12 Characterization and antifungal activity of the modified PMMA denture base acrylic: Nanocomposites impregnated with gold…










CHAPTER
12
Characterization and
antifungal activity of the
modified PMMA denture
base acrylic:
Nanocomposites
impregnated with gold,
platinum, and silver
nanoparticles
Ki Young Nam
Department of Dentistry, College of Medicine, Keimyung University, Daegu, Republic of Korea
12.1 BACKGROUND OF DEVELOPMENT FOR ANTIMICROBIAL
DENTURE ACRYLIC
12.1.1 DENTURE-INDUCED STOMATITIS
Most oro-dental hygiene becomes challenged when pathogenic microbes colonize
the dentine, enamel, dental restorations, and prosthetic materials as well as the
neighboring oral soft tissue. One of the critical materials in dental prosthetic
devices which covers a broad oral tissue area is denture acrylic base. The major
goals of denture treatment include masticator y rehabilitation, speaking, and mid-
facial cosmetics; consequently, improving the wearer’s general or mental health.
PMMA (polymethyl methacrylate)-based acrylic resin has been broad ly applied to
dental materials, especially in denture base processing. PMMA, an economical
alternative to polycarbonate introduced in 1937, is currently the material of choice
for denture bases and it continues to be used because of its favorable working
characteristics, processing ease, accurate fit, stability in the oral environment, and
superior aesthetics with inexpensive equipment. Despite these excellent proper-
ties, there is a need for improvement in the biological aspects. Starting denture
wearing in the oral cavity inevitably leads to changes in the oral environmental
309
Nanobiomaterials in Dentistry. DOI: http://dx.doi.org/10.1016/B978-0-323-42867-5.00012-6
© 2016 Elsevier Inc. All rights reserved.

conditions due to compromised salivary flow (hygienic) effect and encourages
biofilm formations on both the denture prosthetic surface and adjacent mucosa
(
Yildirim et al., 2005). Basically, impressi on surface (intaglio sum) or the tissue
side of denture base should be rough in comparison with the outer, polished
surface (cameo) owing to the absence of the laboratory polishing procedure which
could create a new availab le surface for plaque formation. Local factors (poor
hygiene, prosthetic trauma, an ill-fitting denture by absorption of supporting
tissue) and systemic factors (diabetes mell itus, xerostomia, nutrition deficiency,
etc.) may also contribute to the proliferation of microbes. Denture seating favors
the occurrence of stomatopathy by increasing both the incidences of local injuries
and the contact time of mucosa with microorganisms (
Budtz-Jorgensen and
Bertram, 1970; Ettinger, 1975). About 50% of complete or partial denture wearers
were reported to experience problems related to stomatitis, or they could also harbor
isolated fungi (Budtz-Jorgensen, 1981
). Though microbes could be cleaned out by
saliva flow and swallowed unless they adhere and proliferate, however, once
biofilms formed, they are not easy to be removed because biofilms can be up to
1000 times more resistant to toxicants than the planktonic phase (
Mah et al., 2003).
Chronic denture stomatitis is an erythematous pathogenic condition of the
denture-bearing mucosa and a relatively common disease among denture wearers
with incidences in epidemiological studies of 1167% (
Arendorf and Walker ,
1987
)or2542.4% (McNally et al., 1999). This disease is characterized by
inflamed denture-bearing mucosa, particularly under the upper denture, and
though wearers may complain of a burning sensation, discomfort, or bad taste,
they are occasionally unaware of these problems (
Figure 12.1). One of the major
FIGURE 12.1
Denture-induced stomatitis related with fungal infection in a 67-year-old female partial
denture wearer, an erythematous pathogenic condition of the denture-bearing mucosa is
seen. In this case, inflammation tends to be re-established soon after antifungal
description ceased.
310 CHAPTER 12 Antimicrobial denture nanocomposites

etiological factors in the pathogenesis of this condition is the presence of numer-
ous yeasts, usually Candida albicans (C. albicans) on the fitting surface of the
denture (
Boscato et al., 2009; Oliveira et al., 2009). Candida species are detected
in the oral cavity of 60100% of denture patients (Dagistan et al., 2009
) and
about 60% of removable partia l denture wearers have experienced pathogenic
fungal adhesions (
Ramage et al., 2004). Candidiasis is more common in immuno-
compromised patients and in patients being treated with radiation or chemother-
apy. Once Candida species have colonized, denture polymer surfaces ultimately
can act as a reservoir of infection (
Nikawa et al., 2003; Vanden Abbeele et al.,
2008) and fungal biofilm formation is critical in the pathogenesis of denture
stomatitis (Samaranayake and Nair, 1995; Chandra et al., 2001). The treatments
of fungal stomatitis include denture repair or replacement, pending costs, or
prophylactic measures by the prescription of antifungal drugs. Current antifungal
therapy is application of topical or systemic antifungal agents, including fluco-
nazole and nystatin, etc. (Perezous et al., 2005; Sims et al., 2005; Rowan et al.,
2010). Despite the use of antifungal agents to cure denture stomatitis, infection
often recurs and drug tolerance has been observed in the majority of cases
(Chandra et al., 2001
). Mature denture plaq ues on the fitting surface are associ-
ated with protective biofilms and biofilm-related chronic infections are inherently
difficult to treat and fully eradicate with routine therapy (
Matsuura et al., 1997).
Unlike microbes in the planktonic phase, biofilm displaying phenotypic traits is
known to be notoriously resistant to routine antifungal agents (
Douglas, 2003;
Monteiro et al., 2009).
12.1.2 DEMAND ON ANTIMICROBIAL DENTURE ACRYLIC BASE
With denture wearing, intraoral hygienic states could b e challenged by microbial
adherence on its surface and the ability of microbes to develop biofilms are often
described as a pathogenesis of denture induced oral infection (
Klotz et al., 1985).
Generally, denture cleansing and oral hygiene care are essential for the prevention
of denture stomatitis and patients are generally instructed as follows to avoid
these lesions—avoid: (1) an unfit denture which traumatizes the oral tissue; (2) a
denture that is not thoroughly cleaned; (3) leaving the denture on overnight, etc.
However, for some geriatric or hospitalized patients with physically restricted or
mentally handicapped status, these guidelines might not be adhered to due to dif-
ficulty in rendering appropriate cleaning of the denture and in following strict
instructions as their motor abilities, cognitive or studying capacities may be
reduced (
De Visschere et al., 2006). Chemical-based oral disinfectants, such as
sodium hypochlorite or glutaraldehyde, have been used in denture care. Those
agents could facilitate easy cleansing and would be effective against pathogenic
microbes. However, sodi um hypochlorite is a bleaching agent and may interfere
with the aesthetic s of the prostheses and glutaraldehyde releases toxic vapors,
these bleaching or toxic agents could be stimulants and irritants to oral mucosa or
supporting tissue (
Murdoch-Kinch et al., 1995; Chassot et al., 2006). PMMA
31112.1 Background of Development for Antimicrobial Denture Acrylic

resins have a liquid sorption property due to the high internal energy and polarity
of carboxylic groups in resin (Anusavice, 1996
) and they absorb saliva; also den-
ture immersed in chemical disinfectants might absorb these agents. Therefore, the
risk of released chemical residues in the oral cavity still exists when the denture
is returned.
Dixon et al. (1999) suggested micro wave disinfection of soft denture
base in a 60-Hz microwave oven for 5 min was effective in killing colonized
fungi; however, repeated irradiations significantly affected the hardness of the
material. Syste mic or local antibi otic agents have been prescribed for eliminating
the fungal populations; however, the use of antifungal drugs to treat denture
stomatitis could be limited, as infection is often persistent or resistant (
Chandra
et al., 2001
) and it can cause an overdose of medicine and also is expensive.
Previous studies reported that acrylic soft liners combined with antifungal drugs
could be used in the therapy of denture-ind uced stomatitis (
Williamson, 1968;
Nikawa et al., 2003), there have been the following problem s: short-term dura-
tion, costs of agents used, harmful reaction to older patients, and increased
resistance to apo-lactoferin due to pre-exposure of C. albicans against sub-MIC
(minimal inhibitory concentration) of the antifungal agents (Nikawa et al., 1997
).
Quaternary ammonium compound (
Pesci-Bardon et al., 2006; Beyth et al., 2006)
or silver-zinc zeolite (Casemiro et al., 2008
) as an antimicrobial to incorporate
into denture acrylic or restorative composite resins have been explored, however,
there were some limitations of adverse effects on the mechanical properties of the
composite, difficulties in controlling the release of such agents, and initial cell
adherence (
Ebi et al., 2001). To overcome those denture-related inflammatory
complications, performative and latent antimicrobial denture bases are highly
requested and mandatory (
Saito et al., 1997), however, they are not available in
the market yet. Recently, metal NPs with antimicrobial function have received
considerable attention in diverse fields and there is increas ing interest in the
synthesis of biomaterials holding antimicrobial properties with those particles in
medical and dental fields. Metallic NPs are now being combined with polymers
and other base materials to provide a variety of potential antimicrobial and
antiadhesive applications within the oral cavity (
Allaker and Memarzadeh, 2014).
12.1.3 NOBLE METAL NPs
Metal NPs, under 100 nm in size, have been studied widely due to their unique
physicochemical characteristics with higher catalytic and antimicrobial activity
compared to bulk metals (
Park et al., 2011). Despite the remarkable advances
provided by nanomaterials and nanotechnologies for healthcare, several side
effects have also been revealed. The main health risks related to the use of such
particles consist of cytotoxicity, translocation to undesired cells, unpredictabil ity,
and indeterminate safety concerns. Some metal NPs such as copper, cobalt,
titanium oxide, and silicon oxide showed increased toxicity due to their increased
surface area (
Guzma
´
n et al., 2006), causing inflammat ory effects on cells (Cobb
and Macoubrie, 2004
). Noble nanosized metals such as gold NPs (Au
0
), platinum
312 CHAPTER 12 Antimicrobial denture nanocomposites

NPs (Pt
0
) and silver NPs (Ag
0
) have elicited lots of interest for biomedical
applications because of their biocompatibilities, that is, relatively less toxic to human
cells than other metals, ease of synthesis, surface functionalization, and toxic effects
to bacteria and fungi (
Chwalibog et al., 2010). The biocidal actions of these Au
0
,Pt
0
,
and Ag
0
were reported as they interact directly with various microbes (Pana
´
cek
et al., 2009; Sawosz et al., 2010), therefore they have been explored as an alternative
antimicrobial agent (Sondi and Salopek-Sondi, 2004; Lima et al., 2013) and have
been under the commercial investigation phase recently.
Gold (Au) has a long history of application in the medical field, such as the
killing of bacteria focused on some treatments for nervine, tuberculosis, and
rheumatoid arthritis (
Bhattacharya and Mukherjee, 2008). Au has been exten-
sively investigated in several decades because of its potential applications in
optics, electronics, and catalysis. Medical applications of Au include the use of
sulfurAu compounds as anti-inflammatori es (
Fricker, 1996) and Au compounds
are known to limit the enzymatic activity of liposome in macrophages (
Arceci,
2008
). Au inhibits the proliferation of T cells by modifying the permeability of
mitochondrial membrane (
Weidauer et al., 2007). The success of Au as a catalyst
is a consequence of the manipulation of this metal at the nanometric size, mainly
stabilizing NPs in different inorganic supports such as silica, alumina, and zeolites
(
Lima et al., 2013). Au particles are extensively exploited in organisms because
of their biocompatibility and biologically inert Au
0
can be engineered to possess
chemical or photothermal functionality. Au
0
has a strong affinity toward sulfur of
the sulfhydryl (SH) group through covalent interaction. The Au ion also binds
to thiol groups present in enzymes such as NADH dehydrogenases and disrupts
the respiratory chain facilitat ing the release of active oxygen species and leads to
oxidative stress and significant damage to the cell structures leading to ultimate
cell death (
Feng et al., 2000). Au
0
can be used to coat a wide variety of surfaces
of implants, fabrics for wound treatment, and glass surfaces to maintain hygienic
conditions in the home, hospitals, and other places. Though the predominant anti-
microbial activities and typical binding properties to enhance biomolecular inter-
actions to various bacterial cells have been proposed, few reports are also
available in dental material field because gold was known to have a weaker anti-
microbial effect in comparison with silver and copper.
Platinum (Pt) as a low-reactive noble metal for the organism (
Sawosz et al.,
2010
) has been employed as a catalyst and has been in high demand for use in indus-
trial fields since the early nineteenth century. Pt is used as an alloying agent for
various metal products, including fine wires, noncorrosive containers, medical instru-
ments, as well as dental prostheses such as crowns. The antimicrobial effect of
Pt was revealed through Rosenberg’s study as the inhibition of cell division in
Escherichia coli by the products from Pt electrode (
Rosenberg et al., 1965).
Currently, Pt nanotechnology is being explored for reduction of inflammation
(
Sawosz et al., 2010; Chwalibog et al., 2010), clustering of Pt cations is known to
inactivate microbes by interacting with their enzymes, proteins, or DNA to restrain
cell proliferation or cell division. It also binds to the negatively charged bacterial
31312.1 Background of Development for Antimicrobial Denture Acrylic

cells to change the functionality of the cell membrane or induce chemical inter-
actions, thereby preventing bacterial regeneration and causing bacterial cell disinte-
gration (Onizawa et al., 2009
). Pt
0
is known to scavenge reactive oxygen species and
free radicals from antioxidant responses that can trigger chain reactions and damage
bacteria. For dental applications, Pt
0
incorporation into 4-META/MMA (4-methacry-
loyloxyethyl trimellitic anhydride/methyl methacrylate) adhesives prolonged dentin
bonding durability as compared to the conventional bonding procedures by creating
higher conversion at the interface (
Hoshika et al., 2010, 2011). Pt
0
-resin-based mate-
rial complex improved the biocompatibility as an antioxidant (Ma et al., 2012
)and
addition of Pt
0
to toughen dental porcelain (Fujieda et al., 2012). However, to the
author’s knowledge, no experiment has ever been conducted to explore PMMA
denture—Pt
0
complex for its antimicrobial effects and physical aspects as well as Au
0
.
Silver (Ag) has a long history of use in medicine as an antimicrobial agent. Silver
shows a well-tolerated tissue response and exhibits a very low toxicity profile and is
a highly active compound against a broad spectrum of sessile bacteria and fungi
colonizing on plastic surfaces (
Slawson et al., 1990). The antimicrobial activity of
silver is dependent on silver ions, which bind strongly to electron donor groups in
biological molecules containing sulfur, oxygen, or nitrogen, resulting in defects in
the bacteria cell wall so that cell contents are lost (
Thurman and Gerba, 1989;
Samuel and Guggenbichler, 2004) and ionic silver can interact with the DNA of
bacteria preventing cell reproduction and this may leading to cell death. Ag has been
used in different fields in medicine for many years and Ag
0
, particularly in the
nanosized inorganic particle form, appears to be a more effective means of pro-
phylaxis with its rapid and broad-spectrum efficacy than silver powder (14 μm),
which shows low antimicrobial activity owing to the limited surface (Wright et al.,
1999
). Ag
0
were reported to exhibit greater biocidal action than Au
0
against both
bacterial species (
Herna
´
ndez-Sierra et al., 2008) due to the higher surface activity
(
Ahmad et al., 2013); it has also been more widely studied or processed as compared
to gold and platinum among the noble metals (
Jinhong et al., 2006). Ag
0
has received
attention in recent years because of its sustained silver ions (Ag
1
)release(Volker
et al., 2004
). Polyurethane or silicone, such as a technology of central venous
catheter impregnated by Ag
0
, has been developed and used in clinical studies
(
Samuel and Guggenbichler, 2004). For dental applications, diverse Ag
0
combined
materials such as filling materials, dental cements or sealants, temporary restorations,
coating agents, and adhesives have emerged for the assessment of their antimicrobial
capacity (
Durner et al., 2011; Melo et al., 2013; Ahn et al., 2009).
12.2 PMMA DENTURE NP NANOCOMPOSITE
12.2.1 DENTURE NANOCOMPOSITE BIOMATERIAL
Nanocomposites are defined as the material whose major component is a
polymer and the minor one must have a single dimension below 100 nm. New
314 CHAPTER 12 Antimicrobial denture nanocomposites

technologies require new materials with special combinations of structural and
chemical properties, the surface modification of polymer materials has been a major
challenge in medical applications such as catheters or other devices in the human
body. Nanocomposites have become an active field of study because of large
property changes with very small addition of nanofiller, generally less than 5.0 wt%.
Many polymeric compounds, such as poly (vinyl alcohol), poly (vinylpyrrolidone)
(PVP), poly (ethylene glycol), poly (methacrylic acid), and PMMA have been
proved to be effective protective bases to stabilize NPs. Denture PMMA is an
excellent base for the formulation of nanocomposites including NPs with a proper
tailorability and flexibility to prevent the potential aggregation of particles
(
Sur et al., 2010; Wang et al., 2008). MetalPMMA nanocomposite, a typical
example of using metal NPs as additives in polymer matrix, was reported to improve
the mechanical properties of polymers or antimicrobial activity (Sodagar et al.,
2012; Boomi et al., 2013). Most of the reports of dental polymers containing NPs
have focused on Ag (Ahn et al., 2009; Wady et al., 2012; Monteiro et al., 2012).
As described, the biocidal activity of Au
0
,Pt
0
,andAg
0
against various bacteria
or fungi are well known, but these NPs cannot be applied directly to oral tissue for
therapeutics because concentration-dependent toxicity, necrosis, or apoptosis for Ag
was demonstrated (
Braydich-Stolle et al., 2005). There has been little information
of efficacy after their incorporation to PMMA denture base reported yet. NPs could
act as impurities which change the physicochemical properties in polymers (
Davies
and Rawlings, 1999
) along with varied chemical interactions between the CQO
groups. An optimum amount of NPs combined into polymer materials could be of
critical importance to avoid an adverse effect upon the physicochemical properties
as well as the color stability related to NP impregnation.
12.2.2 PREPARATION OF NPs
A variety of preparation methods have been explored in the last few decades for the
synthesis of NPs with well-known examples including chemical reduction, laser
ablation, gamma irradiation, electron irradiation, photochemical methods, micro-
wave processing, and biological synthetic technique. The most common and popular
technique could be chemical reduction by organic and inorganic reducing agents,
such as sodium citrate, ascorbate, sodium borohydride (NaBH
4
), elemental hydro-
gen, and polyol process. The physical properties of NPs can be influenced by the
size, shape, structure, and compositions, these aspects can be altered or manipulated
by varying either kinetic or thermodynamic variables in the syntheses. NPs should
be stabilized by a protective layer of borohydride ions because salts such as NaCl
shield the negative charges allowing the particles to clump together to form aggre-
gates. To prevent aggregation, NPs can be coated with a polymer, such as PVP, to
inhibit particle aggregation and stabilize the colloidal NPs even when salt is added.
Figure 12.2 schematically shows the prepa ration of Au
0
,Pt
0
, and Ag
0
by
chemical reduction method. Each NP was respectively synthesized by blending
two solutions (A and B) in a homomixer (Homomixer Mark II). Solution A was
31512.2 PMMA Denture NP Nanocomposite

prepared by dissolving PVP in an aqueous solution of hydrogen tetrachloroaurate
(III) for Au
0
, chloroplatinic acid hydrate for Pt
0
, and silver nitrate for Ag
0
.
Solution B was prepared by dissolv ing NaBH
4
in an aqueous PVP solution.
Solution B was added drop-wise into solution A and the mixture was homo ge-
nized at the same speed (3500 rpm) according to the formulation for 5 min. The
chemical process continued for 2 h at room temperature and all chemical reag ents
were used without further purification. TEM (transmission electron micrograph;
Hitachi H 7100, Japan) views identify Au
0
,Pt
0
, and Ag
0
as spherically shape d
particles under 10 nm in a size (
Figure 12.3). The size-dependent physical and
FIGURE 12.2
Schematic diagram for preparation of noble metal (Au, Pt, Ag) nanoparticles and
fabrication of nanocomposite samples.
FIGURE 12.3
TEM of Au
0
,Pt
0
, and Ag
0
prepared in this experiment. Three NPs are shown as
spherically shaped particles measuring under 10 nm in diameter (31000 K).
316 CHAPTER 12 Antimicrobial denture nanocomposites

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CHAPTER12Characterization andantifungal activity of themodified PMMA denturebase acrylic:Nanocompositesimpregnated with gold,platinum, and silvernanoparticlesKi Young NamDepartment of Dentistry, College of Medicine, Keimyung University, Daegu, Republic of Korea12.1 BACKGROUND OF DEVELOPMENT FOR ANTIMICROBIALDENTURE ACRYLIC12.1.1 DENTURE-INDUCED STOMATITISMost oro-dental hygiene becomes challenged when pathogenic microbes colonizethe dentine, enamel, dental restorations, and prosthetic materials as well as theneighboring oral soft tissue. One of the critical materials in dental prostheticdevices which covers a broad oral tissue area is denture acrylic base. The majorgoals of denture treatment include masticator y rehabilitation, speaking, and mid-facial cosmetics; consequently, improving the wearer’s general or mental health.PMMA (polymethyl methacrylate)-based acrylic resin has been broad ly applied todental materials, especially in denture base processing. PMMA, an economicalalternative to polycarbonate introduced in 1937, is currently the material of choicefor denture bases and it continues to be used because of its favorable workingcharacteristics, processing ease, accurate fit, stability in the oral environment, andsuperior aesthetics with inexpensive equipment. Despite these excellent proper-ties, there is a need for improvement in the biological aspects. Starting denturewearing in the oral cavity inevitably leads to changes in the oral environmental309Nanobiomaterials in Dentistry. DOI: http://dx.doi.org/10.1016/B978-0-323-42867-5.00012-6© 2016 Elsevier Inc. All rights reserved. conditions due to compromised salivary flow (hygienic) effect and encouragesbiofilm formations on both the denture prosthetic surface and adjacent mucosa(Yildirim et al., 2005). Basically, impressi on surface (intaglio sum) or the tissueside of denture base should be rough in comparison with the outer, polishedsurface (cameo) owing to the absence of the laboratory polishing procedure whichcould create a new availab le surface for plaque formation. Local factors (poorhygiene, prosthetic trauma, an ill-fitting denture by absorption of supportingtissue) and systemic factors (diabetes mell itus, xerostomia, nutrition deficiency,etc.) may also contribute to the proliferation of microbes. Denture seating favorsthe occurrence of stomatopathy by increasing both the incidences of local injuriesand the contact time of mucosa with microorganisms (Budtz-Jorgensen andBertram, 1970; Ettinger, 1975). About 50% of complete or partial denture wearerswere reported to experience problems related to stomatitis, or they could also harborisolated fungi (Budtz-Jorgensen, 1981). Though microbes could be cleaned out bysaliva flow and swallowed unless they adhere and proliferate, however, oncebiofilms formed, they are not easy to be removed because biofilms can be up to1000 times more resistant to toxicants than the planktonic phase (Mah et al., 2003).Chronic denture stomatitis is an erythematous pathogenic condition of thedenture-bearing mucosa and a relatively common disease among denture wearerswith incidences in epidemiological studies of 1167% (Arendorf and Walker ,1987)or2542.4% (McNally et al., 1999). This disease is characterized byinflamed denture-bearing mucosa, particularly under the upper denture, andthough wearers may complain of a burning sensation, discomfort, or bad taste,they are occasionally unaware of these problems (Figure 12.1). One of the majorFIGURE 12.1Denture-induced stomatitis related with fungal infection in a 67-year-old female partialdenture wearer, an erythematous pathogenic condition of the denture-bearing mucosa isseen. In this case, inflammation tends to be re-established soon after antifungaldescription ceased.310 CHAPTER 12 Antimicrobial denture nanocomposites etiological factors in the pathogenesis of this condition is the presence of numer-ous yeasts, usually Candida albicans (C. albicans) on the fitting surface of thedenture (Boscato et al., 2009; Oliveira et al., 2009). Candida species are detectedin the oral cavity of 60100% of denture patients (Dagistan et al., 2009) andabout 60% of removable partia l denture wearers have experienced pathogenicfungal adhesions (Ramage et al., 2004). Candidiasis is more common in immuno-compromised patients and in patients being treated with radiation or chemother-apy. Once Candida species have colonized, denture polymer surfaces ultimatelycan act as a reservoir of infection (Nikawa et al., 2003; Vanden Abbeele et al.,2008) and fungal biofilm formation is critical in the pathogenesis of denturestomatitis (Samaranayake and Nair, 1995; Chandra et al., 2001). The treatmentsof fungal stomatitis include denture repair or replacement, pending costs, orprophylactic measures by the prescription of antifungal drugs. Current antifungaltherapy is application of topical or systemic antifungal agents, including fluco-nazole and nystatin, etc. (Perezous et al., 2005; Sims et al., 2005; Rowan et al.,2010). Despite the use of antifungal agents to cure denture stomatitis, infectionoften recurs and drug tolerance has been observed in the majority of cases(Chandra et al., 2001). Mature denture plaq ues on the fitting surface are associ-ated with protective biofilms and biofilm-related chronic infections are inherentlydifficult to treat and fully eradicate with routine therapy (Matsuura et al., 1997).Unlike microbes in the planktonic phase, biofilm displaying phenotypic traits isknown to be notoriously resistant to routine antifungal agents (Douglas, 2003;Monteiro et al., 2009).12.1.2 DEMAND ON ANTIMICROBIAL DENTURE ACRYLIC BASEWith denture wearing, intraoral hygienic states could b e challenged by microbialadherence on its surface and the ability of microbes to develop biofilms are oftendescribed as a pathogenesis of denture induced oral infection (Klotz et al., 1985).Generally, denture cleansing and oral hygiene care are essential for the preventionof denture stomatitis and patients are generally instructed as follows to avoidthese lesions—avoid: (1) an unfit denture which traumatizes the oral tissue; (2) adenture that is not thoroughly cleaned; (3) leaving the denture on overnight, etc.However, for some geriatric or hospitalized patients with physically restricted ormentally handicapped status, these guidelines might not be adhered to due to dif-ficulty in rendering appropriate cleaning of the denture and in following strictinstructions as their motor abilities, cognitive or studying capacities may bereduced (De Visschere et al., 2006). Chemical-based oral disinfectants, such assodium hypochlorite or glutaraldehyde, have been used in denture care. Thoseagents could facilitate easy cleansing and would be effective against pathogenicmicrobes. However, sodi um hypochlorite is a bleaching agent and may interferewith the aesthetic s of the prostheses and glutaraldehyde releases toxic vapors,these bleaching or toxic agents could be stimulants and irritants to oral mucosa orsupporting tissue (Murdoch-Kinch et al., 1995; Chassot et al., 2006). PMMA31112.1 Background of Development for Antimicrobial Denture Acrylic resins have a liquid sorption property due to the high internal energy and polarityof carboxylic groups in resin (Anusavice, 1996) and they absorb saliva; also den-ture immersed in chemical disinfectants might absorb these agents. Therefore, therisk of released chemical residues in the oral cavity still exists when the dentureis returned.Dixon et al. (1999) suggested micro wave disinfection of soft denturebase in a 60-Hz microwave oven for 5 min was effective in killing colonizedfungi; however, repeated irradiations significantly affected the hardness of thematerial. Syste mic or local antibi otic agents have been prescribed for eliminatingthe fungal populations; however, the use of antifungal drugs to treat denturestomatitis could be limited, as infection is often persistent or resistant (Chandraet al., 2001) and it can cause an overdose of medicine and also is expensive.Previous studies reported that acrylic soft liners combined with antifungal drugscould be used in the therapy of denture-ind uced stomatitis (Williamson, 1968;Nikawa et al., 2003), there have been the following problem s: short-term dura-tion, costs of agents used, harmful reaction to older patients, and increasedresistance to apo-lactoferin due to pre-exposure of C. albicans against sub-MIC(minimal inhibitory concentration) of the antifungal agents (Nikawa et al., 1997).Quaternary ammonium compound (Pesci-Bardon et al., 2006; Beyth et al., 2006)or silver-zinc zeolite (Casemiro et al., 2008) as an antimicrobial to incorporateinto denture acrylic or restorative composite resins have been explored, however,there were some limitations of adverse effects on the mechanical properties of thecomposite, difficulties in controlling the release of such agents, and initial celladherence (Ebi et al., 2001). To overcome those denture-related inflammatorycomplications, performative and latent antimicrobial denture bases are highlyrequested and mandatory (Saito et al., 1997), however, they are not available inthe market yet. Recently, metal NPs with antimicrobial function have receivedconsiderable attention in diverse fields and there is increas ing interest in thesynthesis of biomaterials holding antimicrobial properties with those particles inmedical and dental fields. Metallic NPs are now being combined with polymersand other base materials to provide a variety of potential antimicrobial andantiadhesive applications within the oral cavity (Allaker and Memarzadeh, 2014).12.1.3 NOBLE METAL NPsMetal NPs, under 100 nm in size, have been studied widely due to their uniquephysicochemical characteristics with higher catalytic and antimicrobial activitycompared to bulk metals (Park et al., 2011). Despite the remarkable advancesprovided by nanomaterials and nanotechnologies for healthcare, several sideeffects have also been revealed. The main health risks related to the use of suchparticles consist of cytotoxicity, translocation to undesired cells, unpredictabil ity,and indeterminate safety concerns. Some metal NPs such as copper, cobalt,titanium oxide, and silicon oxide showed increased toxicity due to their increasedsurface area (Guzma´n et al., 2006), causing inflammat ory effects on cells (Cobband Macoubrie, 2004). Noble nanosized metals such as gold NPs (Au0), platinum312 CHAPTER 12 Antimicrobial denture nanocomposites NPs (Pt0) and silver NPs (Ag0) have elicited lots of interest for biomedicalapplications because of their biocompatibilities, that is, relatively less toxic to humancells than other metals, ease of synthesis, surface functionalization, and toxic effectsto bacteria and fungi (Chwalibog et al., 2010). The biocidal actions of these Au0,Pt0,and Ag0were reported as they interact directly with various microbes (Pana´ceket al., 2009; Sawosz et al., 2010), therefore they have been explored as an alternativeantimicrobial agent (Sondi and Salopek-Sondi, 2004; Lima et al., 2013) and havebeen under the commercial investigation phase recently.Gold (Au) has a long history of application in the medical field, such as thekilling of bacteria focused on some treatments for nervine, tuberculosis, andrheumatoid arthritis (Bhattacharya and Mukherjee, 2008). Au has been exten-sively investigated in several decades because of its potential applications inoptics, electronics, and catalysis. Medical applications of Au include the use ofsulfurAu compounds as anti-inflammatori es (Fricker, 1996) and Au compoundsare known to limit the enzymatic activity of liposome in macrophages (Arceci,2008). Au inhibits the proliferation of T cells by modifying the permeability ofmitochondrial membrane (Weidauer et al., 2007). The success of Au as a catalystis a consequence of the manipulation of this metal at the nanometric size, mainlystabilizing NPs in different inorganic supports such as silica, alumina, and zeolites(Lima et al., 2013). Au particles are extensively exploited in organisms becauseof their biocompatibility and biologically inert Au0can be engineered to possesschemical or photothermal functionality. Au0has a strong affinity toward sulfur ofthe sulfhydryl (SH) group through covalent interaction. The Au ion also bindsto thiol groups present in enzymes such as NADH dehydrogenases and disruptsthe respiratory chain facilitat ing the release of active oxygen species and leads tooxidative stress and significant damage to the cell structures leading to ultimatecell death (Feng et al., 2000). Au0can be used to coat a wide variety of surfacesof implants, fabrics for wound treatment, and glass surfaces to maintain hygienicconditions in the home, hospitals, and other places. Though the predominant anti-microbial activities and typical binding properties to enhance biomolecular inter-actions to various bacterial cells have been proposed, few reports are alsoavailable in dental material field because gold was known to have a weaker anti-microbial effect in comparison with silver and copper.Platinum (Pt) as a low-reactive noble metal for the organism (Sawosz et al.,2010) has been employed as a catalyst and has been in high demand for use in indus-trial fields since the early nineteenth century. Pt is used as an alloying agent forvarious metal products, including fine wires, noncorrosive containers, medical instru-ments, as well as dental prostheses such as crowns. The antimicrobial effect ofPt was revealed through Rosenberg’s study as the inhibition of cell division inEscherichia coli by the products from Pt electrode (Rosenberg et al., 1965).Currently, Pt nanotechnology is being explored for reduction of inflammation(Sawosz et al., 2010; Chwalibog et al., 2010), clustering of Pt cations is known toinactivate microbes by interacting with their enzymes, proteins, or DNA to restraincell proliferation or cell division. It also binds to the negatively charged bacterial31312.1 Background of Development for Antimicrobial Denture Acrylic cells to change the functionality of the cell membrane or induce chemical inter-actions, thereby preventing bacterial regeneration and causing bacterial cell disinte-gration (Onizawa et al., 2009). Pt0is known to scavenge reactive oxygen species andfree radicals from antioxidant responses that can trigger chain reactions and damagebacteria. For dental applications, Pt0incorporation into 4-META/MMA (4-methacry-loyloxyethyl trimellitic anhydride/methyl methacrylate) adhesives prolonged dentinbonding durability as compared to the conventional bonding procedures by creatinghigher conversion at the interface (Hoshika et al., 2010, 2011). Pt0-resin-based mate-rial complex improved the biocompatibility as an antioxidant (Ma et al., 2012)andaddition of Pt0to toughen dental porcelain (Fujieda et al., 2012). However, to theauthor’s knowledge, no experiment has ever been conducted to explore PMMAdenture—Pt0complex for its antimicrobial effects and physical aspects as well as Au0.Silver (Ag) has a long history of use in medicine as an antimicrobial agent. Silvershows a well-tolerated tissue response and exhibits a very low toxicity profile and isa highly active compound against a broad spectrum of sessile bacteria and fungicolonizing on plastic surfaces (Slawson et al., 1990). The antimicrobial activity ofsilver is dependent on silver ions, which bind strongly to electron donor groups inbiological molecules containing sulfur, oxygen, or nitrogen, resulting in defects inthe bacteria cell wall so that cell contents are lost (Thurman and Gerba, 1989;Samuel and Guggenbichler, 2004) and ionic silver can interact with the DNA ofbacteria preventing cell reproduction and this may leading to cell death. Ag has beenused in different fields in medicine for many years and Ag0, particularly in thenanosized inorganic particle form, appears to be a more effective means of pro-phylaxis with its rapid and broad-spectrum efficacy than silver powder (14 μm),which shows low antimicrobial activity owing to the limited surface (Wright et al.,1999). Ag0were reported to exhibit greater biocidal action than Au0against bothbacterial species (Herna´ndez-Sierra et al., 2008) due to the higher surface activity(Ahmad et al., 2013); it has also been more widely studied or processed as comparedto gold and platinum among the noble metals (Jinhong et al., 2006). Ag0has receivedattention in recent years because of its sustained silver ions (Ag1)release(Volkeret al., 2004). Polyurethane or silicone, such as a technology of central venouscatheter impregnated by Ag0, has been developed and used in clinical studies(Samuel and Guggenbichler, 2004). For dental applications, diverse Ag0combinedmaterials such as filling materials, dental cements or sealants, temporary restorations,coating agents, and adhesives have emerged for the assessment of their antimicrobialcapacity (Durner et al., 2011; Melo et al., 2013; Ahn et al., 2009).12.2 PMMA DENTURE NP NANOCOMPOSITE12.2.1 DENTURE NANOCOMPOSITE BIOMATERIALNanocomposites are defined as the material whose major component is apolymer and the minor one must have a single dimension below 100 nm. New314 CHAPTER 12 Antimicrobial denture nanocomposites technologies require new materials with special combinations of structural andchemical properties, the surface modification of polymer materials has been a majorchallenge in medical applications such as catheters or other devices in the humanbody. Nanocomposites have become an active field of study because of largeproperty changes with very small addition of nanofiller, generally less than 5.0 wt%.Many polymeric compounds, such as poly (vinyl alcohol), poly (vinylpyrrolidone)(PVP), poly (ethylene glycol), poly (methacrylic acid), and PMMA have beenproved to be effective protective bases to stabilize NPs. Denture PMMA is anexcellent base for the formulation of nanocomposites including NPs with a propertailorability and flexibility to prevent the potential aggregation of particles(Sur et al., 2010; Wang et al., 2008). MetalPMMA nanocomposite, a typicalexample of using metal NPs as additives in polymer matrix, was reported to improvethe mechanical properties of polymers or antimicrobial activity (Sodagar et al.,2012; Boomi et al., 2013). Most of the reports of dental polymers containing NPshave focused on Ag (Ahn et al., 2009; Wady et al., 2012; Monteiro et al., 2012).As described, the biocidal activity of Au0,Pt0,andAg0against various bacteriaor fungi are well known, but these NPs cannot be applied directly to oral tissue fortherapeutics because concentration-dependent toxicity, necrosis, or apoptosis for Agwas demonstrated (Braydich-Stolle et al., 2005). There has been little informationof efficacy after their incorporation to PMMA denture base reported yet. NPs couldact as impurities which change the physicochemical properties in polymers (Daviesand Rawlings, 1999) along with varied chemical interactions between the CQOgroups. An optimum amount of NPs combined into polymer materials could be ofcritical importance to avoid an adverse effect upon the physicochemical propertiesas well as the color stability related to NP impregnation.12.2.2 PREPARATION OF NPsA variety of preparation methods have been explored in the last few decades for thesynthesis of NPs with well-known examples including chemical reduction, laserablation, gamma irradiation, electron irradiation, photochemical methods, micro-wave processing, and biological synthetic technique. The most common and populartechnique could be chemical reduction by organic and inorganic reducing agents,such as sodium citrate, ascorbate, sodium borohydride (NaBH4), elemental hydro-gen, and polyol process. The physical properties of NPs can be influenced by thesize, shape, structure, and compositions, these aspects can be altered or manipulatedby varying either kinetic or thermodynamic variables in the syntheses. NPs shouldbe stabilized by a protective layer of borohydride ions because salts such as NaClshield the negative charges allowing the particles to clump together to form aggre-gates. To prevent aggregation, NPs can be coated with a polymer, such as PVP, toinhibit particle aggregation and stabilize the colloidal NPs even when salt is added.Figure 12.2 schematically shows the prepa ration of Au0,Pt0, and Ag0bychemical reduction method. Each NP was respectively synthesized by blendingtwo solutions (A and B) in a homomixer (Homomixer Mark II). Solution A was31512.2 PMMA Denture NP Nanocomposite prepared by dissolving PVP in an aqueous solution of hydrogen tetrachloroaurate(III) for Au0, chloroplatinic acid hydrate for Pt0, and silver nitrate for Ag0.Solution B was prepared by dissolv ing NaBH4in an aqueous PVP solution.Solution B was added drop-wise into solution A and the mixture was homo ge-nized at the same speed (3500 rpm) according to the formulation for 5 min. Thechemical process continued for 2 h at room temperature and all chemical reag entswere used without further purification. TEM (transmission electron micrograph;Hitachi H 7100, Japan) views identify Au0,Pt0, and Ag0as spherically shape dparticles under 10 nm in a size (Figure 12.3). The size-dependent physical andFIGURE 12.2Schematic diagram for preparation of noble metal (Au, Pt, Ag) nanoparticles andfabrication of nanocomposite samples.FIGURE 12.3TEM of Au0,Pt0, and Ag0prepared in this experiment. Three NPs are shown asspherically shaped particles measuring under 10 nm in diameter (31000 K).316 CHAPTER 12 Antimicrobial denture nanocomposites chemical properties of NPs, especially the diameter of NPs, could affect theirantibacterial levels, the smaller the particle size, the easier NPs’ penetrationthrough cell membranes and the more they affect intracellular processes in antimi-crobial activity (Campoccia et al., 2013; Kajita et al., 2007) and NPs under 30 nmin size were reported to enhance its catalytic activities including antimicrobialaction (Park et al., 2011).12.2.3 INCORPORATION OF NPs INTO PMMA DENTURE BASEThe pathway of adding NPs into denture PMMA has been shown in two ways:NPs in the form of colloidal solution added to the liquid monomer of acrylic resin(Acosta-Torres et al., 2012; Monteiro et al., 2012) or polymer powder (Wadyet al., 2012; Nam et al., 2012; Li et al., 2014) by volume or weight proportionunder 5% concentration to secure the mechanical aspects. In liquid to liquidmixing, a chemically modified or purified MMA liquid resin should be needed. Inthis experiment, colloidal Au0,Pt0, and Ag0were preliminary combined with thepristine PMMA denture powder (Vertex-SC, Vertex-Dental BV, Netherlands),respectively, according to the range of 0 (control), 10, 50, 100, 200, and 400 ppm(04.0 wt%). Moist denture powders impregnated by colloidal NPs were dehy-drated by natural drying for 120 h at room temperature then thermally desiccatedagain in an oven for 48 h. Dried powders were passed through a sieve (60 mesh)and ground in a ball mill for 1 h to ensur e particle homogenizat ion. ExperimentalPMMA denture NP nanocomposite (PDNC) samples were fabricated as follows:(1) NP- combined powders were mixed with resin monomer (MMA liquid) atdesignated powder/liquid ratio (2.3 g: 0.95 g by wt%); (2) when mixtures reacheda dough stage, they were located and packed into disk holes (20.0 mm 3 2.0 mm)supported by custom-made brass molds sandwiched by two glass frames (5 mmthickness) under 10 kg static pressure; (3) all of the mixtures were self-curedfollowing the manufacturer’s instructions and removed from molds with excessivepolymerized parts trimmed; (4) samples (n 5 320) were immersed for 120 h insterilized distilled water to leach excess residual monomer then finished for 1 hin distilled water using ultrasonic cleaner.12.3 CHARACTERIZATION OF PDNC12.3.1 MICROSTRUCTURE OF PDNCThe microscopy for PDNC samples was performed by field emission electronmicroscope (FE-SEM) and energy dispersion spectroscopy (SEM/EDX; HitachiS-4100 FE-SEM/EDS, Tokyo, Japan) at an accelerated voltage of 20 keV toidentify the presence of NPs in PMMA denture. The unique peak of each metalis clearly shown, which indicated that each NP was successfully loaded andimmobilized into corresponding PMMA resin (Figure 12.4). Optical SEM image31712.3 Characterization of PDNC (31000 K) of PDNC with 400 ppm dose exhibits similar surface texture in com-parison with pristine but some surface cracking and blistering are noted(Figure 12.5).12.3.2 DETERMINATION OF ELUTED ION FROM THE PDNCThe properties of the matrix polymer and its water diffusion characteristics play arole in the release process (Kumar and Munstedt, 2005; Damm et al., 2007) for anti-biotic nanocomposites. However, many literatures have revealed nanocomposites orantibiotic resin polymer releasing no or extremely low cations to medium. Ebi et al.(2001)demonstrated unpolymerized MDPB (12-methacryloyloxydodecylpyridinumbromide) released at extremely low value which is not bacteriostatic against plank-tonic cells.Yu et al. (2008) incorporated Ag0into PMMA denture, but the release ofthese NPs was extremely slow with a very small fraction of silver ion released after54 days.Monteiro et al. (2012) also demonstrated that concentration of Ag0com-bined to the acrylic resin influenced the silver distribution and dispersion in the den-ture polymer, but silver release was not satisfactory, regardless of the immersionFIGURE 12.4The SEM/EDX patterns of metal electrodes. The spectra show the typical peaks of Au, Pt,and Ag that verify successful impregnation of NPs into pristine denture acrylic. Unassignedpeaks originate from polymer or external contaminants.318 CHAPTER 12 Antimicrobial denture nanocomposites period in water. Restricted ion elution related to PDNC could be explained by thefacts that PMMA is a rather hydrophobic polymer which may have generated a bar-rier for water diffusion, and its water uptake may not be sufficient for ion releasefrom each NP strongly entrapped in resin bulk or particle coalescence associated withthe high temperature during resin polymerization (Balan et al., 2008).Cured Au0,Pt0, and Ag0-PDNC disk specimens (20 mm 3 2.0 mm, n 5 75)were put into 100 ml of sterile distilled water and stored at 37C under agitation.The concentration (ppm) of eluted ion was determined at 24 h and 30 days withdistilled water replaced every 24 h. The eluted ion quantity was scored as theamount of ions in the solution per unit of surface area of the disk (cm2) and anatomic absorption spectrophotometer (AAS) and shaking incubator were used.Table 12.1 shows that a very sensitive AAS did not indicate the presenceof NPs in aliquot media from PDNC upto 200 ppm in two immersion timesand the minimum detectable concentrations were calculated as 0.030. 04 pp mat i nitial 24 h and 0.01 ppm at 30 days from PDNC with 400 ppm NPsadded; these values might be from the PDNC surface, not fr om the solidPMMA bulk.FIGURE 12.5SEM images of PDNC containing 4.0% (400 ppm) NPs: (a) Au0, (b) Pt0, and (c) Ag0.Some surface cracking and blistering were observed but overall surface textures weresimilar to those of the control (d) (350).31912.3 Characterization of PDNC 12.4 PHYSICAL PROPERTIES OF PDNC12.4.1 THERMAL STABILITYThermal analysis is a general term used to monitor the change of material properduring the heating process and it has a significant implication as far as polymer fabri-cation processes are concerned (Davy et al., 1997). Denture base as a thermoplasticmaterial can be influenced by temperature variances such as foods in the mouth,cleansing, and particularly the manufacturing stage such as curing and polishingprocesses (Soygun et al., 2013). Several studies assessed the thermal stability ofmodified denture PMMA against heat.Zhang et al. (2012) reported 5 wt% aluminumborate whiskers in the PMMA matrix improved thermal stability of PMMA, andAydogan Ayaz et al. (2013) showed that the thermal stability of PMMA is increasedby the insertion of acrylamide monomer by thermogravimetric (TG) results. As forNPs,Hamedi-Rad et al. (2014) showed that the mean thermal conductivity of PMMAreinforced with nanosilver was significantly higher than the unmodified PMMA.Poor thermal stability of PMMA can lead to a negative influence on physicalproperties of the composites, even if modified denture polymer expresses theantifungal effect. To test PDNC, TG and differential scanning calorimetry (DSC)were carried out simultaneously using a TG-DTA 92 (Setaram, France) with aheating rate of 10C/min from 30C to 600C under nitrogen atmosphere.TG/DSC thermal analysis was used to monitor the polymerization or settingreactions of dental resins and to measure the glass transition temperatures (Tg) ofpolymers (Aydogan Ayaz et al., 2013). Tg is recognized as a critical standard ofamorphous polymeric materials and in the context of denture base polymers as anaid to characterization of PDNC for heat capacity, molecular weight, crosslinking,and stereochemistry. DSC provides a fast method and is generally employed inresearch of the polymerization or setting reactions of dental resins to measure theheat effects of phase transitions of polymers (Aouachria and Bensemra, 2006).Table 12.1 Concentrations of Eluted Ion from Three Experimental PDNC atTwo Designated TimesAmounts of Eluted Ion (ppm)NPs Combined (ppm) Au0-PDNC Pt0-PDNC Ag0-PDNC24 hr 30 days 24 hr 30 days 24 hr 30 days1050100 N.D.200400 0.03 0.01 0.03 0.01 0.04 0.01N.D.: Not Detected.320 CHAPTER 12 Antimicrobial denture nanocomposites Despite adding the NPs, PDNC exhibited similar TG/DSC thermogram to thatof control even with the highest NP dose (400 ppm) during the degradationprocess and NP impregnations did not affect the glass transition phase of thedenture acrylic (Figure 12.6). TG thermograms of Au0,Pt0, and Ag0PDNC wereclosely in accordance with control. Endoth ermic peaks at about 384.7C wasreferred to dissociat ed temperature of pristine PMMA and PDNC samples exhib-ited closely overlapped curves to pristine PMMA with sharp inflections (weightreduction) from 350C to 400C. DSC curves for each PDNC also expressed thesame patterns as those with pristine PMMA at a heating range from 30Cto600C and DSC peaks of three PDNC indicated that the melting points have nosignificant variations as compared to control. Thermal analysis means that eachAu0,Pt0, and Ag0is uniformly immobilized into pri stine denture and NPs werestable at the experimental temperature.12.4.2 FLEXURAL STRENGTHMechanical properties of denture base acrylic are important for the clinicalsituation. Though PMMA has become a common material for producing denturebase acrylic resin since its development, dentures are inherently prone to fracture,which may occur by accidental impact when the denture is outside the mouth, orwhile in function in the mouth due to flexural fatigue as the denture base under-goes repeated masticatory loading. It is important for PDNC to realize that theconversion degree relating to the amount of residual monomer as NPs added,additives may influence the value of sufficient flexural strength to resist fracture.Many reports have already shown that combining with various additives improvesthe properties of polymers with novel functions including fibers (Narva et al.,2005), whiskers (Niu et al., 2010), NPs (Hu et al., 2011; Saladino et al., 2012;Xu et al., 2008), etc. Casemiro et al. (2008)reported the addition of silver zinc-zeolite results in a significant decrease in the flexural and impact strengths ofdenture acrylic; this could increase the possibility of a fracture occurring insideor outside the oral cavity. Among the desirable properties of a denture materialis a possession of an adequate flexural strength, which indicates the resistanceto deformation or fracture of the bulk of the material under a flexural load(Archadian et al., 2000). To evaluate the flexural strength of PDNC, the speci-mens (25 mm 3 2mm3 2 mm, n 5 160) were fabricated according to ISO4049:2000(E). The load was applied perpendicular to the center of the specimenat a crosshead speed of 1 mm/min with a universal testing machine (Model 4200,Instron Inc., USA).Table 12.2 shows all of PDNC within 10200 ppm NPs loadedexhibiting no significant differences from control (P . 0.01) and no statisticallysignificant differences were observed among Au0,Pt0, and Ag0NPs (P . 0.01).However, PDNC with 400 ppm NPs doped showed significantly higher flexuralstrength values than 0200 ppm doses (P , 0.01). Loading inorganic NPs mightplay a role in reinforcement of mechanical denture base structure without negativeeffect to pristine PMMA. This is supported by other reports; the 5 wt% of Ag NPs32112.4 Physical Properties of PDNC 020406080100Weight (%)0 100 200 300 400 500 600Temperature (°C)0 100 200 300 400 500 600Temperature (°C)Control384.7 °CTGDSC102030405060Heat flow (mW)384.7 °CAu°Ag°ControlPt°(a)(b)FIGURE 12.6Comparative TG/DSC curves of control and PDNC groups loading 4.0% (400 ppm) NPsunder nitrogen at 10C/min. (a) Each PDNC sample showed nearly the same TG curve tothat of the control, with sharp inflections from 350 to 450C. (b) DSC curves alsoexpressed similar thermograms from 30 to 600C with the endothermic peak around384.7C along with control.322 CHAPTER 12 Antimicrobial denture nanocomposites incorporation within the acrylic denture base material can improve its viscoelasticproperties (Mahross and Baroudi, 2015) and nanosilver with 2% concentrations toPMMA can improve mean compressive strength significantly higher than that ofthe u nmodified without any adverse effects (Ghaffari et al., 2014).12.4.3 COLOR CHANGEColor stability is an important clinical behavior for denture base resin since itmay provide critical information on the serviceability of this material. Despiteseveral studies determining the efficacy of various antimicrobial composites (Ahnet al., 2009; Wady et al., 2012; Monteiro et al., 2012), there has been little infor-mation in the literature regarding the color stability influenced by NPs eventhough it is an important functional property of dental materia l. Fan et al. (2011)found the amber color of the light-cured dental resins became darker as the Agdose increased andChladek et al. (2011) also experienced that a color changewas significantly dose-dependent for nanosilver concentrations doped in denturesoft liner. Among the factors of discoloration, the oxidative reaction or plasmoneffect (Bohren and Huffman, 1983) of NPs may contribute to these complications.Generally, if ΔEis less than 1.0, chromatic value is deemed to be very slightand the clinically acceptable range is between 1.0 and 2.0 (Johnston and Kao,1989). However, if values were significantly greater than 2.69, which has beenconsidered the perceptibility threshold (Chang et al., 2009), it is no longer withinthe limits of clinical acceptability. Color was measured on the surface of a PDNCdisk sample (20.0 3 2.0 mm, n 5 80) at 24 h from the onset of the curing processand measured by colorimeter (Color-reader®, Minolta CR-10, Japan)in reflectance mode against the white background of an opacity card. The coloris expressed as L(lightness), a(red/green), and b(yellow/blue) values for mea-surement. The value of each PDNC was calculated with the CIE LAB scaleTable 12.2 Flexural Strength Test Values of the Acrylic Resins withIncorporation of the Different Types and Percentages of NPs (Values Given inMean 6 SD MPa) as Compared to Pristine PMMAFlexural Strength (MPa 6 SD)Dose (ppm) Au0-PDNC Pt0-PDNC Ag0-PDNC0 (control) 88.3 6 5.910 87.3 6 7.8 88.7 6 5.2 88.6 6 4.950 88.6 6 5.9 89.3 6 5.9 89.3 6 7.1100 90.1 6 4.6 90.3 6 7.4 89.9 6 6.3200 91.1 6 5.7 92.3 6 7.9 91.8 6 6.8400 98.9 6 3.9a101.1 6 2.8a100.3 6 3.4aaStatistically significant values (P , 0.01).32312.4 Physical Properties of PDNC recommended by Commission Internationale de l’Eclairage (Polyzois et al., 1997).Color change (ΔE) of PDNC was calculated using the followed relationship:ΔE5 ½ðΔLÞ21ðΔaÞ21ðΔbÞ21/2; where ΔL5 L12 L0; Δa5 a12 a0; Δb5 b12 b0(L1,a1,b1: NPs impregnated, L0,a0,b0: pristine).Table 12.3 shows the mean values of observed color differences after the additionof each NP (one-way ANOVA with Tukey’s HSD, P 5 0.01). When compared withcontrol, ΔEwere detected from 6.0423.47 in Au0,2.1719.82 in Pt0,and5.4832.45 in Ag0. The higher dose of NPs incorporated, the significantly greaterΔEthat were expressed (P , 0.01), except Au0between 100 and 400 ppm com-bined (P . 0.01). The highest color change occurred at Ag0-PDNC containing400 ppm combined (ΔE5 32.45 6 1.94) and the lowest color change was observedin Pt0containing 10 ppm (ΔE5 2.17 6 0.12). Pt0showed statistically less ΔEthan Au0and Ag0in every dose from the baseline of control (P , 0.01) (Figure 12.7).Except 10 ppm Pt0incorporated (ΔE5 2.17), all PDNC samples showed rapiddiscoloration and were found to be clinically unacceptable values. Pt0revealed signif-icantly less color change as compared to Au0and Ag0and that could be an advantageon selection of noble NPs as nanoingredients for denture acrylics in future studies.Although the addition of NPs to denture acrylic has an antimicrobialadvantage, it should be challenged for the cosmetic aspect of modified denturePMMA. Further studies are still needed to solve the color change after blendingor dispersion of NPs into the denture base.12.5 ANTIFUNGAL ASSAY12.5.1 ANTIFUNGAL EXPERIMENTVariable methods are available to assess the antifungal activity for dentalmaterials, it should be considered to reproduce the representative denture fungalTable 12.3 The Color Differences (ΔE) of Nanocomposite Specimens withControl (0 ppm) at the Time of 24 h Elapsed from the Onset of CuringProcessΔE(SD)Dose (ppm) Au0-PDNC Pt0-PDNC Ag0-PDNC10 6.04 (0.41)a2.17 (0.12),b5.48 (0.36)a50 15.13 (0.52)c8.19 (0.31)d15.22 (0.69)c100 20.52 (0.92)e11.03 (0.58)f19.81 (1.53)e200 22.56 (1.45)e16.43 (0.82)g27.18 (2.05)h400 23.47 (2.03)e19.82 (1.98)e32.45 (1.94)jClinical acceptable value under the criterion of ΔE(2.69).324 CHAPTER 12 Antimicrobial denture nanocomposites infection because denture stomatitis is typically associated with biofilm formation.Nikawa et al. (2003) have shown that the latent antifungal activity of soft acrylicresin was overpowered at 107cells/ml concentration and other studies have alsoreported that the diagnostic criteria for Candida-associated denture stomatitis wasin the concentration of 107cells/ml (Budtz-Jorgensen, 1981; Chandra et al.,2001). Chandra et al. (2001)reported that the adhesion time of small inoculum(80 μ l) for 90 min on the denture sample and incubation of 72 h were needed tomake a reproducible denture biofilm model for C. albicans. The surface coatingof specimen with synthetic saliva is conducted to mimic the denture-wearingsituation. The adhesion of C. albicans could be enhanced by a saliva-coatedsurface (Edgerton et al., 1993 ) or reduced in saliva-precoated samples becausesaliva coating diminishes the effect of surface roughness and the free energydifference between the materials (Radford et al., 1998; Waters et al., 1997).Though some denture stomatitis is commonly related with Sjo¨gren’s syndromepatients, where salivary flow is absent or minimal, most in vivo dentures wereengaged with saliva to fit the gingival mucosa. As for the amount of fungalsuspension to inoculate on tested samples, the oral microbes would appear to beFIGURE 12.7Photographs of color changings according to NP doses and control. When compared topristine, the colors of samples become darker as the concentration of NPs increased.Pt0-PDNC are observed to be lighter in color than those of Au0and Ag0.32512.5 Antifungal Assay in a stationary phase rather than in a growin g phase, because the nutrition islimited under the antibodies and the antimicrobial enzymes exist in the oralcavity. Baehni and Takeuchi (2003)demonstrated in vitro that microbes insuspension (planktonic phase) are sensitive to lower antiseptic doses than micro-organisms colonized at the surfaces and protected by a biofilm, thus the assaysthrough immersing samples in a large volume of microbial suspension could notreproduce in vivo dentures closely fitting the gingival mucosa.Based on the studies described above, fungal suspension-seeding (American TypeCulture Collection, 66027) was adjusted by an inoculum size of 1 3 107cells/ml, anadhesion time of 90 min with 100 μl inoculum, 72 h incubation, and saliva pre-coating were selected for the present antifungal assay for PDNC. In addition, theincubation medium supplemented with nutritional broth (1.5 ml) was added becauseextending the incubation time in PBS (phosphate buffer solution), which lacks nutri-ents, may influence the ability of C. albicans to adhere and its growth phase.Antifungal effect of PDNC was evaluated through FACS (fluorescence activated cellsorting) with bacterial viability and counting kit (Molecular Probes, OR, USA).Though no golden standard for the antimicrobial assessment of denture base materi-als has been recommended so far, FACS could have an advantage over traditionalcolony-forming unit (CFU) counting or zone of inhibition growth which offers noinformation about the presence of viable cells (Rodriguez and Thornton, 2008).Complemented microscopic evaluation of stained samples can detect both viable andkilled cells, thus it can be used for susceptibility testing of biofilms as for determin-ing the effectiveness of antimicrobial compounds used in the oral cavity. Prior tofungal seeding on PDNC, to confirm the initial sterility of samples, experimentalsamples were sterilized with ethylene oxide gas overnight then they were coated bysynthetic saliva for 1 h. Fungal seed suspensions (1.0 vol.%) with growth medium(1.5 ml) were inoculated on each specimen located in 12-well culture plates. Afterincubation for 72 h at 37C, samples were washed with PBS then the adherent fungiwere detached into PBS by sonication. Cells in PBS were stained with SYTO 9nucleic acid and propidium iodide for live and dead fungal staining. Stained cellswere incubated at room temperature protected from light, then analyzed by flowcytometry analysis. Live or dead fungal cells were assessed by a cytogram ofthe green fluorescence versus red fluorescence and for clear differentiation betweenviable and dead fungi, fluorescent-stained images were observed under fluorescentmicroscope (Olympus BX 51, Tokyo, Japan) (Figure 12.8).12.5.2 ANTIADHERENT EFFECT OF PDNCAntifungal effect s of PDNC were assessed by viable cell counting from retrievedbiofilm suspensions then calculated as the percentage out of the control (one-wayANOVA with Tukey’s HSD, P 5 0.01) as attached fungal cells on PDNC weremostly viable, while dead cells were detected less than 5.0% by fluorescent signalspecific to sta ining. PDNC samples did not express strong fungicidal action ratherthey exhibited the significant antiadherent effect to viable cel ls from 200 to326 CHAPTER 12 Antimicrobial denture nanocomposites 400 ppm NPs loaded whe n compared to control. PDNC above 200 ppm of NPsloaded significantly reduced viable fungal cell adhesion by the rate of 61.0 6 7%in Ag0, 58.6 6 9% in Pt0, and 56.6 6 8% in Au0, respectively. There were nostatistical differences between 200 and 400 ppm loaded and there were no com-parative predominances among Au0,Pt0, and Ag0groups in significant antifungalactivity (Figure 12.9).FIGURE 12.8Microscopic image of fluorescent antibody-stained bacteria for viable cell counting.Yellowish green spots (white arrows) on the merged image indicate double-stained deadcells (3500).FIGURE 12.9Antiadhesion effect (%) of Au0,Pt0, and Ag0-PDNC against C. albicans. As compared withcontrol (0 ppm), all PDNC groups above 200 ppm (2.0%) dose significantly reducedfungal adhesion (P , 0.01).Statistical difference from control (P , 0.01).32712.5 Antifungal Assay 12.5.3 POSSIBLE ANTIADHERENT MECHANISM OF PDNCThe antimicrobial mechanism of denture nanocomposites such as PDNC has notbeen fully determined yet, furthermore, as studies for antimicrobial denture PMMAcontaining NPs have exclusively focused on Ag, there are few data for Pt and Au todate to this author’s knowledge. It should be considered that the antimicrobial effectsor characters reported so far were highly variable literature to literature, even withincontrol groups. This variability could be explained by the differences in experimentalmethod, situation, and instruments, etc. Some researchers have announced nanosilvercomposites that release oxidized Ag ion to medium could cause strong antibacterialactivity (Kassaee et al., 2008; Kong and Jang, 2008) and the composites impregnatedby NPs could act as reservoirs of metallic ions to elute (Hetrick and Schoenfisch,2006). Acosta-Torres et al. (2012) reported PMMAsilver NP disks significantlyreduced by about 75% fungal adherence with facilitated silver ion elution, whichleads to higher antibacterial activity. However, as shown in PDNC elution results(Table 12.1), ion releasing was extremely handicapped owing to the structure of thehydrophobic polymer network whose water uptake may be insufficient to releaseions from NPs fixed in the solid PMMA resin bulk (Nam et al., 2012; Monteiro et al.,2012). In addition, tested denture material Vertex contains a crosslinking agent;ethylene glycol dimethacryl [2-(2-methyl-acryloyloxy) ethyl 2-methyl-acrylate)]ionic movements could be strongly restricted out of crosslinked polymer structure.The possibility of direct contact between the microbes and silver NPs on thesurface of nanocomposites causing a bioc idal act was reported (Ahn et al., 2009),butWady et al. (2012) discovered a denture base acrylic resin containing silverNPs that produced no effect on C. albicans adhesion and biofilm formation.Present antifungal effect of PDNC could be assumed as the “noncontact”interaction between ion and fungi by altered physicochemical interactions or amodified interfacial polarity on PDNC surface. Similarly,Ebi et al. (2001) specu-lated that by certain interaction, antibacterial monomers captured on the resinsurface play a role in inhibiting bacteria under extremely low eluti on. Severalphysicochemical factors (Park et al., 2003; Puri et al., 2008; Campo ccia et al.,2013), such as electrostatic charge, hydrophobicity, or surface free energy, couldinfluence the kinetics of fungal adhesion or behavior on the surface of PDNCsamples. Solid plastic surfaces general ly possess various degrees of negative netsurface charges, similarly, all living bacterial cells as well as yeasts possess a netnegative surface charge (Klotz et al., 1985). Kiremitci-Gumustederelioglu andPesmen (1996)reported bacterial adhesion could be decreased on negativelycharged PMMA/AA (acrylic acid), while increased on positively charged PMMA/DMAEMA (dimethylamino ethyl methacrylate). An antiadherent effect may becorrelated with the complexity of the nanocomposite surface as well as the changein their chemical compositions and synthesis. In PDNC, PVP was combined as acoating and stabilizing ingredient for successful NP dispersion to regulate particlegrowth and prevent their aggregation (Gorup et al., 2011; Monteiro et al., 2012).Some studies suggested that keeping PVP NPs awa y from the biologic surfaces328 CHAPTER 12 Antimicrobial denture nanocomposites produces a low antimicrobial activity (El Badawy et al., 2011; Campoccia et al.,2013; Silva et al., 2013) because fixed PVP NPs play a key role in dominant repul-sive, lower attraction forces. Hydrophobic interaction could also influence the fungaladherence to PDNC surfaces because the electrical forces are minor to the hydropho-bic forces since microbial adherence elicits a considerable amount even in thepresence of repulsive force (Liu and Hurt, 2010; Klotz et al., 1985). Hydrophobicsurfaces also influence bacterial adherence processes by the changed interfacial freeenergy (Liu and Hurt, 2010) and adherence could be developed whenever the sum ofinterfacial tensions is reduced between cells and solid surfaces (Klotz et al., 1985).FIGURE 12.10Schematic representation of theoretical concepts for antiadhesion (repulsive) effect againstfungal cells on PDNC surface (PVP-NPs/PMMA composites) at interfacial gap. Themechanism of action illustrates that incorporating NPs into pristine PMMA denture acrylicmay alter the physicochemical interactions or modify the polarity of the PDNC surface to bemore negatively charged. PDNCs induce the dominant repulsive action against negativelycharged fungal cell wall rather than attractive force. This process is thought to beresponsible for the antiadhesion effect of nanocomposites with nonreleasing ions.32912.5 Antifungal Assay It could be speculated that the antiadherent effect of PDNC could be correlatedwith the complexity of nanocomposite surface as well as the change in its chemicalcomposition or synthetic process. Incorporating above 2.0 wt% (200 ppm) NPs intopristine PMMA may change the physicochemical interactions or modify the polarityof PDNC surface as more negatively charged and that induce the dominant repulsiveforce, that is, antiadherent power, rather than attractive force, that is, sterilizing oneagainst the negatively charged fungal cell wall (Figure 12.10). Regarding the possi-ble toxicities to normal flora and oral epithelial tissues, denture materials should becharacterized by antiadherent surfaces whose primary scope is not the radical actionthat might cause undesirable interactions in oral surroundings but the moderate andpreventive antimicrobial composites. Nevertheless, the present results cannot jumpto this conclusion, because the experiment was an in vitro pattern, short-termanalysis with a sole fungal strain selection. Further studies are still needed, includingunknown interfacial factors, in vivo tests, tests of other strains, and long-termobservations for PDNC to clarify the antifungal mechanism and to be used clinically.12.6 CONCLUSIONSIt is well known that fungal adherence to inert polymers, such as denture acrylicresin, is regarded as an essential prerequisite for colonization. In this chapter, thedevelopment of PDNC, novel nanocomposites for antifungal PMMA-denture basecontaining Au, Pt, and Ag NPs were discussed. 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