The influences of N-acetyl cysteine (NAC) on the cytotoxicity and mechanical properties of Poly-methylmethacrylate (PMMA)-based dental resin

Resin preparation

Self-polymerizing PMMA resin (Unifast Trad, GC, Tokyo, Japan) was prepared by mixing the powder and liquid components for 30 s in accordance with the manufacturer’s recommendations (powder/liquid ratio of 1.0 g/0.5 mL) in a 12-well culture plate. NAC (Sigma; St Louis, MO, USA) was prepared as 1.0 mol/L stock solution in HEPES buffer (Sigma; St Louis, MO, USA), and the pH was adjusted to 7.2 as previously reported (Yamada et al., 2008). To prepare NAC-loaded PMMA resin (0.15, 0.3, 0.6, 0.9 wt%), 13.8, 27.6, 55.2 or 82.8 µL NAC solution was added to the liquid MMA to a final volume of 0.5 mL, and then this NAC-incorporating liquid was mixed with 1.0 g powder. After 30 min of setting, the polymerized resin block was rinsed with ddH₂O once.

Preparation of the extract

The extract of the polymerized resin was prepared by soaking PMMA resin specimens with or without NAC in alpha-modified Eagle’s medium (α-MEM; Gibco BRL Division of Invitrogen, Gaithersburg, Maryland, USA) without serum in the 12-well culture plate at 37 °C in a humid atmosphere of 5% CO₂ for 24 h. According to ISO 10993-12:2007, the ratio between the surface of the sample and the volume of the added medium was 1.25 cm²/mL. The eluent was filtered for sterilization and 10% fetal bovine serum (Gibco, Gaithersburg, Maryland, USA) was added. The extract was stored at 4 °C before use.

Quantification of released NAC with high performance liquid chromatography (HPLC)

HPLC analysis was performed to determine the concentration of released NAC from the experimental PMMA resin containing 0.9 wt% of NAC. The extract was prepared as previously described. HPLC analysis (Model Shimadzu LC-2010A; Shimadzu Corporation, Kyoto, Japan) was performed at 220 nm on an InertSustain C18 column (5 µm, 4.6 mm × 250 mm; GL Sciences, Shinjuku-ku, Tokyo, Japan). The column was maintained at 50 °C and the samples were eluted for 5 min. The mobile phase, at a flow rate of 0.7 mL/min, consisted of methanol and 0.2% phosphoric acid with 100 mM sodium perchlorate. The volume of sample injected was 20 µL. Identification of the analyte, NAC, was made based on the retention time of the NAC peaks registered for the standard solutions. Standard curve was drawn using NAC solution with various concentrations (1, 5, 20, 40, 60, 80 and 100 µg/mL). After the linearity of the standard curve was confirmed by linear regression analysis, the concentration of released NAC from the experimental PMMA resin was calculated using the standard curve. The experiments were repeated for three times, and the mean value was determined as the concentration of released NAC.

Cell cultures

Human dental pulp cells (HDPCs) were isolated from dental pulp tissue of non-carious third molars extracted from young healthy patients (18–25 years old) according to a protocol verbally approved by the Ethics Committee of the Fourth Military Medical University. Briefly, the pulp was harvested, minced and digested in a solution containing 3 mg/mL type I collagenase and 4 mg/mL dispase (Gibco, Gaithersburg, Maryland, USA) at 37 °C for 2 h (Hilkens et al., 2013). Single-cell suspensions were obtained by passing the cells through a 70-µm strainer (BD Falcon, Franklin Lakes, NJ, USA). The cells were cultured in α-MEM (Gibco) supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, Maryland, USA), 100 units/mL penicillin and 100 mg/mL streptomycin. The medium was changed every 3 days. Cells from the second passage were used for both cell attachment assay and cytotoxicity assays.

Cell viability test

Mitochondrial dehydrogenase activity was measured by MTT assay to investigate cell viability. HDPCs were seeded into 96-well culture plates at a density of 5 × 10³ cells/well and incubated at 37 °C and 5% CO₂ for approximately 24 h. When the cells grew to 80% confluence, the medium was replaced by the extract of PMMA resin with or without NAC. After each incubation period (3 days, and 7 days), 20 µL of MTT solution at 5 mg/mL (Sigma-Aldrich, St. Louis, Misouri, USA), disinfected by infiltration, was added to the well, and the plates were incubated for a further 4 h. After disposal of the culture medium, 200 µL dimethyl sulfoxide (DMSO) (Amresco, Solon, Ohio, USA) was added to each well. After gently swirled for 10 min, the absorbance of each well at 490 nm was measured. The blank group contains only complete culture medium without cells. For the control group, cells were cultured in wells without eluent. Relative cell viability was calculated using the following equation: ${\displaystyle \text{Relative cell viability}=\left({\mathrm{OD}}_{\mathrm{experimental}}-{\mathrm{OD}}_{\mathrm{blank}}\right)/\left({\mathrm{OD}}_{\mathrm{control}}-{\mathrm{OD}}_{\mathrm{blank}}\right).}$ The experiments were repeated for three times. Cytotoxicity was rated in accordance with ISO-standard 10993-5 as non-cytotoxic (cell viability higher than 75%), slightly cytotoxic (cell viability ranging from 50% to 75%), moderately cytotoxic (cell viability ranging from 25% to 50%), and severely cytotoxic (cell viability lower than 25%).

The MTT assay was also employed to evaluate the influence of NAC alone on cells. Using HPLC analysis, we confirmed that the concentration of NAC released from the experimental PMMA resin containing NAC was no higher than 0.54 mM. Thus, to exclude the possibility that released NAC may enhance the proliferation of cells, we used 0.54 mM NAC alone to treat the cells and studied its influence on cell viability with MTT assay.

Cell attachment assay

Disc-shaped specimens (10-mm in diameter and 2-mm in thickness) of each subgroup were fabricated using a mould. The paste was filled in the mould and covered with a glass silde. All the specimens were allowed to set for 30 min at 25 °C. All discs were rinsed with ddH₂O once and sterilized by ethylene oxide gas before use. HDPCs were seeded onto the discs at the density of 2.0 × 10⁶ cells/well in a 6-well culture plate. After incubation for 24 h, the samples were rinsed with phosphate-buffered saline (PBS, pH 7.2) to eliminate unattached cells and fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.2 M sodium cacodylate buffer (pH = 7.4). After being freeze-dried and sputter-coated with platinum (E-1030; Hitachi, Tokyo, Japan), the specimens were observed with SEM (Model S-4800; Hitachi, Tokyo, Japan) to examine cell morphology and attachment.

Measurements of degree-of-conversion (DC)

The degree of auto-polymerization conversion of control and experimental resin specimens was measured by Fourier Transform Infrared Spectroscopy (FTIR 8400S; Shimadzu Scientific Instruments, Kyoto, Japan). The resin paste was placed between two polyethylene films and pressed to form a very thin film. The FTIR spectra were collected after 5 min, 10 min, 20 min, 30 min, 1 h, 4 h and 24 h. FTIR spectrum of the uncured mixture was also recorded as reference. For each subgroup, the measurement was repeated for three times.

DC of the resin specimens were calculated using the following equation: ${\displaystyle \mathrm{DC}\left(\%\right)=\left[1-\frac{{\left({\mathrm{Abs}}_{\mathrm{ana}}/{\mathrm{Abs}}_{\mathrm{ref}}\right)}_{\mathrm{cured}}}{{\left({\mathrm{Abs}}_{\mathrm{ana}}/{\mathrm{Abs}}_{\mathrm{ref}}\right)}_{\mathrm{uncured}}}\right]\times 100\%.}$

Abs_ana: the intensity of the carbon–carbon double bond stretching vibration (peak height at 1,638 cm⁻¹);

Abs_ref: the intensity of the symmetric ring stretching at 1,610 cm⁻¹;

Absorbance peak intensity values on the FTIR spectra were calculated using OMNIC 8.0 software (Spectra Tech, USA).

Flexural strength

Flexural strength was tested according to ISO 4049:2009 standard. PMMA resin specimens (2 mm × 2 mm × 25 mm, 12 specimens for each subgroup) were prepared in a rectangular stainless steel mold. After 24 h of setting, the specimens were removed from the mold. The three-point bending test was performed using a universal testing machine (AGS-10kNG, Shimadzu, Kyoto, Japan) at a cross-head speed of 1 mm/min, at a controlled room temperature. The flexural strength (FS) was calculated by the equations: ${\displaystyle \mathrm{FS}=\frac{3PL}{{2wh}^2}}$ where P is the maximum load exerted on the specimen (N), L is the distance between the supports (25 mm), w and h are, respectively, the width (2 mm) and the thickness of the specimen (2 mm). Mean flexural strengths were calculated in MPa (megapascals). Three specimens were tested for each group. After testing, the microstructure of the fractured surfaces obtained from mechanical test were observed with SEM.

Microhardness

A microhardness test (Vickers test) was performed using the specimens after the three point bending test. Five half-bars from each subgroup were tested. Vickers microhardness was measured using a microhardness tester (HX-1000TM; Taiming, Shanghai, China). The indenter point was kept on the surface for 35 s with 25 g load. Three indentations were made on each specimen.

Surface roughness

Three half-bars from each subgroup tested by flexural strength testing were randomly chosen for analysis of surface roughness. The upper and lower surfaces of the specimens were polished with SiC grinding paper (320-grit, 600-grit, 1,200-grit and 2,000-grit) under running water on a polishing machine (UNIPOL-830; Shenyang Kejing Automation, Kejing, China). Specimens were ultrasonically (Vitasonic II; VITA Zahnfabrik, Berlin, Germany) cleaned for 10 min before testing. The surface roughness was analyzed with a 3D non-contact optical profilometer (PS50 Optical Profilometer; Nanovea, Irvine, California, USA). This device allowed a scanning over an area of 2 mm in length (x-axis) and 2 mm in width (y-axis). Surface roughness (Ra) was calculated for each scanned area.

Statistical analysis

The results for cell viability, degree of conversion, flexural strength, microhardness and surface roughness were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s test. SPSS 16.0 software (SPSS Inc, Chicago, Illinois, USA) was used for the statistical analysis. Statistical significance was preset at p = 0.05 for all tests.

Quantification of NAC elution using HPLC

Figure 1 shows representative chromatograms for NAC standard solution (0.1 mg/mL) and eluted NAC from experimental specimen containing 0.9 wt% of NAC. Identification of the analyte, NAC, was made based on the retention time of the NAC peaks registered for the standard solutions. The relationship obtained for the linear peak area (A) and concentration (c) for the analyte (NAC) was: A = 27053c + 48133, with a correlation coefficient R² = 0.99859. According to the calibration curve, the concentration of NAC released from the experimental resin containing 0.9 wt% of NAC was calculated as 0.54 ± 0.02 mM.

MTT assay

MTT assay revealed that, as compared to control group, cell viability at day 3 and day 7 was remarkably lower in the culture with PMMA extract (Fig. 2A). NAC exhibited protective effects on the cytotoxicity of PMMA in a concentration-dependent manner. Cultures with the extract of PMMA incorporating NAC showed higher cell viability than untreated PMMA resin. At day 7, relative cell viability was 77% for PMMA resin containing 0.15 wt.% NAC, which is more than 10-fold of that in subgroup 0 wt% (p < 0.05) (Fig. 2A). PMMA resin containing 0.9 wt% NAC presented the highest relative cell viability, reaching 95.6% at day 3 and 97.6% at days 7 (Fig. 2A). Figure 2B showed that 0.54 mM of NAC alone, based on results of HPLC, induced no significant influence on the viability of HDPCs (p > 0.05).

Cell attachment assay

Figure 2C shows the attachment of HDPCs on the surfaces of specimens at day one. In subgroup 0 wt% and subgroup 0.15 wt%, cells grew poorly. Cells with round or collapsed appearances were observed in these subgroups. In subgroup 0.3 wt%, 0.6 wt% and 0.9 wt%, HDPCs attached and spreaded well on the specimens, exhibiting spindle- or polygonal-shapes. The number of adhering cells increased as the concentration of NAC increased in the experimental PMMA resins. The surface of subgroup 0.9 wt% was almost fully covered by HDPC cells.

Degree of conversion

DC was measured from 5 min to 24 h after the liquid MMA was mixed with PMMA powder (Fig. 3). During the first 10 min curing period, DC increased dramatically. At 10 min, the DCs of the control and experimental subgroups ranged from 41.0% to 58.8%. Experimental subgroups containing NAC showed significant lower DC values as compared to control subgroup (p < 0.05). After 10 min, DC increased in a relatively slow manner. At 24 h, the DC values of the five experimental subgroups were all higher than 72.0%. In particular, the DC for experimental PMMA resin containing 0.15 wt% NAC reached 75.2% after 24 h of setting, showing no significant difference to the control PMMA resin without NAC (p > 0.05), whereas experimental PMMA resin with 0.3 wt% or higher concentration of NAC revealed slightly but statistically compromised DC values.

Flexural strength testing

The flexural strength (FS) of specimens in various subgroups after 24 h of setting are shown in Fig. 4A. Adding NAC to PMMA resin induced a negative influence on the flexural strength of the material. Although the experimental resin containing 0.15 wt% NAC showed similar flexural strength (91.0 ± 6.2 MPa) to that of control group (95.9 ± 5.8 MPa), subgroups with higher concentration of NAC exhibited significantly compromised flexural strength, and the PMMA with 0.9 wt% showed the lowest flexural strength of 72.8 MPa (p < 0.05).

Microhardness

Figure 4B showed the results of microhardness test after 24 h of setting. The trend of surface microhardness was similar to that of flexural strength. The incorporation of 0.15 wt% NAC had no significantly adverse effects on the microhardness of PMMA resin (p > 0.05 as compared to control group without NAC). However, when the concentration of incorporated NAC was further increased, the microhardness of the materials showed a significantly reduced value.

Surface roughness

Figure 4C show the mean Ra values of the specimens. The mean Ra values was 0.60 µm and 0.86 µm for subgroup 0.15 wt% and 0.3 wt%, respectively, which is not significantly different from that of control group. However, other subgroups with higher concentrations of NAC revealed significantly higher Ra values, indicating that adding NAC beyond the concentration of 0.3% has negative influences on the surface roughness of PMMA resin.

SEM of fractured surfaces

The SEM image of the fractured surfaces for each subgroup is presented in Fig. 4D. As can be seen, PMMA resin without NAC showed a relatively smooth appearance. The fractured surface of experimental resin containing 0.15 wt% NAC was similar to that of control group. However, for the subgroup with 0.3 wt% or higher concentration of NAC, pit-like internal defects can be clearly seen. Higher concentration of NAC incorporation is accompanied with greater number of internal defects.