**2. Materials and Methods**

### *2.1. Synthesis of Urethane-acrylate (UA)*

As shown in Figure 2, UAs were prepared from the reaction between the isocyanate groups of isophorone diisocyanate (IPDI) in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as catalyst, and hydroxyl groups of polyols to achieve urethane prepolymer end-capped with isocyanates, followed by the addition of hydroxyethylmethacrylate (HEMA). In this study, UA prepared from polyols by using polytetramethylene ether glycol with a Mw of 650 (PTMEG 650) is denoted as UAT65, while UA prepared from polycarbonate polyol with a Mw of 500 (PCPO 500) is denoted as UAC50. The introduction of acrylate group was prepared from using HEMA as reagent in the molar ratio (2/1/1) of IPDI/polyol/HEMA. The synthesis of UAs was monitored by using IR spectrum (Jasco 4600 FT-IR Spectrophotometer with a Jasco ATR Pro 450-S accessory, Jasco, Tokyo, Japan). The molecular weights were measured by using gel permeation chromatography (GPC) with samples dissolved in 1 wt % THF. (Waters chromatography system, two Waters Styragel linear columns, and polystyrene as the standard with Mw 104, 2560, 7600, 18,000, and 37,900 by using THF as eluent at the flow rate of 1 mL/min.)

*Polymers* **2019**, *11*, x FOR PEER REVIEW 4 of 21

**Figure 2.** The preparation of urethane acrylate (UA). **Figure 2.** The preparation of urethane acrylate (UA).

#### *2.2. Preparation of Antibacterial Sealer Based on UA 2.2. Preparation of Antibacterial Sealer Based on UA*

The UA samples (UAT65 and UAC50) were dissolved in acetone at 80 °C and mixed with nanoscale silicate materials (Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP) to prepare UA-based composites. The preparation and characterization of Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP were performed according to the literature reported by our groups [21]. The sealers were then prepared by the introduction of a diluent, tripropylene glycol diacrylate (TPGDA), and an initiator containing camphorquinone (CQ) as a photosensitizer with ethyl-4-dimethylaminobenzoate (EDMA) for binary initiation processes with a ratio of CQ/EDMAB = 1/2 (*w/w*), together with 0.5 wt % azobisisobutyronitrile (AIBN) for ternary initiation processes. The characterizations of composites were carried out by using the thermogravimetric analysis (TGA; TGA Q50 TA instrument, TA Instrument, New Castle, DE, USA) to investigate the inorganic contents. For the curing process, the sealer samples were first cast into Teflon molds in the dimension of 20-mm long, 2-mm wide, and subsequently cured with a light emitting diode curing unit at an intensity of 800 mW/cm2 for 40 s from the coronal aspect (SmartLite, Dentsply, PA). The degrees of curing conversions were investigated by using FT-IR to calculate the peak area transition at 1638 cm−1 of aliphatic C=C double bond of acrylate group according to the following equation (1) [30]: The UA samples (UAT65 and UAC50) were dissolved in acetone at 80 ◦C and mixed with nanoscale silicate materials (Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP) to prepare UA-based composites. The preparation and characterization of Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP were performed according to the literature reported by our groups [21]. The sealers were then prepared by the introduction of a diluent, tripropylene glycol diacrylate (TPGDA), and an initiator containing camphorquinone (CQ) as a photosensitizer with ethyl-4-dimethylaminobenzoate (EDMA) for binary initiation processes with a ratio of CQ/EDMAB = 1/2 (*w*/*w*), together with 0.5 wt % azobisisobutyronitrile (AIBN) for ternary initiation processes. The characterizations of composites were carried out by using the thermogravimetric analysis (TGA; TGA Q50 TA instrument, TA Instrument, New Castle, DE, USA) to investigate the inorganic contents. For the curing process, the sealer samples were first cast into Teflon molds in the dimension of 20-mm long, 2-mm wide, and subsequently cured with a light emitting diode curing unit at an intensity of 800 mW/cm<sup>2</sup> for 40 s from the coronal aspect (SmartLite, Dentsply, PA). The degrees of curing conversions were investigated by using FT-IR to calculate the peak area transition at 1638 cm−<sup>1</sup> of aliphatic C=C double bond of acrylate group according to the following equation (1) [30]:

$$\text{Degree of conversion }\% \text{ (DC\%)} = \left[1 - \frac{[A\_{1638}]\_{\text{after during}}}{[A\_{1638}]\_{\text{before during}}}\right] \times 100\% \tag{1}$$

#### *2.3. Curing Condition and Depth Test 2.3. Curing Condition and Depth Test*

To evaluate the feasibility of the sealers for root canal, the flow and viscosity tests were carried out for UA resins according to ISO 6876:2001. Based on the specifications, the mixture (0.05 mL) of UA resin and TPGDA (UAT65/TPGDA or UAC50/TPGDA) was placed on the center of glass plate using a graduated syringe. After the initial mixing for 180 s (±5 s), another glass plate was placed on top of the sealer, following a weight providing a total mass of 120 g (±2 g). After 10 min, the weight was removed in order to measure the maximum and minimum diameter of the compressed disks of To evaluate the feasibility of the sealers for root canal, the flow and viscosity tests were carried out for UA resins according to ISO 6876:2001. Based on the specifications, the mixture (0.05 mL) of UA resin and TPGDA (UAT65/TPGDA or UAC50/TPGDA) was placed on the center of glass plate using a graduated syringe. After the initial mixing for 180 s (±5 s), another glass plate was placed on top of the sealer, following a weight providing a total mass of 120 g (±2 g). After 10 min, the weight was removed in order to measure the maximum and minimum diameter of the compressed disks of the

the sealers [31]. Apart from that, the viscosities of the sealers were measured by using a syringe-based viscometer based on Instron 3360 series universal testing system [32]. In Figure 3, the measurement

sealers [31]. Apart from that, the viscosities of the sealers were measured by using a syringe-based viscometer based on Instron 3360 series universal testing system [32]. In Figure 3, the measurement of maximal curing increment thickness for resin composites was conducted by using a ISO 4049:2009 method [33]. The curing process was performed with a 2 × 2 × 20 mm re-usable stainless-steel mold with a top cover to prevent materials' leakage. After curing process under a light source of halogen lamp for 40 s, all samples were kept in saturated humidity at 37 ◦C for 24 h to measure the curing depths. The mechanical properties were also measured by using the Universal Testing Instrumentals according to ASTM D412-98a with a stretching rate at 100 mm/min. The tensile tests were conducted for five times on an average. *Polymers* **2019**, *11*, x FOR PEER REVIEW 5 of 21 of maximal curing increment thickness for resin composites was conducted by using a ISO 4049:2009 method [33]. The curing process was performed with a 2 × 2 × 20 mm re-usable stainless-steel mold with a top cover to prevent materials' leakage. After curing process under a light source of halogen lamp for 40 s, all samples were kept in saturated humidity at 37 °C for 24 h to measure the curing depths. The mechanical properties were also measured by using the Universal Testing Instrumentals according to ASTM D412-98a with a stretching rate at 100 mm/min. The tensile tests were conducted for five times on an average.

**Figure 3.** The test method for curing depths according to ISO 4049:2009 [33]. **Figure 3.** The test method for curing depths according to ISO 4049:2009 [33].

#### *2.4. Biocompatibility Test 2.4. Biocompatibility Test*

Biocompatibility tests were performed by using the ISO 10993-5 regulation through Alamar Blue assay and lactate dehydrogenase (LDH) assay [34]. Alamar Blue assay was carried out according to the following steps: first, 3T3 cells were cultured under 10% fetal bovine serum (FBS) Dulbecco's modified eagle's medium (DMEM) at 37 °C in the presence of 5% CO2. All Cells were subcultured twice before the following experiment. In the next step, the specimens were polymerized under photo-polymerization machine on top of a round Teflon mold, which is 8 mm in diameter and 2 mm in height, in a distance of 0.5 cm for 40 s. After the polymerization, the UA composites were then removed from the mold, sterilized by UV-light for 24 h, and soaked in fresh medium at 37 °C for 24 h. In the final step, the medium containing 3T3 cells were added to 96-well plates with a cell number of 104/well. After the cells fully adhered on the plate, the original cell culture medium was replaced by the extract material. The cell incubation for Alamar blue assay cell activity analysis was conducted at 37 °C for 1, 3, and 7 days in the presence of 5% CO2. Moreover, the evaluation of LDH assay for cytotoxicity were also conducted for 1, 3, and 7 days by using 3T3 cells under similar culture condition in 10% FBS DMEM at 37 °C in the presence of 5% CO2. Biocompatibility tests were performed by using the ISO 10993-5 regulation through Alamar Blue assay and lactate dehydrogenase (LDH) assay [34]. Alamar Blue assay was carried out according to the following steps: first, 3T3 cells were cultured under 10% fetal bovine serum (FBS) Dulbecco's modified eagle's medium (DMEM) at 37 ◦C in the presence of 5% CO2. All Cells were subcultured twice before the following experiment. In the next step, the specimens were polymerized under photo-polymerization machine on top of a round Teflon mold, which is 8 mm in diameter and 2 mm in height, in a distance of 0.5 cm for 40 s. After the polymerization, the UA composites were then removed from the mold, sterilized by UV-light for 24 h, and soaked in fresh medium at 37 ◦C for 24 h. In the final step, the medium containing 3T3 cells were added to 96-well plates with a cell number of 104/well. After the cells fully adhered on the plate, the original cell culture medium was replaced by the extract material. The cell incubation for Alamar blue assay cell activity analysis was conducted at 37 ◦C for 1, 3, and 7 days in the presence of 5% CO2. Moreover, the evaluation of LDH assay for cytotoxicity were also conducted for 1, 3, and 7 days by using 3T3 cells under similar culture condition in 10% FBS DMEM at 37 ◦C in the presence of 5% CO2.
