*2.5. Antibacterial Test*

*2.5. Antibacterial Test*  The antibacterial activity of UAT65/TPGDA or UAC50/TPGDA with various Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP contents was performed according to the Japanese Industrial Standard as shown in Figure 4 (JIS Z 2801-2000) [32]. Gram-positive *E. faecalis* (ATCC 29212, Super Laboratory Co., Taiwan) was the bacterial strain cultivated in brain heart infusion (BHI) broth. The number of bacterial suspensions was adjusted to 104 colony-forming units per milliliter (CFU/mL). The antibacterial test was carried out by the following steps. First, colonies were added into 5 mL HBI broth, cultivated at 37 °C for 16–18 h. The medium of bacteria fluid of lysogeny broth (LB) was replaced by phosphate-buffered saline (PBS) by using the centrifugation for three times (8,000 xg, 3 min), and diluted to the concentration of 10−5–10−7 (CFU/mL). Subsequently, the as-prepared The antibacterial activity of UAT65/TPGDA or UAC50/TPGDA with various Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP contents was performed according to the Japanese Industrial Standard as shown in Figure 4 (JIS Z 2801-2000) [32]. Gram-positive *E. faecalis* (ATCC 29212, Super Laboratory Co., Taiwan) was the bacterial strain cultivated in brain heart infusion (BHI) broth. The number of bacterial suspensions was adjusted to 104 colony-forming units per milliliter (CFU/mL). The antibacterial test was carried out by the following steps. First, colonies were added into 5 mL HBI broth, cultivated at 37 ◦C for 16–18 h. The medium of bacteria fluid of lysogeny broth (LB) was replaced by phosphate-buffered saline (PBS) by using the centrifugation for three times (8,000 xg, 3 min), and diluted to the concentration of 10−5–10−<sup>7</sup> (CFU/mL). Subsequently, the as-prepared suspension was spread onto the BHI agar plates

suspension was spread onto the BHI agar plates (10 μL) and incubated cells at 37 °C for 16–18 h. The 0.5 McFarland (108 CFU/mL) bacterial fluid was produced by using *E. faecalis* (ATCC 29212). (10 µL) and incubated cells at 37 ◦C for 16–18 h. The 0.5 McFarland (10<sup>8</sup> CFU/mL) bacterial fluid was produced by using *E. faecalis* (ATCC 29212). Specimens were shaped to square (5 cm × 5 cm), wiped with alcohol, and sterilized by the exposure of UV-light for 24 h. Then, 400 µL bacterial fluid (10<sup>4</sup> CFU/mL) was added to the specimens, covered with a sterile square PE film (4 cm <sup>×</sup> 4 cm) and incubated at 37 ◦C under relative humidity 90% for 24 h. After bacterial adhesion or proliferation, the specimens were rinsed for three times by using PBS and then transformed to a new 24-well plate. Each specimen was soaked in 2.5% glutaraldehyde and reacted at 4 ◦C for 1 h, followed by the removal of glutaraldehyde by rinsing with PBS for three times. Before the investigation of morphology for the evaluation of antibacterial activity, the samples were prepared by using ethanol-wet bonding technique [35]. The freeze-drying processes were conducted for 24 h by the increased concentration of (*w*/*w*) ethanol from 30%, 50%, 70%, 90%, 95%, 99% to 100%. In addition, the statistical analysis was performed by Statistical Analysis Software (SAS). One-way analysis of variance (ANOVA) was used to analyze the difference between bacterial groups, and Duncan's multiple tests were used to distinguish various bacterial groups. The *p*-value <0.05 is regarded as statistically significant. *Polymers* **2019**, *11*, x FOR PEER REVIEW 6 of 21 with a sterile square PE film (4 cm × 4 cm) and incubated at 37 °C under relative humidity 90% for 24 h. After bacterial adhesion or proliferation, the specimens were rinsed for three times by using PBS and then transformed to a new 24-well plate. Each specimen was soaked in 2.5% glutaraldehyde and reacted at 4 °C for 1 h, followed by the removal of glutaraldehyde by rinsing with PBS for three times. Before the investigation of morphology for the evaluation of antibacterial activity, the samples were prepared by using ethanol-wet bonding technique [35]. The freeze-drying processes were conducted for 24 h by the increased concentration of (*w/w*) ethanol from 30%, 50%, 70%, 90%, 95%, 99% to 100%. In addition, the statistical analysis was performed by Statistical Analysis Software (SAS). One-way analysis of variance (ANOVA) was used to analyze the difference between bacterial groups, and Duncan's multiple tests were used to distinguish various bacterial groups. The *p*-value <0.05 is regarded as statistically significant.

**Figure 4.** The process of antibacterial evaluation. **Figure 4.** The process of antibacterial evaluation.

#### **3. Results and Discussion 3. Results and Discussion**

#### *3.1. Synthesis of Urethan-acrylate (UA) 3.1. Synthesis of Urethan-acrylate (UA)*

The preparation of urethane acrylate was monitored by using the IR spectra as shown in Figure 5. For the spectrum of the initial mixture of IPDI and PCPO 500, two distinct peaks at 2260 and 1742 cm-1 were present for isocyanate, and carbonyl group of carbonate ester, respectively. After 2 h into the reaction, a newly emerged shoulder at 1718 cm−1 was observed, indicating the formation of urethane carbonyl group [36,37]. Moreover, the urethane prepolymer end-capped with isocyanates was further reacted with HEMA to provide the product of UAC50 as evident by the near disappearance of isocyanate group and the formation of a new adsorption peak at 1638 cm−1 of vinyl group. The additional TPGDA in the mixture of UAC50/TPGDA = 70/30 (*w/w*) resulted in a stronger absorption intensity of vinyl group. The weight average molecular weights were measured by using GPC, leading to the results of 3320 g/mol for UAC50 and 3620 g/mol for UAT65. The preparation of urethane acrylate was monitored by using the IR spectra as shown in Figure 5. For the spectrum of the initial mixture of IPDI and PCPO 500, two distinct peaks at 2260 and 1742 cm−<sup>1</sup> were present for isocyanate, and carbonyl group of carbonate ester, respectively. After 2 h into the reaction, a newly emerged shoulder at 1718 cm−<sup>1</sup> was observed, indicating the formation of urethane carbonyl group [36,37]. Moreover, the urethane prepolymer end-capped with isocyanates was further reacted with HEMA to provide the product of UAC50 as evident by the near disappearance of isocyanate group and the formation of a new adsorption peak at 1638 cm−<sup>1</sup> of vinyl group. The additional TPGDA in the mixture of UAC50/TPGDA = 70/30 (*w*/*w*) resulted in a stronger absorption intensity of vinyl group. The weight average molecular weights were measured by using GPC, leading to the results of 3320 g/mol for UAC50 and 3620 g/mol for UAT65.

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**Figure 5.** FTIR spectra of UAC50 and UAC50/TPGDA. **Figure 5.** FTIR spectra of UAC50 and UAC50/TPGDA. **Figure 5.** FTIR spectra of UAC50 and UAC50/TPGDA.

#### *3.2. Preparation and Thermal Properties of UA Composites 3.2. Preparation and Thermal Properties of UA Composites 3.2. Preparation and Thermal Properties of UA Composites*

The determination of the processing effectiveness and the quantitative fillers within the matrix are usually investigated by using thermogravimetric analysis (TGA) for the polymer composites [38]. The UA composites were prepared by mixing 5 wt % or 10 wt % in total composites by using the nanometer-thin silicate platelets immobilized with nanoparticles such as AgNPs (Ag@NSP), ZnONPs (ZnO@NSP), or AgNPs together with ZnONPs (Ag/ZnO@NSP) [20,22,25,39,40]. In TGA thermograms (Figure 6), the UA composites exhibited 5% weight loss at about 300 °C, indicating that good thermal stability was available for the following tests. Moreover, the addition of nanoparticles immobilized on the nanometer-thin silicate platelets provided higher char yield (%) dependent on the addition of inorganic contents. The determination of the processing effectiveness and the quantitative fillers within the matrix are usually investigated by using thermogravimetric analysis (TGA) for the polymer composites [38]. The UA composites were prepared by mixing 5 wt % or 10 wt % in total composites by using the nanometer-thin silicate platelets immobilized with nanoparticles such as AgNPs (Ag@NSP), ZnONPs (ZnO@NSP), or AgNPs together with ZnONPs (Ag/ZnO@NSP) [20,22,25,39,40]. In TGA thermograms (Figure 6), the UA composites exhibited 5% weight loss at about 300 ◦C, indicating that good thermal stability was available for the following tests. Moreover, the addition of nanoparticles immobilized on the nanometer-thin silicate platelets provided higher char yield (%) dependent on the addition of inorganic contents. The determination of the processing effectiveness and the quantitative fillers within the matrix are usually investigated by using thermogravimetric analysis (TGA) for the polymer composites [38]. The UA composites were prepared by mixing 5 wt % or 10 wt % in total composites by using the nanometer-thin silicate platelets immobilized with nanoparticles such as AgNPs (Ag@NSP), ZnONPs (ZnO@NSP), or AgNPs together with ZnONPs (Ag/ZnO@NSP) [20,22,25,39,40]. In TGA thermograms (Figure 6), the UA composites exhibited 5% weight loss at about 300 °C, indicating that good thermal stability was available for the following tests. Moreover, the addition of nanoparticles immobilized on the nanometer-thin silicate platelets provided higher char yield (%) dependent on the addition of inorganic contents.

**Figure 6. T**hermogravimetric analysis (TGA) thermograms of UAC50- and UAC50-based composites. **Figure 6. T Figure 6.** Thermogravimetric analysis (TGA) thermograms of UAC50- and UAC50-based composites. hermogravimetric analysis (TGA) thermograms of UAC50- and UAC50-based composites.

#### *3.3. Curing Conditions and Depths for UA Composites* Moreover, viscosity is another important factor for the root canal sealer to establish connection

molecular weight when compared with the UA resins.

*3.3. Curing Conditions and Depths for UA Composites* 

The endodontic sealers are required to exhibit several properties to meet the desired performance. The flow behavior is one of the most important factors for sealers to penetrate into small irregularities of the root canal system and dentinal tubules as shown in Figure 7. According to the regulation of ISO 4049:2009, the flow diameter should be over 20 mm after compression under a weight disc for 10 min. The tests were carried out by the introduction of difunctional TPGDA in order to increase flow diameter and crosslinking density. As a result, the samples with higher ratio of TPGDA exhibited larger flow diameter, such as UAT65/TPGDA = 80/20, 70/30, or 60/40; or UAC50/TPGDA = 70/30 or 60/40, since the difunctional TPGDA monomer is a diluent with a lower molecular weight when compared with the UA resins. between the root canal, periodontal ligament, and the apical foramen. According to ISO 4049:2009, the viscosity should range from 1000 to 2000 cp to meet the desired operation [32]. In Figure 8, UAT65/TPGDA (70/30) and UAC50/TPGDA (70/30) were the samples of choice for further investigations since the addition of the optimized TPGDA content would achieve the viscosity range mentioned above. It is important to note that flow and viscosity properties depends on not only the ratios between UA resins and TPGDA but the use of polyols such as PTMEG or PCPO. The PCPObased UA resins exhibited somewhat lower flow diameter and higher viscosity under the same ratio of UA/TPGDA composition. This is because the incorporation of the PCPO-based polyol with carbonate groups brought about more hydrogen bonding interactions with the urethane linkages. This would restrain the molecular mobility of the UA segments [41,42].

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The endodontic sealers are required to exhibit several properties to meet the desired performance. The flow behavior is one of the most important factors for sealers to penetrate into small irregularities of the root canal system and dentinal tubules as shown in Figure 7. According to the regulation of ISO 4049:2009, the flow diameter should be over 20 mm after compression under a weight disc for 10 min. The tests were carried out by the introduction of difunctional TPGDA in order to increase flow diameter and crosslinking density. As a result, the samples with higher ratio of

**Figure 7.** Flow analysis of UA resins with various weight ratios of UAT65/tripropylene glycol diacrylate (TPGDA) or UAC50/TPGDA according to ISO 4049:2009. **Figure 7.** Flow analysis of UA resins with various weight ratios of UAT65/tripropylene glycol diacrylate (TPGDA) or UAC50/TPGDA according to ISO 4049:2009.

Moreover, viscosity is another important factor for the root canal sealer to establish connection between the root canal, periodontal ligament, and the apical foramen. According to ISO 4049:2009, the viscosity should range from 1000 to 2000 cp to meet the desired operation [32]. In Figure 8, UAT65/TPGDA (70/30) and UAC50/TPGDA (70/30) were the samples of choice for further investigations since the addition of the optimized TPGDA content would achieve the viscosity range mentioned above. It is important to note that flow and viscosity properties depends on not only the ratios between UA resins and TPGDA but the use of polyols such as PTMEG or PCPO. The PCPO-based UA resins exhibited somewhat lower flow diameter and higher viscosity under the same ratio of UA/TPGDA composition. This is because the incorporation of the PCPO-based polyol with carbonate groups brought about more hydrogen bonding interactions with the urethane linkages. This would restrain the molecular mobility of the UA segments [41,42].

<sup>5</sup>UAC50/TPGDA

<sup>6</sup>UAC50/TPGDA

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**Figure 8.** Viscosity analysis of UA resins with various weight ratios of UAT65/TPGDA or UAC50/TPGDA according to ISO 4049:2009. **Figure 8.** Viscosity analysis of UA resins with various weight ratios of UAT65/TPGDA or UAC50/TPGDA according to ISO 4049:2009.

The curing conditions of UAT65/TPGDA (70/30) or UAC50/TPGDA (70/30) were carried out by using ternary initiation processes for photo crosslinking. The degree of acrylate conversion (DC%) would be based on the peak area at 1638 cm−1 of aliphatic C=C double bond of acrylate content as a function of various initiator concentrations (1, 3, 6, and 9 phr) before and after curing (Table 1). Optimum mechanical properties with a tensile strength of 56.15 ± 3.26 MPa and a Young's modulus of 361.88 ± 32.38 MPa were achieved for the sample UAT65/TPGDA (70/30) cured with 3 phr initiator concentration. It is important to note that the DC% values were close to 70% for the above-mentioned samples. This is because the low conversion degree of acrylate functional groups with a low concentration initiator resulted in insufficient crosslinking density for obtaining good mechanical performance, whereas the excessive photoinitiators would provide the localized absorption and crosslinking density over the percolation threshold, leading to the adverse effect on the mechanical The curing conditions of UAT65/TPGDA (70/30) or UAC50/TPGDA (70/30) were carried out by using ternary initiation processes for photo crosslinking. The degree of acrylate conversion (DC%) would be based on the peak area at 1638 cm−<sup>1</sup> of aliphatic C=C double bond of acrylate content as a function of various initiator concentrations (1, 3, 6, and 9 phr) before and after curing (Table 1). Optimum mechanical properties with a tensile strength of 56.15 ± 3.26 MPa and a Young's modulus of 361.88 ± 32.38 MPa were achieved for the sample UAT65/TPGDA (70/30) cured with 3 phr initiator concentration. It is important to note that the DC% values were close to 70% for the above-mentioned samples. This is because the low conversion degree of acrylate functional groups with a low concentration initiator resulted in insufficient crosslinking density for obtaining good mechanical performance, whereas the excessive photoinitiators would provide the localized absorption and crosslinking density over the percolation threshold, leading to the adverse effect on the mechanical properties [43].

properties [43]. **Table 1.** Mechanical properties and DC% for the UAT65/TPGDA (*w/w* = 70/30) and TPGDAC50/TPGDA (*w/w* = 70/30) samples after curing with different dosages of photoinitiator. **Entry UA Formulation Photoinitiator (phr 1) Tensile Strength (MPa) Young's Modulus (MPa) DC 2 (%)**  Since the acrylate-based photopolymerization depends deeply on the sealer transparency, the curing depths of UAT65/TPGDA resin (*w*/*w* = 70/30) and UAC50/TPGDA resin (*w*/*w* = 70/30) were assessed by using various contents of antibacterial NSPs immobilized with nanoparticles including Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP (Figures 9–11). It is reported that the photo crosslinking activity relies on the use of different types of metal nanoparticles and the rate of photo crosslinking, and the DC% also varies with the additives under similar processing condition [44]. In this study, the curing depths decreased with increasing content of antibacterial NSP agents.

<sup>1</sup>UAT65/TPGDA (*w/w* = 70/30) 1 51.71 ± 3.03 362.81 ± 37.10 46.27 ± 3.07 <sup>2</sup>UAT65/TPGDA (*w/w* = 70/30) 3 56.15 ± 3.26 361.88 ± 32.38 64.91 ± 1.06 <sup>3</sup>UAT65/TPGDA (*w/w* = 70/30) 6 45.96 ± 2.22 33.49 ± 1.65 67.58 ± 2.27 <sup>4</sup>UAT65/TPGDA (*w/w* = 70/30) 9 36.78 ± 1.65 1.92 ± 0.42 70.35 ± 1.76 These UA composite composed of various antibacterial nanomaterials are denoted as Ag@NSP-n, ZnO@NSP-n, or Ag/ZnO@NSP-n, where the "n" is denoted as the parts per million (ppm) to the total weight of UAT65/TPGDA or UAC50/TPGDA. According to the regulation of ISO 4049:2009, the curing depth should be larger than 10 mm. As a result, the limitation for the addition of a maximum amount of Ag@NSP is 500 ppm (Ag@NSP-500) as shown in Figure 9. Similar results were also obtained for the composites incorporated with ZnO@NSP or Ag/ZnO@NSP (Figures 10 and 11). This indicates that the presence of different nanomaterials such as Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP in the UA composites did not influence curing depths much.

(*w/w* = 70/30) 1 46.30 ± 3.18 1072.25 ± 46.54 57.89 ± 0.41

(*w/w* = 70/30) 3 60.54 ± 4.72 1042.02 ± 39.62 72.44 ± 1.57


**Table 1.** Mechanical properties and DC% for the UAT65/TPGDA (*w*/*w* = 70/30) and TPGDAC50/TPGDA (*w*/*w* = 70/30) samples after curing with different dosages of photoinitiator. <sup>7</sup>UAC50/TPGDA

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1 : parts per hundred; <sup>2</sup> : after the exposure under UV light for 40 s. indicates that the presence of different nanomaterials such as Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP

in the UA composites did not influence curing depths much.

**Figure 9.** Curing depths of UAT65/TPGDA resins (*w/w* = 70/30) and UAC50/TPGDA resins (*w/w* = 70/30) with various Ag@NSP contents (ppm). **Figure 9.** Curing depths of UAT65/TPGDA resins (*w*/*w* = 70/30) and UAC50/TPGDA resins (*w*/*w* = 70/30) with various Ag@NSP contents (ppm).

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**Figure 10.** Curing depths of UAT65/TPGDA resins (*w/w* = 70/30) and UAC50/TPGDA resins (*w/w* = 70/30) with various ZnO@NSP contents (ppm). **Figure 10.** Curing depths of UAT65/TPGDA resins (*w*/*w* = 70/30) and UAC50/TPGDA resins (*w*/*w* = 70/30) with various ZnO@NSP contents (ppm). **Figure 10.** Curing depths of UAT65/TPGDA resins (*w/w* = 70/30) and UAC50/TPGDA resins (*w/w* = 70/30) with various ZnO@NSP contents (ppm).

**Figure 11.** Curing depths of UAT65/TPGDA resins (*w/w* = 70/30) and UAC50/TPGDA resins (*w/w* = **Figure 11.** Curing depths of UAT65/TPGDA resins (*w/w* = 70/30) and UAC50/TPGDA resins (*w/w* = 70/30) with various Ag/ZnO@NSP contents (ppm). **Figure 11.** Curing depths of UAT65/TPGDA resins (*w*/*w* = 70/30) and UAC50/TPGDA resins (*w*/*w* = 70/30) with various Ag/ZnO@NSP contents (ppm).

#### 70/30) with various Ag/ZnO@NSP contents (ppm). *3.4. Biocompatibility Analysis*

*3.4. Biocompatibility Analysis*  Grossman [45] advocated that an ideal root canal filling material should not irritate periradicular tissues. In addition, Faccioni et al. found that root canal materials with metal ions might influence cell metabolism and differentiation [46]. Other studies found that the incomplete photopolymerization reaction resulted in the release of uncured monomers and initiators, which *3.4. Biocompatibility Analysis*  Grossman [45] advocated that an ideal root canal filling material should not irritate periradicular tissues. In addition, Faccioni et al. found that root canal materials with metal ions might influence cell metabolism and differentiation [46]. Other studies found that the incomplete photopolymerization reaction resulted in the release of uncured monomers and initiators, which Grossman [45] advocated that an ideal root canal filling material should not irritate periradicular tissues. In addition, Faccioni et al. found that root canal materials with metal ions might influence cell metabolism and differentiation [46]. Other studies found that the incomplete photopolymerization reaction resulted in the release of uncured monomers and initiators, which would affect the mitochondrial enzyme activity [47,48]. Therefore, the concentration of antibacterial agent in sealers

would affect the mitochondrial enzyme activity [47,48]. Therefore, the concentration of antibacterial

would affect the mitochondrial enzyme activity [47,48]. Therefore, the concentration of antibacterial

depends deeply on the biocompatibility of composites. In this study, the tests of Alamar Blue assay and LDH assay were conducted for the biocompatibility tests for UAT65/TPGDA (*w*/*w* = 70/30) and UAC50/TPGDA resins (*w*/*w* = 70/30) with various concentrations of antimicrobial agents such as Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP as shown in Figures 12 and 13, respectively.

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**Figure 12.** Alamar blue assay of (**a**) UAT65/TPGDA (*w/w* = 70/30) and (**b**) UAC50/TPGDA (*w/w* = 70/30) resins with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP (p < 0.05). **Figure 12.** Alamar blue assay of (**a**) UAT65/TPGDA (*w*/*w* = 70/30) and (**b**) UAC50/TPGDA (*w*/*w* = 70/30) resins with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP (*p* < 0.05).

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**Figure 13.** Lactate dehydrogenase (LDH) assay of (**a**) UAT65/TPGDA (*w/w* = 70/30) and (**b**) UAC50/TPGDA (*w/w* = 70/30) with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP (*p* < 0.05). *3.5. Antibacterial Analysis*  **Figure 13.** Lactate dehydrogenase (LDH) assay of (**a**) UAT65/TPGDA (*w*/*w* = 70/30) and (**b**) UAC50/TPGDA (*w*/*w* = 70/30) with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP (*p* < 0.05).

The antibacterial activities of the composites based on UAC50/TPGDA (*w/w* = 70/30) with nanoparticles-on-platelet nanohybrids (Ag@NSP, ZnO@NSP, Ag/ZnO@NSP) were analyzed by treating the composites with Gram-positive *E. faecalis* (ATCC 29212) according to Japanese Industrial Standard (JIS Z 2801-2000) as shown in Table 2. According to the biocompatibility tests in previous section, the use of antibacterial NSP agents should not be higher than 100 ppm for UAC50/TPGDA (*w/w* = 70/30) resins. Indeed, the AgNPs-based composites with 75 ppm (Ag@NSP-75) or 100 ppm (Ag@NSP-100) inorganic additives exhibited antibacterial activity. However, ZnONPs-based composites even with 1000 ppm (ZnO@NSP-1000) were not able to exhibit antibacterial activity. In fact, the antibacterial activity was feasible only when the use of high concentration of ZnONPs over 2000 ppm (ZnO@NSP-2000 and ZnO@NSP-3000), indicating that the Ag@NSP-based composites In the Alamar blue assay test, both pristine resins of UAT65/TPGDA (*w*/*w* = 70/30) (Figure 12a) and UAC50/TPGDA (*w*/*w* = 70/30) (Figure 12b) were substantially free of cytotoxicity. As the antimicrobial agents were incorporated into the resins to form UA composites, reduced metabolic activities were observed, as shown in Figure 12. Furthermore, the composite with Ag@NSP exhibited the poorest biocompatibility when compared to other samples, especially in the example for both UAT65/TPGDA and UAC50/TPGDA incorporated with 500 ppm additives. The metabolic activities were higher than 70% for all the UA composites with 100 ppm or less than 100 ppm antibacterial NSP agents. This

would be better candidates for the root canal sealer applications. In addition, the simultaneous immobilization of AgNPs and ZnONPs on silicate platelets, i.e., Ag/ZnO@NSP, led to much better antimicrobial results even for the composite with an antibacterial agent concentration as low as 50

ppm (Ag/ZnO@NSP-50).

indicates that good biocompatibility could be achieved with the addition of a certain content of the antimicrobial agents such as Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP to the composites.

In the LDH assay test, the cytotoxicity is also dependent on the addition of various concentrations of Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP to the composites (Figure 13). Given the fact that UA composites with 100 ppm would exhibit good biocompatibility, the cytotoxicity of the UA composites with 100 ppm antimicrobial agents on 3T3 cells was investigated and observed in the following order: Ag@NSP-100 > Ag/ZnO@NSP-100 > ZnO@NSP-100. This implies that the composites comprising AgNPs would exhibit poor cytotoxicity performance. In addition, the cytotoxicity of the composites based on the UAC50/TPGDA (*w*/*w* = 70/30) are lower than that of the composites based on UAT65/TPGDA (*w*/*w* = 70/30), especially in the example for both UAT65/TPGDA (~90% of control) and UAC50/TPGDA (~80% of control) incorporated with 500 ppm Ag@NSP. As a matter of fact, polycarbonate-based polyurethanes (PUs) with better biocompatibility correspond to the weaker immune response when compared with polyether-based PUs according to the literature [49]. This is because the α-carbon atoms of the polyether-based PUs (such as UAT65) are highly susceptible to oxidation by oxygen radicals to form esters, which result in unstable chemical structures for polymers [50]. As a result, the carbonate containing UAC50/TPGDA system would be the material of choice for dental root canal sealers instead of the ether-containing UAT65/TPGDA resins.

#### *3.5. Antibacterial Analysis*

The antibacterial activities of the composites based on UAC50/TPGDA (*w*/*w* = 70/30) with nanoparticles-on-platelet nanohybrids (Ag@NSP, ZnO@NSP, Ag/ZnO@NSP) were analyzed by treating the composites with Gram-positive *E. faecalis* (ATCC 29212) according to Japanese Industrial Standard (JIS Z 2801-2000) as shown in Table 2. According to the biocompatibility tests in previous section, the use of antibacterial NSP agents should not be higher than 100 ppm for UAC50/TPGDA (*w*/*w* = 70/30) resins. Indeed, the AgNPs-based composites with 75 ppm (Ag@NSP-75) or 100 ppm (Ag@NSP-100) inorganic additives exhibited antibacterial activity. However, ZnONPs-based composites even with 1000 ppm (ZnO@NSP-1000) were not able to exhibit antibacterial activity. In fact, the antibacterial activity was feasible only when the use of high concentration of ZnONPs over 2000 ppm (ZnO@NSP-2000 and ZnO@NSP-3000), indicating that the Ag@NSP-based composites would be better candidates for the root canal sealer applications. In addition, the simultaneous immobilization of AgNPs and ZnONPs on silicate platelets, i.e., Ag/ZnO@NSP, led to much better antimicrobial results even for the composite with an antibacterial agent concentration as low as 50 ppm (Ag/ZnO@NSP-50).

The direct contact of UAC50/TPGDA composites with the bacterial population could be visualized by using scanning electron microscopy (SEM) as shown in Figure 14. According to the investigation above, this study was conducted by using *E. faecalis* on the surfaces of UA/TPGDA composites with 50 ppm Ag@NSP, 75 ppm Ag@NSP, and 50 ppm Ag/ZnO@NSP after 6h and 24h. For the UAC50/TPGDA resin without the use of antibacterial agents, a rapid growth of bacterial in number was observed as shown in Figure 14a,e. For the composite (Ag@NSP-50) with 50 ppm of Ag@NSP, a certain amount of bacteria appeared first after 6h (Figure 14b). Subsequently, these bacteria increased in number and aggregated after 24 h (Figure 14f). For the composite (Ag@NSP-75) with 75 ppm of Ag@NSP, a certain amount of bacteria appeared to be aggregated and deformed after 6h, and subsequently these bacteria remained aggregated without the sign of number increase as shown in Figure 14c,g, respectively. It is likely that the nanoparticles can re-charge from NSPs to inactivate and rupture bacterial aggregates [51].

It was reported that the combined use of different types of nanoparticles could adsorb onto the cytoderm of the bacteria and even penetrate the cytomembrane to disturb the normal function of cells, leading to cell apoptosis [50]. This is evidenced by the presence of only 50 ppm Ag/ZnO@NSP in the UAC50/TPGDA composite capable of exhibiting a satisfactory antibacterial effect against *E. faecalis* (Table 2). For the composites (Ag/ZnO@NSP-50) with 50 ppm of Ag/ZnO@NSP, once again a certain amount of bacteria appeared to be aggregated and deformed after 6h, and subsequently these bacteria remained aggregated without the sign of number increase as shown in Figure 14d,f. The

**4. Conclusions** 

simultaneous immobilization of AgNPs and ZnONPs on silicate platelets could not only enhance the antibacterial activities and reduce the dose of AgNPs, but act as a promoter in the antibacterial effect for the Ag/ZnO@NSP-based composites as well. *Polymers* **2019**, *11*, x FOR PEER REVIEW 18 of 21

**Figure 14.** SEM images (2500x) of *E. faecalis* on the surfaces of UAC50/TPGDA = (*w/w* = 70/30) with various antibacterial agents: control (**a**,**e**); 50 ppm Ag@NSP (**b**,**f**); 75 ppm Ag@NSP (**c**,**g**); 50 ppm Ag/ZnO@NSP (**d**,**h**) for 6 h and 24 h, respectively. **Figure 14.** SEM images (2500x) of *E. faecalis* on the surfaces of UAC50/TPGDA = (*w*/*w* = 70/30) with various antibacterial agents: control (**a**,**e**); 50 ppm Ag@NSP (**b**,**f**); 75 ppm Ag@NSP (**c**,**g**); 50 ppm Ag/ZnO@NSP (**d**,**h**) for 6 h and 24 h, respectively.

The purpose of this study is to develop a new root canal sealer with a biocompatible resin based on a macrodiol of polycarbonate (PCPO) that could be prepared through the utilization of carbon dioxide as feedstock. With good biocompatibility and biostability, these carbonate-containing urethane acrylate resins would be able to exhibit superior performance to the reference, polyether (PTMEG)-based resins in the preparation of root canal obturation sealers. The successful

the PCPO-based urethane acrylate was selected to be the resin sealer matrix. Moreover, the incorporation of ZnONPs and AgNPs simultaneously immobilized on silicate platelets into the


**Table 2.** Antibacterial activity of the composites based on UAC50/TPGDA (*w*/*w* = 70/30) resins with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP.

(1): composites using UAC50 as polymer matrix; (2): "#" indicating the inhibition of bacteria; "×" indicating the growth of bacteria.

#### **4. Conclusions**

The purpose of this study is to develop a new root canal sealer with a biocompatible resin based on a macrodiol of polycarbonate (PCPO) that could be prepared through the utilization of carbon dioxide as feedstock. With good biocompatibility and biostability, these carbonate-containing urethane acrylate resins would be able to exhibit superior performance to the reference, polyether (PTMEG)-based resins in the preparation of root canal obturation sealers. The successful incorporation of nanoparticles immobilized on nanoscale platelets in the resin matrix resulted in the root canal obturation sealers with satisfactory biocompatibility and antibacterial effect. As a result, the PCPO-based urethane acrylate was selected to be the resin sealer matrix. Moreover, the incorporation of ZnONPs and AgNPs simultaneously immobilized on silicate platelets into the PCPO-based urethane acrylates would not only enhance the antibacterial activities, but also serve as a promoter in the antibacterial effect. Based on the above, the UAC50/TPGDA (70/30 = *w*/*w*) resin with 50 ppm Ag/ZnO@NSP has a great potential as an antibacterial root canal sealer.

**Author Contributions:** Conceptualization, H.-H.C., R.-J.J., and C.-P.L.; methodology, Y.-T.T.; validation, H.-H.C. and C.-P.L.; data curation, S.-W.H. and Y.-F.K.; formal analysis, Y.-C.H., H.-H.C. and Y.-T.T.; writing—original draft preparation, Y.-T.T.; writing—review and editing, C.-L.Y. and C.-H.W.; supervision, J.-J.L. and C.-P.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the "Advanced Research Center for Green Materials Science and Technology" from The Featured Area Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (108L9006) and the Ministry of Science and Technology in Taiwan (MOST 108-3017-E-002-002, MOST 106-2221-E-002-189-MY3).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


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