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Tissue conditioner (TC) has widely been used to treat abused mucosal tissues underlying ill-fitting acrylic dentures as temporary expedients. TC is one of short-term used denture soft liners approximately for 2 weeks and formed in situ from the mixture of polyethylmethacrylate (PEMA) and ester-based liquid plasticizer in ethyl alcohol solution without an acrylic monomer. However, loss of plasticizer can lead to gradual hardening with the roughened surface with time-elapsed after the onset of curing. Therefore, there can be major complications due to subsequent microbial colonization or inevitable biofilm formation on TC surface (1). Colonization of microorganisms as streptococci, candida or staphylococci has been reported in patients wearing removal prostheses. Such pathogens are capable of initiating infectious diseases as denture stomatitis, or even pharyngeal and respiratory infections particularly in elderly and medically compromised individuals (2). Chemical or mechanical cleansing could cause physical damages to the liners (3) and a combination of antiseptic drugs into TC was associated with problems such as; the short-term durations, harmful reactions in the elderly patients, drug resistance and costs (4). With the advancement in nanocomposite technology, numerous trials have been conducted by blending silver nanoparticles (AgNP) into PEMA to assign an intrinsic antimicrobial function (5-7). Though AgNP are as considered a promising alternative to antibiotics for multidrug-resistant pathogens, their toxicology remain largely unknown or controversial (8, 9) and metallic nanoparticles such as AgNP are technically difficult to disperse, as nanosized particles tend to aggregate. Recently, AgNP-doped chitosan, a polysaccharide biopolymer, was introduced to exert little cytotoxicity with slow silver release from the matrix (10, 11). Several pieces of research have focused on the synergistic effect between silver ions and chitosan; silver ions improve antibacterial effect and chitosan stabilizes AgNP or prevents their agglomerations (12, 13). The unique catalytic and surface characteristics of chitosan play a pivotal role in enhancing the antibacterial effect of silver (14, 15) and compared to uncoated AgNP, the nanosilver-chitosan coupling is known to be less toxic to gingival fibroblasts (16). The development of intrinsic antimicrobial denture soft liner is mandatory to prevent soft liner-related inflammations. Present study demonstrated the synthesis of silver-chitosan nanocomplex and antibacterial effect of modified PEMA liner incorporated with Ag-chi. In addition, biocompatibility and mechanical validities were characterized for a challenge to clinical use.

Stable formation of Ag-chi nanocomplex was observed by a characteristic plasmon band of AgNP indicated at 412 nm wavelength in UV–vis absorption spectra (Figure 2). Compared to control, the modified TC with 5.0% and 7.0% Ag-chi significantly reduced bacterial adhesion by 59.1 (±13.7)% and 66.1 (±9.8)% in S. mutans, and by 52.6 (±9.1)% and 67.2 (±6.7)% in S. sobrinus respectively after 24 hour incubation (p<0.01) (Figure 3). There was no statistical difference in antibacterial effect between 5.0% and 7.0% groups and no significant bacterial susceptibility between the two streptococci was observed (p>0.01). MTS result showed none of Ag-chi-TC induced cytotoxicity to HGF-1 (>60% of cell viability), and 5.0% and 7.0% groups exhibited the statistically lower cell viability from negative control in the two incubation periods (Figure 4). The extent of leaching of silver ion ranged 0.009 to 0.051 ppm with a dose-dependent of Ag-chi amounts and gradual decrease of silver ion were observed in all of groups as a function of immersion times from 24 hours to 14 days in water (Figure 5). SEM topography of Ag-chi-TC exhibited similar surface texture with some enlarged blisters or increased porosities as compared to control (Figure 6). Ag-chi did not affect the UTS of pristine TC regardless of its blending doses and storage intervals (Table 1), (p>0.01).

Figure 2. 
UV-vis spectra of chitosan with and without AgNP. The peak at 298 nm indicates nitrogen transition of noncovalent electron pair in O₂ of chitosan, the peak at 412 nm indicates the surface plasmon band of AgNP.

Figure 3. 
Anti-adherent effect of Ag-chi-TC against two streptococci after 24-hour incubation. As compared to control, 5.0 and 7.0% groups significantly reduced bacterial adhesion (p<0.01). Different letter indicates a statistical difference (p<0.01).

Figure 4. 
Cell viability (MTS assay) of HGF-1 cells cultured with extracts from of Ag-chi-TC for 24 and 72 hour. HGF-1 viabilities above 60% were rated as non-cytotoxic based on the activity of control negative containing cell culture medium only. Different letter indicates a statistical difference (p<0.01).

Figure 5. 
Average concentration of released silver ion from (A) 7.0, (B) 5.0, (C) 3.0 and (D) 1.0% of Ag-chi-TC as a function of immersion time elapsed in water from 24 hours to 14 days.

Figure 6. 
SEM topography (×100) on 7.0% Ag-chi-TC (A) and pristine TC (B). Similar surface texture is seen in gross, some enlarged blisters may be related to Ag-chi additives.

The stable molecular structure of chitosan is presented in Fgure 7, and the lone-pair electrons in chitosan polysaccharide are known to be easily bound to a metal surface by nitrogen and oxygen atom. The aqueous chitosan in this study was protonated with positive charges and chelated onto the surfaces of AgNP due to presence of active amino (-NH₂) and hydroxyl (-OH) functional groups. The couplings may lead to the stabilization of the nanoparticles to be stabilized or the protection of nanoparticles from their aggregations (18). UV-Vis spectrum of synthesized Ag-chi is presented in Figure 2, the peak at 298 nm is nitrogen transition of noncovalent electron pair in O₂ of chitosan and the peak at 412 nm is a typical plasmon band of spherical AgNP, thereby implying a stable conversion of AgNO₃ to AgNP synthesized by in situ reduction with chitosan. Dental composites or PMMA containing nanosilver could act a role of the reservoir for silver ions and demonstrate a strong bactericidal activity with active elution of oxidized silver ion into the medium (19, 20). Within 24-hours of incubation, Ag-chi-TC did not exhibit any biocidal action rather provided a significant anti-adherent effect at above 5.0% Ag-chi compared to control (Figure 3). Ag-chi-TC showed evident silver ion release in water as a function of immersion in a dose-dependent manner to the concentration of additives with the maximal 0.051 ppm in 7.0%. Eluted values were proportionally decreased overtime and restricted ion elution were related to plasticizers in Coe soft, usually leached out in the saliva over a period of time and a hydrophobic character of PEMA. Reductions in softness of this material may entrap silver ion due to its insufficient water uptake for ion release from PEMA bulk. Unlike the elution test for Ag-chi-TC immersed in 50 cc of water, practical leaching within the oral cavity may be restricted due to lower saliva flow (21, 22). In the present antimicrobial assay, a small volume of broth (1 mL) and bacterial suspension (100 μL) were inoculated on specimens with synthetic saliva-coated before incubation to mimic a clinical TC use. Denture stomatitis is commonly related to the Sjögren’s syndrome where the salivary flow is absent or minimal and as most of in vivo dentures are closely engaged to fit the gingival mucosa. Generally, microbes in suspension (planktonic phase) are sensitive to lower antiseptic doses than colonized by a biofilm (23), thus, it is speculated that the anti-adherent effect was probably due to the direct contact (24) between streptococci and the surface of Ag-chi-TC. In present study, chitosan was utilized to control AgNP agglomeration and even distribution of agents on the specific surface area is critical to guarantee a stable role of contact inhibitor. Biologic and toxicological aspects of denture materials are important in relation to their clinical usage along with antimicrobial effect. A polysaccharide biopolymer chitosan was doped to silver to exert little cytotoxicity with slow silver release from the matrix (10, 11); however, minute silver leaching may cause adverse reactions such as allergies and local chemical irritation on oral mucosa. In MTS assay, control negative (culture medium only) group was designated to exclude the influence of plasticizers in the structure of TC. The significantly lower cell viabilities at 5.0 and 7.0% groups from control may be related to influence of silver, however, there was little influence to HGF-1 proliferation in maximal 0.051 ppm silver ion eluted. Other studies similarly reported that addition of 1.0% of AgNP to MTA (mineral trioxide aggregate) did not alter its biocompatibility in an animal study (25) and fibrin sponge dispersed by 47 ppm, 23 ppm of AgNP was non-toxic response to tissue, especially under low concentration (26).

Figure 7. 
The most stable molecular structure of chitosan calculated by HyperChem PM3 method (HyperChem Professional 8.0, Hypercube Inc., Gainsville, FL, USA).

Ag-chi particles, an extrinsic element, could act as heterogeneous nuclei or impurities and this might influence or jeopardize the PEMA matrix, thereby compromising the functionality of pristine TC. UTS provides information on the ultimate strength of rubber in tension, which is one of the fundamental properties of the elastomer. In all the tested samples, UTS were significantly decreased at 7-day storage due to the loss of plasticizer. As compared to control, UTS values were not influenced by Ag-chi incorporation at two storage periods (Table 1), though some enlarged blisters or porosities are detected in Ag-chi 7.0% (Figure 6). Present results corroborate with other studies for the incorporation of some functional agents into PEMA; the addition of nystatin into Dura conditioner^Ⓡ at concentrations below 1,000,000 units did not affect the tensile strength (27) and 10% silver-zeolite incorporation did not alter the dynamic viscoelastic properties of Shofu tissue conditioner^Ⓡ (28).

Within the limitations of this study, it is proposed that modified TC loaded Ag-chi can be a candidate as a denture biomaterial with mechanical validity. Nevertheless, evident conclusions on the antimicrobial mechanism of modified TC cannot be achieved because there is still no consensus in the literature for bacterial inhibition in silver-containing compounds. In addition, to guarantee the stable role of contact inhibitor, the even distribution of agents on the specific surface area should be emphasized. Herein, aqueous chitosan was utilized to control AgNP agglomeration and blending of Ag-chi to TC was performed by powder to powder protocol. A futuristic trial on the combination of Ag-chi into TC monomer is considered to an alternative protocol for improved its dispersion as well as homogenized distribution. Further studies including in vivo, multi-brand, multi-strain, and multifactor assays are also necessitated for clinical applications.

Damaged tissue conditioners can induce microorganism development that can threaten the health of the dentures wearer. Tissue conditioner with 5.0% silver-chitosan nanocomplex is proposed as an appropriate carrier for the prevention of stomatitis and this composition did not influence the consistency of pristine PEMA. Based on the outcomes, modified tissue conditioner loading nanosilver-chitosan is suggested as a new denture biomaterial.