2.1

Synthesis of MSNs and loading of AgSD

First, MSNs were prepared by a modified Stöber method . Briefly, 5.5 mmol of cetyltrimethylammonium bromide (CTAB, Sigma–Aldrich) was dissolved in 960 ml of dH ₂ O and 7 ml of 2 M NaOH at 80 °C. Next, 67 mmol of tetraethyorthosilicate (TEOS, Sigma–Aldrich) was added quickly, and the mixture was vigorously stirred for 2 h at 80 °C. The as-prepared MSNs were separated from the white solution by centrifugation at 8000 rpm for 3 min and were then washed 3 times with water/ethanol solutions using re-dispersion/centrifugation cycles and finally dried at 70 °C overnight. To remove the surfactant CTAB, the as-prepared MSNs were heat treated at 550 °C for 5 h under air flow.

The Ag-loaded MSNs (Ag-MSNs) were obtained by loading AgSD into the MSNs. MSNs were dispersed at a concentration of 1 mg/ml in a silver-sulfadiazine solution (1 mg/ml AgSD in acidified acetone–ethanol mixture (1:1)) and were then stirred for 6 h. Ag-MSNs were separated from the AgSD solution similarly to the procedure mentioned above and were then washed several times with water/ethanol and finally dried at 50 °C overnight. The particle size, morphology and elemental composition of the samples were investigated via transmission electron microscopy (TEM; 7100, JEOL, Peabody, MA, USA) with energy-dispersive spectroscopy (EDS, Oxford, Abingdon, UK). The pore volume and pore size were analyzed from N ₂ adsorption–desorption measurements at −196.15 °C using an automated analyzer (Quadrasorb SI, Quantachrome Instruments Ltd., Boynton Beach, FL, USA) on the basis of the non-local density functional theory method. The specific surface area was calculated according to the Brunauer–Emmett–Teller (BET) method. The amounts of AgSD loaded into the MSNs were measured using a thermogravimetric analyzer (Thermo Plus Evo II, Rigaku, Tokyo, Japan) with a heating rate of 5 °C min ⁻¹ up to 600 °C under nitrogen flow and UV/vis spectrometry with a calibration curve at the maximum absorbance of 297 nm . To confirm the accuracy of the UV/vis spectrometry assay compared to that of thermogravimetric analysis for detecting loaded AgSD amounts, further experiments using the UV/vis spectrometry assay were conducted.

2.2

Incorporation of MSNs or Ag-MSNs into PMMA

Commercially available, chemically activated PMMA products (Orthocryl resin, Dentaurum, Ispringen, Germany; lot numbers: 452986B for powder and 450128 for liquid) were chosen both to avoid causing thermal damage to the loaded silver-sulfadiazine during heat polymerization and for the ease of fabricating these specimens. Nanoparticles were prepared in quantities of 0.5, 1.0, 2.5, and 5.0% in weight % relative to PMMA powder. After the nanoparticles were dispersed in MMA liquid under sonication (Wise clean, Daehan Science, Seoul, Korea) at the above determined conditions, PMMA powder was mixed with the liquid at a ratio of 1.2:1, powder (g) to liquid (ml). After polymerization at 40 °C and 2.2 bar for 30 min (MultiCure, Vertex, ZEIST, Netherlands), bar-shaped specimens (1.4 × 3.0 × 18 mm) were prepared for mechanical testing by cutting the cured blocks (4 × 15 × 20 mm) and then polishing them with up to 2400 grit SiC paper. Disk specimens (ø = 11.5 mm and d = 1.5 mm) were prepared for other investigations and polished with up to 800 grit SiC paper. All specimens were sterilized with ethylene oxide (EO) gas using a Person EO50 system (Person medical, Gunpo-si, Gyeonggi-do, Korea).

2.3

Characterization of PMMA incorporating Ag-MSNs

The surface characteristics and composition of PMMA incorporating Ag-MSNs were characterized by scanning electron microscopy (SEM, Sigma 500, ZEISS, Jena, Germany) at 3 kV and EDS (Ultradry, Thermo fisher, Waltham, MA, USA) at 15 kV. The release of silver ions from a disk specimen immersed in yeast-malt (YM) broth (Difco, BD, Franklin Lakes, NJ, USA) (2 ml) was analyzed at predetermined times (1, 3, and 24 h; 2, 4, 7, 14, and 28 days) using inductively coupled plasma atomic emission spectrometry (ICP-AES, Optima 4300 DV, PerkinElmer, n = 3) after changing the medium. Surface energy (Phoenix 300, SEO, Suwonsi, Gyunggido, Korea) was determined according to the Owens–Wendt method using the contact angles from water and ethylene glycol (both 5 μl, n = 5; Phoenix 300, SEO, Suwonsi, Gyunggido, Korea), and roughness was measured (Ra, SJ-400, Mitutoyo, Japan) at a speed of 0.5 mm/s with 4.0-mm scans (n = 5). These measurement procedures are described in detail elsewhere .

2.4

Mechanical properties

The prepared bar specimens were positioned for 3 point flexural tests on an Instron 5966 machine (MA, USA) with a 500-N load cell at a span length of 14 mm. The flexural strength and elastic modulus were determined at a crosshead speed of 1.0 mm/min (n = 10) . The Vickers hardness (HM-221, Mitutoyo, Tokyo, Japan) was measured with 300 gf (2.94 N) for 30 s in five different spots on each specimen, and these values were averaged (n = 5).

2.5

Microbial anti-adhesive study

C. albicans (ATCC 10231, USA) and S. oralis (ATCC 9811) were grown according to the manufacturer’s protocol; then, 100 μl of 6 × 10 ⁷ /ml of microbes in the log phase of growth was seeded on the surface of a disk specimen. After 1 h of culture at 37 °C with 5% of CO ₂ to allow attachment to the specimen, non-adherent microbes were removed by washing the specimen three times with PBS. After incubating C. albicans and S. oralis in 12-well plates for 12 or 3 h with 2 ml of the proper culture medium per well, 10% of PrestoBlue (Molecular Probes, USA) was added to each well, and the plates were incubated for an additional 1.5 h. Liquid (100 μl) was transferred to a 96-well plate, and the absorbance was read at 570 and 600 nm using a microplate reader (BioTek, Winooski, VT, USA). The absorbance values at 570 nm were normalized to those at 600 nm for each well and were used to quantify the attached microbes. To confirm the correlation between the normalized absorbance determined by the PrestoBlue assay and the microbe numbers, the serial colony forming counting unit (CFU as log unit) method was used to construct a trend line, which showed a linear correlation (R ² = 0.98) . To confirm the PrestoBlue results from the 3-h microbial cultures, SEM (Sigma 500) was taken for adherent C. albicans on PMMA as representative between the two microbial species after fixing with 4% paraformaldehyde for 10 min. In addition, to check the live and dead states of the adherent C. albicans, a live and dead yeast viability kit (ThermoFisher scientific, Waltham, MA, USA) was performed according to the manufacturer’s protocol, and images were visualized by confocal microscopy (LSM 510, Zeiss, Switzerland). Live and dead cells were artificially visualized in green and red, respectively. Representative images or means (n = 3) with standard deviations (SDs) were recorded after independent experiments were performed in triplicate.

2.6

Long-term microbial anti-adhesive study

Disk specimens were aged in a humidified incubator at 37 °C with 5% CO ₂ for up to 28 days in YM broth. After aging, the specimens were gathered at the determined time points described for the release test, washed with PBS (Welgene) three times, and dried in a vacuum desiccator; then, EO gas sterilization was performed. The microbial anti-adhesive test was performed with C. albicans under the conditions described above (n = 5). Representative means and SDs were recorded after independent experiments were performed in triplicate.

2.7

Recharging ability

To recharge the resin (aged for 28 days) with AgSD, each disk specimen was incubated for 1 h in a AgSD-suspended solution prepared by ultrasound dispersion of 10 mg of AgSD powder per 1 ml of ethanol (Sigma, St. Louis, MO, USA). Different than the AgSD in acetone–ethanol mixture used for initial loading, recharging was performed with ethanol to avoid high chemical degradation of PMMA in acetone. The recharged specimens were washed with water/ethanol to remove any weakly adsorbed AgSD and were finally dried at 37 °C in a vacuum chamber before the release and long-term microbial anti-adhesive studies (∼28 days) were performed. The amount of recharged AgSD was investigated using UV/vis spectrometry with a calibration curve at the maximum absorbance of 297 nm . The concentration of silver ions released from the recharged specimens and the long-term microbial anti-adhesive activity were analyzed using ICP-AES and an attachment study, as previously described.

2.8

Cytotoxicity test

Immortalized human oral keratinocytes (IHOKs) were chosen as representative human keratinocytes in this study . After cells were seeded (1 × 10 ⁴ cells/96-well plate) and incubated at 37 °C in a humidified atmosphere of 5% CO ₂ and 95% air, one part of extract from the specimens was added to one part of 2X DMEM/F-12(3:1) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). After 24 h of incubation, a water-soluble tetrazolium salt (WST) assay was performed according to previously described methods using a wavelength of 450 nm .

2.9

Statistical analysis

The data are expressed as the mean ± SD of at least three independent experiments. Statistical significance was evaluated by a one-way analysis of variance with a Tukey post hoc test using SPSS (Version 21.0; SPSS, Chicago, IL). A value of P < 0.05 was considered statistically significant.

2.1

Synthesis of MSNs and loading of AgSD

First, MSNs were prepared by a modified Stöber method . Briefly, 5.5 mmol of cetyltrimethylammonium bromide (CTAB, Sigma–Aldrich) was dissolved in 960 ml of dH ₂ O and 7 ml of 2 M NaOH at 80 °C. Next, 67 mmol of tetraethyorthosilicate (TEOS, Sigma–Aldrich) was added quickly, and the mixture was vigorously stirred for 2 h at 80 °C. The as-prepared MSNs were separated from the white solution by centrifugation at 8000 rpm for 3 min and were then washed 3 times with water/ethanol solutions using re-dispersion/centrifugation cycles and finally dried at 70 °C overnight. To remove the surfactant CTAB, the as-prepared MSNs were heat treated at 550 °C for 5 h under air flow.

The Ag-loaded MSNs (Ag-MSNs) were obtained by loading AgSD into the MSNs. MSNs were dispersed at a concentration of 1 mg/ml in a silver-sulfadiazine solution (1 mg/ml AgSD in acidified acetone–ethanol mixture (1:1)) and were then stirred for 6 h. Ag-MSNs were separated from the AgSD solution similarly to the procedure mentioned above and were then washed several times with water/ethanol and finally dried at 50 °C overnight. The particle size, morphology and elemental composition of the samples were investigated via transmission electron microscopy (TEM; 7100, JEOL, Peabody, MA, USA) with energy-dispersive spectroscopy (EDS, Oxford, Abingdon, UK). The pore volume and pore size were analyzed from N ₂ adsorption–desorption measurements at −196.15 °C using an automated analyzer (Quadrasorb SI, Quantachrome Instruments Ltd., Boynton Beach, FL, USA) on the basis of the non-local density functional theory method. The specific surface area was calculated according to the Brunauer–Emmett–Teller (BET) method. The amounts of AgSD loaded into the MSNs were measured using a thermogravimetric analyzer (Thermo Plus Evo II, Rigaku, Tokyo, Japan) with a heating rate of 5 °C min ⁻¹ up to 600 °C under nitrogen flow and UV/vis spectrometry with a calibration curve at the maximum absorbance of 297 nm . To confirm the accuracy of the UV/vis spectrometry assay compared to that of thermogravimetric analysis for detecting loaded AgSD amounts, further experiments using the UV/vis spectrometry assay were conducted.

2.2

Incorporation of MSNs or Ag-MSNs into PMMA

Commercially available, chemically activated PMMA products (Orthocryl resin, Dentaurum, Ispringen, Germany; lot numbers: 452986B for powder and 450128 for liquid) were chosen both to avoid causing thermal damage to the loaded silver-sulfadiazine during heat polymerization and for the ease of fabricating these specimens. Nanoparticles were prepared in quantities of 0.5, 1.0, 2.5, and 5.0% in weight % relative to PMMA powder. After the nanoparticles were dispersed in MMA liquid under sonication (Wise clean, Daehan Science, Seoul, Korea) at the above determined conditions, PMMA powder was mixed with the liquid at a ratio of 1.2:1, powder (g) to liquid (ml). After polymerization at 40 °C and 2.2 bar for 30 min (MultiCure, Vertex, ZEIST, Netherlands), bar-shaped specimens (1.4 × 3.0 × 18 mm) were prepared for mechanical testing by cutting the cured blocks (4 × 15 × 20 mm) and then polishing them with up to 2400 grit SiC paper. Disk specimens (ø = 11.5 mm and d = 1.5 mm) were prepared for other investigations and polished with up to 800 grit SiC paper. All specimens were sterilized with ethylene oxide (EO) gas using a Person EO50 system (Person medical, Gunpo-si, Gyeonggi-do, Korea).

2.3

Characterization of PMMA incorporating Ag-MSNs

The surface characteristics and composition of PMMA incorporating Ag-MSNs were characterized by scanning electron microscopy (SEM, Sigma 500, ZEISS, Jena, Germany) at 3 kV and EDS (Ultradry, Thermo fisher, Waltham, MA, USA) at 15 kV. The release of silver ions from a disk specimen immersed in yeast-malt (YM) broth (Difco, BD, Franklin Lakes, NJ, USA) (2 ml) was analyzed at predetermined times (1, 3, and 24 h; 2, 4, 7, 14, and 28 days) using inductively coupled plasma atomic emission spectrometry (ICP-AES, Optima 4300 DV, PerkinElmer, n = 3) after changing the medium. Surface energy (Phoenix 300, SEO, Suwonsi, Gyunggido, Korea) was determined according to the Owens–Wendt method using the contact angles from water and ethylene glycol (both 5 μl, n = 5; Phoenix 300, SEO, Suwonsi, Gyunggido, Korea), and roughness was measured (Ra, SJ-400, Mitutoyo, Japan) at a speed of 0.5 mm/s with 4.0-mm scans (n = 5). These measurement procedures are described in detail elsewhere .

2.4

Mechanical properties

The prepared bar specimens were positioned for 3 point flexural tests on an Instron 5966 machine (MA, USA) with a 500-N load cell at a span length of 14 mm. The flexural strength and elastic modulus were determined at a crosshead speed of 1.0 mm/min (n = 10) . The Vickers hardness (HM-221, Mitutoyo, Tokyo, Japan) was measured with 300 gf (2.94 N) for 30 s in five different spots on each specimen, and these values were averaged (n = 5).

2.5

Microbial anti-adhesive study

C. albicans (ATCC 10231, USA) and S. oralis (ATCC 9811) were grown according to the manufacturer’s protocol; then, 100 μl of 6 × 10 ⁷ /ml of microbes in the log phase of growth was seeded on the surface of a disk specimen. After 1 h of culture at 37 °C with 5% of CO ₂ to allow attachment to the specimen, non-adherent microbes were removed by washing the specimen three times with PBS. After incubating C. albicans and S. oralis in 12-well plates for 12 or 3 h with 2 ml of the proper culture medium per well, 10% of PrestoBlue (Molecular Probes, USA) was added to each well, and the plates were incubated for an additional 1.5 h. Liquid (100 μl) was transferred to a 96-well plate, and the absorbance was read at 570 and 600 nm using a microplate reader (BioTek, Winooski, VT, USA). The absorbance values at 570 nm were normalized to those at 600 nm for each well and were used to quantify the attached microbes. To confirm the correlation between the normalized absorbance determined by the PrestoBlue assay and the microbe numbers, the serial colony forming counting unit (CFU as log unit) method was used to construct a trend line, which showed a linear correlation (R ² = 0.98) . To confirm the PrestoBlue results from the 3-h microbial cultures, SEM (Sigma 500) was taken for adherent C. albicans on PMMA as representative between the two microbial species after fixing with 4% paraformaldehyde for 10 min. In addition, to check the live and dead states of the adherent C. albicans, a live and dead yeast viability kit (ThermoFisher scientific, Waltham, MA, USA) was performed according to the manufacturer’s protocol, and images were visualized by confocal microscopy (LSM 510, Zeiss, Switzerland). Live and dead cells were artificially visualized in green and red, respectively. Representative images or means (n = 3) with standard deviations (SDs) were recorded after independent experiments were performed in triplicate.

2.6

Long-term microbial anti-adhesive study

Disk specimens were aged in a humidified incubator at 37 °C with 5% CO ₂ for up to 28 days in YM broth. After aging, the specimens were gathered at the determined time points described for the release test, washed with PBS (Welgene) three times, and dried in a vacuum desiccator; then, EO gas sterilization was performed. The microbial anti-adhesive test was performed with C. albicans under the conditions described above (n = 5). Representative means and SDs were recorded after independent experiments were performed in triplicate.

2.7

Recharging ability

To recharge the resin (aged for 28 days) with AgSD, each disk specimen was incubated for 1 h in a AgSD-suspended solution prepared by ultrasound dispersion of 10 mg of AgSD powder per 1 ml of ethanol (Sigma, St. Louis, MO, USA). Different than the AgSD in acetone–ethanol mixture used for initial loading, recharging was performed with ethanol to avoid high chemical degradation of PMMA in acetone. The recharged specimens were washed with water/ethanol to remove any weakly adsorbed AgSD and were finally dried at 37 °C in a vacuum chamber before the release and long-term microbial anti-adhesive studies (∼28 days) were performed. The amount of recharged AgSD was investigated using UV/vis spectrometry with a calibration curve at the maximum absorbance of 297 nm . The concentration of silver ions released from the recharged specimens and the long-term microbial anti-adhesive activity were analyzed using ICP-AES and an attachment study, as previously described.

2.8

Cytotoxicity test

Immortalized human oral keratinocytes (IHOKs) were chosen as representative human keratinocytes in this study . After cells were seeded (1 × 10 ⁴ cells/96-well plate) and incubated at 37 °C in a humidified atmosphere of 5% CO ₂ and 95% air, one part of extract from the specimens was added to one part of 2X DMEM/F-12(3:1) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). After 24 h of incubation, a water-soluble tetrazolium salt (WST) assay was performed according to previously described methods using a wavelength of 450 nm .

2.9

Statistical analysis

The data are expressed as the mean ± SD of at least three independent experiments. Statistical significance was evaluated by a one-way analysis of variance with a Tukey post hoc test using SPSS (Version 21.0; SPSS, Chicago, IL). A value of P < 0.05 was considered statistically significant.