Jurnal Paper_Development of novel antimicrobial acrylic denture modified with copper nanoparticles.pdf

J Prosthodont Res. 2023; **(**): ****–**** Original...

J Prosthodont Res. 2023; **(**): ****–**** Original Journal of Prosthodontic Research article Development of novel antimicrobial acrylic denture modified with copper nanoparticles Sebastián Correa a, Loreto Matamala b, Juan Pablo González a, Mónica de la Fuente a, Hetiel Miranda a, Bruno Olivares a, Miguel Maureira a, Amaru Agüero a, Leyla Gómez c, Ximena Lee d, Madeleine Urzúa d, Cristian Covarrubias a,* aLaboratory of Nanobiomaterials, Institute for Research in Dental Sciences, Faculty of Dentistry, University of Chile, Santiago, Chile, b Department of Conservative Dentistry, Faculty of Dentistry, University of Chile, Santiago, Chile, c Laboratory of Microbiology, Department of Pathology and Oral Microbiology, University of Chile, Chile, d Public Health, Institute for Research in Dental Sciences, Faculty of Dentistry, University of Chile, Santiago, Chile Abstract Purpose: This study aimed to synthesize heat-cured poly(methyl methacrylate) (PMMA) acrylic formulated with copper nanoparticles (nCu) for producing dentures with antimicrobial properties and ability to prevent denture stomatitis (DS). Methods: nCu/PMMA nanocomposites were prepared through in situ formation of nCu into methyl methacrylate (MMA). The fabricated material was characterized using scanning electron microscopy, spectroscopy (energy-dispersive X-ray, attenuated total reflectance–Fourier-transform infrared, and X-ray photoelectron spectroscopy), X-ray diffraction analysis, and mechanical flexural tests (ISO 20795-1:2008). Antimicrobial activity against Candida albicans and oral bacteria was determined. MTS assay (ISO 10993-5:2009) and copper release experiments were conducted to assess cytotoxicity. In the clinical trial, participants wearing nCu/PMMA (n=25) and PMMA (n=25) dentures were compared; specifically, DS incidence and severity and Candida species proliferation were assessed for 12 months. Data were analyzed using analysis of variance with Tukey’s post hoc test (α=0.05). Results: nCu/PMMA nanocomposite loaded with 0.045% nCu exhibited the maximum antimicrobial activity against C. albicans and other oral bacteria without producing cytotoxicity in the wearer. nCu/PMMA dentures retained their me- chanical and aesthetic properties as well as inhibited the growth of Candida species on both denture surface and patient palate. DS incidence and severity were lower in the nCu/PMMA denture group than in the PMMA denture group. Conclusions: PMMA acrylic produced with copper nanotechnology is antimicrobial, biocompatible, and aesthetic and can reduce DS incidence. Thus, this material may act as a novel preventive alternative for oral infections associated with denture use. Keywords: Antimicrobial PMMA, Copper nanoparticles, Candida albicans, Denture stomatitis, Dental nanotechnology Received 2 September 2022, Accepted 18 April 2023, Available online 20 May 2023 1. Introduction unique oral microenvironment produced by denture use. The denture fitting surface has been demonstrated to act as a reservoir The rapid increase in the global elderly population has present- for adherent Candida species and other microorganisms, which are ed various challenges pertaining to oral health, including prosthetic the major causative agents of denture stomatitis (DS)[4,5]. DS is one replacement of missing teeth. Removable and implant-supported of the most common conditions affecting denture wearers and is dentures remain a common solution for the rehabilitation of elderly characterized by chronic inflammation and erythema of the oral mu- edentulous patients. Although dentures improve the quality of cosa. Epidemiological studies have indicated that the prevalence life, masticatory function, and psychological status of patients, of DS ranges from 15% to >70%, generating symptoms of variable the accumulation of oral biofilms on the denture surface constitutes severity, from asymptomatic to considerable discomfort, altered a problem of special concern. The proliferation of microorganisms taste, burning sensation, dysphagia, and pain. Although proper on dentures can lead to acrylic staining, prosthetic deterioration, hygiene practices for dentures are of paramount importance in pre- bad breath, calculus formation, and mucosal infections. Candida venting DS, patients’ habits and attitude do not necessarily lead to is a commensal yeast that inhabits different zones of the oral cav- compliance with denture cleansing recommendations. Moreover, ity and can be transformed into an opportunistic pathogen in the hygiene practices may affect the color and mechanical stability of prosthetic acrylic implants. DOI: https://doi.org/10.2186/jpr.JPR_D_22_00227 To overcome these limitations, strategies employing acryl- *Corresponding author: Cristian Covarrubias, Olivos 943, Independencia, Santiago, Chile. E-mail address: [email protected] ics with antimicrobial properties are currently being explored for preventing the proliferation of microorganisms, ultimately reduc- Copyright: © 2023 Japan Prosthodontic Society. All rights reserved. 2 S. Correa, et al. / J Prosthodont Res. 2023; **(**): ****–**** ing the incidence of DS. Several studies on site-specific detection Tokyo, Japan). The elemental composition of nCu was determined of Candida species have proven that DS is associated with greater using energy-dispersive X-ray (EDX) spectroscopy (Aztec, Oxford growth of yeast on the denture acrylic surface than on the palate of Instruments, Pcl., High Wycombe, UK) coupled with SEM. In addition, wearer[10–13], implying that DS treatment should preferably focus we examined the nCu/PMMA nanocomposite using BS-SEM and EDX on the denture material. Thus, the effects of polymer coating or spectroscopy. The PMMA polymer structure was confirmed using at- incorporating several types of antimicrobial substances into dental tenuated total reflectance–Fourier-transform infrared spectroscopy acrylic, such as quaternary ammonium monomers or silver/zinc– (ATR–FTIR; Cary 630, Agilent Technologies, Inc., Santa Clara, CA). For modified zeolites[16,17], have been explored. surface chemical characterization of the nCu/PMMA nanocomposite, X-ray photoelectron (XPS) spectroscopy was performed (PHI 1257, Recently, the use of nanoparticles with antimicrobial activity Physical Electronics Inc., Chanhassen, MN, USA). We evaluated the has gained increasing attention. Nanosized metal particles ranging mechanical properties of the acrylics using a flexural test according in diameter from 1 to 100 nm show a larger surface area, greater to ISO 20795-1:2008 on a micro-test tensile machine (Deben Research reactivity, and stronger antimicrobial potential. In principle, the Ltd., Suffolk, UK) using a 100 N load cell. incorporation of small amounts of nanoparticles into polymers may produce nanocomposites with potent antimicrobial activity without 2.2. Antimicrobial activity significantly altering their other properties. Poly(methyl methacry- late) (PMMA) doped with silver nanoparticles (nAg) exhibits excellent We evaluated the surface antimicrobial activity of the nCu/ antimicrobial activity[19,20]. However, the resulting grayish color of PMMA acrylics against C. albicans (ATCC 90029). We applied a modi- the material is a major aesthetic concern and may incur relatively fied antimicrobial test based on the ISO 22196:2011 to assess surface high costs depending on the silver content. In this context, copper antimicrobial activity of the materials[27,28]. Specimens of neat nanoparticles (nCu) offer an attractive alternative for antimicrobial acrylic (PMMA) and nCu/PMMA nanocomposites (4 mm in height denture design. Copper presents broad-spectrum antimicrobial ac- × 10 mm in diameter) were incubated in test tubes with 1 mL of a tivity, and its antibacterial and anti-inflammatory properties have 0.5 McFarland C. albicans suspension (Sabouraud chloramphenicol been known since ancient times. Currently, advances in nano- broth) for 24 h. Thereafter, the acrylics were washed with a 0.9 wt% technology have garnered much interest to exploit the antimicrobial NaCl solution, followed by 1% Tween 80, to remove yeast growing on properties of copper. In dentistry, the antimicrobial activity of nCu the surface. Samples of 100 μL were collected from the suspension, has been demonstrated against periodontal, cariogenic, and diluted, and plated on Sabouraud agar. After 48 h of aerobic incuba- endodontic bacteria, implying its potential use in dental practice. tion at 37°C, we counted the colonies and calculated the number of colony-forming units (CFUs) in each milliliter. Additionally, we ex- In the present study, we introduce, for the first time, antimicro- amined the biofilms formed on the acrylic surfaces using SEM. After bial acrylic dentures fabricated with nCu. We hypothesized that con- incubation, we fixed the adherent yeasts by immersing the samples trolled incorporation of nCu into PMMA can enable the production of in glutaraldehyde, dehydrated them in an ethanol series, dried them dentures with antimicrobial activity (specifically against C. albicans), in supercritical CO2 (Tousimis, Autosamdri-815), and gold-coated which may reduce DS incidence without altering the biocompatibil- them prior to SEM visualization. Eight representative micrographs ity and mechanical or aesthetic properties of the acrylic. This work were acquired from the specimen area at magnifications ranging involved the synthesis of nanocomposites and in vitro assessment from ×200 to ×1,000 with 8 kV voltage. of their biological properties as well as a preliminary clinical trial on dentures fabricated with nCu/PMMA nanocomposites. In addition, we assessed the antibacterial activity of an opti- mized nCu/PMMA acrylic composition, specifically against strains of 2. Materials and Methods Streptococcus mutans (ATCC 25175), Aggregatibacter actinomycetem- comitans (ATCC 43718), and Staphylococcus aureus (ATCC 25923). The 2.1. Synthesis and characterization of the nCu/PMMA nanocomposites inhibitory capacity of the nCu/PMMA nanocomposite against these microorganisms was calculated using the following formula: We produced dental acrylic nanocomposites containing nCu nanoparticles using an in situ preparation method[25,26]. Briefly, CFUPMMA − CFUnCu / PMMA we added 100 µL of 1.8%–14.5% w/v copper acetate [Cu(CH3COO)2] Inhibitory capacity ( % ) = x 100 CFUPMMA aqueous solution to 2 mL of methyl methacrylate (MMA) monomer (Acryl BH Fluid, GDF, Rosbach Vor der Höhe, Germany), which had been previously mixed with 1.5 mL of 95% ethanol. The mixture was where CFUPMMA and CFUnCu/PMMA is the number of CFUs grown on the maintained under constant stirring at 55°C–65°C until formation of a surface of PMMA and nCu/PMMA nanocomposite, respectively. reddish nCu suspension. Thereafter, the nCu monomer suspension was mixed with heat-cured acrylic powder (Acryl BH Pulver, pink 2.3. Biocompatibility veined, GDF, Rosbach Vor der Höhe, Germany) at the monomer/ acrylic ratio of 1:2.5 (w/w). The mixture was transferred to stainless We assessed the cytotoxicity of the acrylics following a test pro- steel molds (4 mm in height × 10 mm in diameter), hand pressed, and tocol for direct contact indicated for the in vitro cytotoxicity of medi- finally heat-cured in brass dental flasks at 95°C for 1.5 h. cal devices [ISO 10993-5:2009(E)]. First, we cultured human gingival stem cells (HGSCs). Then, we assessed the materials by measuring cell We confirmed the presence of nCu nanoparticles in the viability using the MTS colorimetric assay (CellTiter 96® AQueous One monomer based on surface plasmon resonance determined by ul- Solution Cell Proliferation Assay; Promega Corp., Madison, WI) at 490 traviolet–Visible (UV–vis) spectrophotometry (UV2, ATI Unicam, Inc., nm after 1, 7, and 10 days of incubation at 37°C. Cambridge, UK). We then analyzed the nanoparticles using backscat- tered scanning electron microscopy (BS-SEM; JSM-IT300LV, JEOL Ltd.,  S. Correa, et al. / J Prosthodont Res. 2023; **(**): ****–**** 3 2.4. Copper release measurement randomized design. The release of copper ions from nCu/PMMA nanocomposites 2.5.5. Evaluation: Microbiology and DS examination was measured at different time intervals using the HI-4108 copper combination ion-selective electrode (Hanna Instruments Inc., Woon- We evaluated the presence and severity of DS and proliferation socket, RI, USA) after incubating the acrylics in 20 mL of artificial of C. albicans in each patient. We performed the first clinical examina- saliva (pH 6.5) at 37°C for 35 days. tion and microbiological sampling during initial denture placement at the baseline visit. We repeated the clinical examination together 2.5. Preliminary clinical trial with additional microbiological sampling during subsequent tests of 2.5.1. Approval by ethics committee denture adherence at 4, 7, and 12 months. DS was identified during clinical examination by categorizing the presence of clinical signs of The local ethics committee of the authors’ university approved disorder in the hard palate adjacent to the upper denture as “Yes” or the present single-blind randomized controlled trial (no. 11/2017). “No.” We subcategorized the severity of DS according to Newton’s We recruited patients from the clinic of dentistry at the authors’ classification as type I (pin-point hyperemic lesions or localized university. simple inflammation), type II (diffuse erythema confined to the mu- cosa contacting the denture or generalized simple inflammation), 2.5.2. Participant selection and study design or type III (granular surface or inflammatory papillary hyperplasia). We collected (using sterile cotton swabs) microbiological samples Patients requiring both maxillary and mandibular complete for analysis from the following three zones: (1) palatal mucosa, (2) dentures were enrolled in the trial. The participants were recruited denture surface area of approximately 4 cm2 in contact with the between November 11, 2017, and December 20, 2017. palatal mucosa, and (3) unstimulated saliva. The swab specimens were placed in Falcon tubes containing sterile PBS (pH 7.4) and Inclusion criteria: Candidate participants included (1) edentulous then transported to the laboratory at 4°C. In the laboratory, 100 μL patients with an indication for full dentures, (2) healthy participants dilutions were plated on Sabouraud chloramphenicol agar and incu- or patients with mild systemic disease (ASA II), or (3) individuals bated at 37°C for 48 h. After incubation, the CFUs of Candida species willing to visit the clinic for denture adjustment as recall patients. were counted. All participants were required to provide written informed consent prior to any study procedure. 2.6. Statistical analysis Exclusion criteria: Participants with a history of allergies to the Mechanical properties, cell viability, and antimicrobial activity ingredients of the denture base material; participants presenting are expressed as mean±SD. There were nine samples per group at with disabilities that did not permit regular dental treatment; and each time point. Means between groups were compared using participants with a history of antibiotic, antifungal, or steroidal drug multiple-sample one-way analysis of variance (ANOVA). We ap- use up to 15 days prior to providing a sample were excluded. We plied one-way ANOVA to determine significant differences, followed employed WinEpi 2.0 to determine the required sample size using an by Tukey’s post hoc test to determine significant between-group expected prevalence of DS of 22%, a level of confidence of 95%, and differences. To analyze the results of the preliminary clinical trial, a statistical power of 80%; we also assumed a rate of loss to follow-up we assessed the differences between the experimental and control of 10%. The software suggested a sample size of 50 participants. The groups using a paired t-test. The differences were considered signifi- enrolled participants were assigned using simple randomization to cant at P < 0.05. either group 1 (control group), including 25 participants who wore conventional PMMA acrylic dentures, or group 2 (experimental 3. Results group), including 25 participants who wore nCu/PMMA nanocom- posite dentures. 3.1. Synthesis and characterization of nCu/PMMA nanocomposites 2.5.3. Denture fabrication We confirmed the nanometric nature of the nCu particles in the MMA monomer and obtained a characteristic surface plasmon reso- The Beraudent Dental Laboratory (Santiago, Chile) manu- nance absorption peak at 593 nm (Fig. 1a) (which is a unique optical factured both the conventional and nCu/PMMA nanocomposite property characterizing metal particles with nanometric dimen- dentures. The nCu/PMMA nanocomposite dentures were fabricated sions). The BS-SEM image (Fig. 1b) revealed metallic nCu embedded using nanotechnology patented by the authors’ university, derived in the organic monomer as bright particles with sizes ranging from from the developed and tested processes. approximately 30 to 150 nm. EDX elemental mapping confirmed the presence of copper, carbon, and oxygen in the structures of nCu and 2.5.4. Intervention: Installation of the nCu/PMMA denture MMA (Figs. 1c-e). The concentration of copper in the monomer was 0.6% by weight (Table 1). The intervention involved the use of the nCu/PMMA nanocom- posite dentures in group 2 patients. Prior to denture installation, we Moreover, BS-SEM allowed us to visualize nCu in the PMMA evaluated the technical quality of the dentures, particularly their polymer matrix of the Cu/PMMA nanocomposite prepared with extension, retention, support, stability, and soft and hard tissue relief 0.045% wt. copper (Fig. 2a). EDX mapping revealed that copper zones. Members of the research team calibrated the dentures ac- was homogeneously distributed (Fig. 2b). The measured copper cording to a technical evaluation questionnaire comprising dichoto- content was of 0.04% wt. (Table 1), consistent with the copper mous response items. The participants were blinded to their group concentration used in nanocomposite synthesis. Further, the Cu/ assignment; therefore, the clinical trial followed a single-blinded PMMA nanocomposite displayed elemental portions of carbon and 4 S. Correa, et al. / J Prosthodont Res. 2023; **(**): ****–**** Table 1. Energy-dispersive X-ray elemental analysis of the poly (methyl meth- acrylate) (PMMA) monomer containing the in situ-synthesized nCu and of the nCu/PMMA nanocomposite prepared with 0.045% wt. copper. Data are pre- sented as mean±standard deviation. Element content (% wt.) C O Cu nCu/MMA monomer 85.70 ± 0.30 13.70 ± 0.30 0.60 ± 0 nCu/PMMA acrylic 66.80 ± 0.10 33.16 ± 0.20 0.04 ± 0.01 cm-1) bonds were observed in the spectrum of the nCu/PMMA nano- composite. Flexural tests on the mechanical properties of the nCu/ PMMA nanocomposite (Table 2) revealed that the bending strength and bending modulus of nCu/PMMA were not significantly different from those of PMMA. 3.2. Antimicrobial activity We determined the antifungal activity (specifically against C. al- bicans) of the nCu/PMMA nanocomposites (Fig. 3). The nCu content infused in the PMMA matrix conferred all acrylic nanocomposites with antifungal properties. The maximum reduction in the number of adherent yeast CFUs located on the acrylic surface was achieved using 0.045% wt. nCu. The SEM images (Figs. 3b and c) displayed abundant C. albicans growth (with hyphal morphology) on the conventional PMMA acrylic surface compared with near complete absence of yeasts on the surface of the 0.045% wt. nCu/PMMA nanocomposite. Overall, the optimized nanocomposite exhibited a greater inhibition capacity against the representative pathogenic bacteria (Table 3). 3.3. Biocompatibility As shown in Figure 4, the viability of HGSCs on the 0.045% wt. nCu/PMMA nanocomposite did not differ significantly from that of HGSCs on the neat PMMA and that of control cells without material. 3.4. Copper release The copper release experiments conducted using artificial saliva (Fig. 5) indicated that after 35 days of incubation, the 0.055% and Fig. 1. Structural and compositional characterization of copper nanopar- 0.045% wt. nCu/PMMA nanocomposites produced the maximum ticles synthesized into the methyl methacrylate monomer, as visualized by copper concentrations of 3.8 × 10 −3 and 5.9 × 10 −3 μg·mL-1, respec- (a) surface plasmon resonance absorption spectrum and photography, (b) tively. backscattered scanning electron microscopy, and (c-e) energy-dispersive X- ray elemental mapping 3.5. Preliminary clinical trial Initially, we enrolled 45 participants aged 45–80 years. Of the oxygen, originating from the PMMA polymer composition (Figs. 2c initial 45 participants, 20 dropped out for several reasons prior to the and d). XRD analysis revealed the amorphous pattern of the PMMA first follow-up visit (at the 4th month after the baseline visit). A total of polymer in both PMMA and Cu/PMMA acrylics (Fig. 2e). In addition, 25 patients completed the entire 12-month protocol: 12 patients who the XRD pattern of the Cu/PMMA matrix exhibited a peak at 2θ = wore the conventional PMMA denture (2 males and 10 females) and 43.3°, corresponding to the (111) plane of zerovalent copper (JCPDS 13 patients who wore the experimental 0.045% wt. nCu/PMMA den- 04-0836). Furthermore, we performed surface-sensitive XPS analysis tures (6 males and 7 females). Figures 6a and b shows a photograph (Fig. 2f) to determine whether copper was present on the surface of of a typical post-use nCu/PMMA denture, which does not exhibit the nanocomposite material. The 2p1/2, 2p3/2, and 3p photoelectron aesthetic differences compared to a conventional PMMA denture. lines from copper exhibit very low intensities and thus cannot be dis- The CFU counts of Candida species measured in the saliva, on the tinguished from the signal noise. FT-IR analysis (Fig. 2g) showed that palate, and on the denture surface of the participants are plotted in the characteristic vibrations of the PMMA structure corresponding Figures 6c-e. No significant differences in CFU counts in the saliva to the C–C–O (482 cm-1), C–O–C (1,151 and 1,244 cm-1), C=O (767 and samples were noted between group 1 (wearing PMMA dentures) 1,732 cm-1), O–CH3 (1,450 cm-1), and C–H (847, 1,364, 2,958, and 3000 and group 2 (wearing nCu/PMMA dentures) patients. However, we  S. Correa, et al. / J Prosthodont Res. 2023; **(**): ****–**** 5 Fig. 2. Structural and compositional characterization of 0.045% wt. nCu/PMMA nanocomposite, as visualized by (a) backscattered scanning electron mi- croscopy, (b-d) energy-dispersive X-ray elemental mapping, (e) X-ray diffraction patterns, (f) X-ray photoelectron spectroscopy, and (g) and attenuated total reflectance with Fourier-transform infrared spectroscopy Table 2. Flexural strength and modulus of the poly (methyl methacrylate) with type I DS (Fig. 6i), and group 1 patients who presented with (PMMA) monomer and nCu/PMMA nanocomposite prepared with 0.045% type II DS (Fig. 6j) at 7 month after denture installation. wt. copper. Data are presented as mean±standard deviation, P < 0.05 (n=5 per group). 4. Discussion PMMA nCu/PMMA P Value Flexural strength [GPa] 2.8 ± 4 3.1 ± 5 > 0.05 Fungal biofilm formation on denture acrylics and occurrence of Flexural modulus [GPa] 7.9 ± 8 7.1 ± 9 > 0.05 DS represents the major problems for denture users. Currently, the available dental acrylics do not possess antimicrobial properties, which can prevent the growth of microorganisms on the denture surface, thereby reducing the risk of mucosal infections. In the observed significantly lower counts of Candida species on the palate present study, we exploited the antimicrobial properties of nCu to and denture surfaces of group 2 patients at the 7-month visit. At 12 develop a novel biocompatible acrylic nanocomposite with unique months, CFU count in samples obtained from the palate or denture antifungal and mechanical properties. surface of group 2 patients was near zero, unlike the relatively high count of yeasts in the samples of group 1 patients. Figure 6f presents 4.1. Synthesis and characterization of nCu/PMMA nanocomposites the incidence of DS among the participants. The incidence of DS was lower in group 2 than in group 1. No DS was detected in the group 2 Our BS-SEM/EDX analysis indicated that the nCu/PMMA nano- patients at 7 months, and at 12 months, the incidence of DS in group composite presented a relatively uniform distribution of metal 2 was ~55% lower than that in group 1. The severity of DS among the nanoparticles in the acrylic matrix, which we expected to find owing participants is presented in Figure 6g. Group 1 patients presented to our in situ fabrication method characterized by improved nanopar- with type I, type II, or type III DS, whereas group 2 patients only pre- ticle dispersion, interfacial compatibility, and particle size control. sented with type I or type II DS. Figure 6 also shows representative Similarly, our XRD analysis of the nanocomposite showed reflection photographs of group 2 patients who exhibited a generally clinically associated with the (111) plane of zero-valent copper, indicating that healthy palatal mucosa (Fig. 6h), group 2 patients who presented the nanoparticles were not oxidized during the composite forma- 6 S. Correa, et al. / J Prosthodont Res. 2023; **(**): ****–**** Fig. 4. Cytocompatibility of poly(methyl methacrylate) (PMMA) and nCu- modified PMMA acrylics. MTS cell viability of human gingival stem cells cul- tured on neat PMMA and 0.045% wt. nCu/PMMA nanocomposite at differ- ent incubation times. Controls correspond to cells cultured without acrylic materials. Fig. 3. Antimicrobial activity of poly(methyl methacrylate) (PMMA) and nCu- modified PMMA acrylics against Candida albicans after 48 h of incubation, as assessed based on (a) the number of colony-forming units (CFUs) of adherent C. albicans on the nCu/PMMA acrylic surface, (b) scanning electron micro- graphs of the PMMA surface, and (c) scanning electron micrographs of the 0.045% wt. nCu/PMMA nanocomposite surface Table 3. Surface inhibitory capacity of 0.045% wt. nCu/poly (methyl methac- rylate) nanocomposite against several pathogenic microorganisms. Data are presented as mean±standard deviation. Inhibitory SD capacity (%) Candida albicans (Yeast) 95 0.4 Streptococcus mutans (Gram positive) 97 0.8 Fig. 5. Release of copper ions from nCu-modified poly(methyl methacrylate) Aggregatibacter actinomycetemcomitans (Gram negative) 88 2.0 (PMMA) acrylics. Cumulative release profiles of copper ions from the nCu/ Staphylococcus aureus (Gram positive) 94 1.5 PMMA nanocomposites immersed in artificial saliva (pH 6.5) for 35 days. PMMA, as determined by our FT-IR analysis. Moreover, the spectrum tion. Although copper tends to be surface-oxidized in air, the MMA of the nanocomposite did not exhibit new bands corresponding to organic molecules associated with the surface of nCu can act as a the possible bonds formed between the metal nanoparticles and protective layer preventing nCu oxidation[32–34]. Surface-sensitive polymer chains, indicating that the interfacial interactions occurred XPS analysis showed copper peaks with an intensity comparable to through weak physical forces. Furthermore, the flexural mechanical that of signal noise, suggesting that the surface copper concentra- properties of our experimental acrylic were equivalent to those of tion was below the XPS detection limit (~0.1 atomic%), in agree- conventional dental acrylic, unlike the reduced flexural strength ment with the low bulk content of copper incorporated into the reported for acrylics formed with nAg. These differences can PMMA matrix (