times. The Kolmogorov–Smirnov normality test, the Mauchly sphericity test and Levene homoscedasticity test were performed. The Kolmogorov–Smirnov test indicated that the normality assumption is not fulfilled for both groups at all times, obtaining P values from <0.01 to 0.2. The Mauchly test of sphericity showed that the assumption of sphericity for the time variable is not met either (Mauchly’s W 0.011, df 9, P < 0.001) and that the same was observed with the Levene homoscedasticity test; only at 24 h was equality of variances identified in both groups (P = 0.152). To determine the difference of the dependent variable with respect to the group, an intersubject effects test was used (𝛼 = 0.05).

Fig. 1.

TEM image of the synthetized AgNPs (a, a′), DLS test result (b). UV–Vis spectra of the AgNPs (c). The values obtained by the analysis of AAS shows an increase in Ag release over time with a higher release speed in the first two days; from the second to the seventh day, it shows stable release (d).

3. Results

3.1. Synthesis and characterization of AgNPs

The process of filtering and drying the AgNPs allows the release of ions and residue of the colloidal solution, as well as the smaller particles. Figures 1a and a′ shows a TEM image of redispersed AgNPs, showing an irregular and quasispherical shape with a narrow size distribution with size ranges from 2 to 17 nm. The DLS analysis (Fig. 1b) showed an average size of the colloidal dispersion of 25 nm. Figure 1c shows the absorption spectrum in the UV/visible region of AgNPs, where a characteristic band with a peak at 400 nm wavelength was observed; suggesting the reduction of Ag⁺ to AgNPs.

3.2. Silver release of PMMA/AgNPs

Figure 1d shows a comparison of the solubility of silver ions (Ag⁺) at different times: 24 h, 48 h, 7 d, and 30 d, for the PMMA/AgNP specimens with the highest release of Ag⁺ at 30 d with an average of 1.2 μg/mL (Table 1). This result represents 38% of the total silver deposited on the polymeric surface (3.1 μg). However, it was demonstrated that, in the first 48 h of immersion of the specimens, the AgNPs released Ag⁺ more quickly that at 7 and 30 d.

3.3. Biofilm formation

Descriptive statistics of the Live/Dead bacterial relationship were performed in both groups and at their different times. Higher means (Live/Dead bacterial relationship) for the control group at different times were observed when compared with those of the experimental group (Table 2).

S. aureus biofilm formation increased from the beginning of formation at the 6 h period according to the count of viable cells in both study groups, maintaining constant growth up to 72 h. In the control group, there was an increase in AFU of 115% between 6 and 12 h; from 12 to 24 h, AFU increased by another 115%; from 24 to 48 h, AFU increased by 74%; and, finally, from 48 to 72 h, AFU increased by 57%. For the experimental group of PMMA with AgNPs, there was an increase in AFU of 98% between 6 and 12 h; from 12 to 24 h, AFU increased by 40%; from 24 to 48 h, AFU increased by 46%; and, finally, from 48 to 72 h, AFU increased by 58%.

Table 1
Analysis of Ag release in PMMA specimens

PMMA/AgNP

Time Silver release (ppm) Total silver (%) Release speed (μg/day)

24 h 0.162 ± 0.029 4.5 0.162

48 h 0.492 ± 0.081 13.6 0.246

7 days 0.603 ± 0.069 16 0.086

30 days 1.206 ± 0.767 33 0.040

	PMMA/AgNP
24 h	0.162 ± 0.029	4.5	0.162
48 h	0.492 ± 0.081	13.6	0.246
7 days	0.603 ± 0.069	16	0.086
30 days	1.206 ± 0.767	33	0.040

Table 2

Descriptive statistics (live/dead bacterial relationship)

Hours	Groups			Statistic
6	Control	Mean		2.3022
		Confidence interval for the mean at 95%	Lower limit	1.5849
			Upper limit	3.0195
		Standard deviation		3.44420
	Experimental	Mean		2.0811
		Confidence interval for the mean at 95%	Lower limit	1.9825
			Upper limit	2.1797
		Standard deviation		0.47366

12	Control	Mean		2.6937
		Confidence interval for the mean at 95%	Lower limit	2.5876
			Upper limit	2.7999
		Standard deviation		0.50980
	Experimental	Mean		1.3695
		Confidence interval for the mean at 95%	Lower limit	1.2971
			Upper limit	1.4418
		Standard deviation		0.34725

24	Control	Mean		2.5715
		Confidence interval for the mean at 95%	Lower limit	2.4813
			Upper limit	2.6617
		Standard deviation		0.43316
	Experimental	Mean		2.1265
		Confidence interval for the mean at 95%	Lower limit	2.0467
			Upper limit	2.2063
		Standard deviation		0.38328

48	Control	Mean		2.4722
		Confidence interval for the mean at 95%	Lower limit	2.3783
			Upper limit	2.5661
		Standard deviation		0.45100
	Experimental	Mean		2.4268
		Confidence interval for the mean at 95%	Lower limit	2.2557
			Upper limit	2.5979
		Standard deviation		0.82162

72	Control	Mean		2.4936
		Confidence interval for the mean at 95%	Lower limit	2.4193
			Upper limit	2.5680
		Standard deviation		0.35705
	Experimental	Mean		2.1327
		Confidence interval for the mean at 95%	Lower limit	1.9982
			Upper limit	2.2673
		Standard deviation		0.64615

Table 3

Growth of the values of AFU (Arbitrary Fluorescence Units) of Live and Dead biofilm S. aureus in the five evaluated time periods in the control group (without AgNP) and in the experimental group (with AgNP)

Group		6 h	12 h	24 h	48 h	72 h
Control	Mean live	13.11	28.38	61.16	106.54	167.45
	SD	1.05	3.09	11.17	4.9	13.78
	Mean dead	6.86	16.49	24.5	42.87	49.63
	SD	0.38	4.91	8.55	3.47	6.77
Experimental	Mean	7.33	14.55	20.45	29.91	47.4
	SD	1.6	1.91	2.9	3.75	11.67
	Mean dead	5.17	8.4	12.1	18.1	25.29
	SD	1.06	1.29	1.56	4.96	4.76

SD: Standard deviation.

The values of AFU by formation period are presented in Table 3, and the values of the AFU of viable and dead cells in the control group and in the experimental group by evaluation period are shown in Fig. 2a, b. Table 4 provides the significant differences in AFU between the control and experimental groups for each of the periods evaluated. The intersubject effects test showed a statistically significant difference in the V/R relation with respect to the groups (F = 30.343, df 1, P < 0.001). The lower limit test showed a borderline difference of the different times in each group (F = 3.487, P = 0.06) and a statistically significant difference in the interaction of the groups at the different times (F = 8.308, P = 0.004).

Fig. 2.

Growth curve of live and dead S. aureus biofilm cells on the surface of the control group (a) and on the surface of the experimental group (b).

Table 4

Mean differences between the control and experimental groups at 6, 12, 24, 48, and 72 h, and the decrease in AFU (Live bacteria) at each time with respect to the immediately previous period

Time (h)	Difference (Exp–Ctrl)	95% CI of the difference
6	−5.78	−7.52 to −4.04
12	−13.83	−17.14 to −10.52
24	−40.71	−51.21 to −30.2
48	−76.63	−82.32 to −70.95
72	−120.05	−136.48 to −103.61

Table 5

Description of the architecture of the S. aureus biofilm at 6, 12, 24, 48, and 72 h

Evaluation period	Observed surface	Morphology and characteristics of the biofilm
6 h	Control Fig. 3(a)	Defined coccoid shapes, isolated cells, clusters with conglomerate focalizations, intense and dynamic metabolic activity with high cell fluorescence.
	Experimental Fig. 3(f)	There are no focal zones of primary settlement, well-defined coccoid shapes with poor growth. No clusters in conglomerates.

12 h	Control Fig. 3(b)	Well-defined coccoid shapes of intense growth and the presence of isolated cells, in pairs, and starting growth in clusters.
	Experimental Fig. 3(g)	Defined coccoid shapes, clusters of lower fluorescence compared to the control surface.

24 h	Control Fig. 3(c)	Biofilm formation was established, indicating high metabolic activity. Growth was uniform without isolated shapes. The total area was a conglomerate of grouped bacteria.
	Experimental Fig. 3(h)	The bacteria spread into small clusters. This corresponds to a phase of settlement, with little growth. A few bacteria were observed with red fluorescence.

48 h	Control Fig. 3(d)	Focuses of coccoid bacteria concentrated with an intense green fluorescence were identified. A large number of complex clusters were observed, indicating structural maturity of the biofilm and diverse metabolism depending on the fluorescence.
	Experimental Fig. 3(i)	Bacteria were observed in the total area. Light clusters of bacteria were more abundant and were less separated from each other.

72 h	Control Fig. 3(e)	Focuses were observed of coccoid bacteria that became denser with intense green fluorescence in the center, indicating a well-structured biofilm and an increase in size and complexity.
	Experimental Fig. 3(j)	Coccoid shapes were observed without good definition, with poor growth, and a decrease in the intensity of the fluorescence. No clustering was observed.

Table 6

Description of the architecture of the S. aureus biofilm in the 3D reconstruction between study groups at 6, 12, 24, 48, and 72 h

Evaluation period	Observed surface	f3D reconstruction at 45° angulation
6 h	Control Fig. 4(a)	The basal layer showed uniform thickness. Isolated and separated cells were observed, demonstrating a phase of bacterial settlement in process.
	Experimental Fig. 4(f)	The basal layer continued to be thin, without the presence of clusters. The bacteria were distributed in isolation or in pairs, viable and non-viable mixed cells were observed.

12 h	Control Fig. 4(b)	The continuous basal layer exhibited uniform thickness, and isolated and separated cells were observed with a bacterial-settlement phase in process.
	Experimental Fig. 4(g)	The basal layer grew in thickness with respect to 6 h, and no conglomerates were observed.

24 h	Control Fig. 4(c)	The basal layer continued to be uniform, conglomerate bacteria were observed, the thickness of the biofilm was greater, and few non-viable bacteria were observed.
	Experimental Fig. 4(h)	The basal layer grew in thickness with respect to 12 h, no conglomerates were observed, and the bacteria were distributed into small clusters A minimal amount of non-viable bacteria continued to be observed.

48 h	Control Fig. 4(d)	The basal layer increased after 24 h. Cluster structures and medium-density conglomerates with non-viable bacteria were observed.
	Experimental Fig. 4(i)	The basal layer decreased slightly in thickness with respect to the 24 h, and few non-viable bacteria were observed.

72 h	Control Fig. 4(e)	There was no significant increase in thickness with respect to 24 h. Conglomerates in the form of dense granules and smaller cluster structures with non-viable bacteria were observed
	Experimental Fig. 4(j)	The basal layer decreased in thickness and was less dense and more opaque compared to 48 h. The characteristics were more similar to those of the 12 h biofilm than to the 24 or the 48 h biofilm, which indicates its decrease.

According to the images obtained with CLSM of each of the specimens analyzed, notable differences were observed in the groups of PMMA specimens with and without AgNPs. On the surface of the control group specimens, a bacterial biofilm of S. aureus was developed in vitro in a phase of highly dynamic reproductive growth and high metabolic activity, during which, as in the evaluated 6, 12, 24, 48, and 72 h time periods, complex structures were observed that revealed areas of maturity in the biofilm. From the CSLM images and by quantification of the fluorescence, which is proportional to the metabolic activity of the cells in the formed biofilm, these biofilms were found to develop homogeneously in a growth phase with high metabolic activity, with some areas of greater structural maturity. Tables 5 and 6 present a detailed description of the biofilm architecture. The comparison of the biofilm images during each of the evaluated time periods is illustrated in Fig. 3, and the 3D reconstruction of the biofilm between the control and the experimental groups in Fig. 4. The images showed that growth of the biofilm on the acrylic resin surface was inhibited, presumably because dead cells, or cells with low metabolic activity were scarce.

Fig. 3.

Images obtained with CLSM of S. aureus biofilm formation (co-localization live/dead cells) on the surface of PMMA without AgNPs (control group) at 6 h (a), 12 h (b), 24 h (c), 48 h (d), 72 h (e), and with AgNPs (experimental group) at 6 h (f), 12 h (g), 24 h (h), 48 h (i), and 72 h (j).

Fig. 4.

3D reconstruction of S. aureus biofilm formation (co-localization live/dead cells) on the surface of PMMA without AgNPs (control group) at 6 h (a), 12 h (b), 24 h (c), 48 h (d), 72 h (e) and with AgNPs (experimental group) at 6 h (f), 12 h (g), 24 h (h), 48 h (i), and 72 h (j).

4. Discussion

Biofilm formation on the surface of polymeric appliances is a major concern in medicine and dentistry. NPs that cover the surface or volume of the biomaterial are being used to eliminate pathogenic bacteria, even in microenvironments as complex as the oral cavity [35]. The results of this preliminary in vitro research demonstrated reduced growth and maturation of S. aureus biofilm on a PMMA surface impregnated with AgNPs for use in dentistry. The differences between the control group without AgNPs and the experimental group with AgNPs were statistically significant (P < 0.05) at 6, 12, 24, 48, and 72 h, revealing promising results for future clinical dental application.

Previous studies have shown that AgNPs deposited by in situ polymerization are weakly attached to polymer chains. Silver in aqueous solution can be released by absorption in the porous polymer with an antimicrobial effect, with 0.03% wt of silver deposited in the PMMA matrix [30,36]. In the present study, only 0.001%wt was used with antibiofilm properties. Further, the release of Ag⁺ over time was lower than the values of cytotoxicity, inflammation, and genotoxicity widely reported [31]. Therefore, future studies should analyze the agglomerates, the kind of chemical union of nanoparticles and polymer chains, and the thickness of the surface coating with well-established surface techniques. In situ polymerization appears to be an effective and straightforward AgNP method that can bring benefits in medical and dental areas where PMMA is used [37].

The delivery of Ag⁺ from AgNPs is a determining factor of their activity. Previous studies have reported that AgNPs with a size of 50 nm or less are active against a wide range of bacteria and fungi, while 25-nm AgNPs have reported efficacy against viruses and have been proposed for antibacterial, antifungal, and antiviral uses [38,39]. In the present study, the average size of AgNPs added to the surface of PMMA was from 25 nm; therefore, our results suggest an antimicrobial response for AgNP synthesis with narrow particles ranges, though not a completely homogeneous solution. Cheng et al. reported that 3-nm AgNPs were dispersed throughout the polymer matrix, suggesting that the ideal size is in the range of 2 to 5 nm [40]. In contrast, Farhadian et al. used a 40-nm NP size and Riau et al. evaluated 90-nm NPs, both reporting effective killing of microorganisms [41,42].

AgNPs have been reported to be an effective microbicidal agent because of their high surface area-to-volume ratios, which allow them to adhere to the membrane, affecting permeability, inducing cellular structural changes, and giving rise to cell death [30,34]. Our findings are consistent with those of previously published studies demonstrating that AgNPs inhibit the biofilm growth of different microorganisms [43,44]. AgNPs have been reported to inhibit the growth of oral bacteria at very low concentrations without adverse effects and to have strong antimicrobial activity in the planktonic phase and in the subsequent biofilm formation of the cariogenic bacteria [13,22]. Ghorbanzadeh et al. reported the strong antibacterial activity of AgNPs incorporated into PMMA as a new dental acrylic resin against Escherichia coli one of the studies that showed that AgNPs exert antimicrobial activity on pathogenic microorganisms [35]. The focus of this study was on an S. aureus biofilm in a dynamic model with essential factors such as hydrodynamic flow, temperature, and nutrients that are controlled by using the CDC biofilm reactor; this provided controllable conditions for biofilm formation and the evaluation of antimicrobial particles. Studies have reported the antimicrobial effect of AgNPs on S. aureus, S. mutans and Candida albicans in dental materials [5]. In this study, we evaluated S. aureus biofilm formation because evidence exists for the bactericidal effect of dental materials with AgNPs on S. aureus colony counts [45,46]. Previous studies have reported that the prevalence of S. aureus colonization in patients receiving dental treatment was 42% [47], which is in the range of populations in the USA and Europe [48]. S. aureus is considered one of several opportunistic microbial pathogens associated with systemic diseases [49] and has more affinity to stainless steel and the adhesion forces of the biofilm depending on the type of dental materials tested and the surface characteristics of the material [50].

The system used to evaluate the biofilm formation in this study was the CDC biofilm reactor, which has been previously evaluated with other microorganisms such as Candida albicans [34,51], Streptococcus mutans [52] and S. aureus [53]; these customized bioreactors provide a sterile simulated environment to support biofilm formation and allow the evaluation of the biological response of dental materials [54]. Authors such as Rudney et al. and Li et al. were able to develop an oral biofilm over the surfaces of dental materials using a bioreactor [55,56].

The CLSM images showed an S. aureus biofilm grown on the surface of PMMA without AgNPs. Maturing forms conglomerated and coccoid groups with high metabolic activity increased as these communities developed. In the experimental group, we observed dispersed cells with different degrees of viability and with a decreased metabolism, demonstrating that AgNPs incorporated into PMMA exhibited excellent antibiofilm properties as evidenced by the CLSM micrographs. The findings were consistent with those of Oei et al. who reported excellent antimicrobial properties against the growth of planktonic bacterial-cell growth by incorporating AgNPs into acrylic resin materials [57]. Goswami et al. who studied the effect of AgNPs on biofilm production of different bacteria, reported that AgNPs are capable of inhibiting 89% of biofilms formed by S. aureus [58]. Other models, such as that reported by Ramage et al. suggest that the highest metabolic activity of a microbial biofilm occurred at 12 h, while, in our study, this was observed at 24 h [59]. The difference was probably due to the flow rate, agitation, and temperature used in the CDC biofilm reactor and to the growth conditions of each of the studied microorganisms. The fabrication method used in this study of incorporating NPs into the surface of the PMMA and into the final layers by adding them to the monomer demonstrated in the images obtained by CLSM that inhibition of the S. aureus biofilm was uniform on the entire surface of the PMMA specimens, preventing the accumulation of AgNPs in localized zones.

As used in dentistry, PMMA is widely used for elaborate prostheses, denture base and orthodontic-orthopedic appliances, is a material with a surface with micro- and nanoroughness that provides microretentive spaces for accumulate a large amount of dental biofilm [60], this reservoir of microorganism is the main cause of diseases such as denture stomatitis and for potential respiratory pathogens [23,61]. Studies suggest that oral cavity is a reservoir of S. aureus, besides C. albicans in denture wearers; also are present in biofilms in conditions such as angular chelitis, cystic fibrosis and diabetic foot ulcers [62,63]. One of the most abundant species in denture stomatitis is P. aeruginosa followed by S. pneumoniae and S. aureus [23], Short et al. showed that S. aureus pushes C. albicans towards a more virulent genotype [64]; even S. aureus, S. epidermidis and Pseudomonas aeruginosa are frequently implicated in polymicrobial infections [65,66].

In addition, and possibly despite the hygiene of the patient, the bristles of the toothbrush do not manage to penetrate these porosities. In this regard, surface modifications and antibacterial nano-additive biomaterials incorporated into biomaterials provide numerous advantages for the elimination of biofilm [67]. In our study, AgNPs incorporated into PMMA significantly reduced the biofilm formation of S. aureus. Thus, our results may be a step toward in the development of novel dental materials with an antimicrobial effect.

Several studies have evaluated the effect of AgNPs as an antibacterial agent, but the long-term effect of AgNPs on PMMA and evaluate their antibacterial effect on other pathogenic microorganisms in the oral cavity require further investigation. We evaluated biofilm growth in diverse and dynamic environments where factors such as fluid shear are important. The in vitro control of all possible variables is an essential factor for the appropriate study of bacterial biofilm. One of the limitations of this study is that only single species biofilms was evaluate, in further studies, it is necessary to evaluate other species of biofilm to confirm the antibacterial effect of the AgNPs. In this respect, the proposed model permits the control of different factors that favor the study of the biofilm that is developing on the PMMA at different stages. Our results confirm the ability of S. aureus to infect the PMMA surface and are consistent with those of other studies that have reported the considerable capacity of S. aureus strains to adhere to dental alloys, as well as to epithelial cells [68].

5. Conclusion

AgNPs incorporated by a straightforward method into the surface of PMMA conventionally used to manufacture orthodontic appliances provide a significant inhibitory effect on the formation, growth, and maturation of S. aureus biofilm, preventing the settlement and development of bacteria.

Footnotes

Acknowledgements

None to report.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This study was in part supported by Fondo de Apoyo a la Investigación 2014, C14-FAI-04-52.52 and PROMEP/103.5/13/6575. CONACYT for scholarships 234478 and materials characterization laboratory of the Faculty of Sciences of the UASLP.

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	PMMA/AgNP
Time	Silver release (ppm)	Total silver (%)	Release speed (μg/day)
24 h	0.162 ± 0.029	4.5	0.162
48 h	0.492 ± 0.081	13.6	0.246
7 days	0.603 ± 0.069	16	0.086
30 days	1.206 ± 0.767	33	0.040

Antibiofilm properties of silver nanoparticles incorporated into polymethyl methacrylate used for dental applications

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Material and methods

2.1. Synthesis of AgNPs

2.2. Preparation of PMMA/AgNP specimens

2.3. Characterization of AgNPs and silver release quantification

2.4. Strains and culture reactor conditions

2.5. S. aureus biofilm evaluation

2.6. Statistical analysis

3.1. Synthesis and characterization of AgNPs

3.2. Silver release of PMMA/AgNPs

3.3. Biofilm formation

Table 1 Analysis of Ag release in PMMA specimens PMMA/AgNP Time Silver release (ppm) Total silver (%) Release speed (μg/day) 24 h 0.162 ± 0.029 4.5 0.162 48 h 0.492 ± 0.081 13.6 0.246 7 days 0.603 ± 0.069 16 0.086 30 days 1.206 ± 0.767 33 0.040

5. Conclusion

Footnotes

Acknowledgements

Conflict of interest

Funding

References

Table 1
Analysis of Ag release in PMMA specimens

PMMA/AgNP

Time Silver release (ppm) Total silver (%) Release speed (μg/day)

24 h 0.162 ± 0.029 4.5 0.162

48 h 0.492 ± 0.081 13.6 0.246

7 days 0.603 ± 0.069 16 0.086

30 days 1.206 ± 0.767 33 0.040