Composite resin reinforced with silver nanoparticles–laden hydroxyapatite nanowires for dental application

Abstract

Objective

The object is to find a functional one-dimensional nanofibrous filler for composite resin, which is able to provide both efficient reinforcement and high antibacterial activity.

Methods

Hydroxyapatite (HA) nanowires were synthesized via hydrothermal technique using calcium oleate as the precursor. Polydopamine (PDA)–coated HA (HA–PDA) nanowires were prepared by soaking HA nanowires in dopamine (DA) aqueous solution. Silver nanoparticles (AgNPs)–laden HA (HA–PDA–Ag) nanowires were prepared via reduction reaction by adding silver nitrate and glucose into HA–PDA suspensions in DI water. The resulted HA–PDA–Ag nanowires were then mixed into Bis-GMA/TEGDMA (50/50, w/w) at 4–10 wt.%, thermal-cured, and submitted to characterizations including mechanical properties, interfacial adhesion between filler and resin matrix, distribution of HA nanowires and AgNPs, as well as silver ion release, cytotoxicity and antibacterial activity.

Results

HA–PDA–Ag nanowires were readily obtained and the loading amounts of AgNPs could be controlled by adjusting the feeding doses of silver nitrate and HA–PDA nanowires. Benefiting from the PDA surface layer, HA–PDA–Ag nanowires could disperse well in composite resin and form good interfacial adhesion with the resin matrix. In comparison with neat resin, significant increases in flexural strength and modulus of cured composites were achieved at the addition amounts of HA–PDA–Ag nanowires being 6–8 wt.%. The distribution of AgNPs was homogeneous throughout the resin matrix in all designs, which endowed the composites with high antibacterial activity against streptococcus mutans. Continuous silver ion release from composites was detected, however, it was determined the composites would have insignificant cytotoxicity based on the proliferation of L929 fibroblasts in extracts of HA–PDA–Ag nanowires.

Significance

The finding proved that HA–PDA–Ag nanowires could serve as functional nanofillers for composite resins, which should help much in developing materials for satisfactory long-term clinical restorations.

Introduction

Composite resins are playing irreplaceable roles in dental restorations benefiting from their multiple advantages such as good maneuverability, excellent esthetics and acceptable biocompatibility . In decades, the organic components of composite resins remained almost unchanged, which are mainly methacrylate-type resins like bisphenol A-glycidyl methacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) . While numerous attempts have been kept being carried out in relating to inorganic fillers for the purpose to develop high performance composite resins . To achieve satisfactory long-term clinical restorations, composite resins are required to have enough mechanical properties, low polymerization shrinkage and high wear resistance, as well as antibacterial activity etc. These requirements are normally met via the introduction of diverse functionalized fillers .

Conventional inorganic fillers for composite resins are mainly particular barium glass, silica and zirconium dioxide powders . To the present, their sizes have developed from microhybrid, to microfill and further to nanofill . However, within the last decade, fibrous fillers for composite resins have attracted much attention because one-dimensional fillers displayed unique advantages as reinforcements in comparison with particular fillers . Due to the effects of fiber bridging and pullout, it was reported that glass fibers or whiskers were able to transfer load more efficiently than particular fillers . Similarly, hydroxyapatite (HA) whiskers were demonstrated to contribute much in improving mechanical properties of composite resins via mechanisms including whisker pullout, crack deflection, bridging and pinning . In some other reports, the use of fibrous fillers was thought able to reduce polymerization shrinkage benefiting from the overlapping of the fibers .

Among various fibrous fillers, nanofibers were especially deemed as promising fillers for composite resins because of their high specific surface area, high surface free energy and high aspect ratio . These features made them able to effectively improve the flexural strength and the fracture toughness of composite resins. For instance, Guo et al. mixed zirconia–silica (ZS) or zirconia–yttria–silica (ZYS) ceramic nanofibers into Bis-GMA resin to obtain dental composites with improved mechanical properties. Chen et al. found that the mechanical properties of dental composites could be improved with the incorporation of only a small mass fraction of HA nanofibers. Moreover, the incorporation of nanofibrous fillers was reported able to reduce polymerization shrinkage and to improve wear resistance at the same time .

Antibacterial function of composite resins was helpful in controlling secondary caries adjacent to the filling, which was reported the main cause for numerous restoration failures . Antibacterial composite resins were usually achieved by introducing fluoride-releasing fillers like strontium fluoride (SrF 2 ) or ytterbium trifluoride (YbF 3 ) , quaternary ammonium or silver-containing fillers . Due to the universal antibacterial activity of silver ions against both gram-positive and gram-negative bacteria, numerous studies have been carried out by introducing fillers like silver ion doped silica and silica glass into composite resins . In addition to released silver ions, silver nanoparticles (AgNPs) could also endow composite resins with antibacterial activity by mixing AgNPs .

In considering the addition of nano-scaled fillers into composite resins, however, a primary difficulty was to get their homogeneous dispersion in the polymeric matrixes . To achieve homogeneous dispersion of AgNPs in dental resins, some studies used silver 2-ethylhexanoate as the precursor and AgNPs formed in situ with excellent dispersibility . While silane coupling agent treatment and surface grafting modification were the two most applied strategies to ameliorate this problem for inorganic nanofillers . For instance, SiO 2 nanofibers were treated with γ-methacryloxypropyltrimethoxysilane and mixed with Bis-GMA/TEGDMA resin , HA nanofibrous fillers were treated with glycoxalic acid and added into dental resin . The effect of these surface modifications on improving dispersibility and interfacial adhesion was positive, but still having limitations for practical uses . In recent years, surface modification on nano-scaled fillers with mussel-inspired dopamine (DA) was highlighted in preparing organic-inorganic composites . It was suggested that the good affinity of polydopamine (PDA) modified fillers to resin matrixes resulted from the strong bonding between DA units and resin macromolecules via hydrogen bonding or π–π interaction, which enhanced both the dispersion of fillers and the effective interfacial stress transfer between different phases . Interestingly, the catechol group in DA was reported able to reduce silver ions into AgNPs and bind the nanoparticles firmly .

Therefore, in this study, novel functional nanofibrous filler was proposed and tested for preparing high performance antibacterial composite resin. The idea was to produce HA nanowires via hydrothermal technique, followed by DA modification and loading of AgNPs. These AgNPs–laden HA nanowires were mixed into Bis-GMA/TEGDMA resin to get cured composites. The dispersions of both HA nanowires and AgNPs in composite resins were supposed homogeneous in viewing of the features of PDA surface modification. Afterwards, evaluations on mechanical properties, silver ion release, cytotoxicity and antibacterial activity were carried out. Targeting for clinical applications, the favorable hypothesis of the present study is that the one-dimensional AgNPs–laden HA nanowires are able to serve as efficient reinforcement for composite resin and endow composite resin with high antibacterial activity.

Materials and methods

Materials

Dental resins as Bis-GMA and TEGDMA, as well as polyvinyl pyrrolidone (PVP, molecular weight 1,300,000) were purchased from Sigma–Aldrich and used directly. Benzoyl peroxide (BPO) was also purchased from Sigma–Aldrich and used after recrystallization. Dopamine hydrochloride (DA·HCl) and tris(hydroxymethyl aminomethane) (Tris) were purchased from Alfa Aesar. Other reagents required for the study, including oleic acid, calcium chloride (CaCl 2 ), sodium dihydrogen phosphate dihydrate (NaH 2 PO 4 ·2H 2 O), silver nitrate (AgNO 3 ), sodium hydroxide (NaOH), ammonium hydroxide (NH 3 ·H 2 O) and glucose, were purchased from Beijing Chemical Works (China) and used without further purification.

Synthesis of HA nanowires

HA nanowires were synthesized by using calcium oleate as the precursor via hydrothermal process . Initially, CaCl 2 (0.44 g) and NaOH (2 g) were dissolved in DI water (40 mL), respectively. Then, the aqueous solutions of CaCl 2 and NaOH were added dropwise into an ethanol (24 g) solution containing oleic acid (24 g) one after another under continuous stirring. Subsequently, 20 mL aqueous solution of NaH 2 PO 4 ·2H 2 O (0.48 g) was added dropwise into the system under continuous stirring. Thereafter, the resulting mixture was transferred into a Teflon-lined stainless steel autoclave (200 mL) and heated at 180 °C for 24 h. After the reaction, the obtained suspension was centrifuged and the precipitates were washed three times with ethanol and DI water, followed by freeze-drying to get HA nanowires.

Preparation of AgNPs–laden HA nanowires

Before the loading of AgNPs, HA nanowires were surface modified via the oxidized self-polymerization of dopamine to generate an active PDA coating. Briefly, freshly made HA nanowires (0.2 g) were dispersed in 100 mL Tris solution (10 mM) with pH being adjusted to 8.5 using HCl solution. Dopamine hydrochloride was then added into the system to obtain a DA aqueous solution of 2 mg mL −1 . The coating reaction was performed at room temperature under continuous stirring for 48 h. Subsequently, the modified HA nanowires (HA–PDA) were separated, washed three times with ethanol and DI water, and freeze-dried.

Tollens’ reagents were pre-prepared by dissolving different amounts of AgNO 3 in DI water (0.1 g/L, 0.5 g/L, 1.0 g/L) with the addition of NH 3 ·H 2 O to get transparent solutions. To the solutions, PVP (5 wt.%) was dissolved and acted as dispersant. Then, HA–PDA nanowires (0.3 g) were dispersed into the solutions (100 mL), followed by the addition of glucose (glucose: Ag + = 0.6 in mole) to reduce silver ions. The reaction was performed at room temperature for 8 h under continuous stirring. Subsequently, the AgNPs–laden HA nanowires (HA–PDA–Ag) were washed three times with ethanol and DI water, and freeze-dried. HA–PDA–Ag samples made from Tollens’ reagents with different concentrations of AgNO 3 were termed as HA–PDA–Ag-0.1, HA–PDA–Ag-0.5 and HA–PDA–Ag-1.0, accordingly.

Preparation of Bis-GMA/TEGDMA composites

Bis-GMA/TEGDMA (50/50, w/w) composites containing different mass fractions (0, 4, 6, 8 or 10 wt.%) of HA–PDA–Ag-1.0 were prepared via thermocuring with the addition of BPO (0.5 wt.%). Initially, HA–PDA–Ag-1.0 nanowires were dispersed in low viscous TEGDMA with the aid of ultrasonication. The suspensions were then mixed with viscous Bis-GMA, in which, BPO was dissolved in advance. Subsequently, the resin/filler mixtures were transferred into aluminum alloy molds, vacuum-degassed and thermal cured for 12 h at 120 °C. The thermocuring temperature of the resin mixture was determined by using a DSCQ20 differential scanning calorimetry (TA instruments, USA).

Flexural properties

Flexural strength ( Fs ) and flexural modulus ( Ey ) of Bis-GMA/TEGDMA composites were measured by the three-point bending test on a universal test machine (Instron 1121, UK) according to ISO 10477:92 standard. After the thermocuring, beam-shaped composite specimens were retrieved from the molds and carefully polished to the size of 25 × 2 × 2 mm (l × w × h) with 600 grit silicon carbide paper before the test. Fs and Ey were calculated from the following formulae:

Fs = 3 Fl /2 bh 2
Ey = l 3 F 1 /4 fbh 3

where F is the applied load ( N ) at the highest point of the load deflection curve, l is the span length (20 mm), b is the width of the test specimen, and h is its thickness, F 1 is the load ( N ) at a convenient point in the straight line portion of the trace, f is the deflection (mm) of the test specimen at load F 1 . Eight replicate samples were tested for each kind of composite specimen for averaging.

Characterizations

Crystal structures of prepared HA, HA–PDA and HA–PDA–Ag nanowires were analyzed by X-ray diffractometer (XRD, D/Max2500VB2+, Rigaku, Japan) using Cu Kα radiation with a fixed incidence of 1° at a 2 θ scanning rate of 10°/min in the range of 5–90°. Surface chemical compositions of all the nanowires were determined by X-ray photoelectron spectroscopy (XPS), which were performed on an ESCA Lab250 XPS spectrometer (Thermo Electron Corporation, USA) with an Al Kα X-ray source (1486.6 eV photons) under vacuum (10 −8 Torr or lower) using an incidence angle of 45° at a power of 150 W. In order to compensate for surface charging effects, all binding energies (BEs) were referenced to the C 1s hydrocarbon peak at 285 eV. Amounts of PDA coating and AgNPs loading onto HA nanowires were determined by thermal gravity analysis (TGA) using a Q50 thermogravimetric analyzer (TA instruments, USA) in nitrogen atmosphere from room temperature to 800 °C at a heating rate of 20 °C/min.

Morphology observations were conducted both on scanning electron microscope (SEM, S4800, Hitachi, Japan) at an accelerating voltage of 15 kV and transmission electron microscope (TEM, H-800, Hitachi, Japan) at an accelerating voltage of 200 kV, intending to confirm the synthesis of nanowires including HA, HA–PDA and HA–PDA–Ag. Before the SEM observation, samples were sputter-coated with platinum (30 mA, 30 s) using a sputter-coater (Polaron E5600, USA). Morphology of representative fracture surfaces of Bis-GMA/TEGDMA composites containing different mass fractions of HA–PDA–Ag nanowires were similarly examined using SEM. The distribution of HA–PDA–Ag in the resin matrix was illustrated by calcium and silver element mapping, which were performed under the same parameters as SEM observation with an exposure time of 180 s.

Silver ion release

Release behaviors of silver ions were conducted for both HA–PDA–Ag nanowires and Bis-GMA/TEGDMA composites containing HA–PDA–Ag nanowires. In brief, 0.5 g HA–PDA–Ag nanowires or 1.0 g Bis-GMA/TEGDMA composite specimens were soaked in 50 mL phosphate buffered saline (PBS, pH 7.4) and incubated at 37 °C with continuous agitation (60 rpm). The liquids were collected at predetermined time points and submitted to inductively coupled plasma optical emission spectrometer (ICP, ICPS-7500, Shimadzu, Japan) measurements to quantify the released silver ions. At the same time, fresh PBS was added to continue the release experiment. Three independent experiments were performed for averaging.

Cytotoxicity assay

L929 fibroblasts (purchased from Cell Culture Center, Peking Union Medical College, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, PAA, Germany), 100 IU/mL penicillin (Sigma), and 100 mg/mL streptomycin (Sigma). The culture was maintained with 5% CO 2 at 37 °C and saturated humidity until 80% confluence prior to use.

Referring to ISO 10993-12:200, nanowires including HA, HA–PDA and HA–PDA–Ag (2.0 g) were soaked in DMEM (10 mL) for 24 h, and the extracts were used for cell culture. Before the soaking, all the nanowires were kept in 70% ethanol with exposure to ultraviolet light for 2 h, and then washed three times with PBS. To each well of 96-well plates, 1 × 10 4 cells were seeded and incubated with various extracts at 37 °C in a humidified atmosphere with 5% CO 2 , using DMEM as a negative control and phenol solution (6.4 g L −1 ) as a positive control. The media were refreshed every two days. Cell proliferation was analyzed using Cell Counting Kit-8 (CCK-8, Beyotime, China). Briefly, at 1, 3, 5, and 7 days, 20 μL of CCK-8 solution was added into each well and incubated at 37 °C for 4 h, and then the OD values were measured using a microreader (Bio-Rad 680) at a wavelength of 450 nm.

Antibacterial activity

The antibacterial activities of Bis-GMA/TEGDMA composite resins containing different mass fractions of HA–PDA–Ag nanowires were evaluated using pathogenic bacterial strains of streptococcus mutans (S.M., from Tianjin Medical University) by live/dead assay. The S.M. suspension was diluted to ∼1 × 10 7 colony-forming unit (CFU) per milliliter by adding Brain Heart Infusion (BHI, Beijing Solarbio Science & Technology Co., Ltd.) broth. The inoculum of bacterial suspension (2 μL) was instilled onto the surfaces of composite resin specimens and incubated for 24 h at 37 °C. Subsequently, the specimens were stained with acridine orange/ethidium bromide (AO/EB), and observed under laser confocal scanning microscope (LCSM, TCS SP8, Leica).

Statistical analysis

The results of flexural strength and modulus were represented as mean ± standard deviation for n = 8. All biological quantitative data were represented as mean ± standard deviation for n = 3. Statistical analysis was made based on t-tests and a difference between groups of *p < 0.05 was considered significant and **p < 0.01 was considered highly significant.

Materials and methods

Materials

Dental resins as Bis-GMA and TEGDMA, as well as polyvinyl pyrrolidone (PVP, molecular weight 1,300,000) were purchased from Sigma–Aldrich and used directly. Benzoyl peroxide (BPO) was also purchased from Sigma–Aldrich and used after recrystallization. Dopamine hydrochloride (DA·HCl) and tris(hydroxymethyl aminomethane) (Tris) were purchased from Alfa Aesar. Other reagents required for the study, including oleic acid, calcium chloride (CaCl 2 ), sodium dihydrogen phosphate dihydrate (NaH 2 PO 4 ·2H 2 O), silver nitrate (AgNO 3 ), sodium hydroxide (NaOH), ammonium hydroxide (NH 3 ·H 2 O) and glucose, were purchased from Beijing Chemical Works (China) and used without further purification.

Synthesis of HA nanowires

HA nanowires were synthesized by using calcium oleate as the precursor via hydrothermal process . Initially, CaCl 2 (0.44 g) and NaOH (2 g) were dissolved in DI water (40 mL), respectively. Then, the aqueous solutions of CaCl 2 and NaOH were added dropwise into an ethanol (24 g) solution containing oleic acid (24 g) one after another under continuous stirring. Subsequently, 20 mL aqueous solution of NaH 2 PO 4 ·2H 2 O (0.48 g) was added dropwise into the system under continuous stirring. Thereafter, the resulting mixture was transferred into a Teflon-lined stainless steel autoclave (200 mL) and heated at 180 °C for 24 h. After the reaction, the obtained suspension was centrifuged and the precipitates were washed three times with ethanol and DI water, followed by freeze-drying to get HA nanowires.

Preparation of AgNPs–laden HA nanowires

Before the loading of AgNPs, HA nanowires were surface modified via the oxidized self-polymerization of dopamine to generate an active PDA coating. Briefly, freshly made HA nanowires (0.2 g) were dispersed in 100 mL Tris solution (10 mM) with pH being adjusted to 8.5 using HCl solution. Dopamine hydrochloride was then added into the system to obtain a DA aqueous solution of 2 mg mL −1 . The coating reaction was performed at room temperature under continuous stirring for 48 h. Subsequently, the modified HA nanowires (HA–PDA) were separated, washed three times with ethanol and DI water, and freeze-dried.

Tollens’ reagents were pre-prepared by dissolving different amounts of AgNO 3 in DI water (0.1 g/L, 0.5 g/L, 1.0 g/L) with the addition of NH 3 ·H 2 O to get transparent solutions. To the solutions, PVP (5 wt.%) was dissolved and acted as dispersant. Then, HA–PDA nanowires (0.3 g) were dispersed into the solutions (100 mL), followed by the addition of glucose (glucose: Ag + = 0.6 in mole) to reduce silver ions. The reaction was performed at room temperature for 8 h under continuous stirring. Subsequently, the AgNPs–laden HA nanowires (HA–PDA–Ag) were washed three times with ethanol and DI water, and freeze-dried. HA–PDA–Ag samples made from Tollens’ reagents with different concentrations of AgNO 3 were termed as HA–PDA–Ag-0.1, HA–PDA–Ag-0.5 and HA–PDA–Ag-1.0, accordingly.

Preparation of Bis-GMA/TEGDMA composites

Bis-GMA/TEGDMA (50/50, w/w) composites containing different mass fractions (0, 4, 6, 8 or 10 wt.%) of HA–PDA–Ag-1.0 were prepared via thermocuring with the addition of BPO (0.5 wt.%). Initially, HA–PDA–Ag-1.0 nanowires were dispersed in low viscous TEGDMA with the aid of ultrasonication. The suspensions were then mixed with viscous Bis-GMA, in which, BPO was dissolved in advance. Subsequently, the resin/filler mixtures were transferred into aluminum alloy molds, vacuum-degassed and thermal cured for 12 h at 120 °C. The thermocuring temperature of the resin mixture was determined by using a DSCQ20 differential scanning calorimetry (TA instruments, USA).

Flexural properties

Flexural strength ( Fs ) and flexural modulus ( Ey ) of Bis-GMA/TEGDMA composites were measured by the three-point bending test on a universal test machine (Instron 1121, UK) according to ISO 10477:92 standard. After the thermocuring, beam-shaped composite specimens were retrieved from the molds and carefully polished to the size of 25 × 2 × 2 mm (l × w × h) with 600 grit silicon carbide paper before the test. Fs and Ey were calculated from the following formulae:

Fs = 3 Fl /2 bh 2
Ey = l 3 F 1 /4 fbh 3

where F is the applied load ( N ) at the highest point of the load deflection curve, l is the span length (20 mm), b is the width of the test specimen, and h is its thickness, F 1 is the load ( N ) at a convenient point in the straight line portion of the trace, f is the deflection (mm) of the test specimen at load F 1 . Eight replicate samples were tested for each kind of composite specimen for averaging.

Characterizations

Crystal structures of prepared HA, HA–PDA and HA–PDA–Ag nanowires were analyzed by X-ray diffractometer (XRD, D/Max2500VB2+, Rigaku, Japan) using Cu Kα radiation with a fixed incidence of 1° at a 2 θ scanning rate of 10°/min in the range of 5–90°. Surface chemical compositions of all the nanowires were determined by X-ray photoelectron spectroscopy (XPS), which were performed on an ESCA Lab250 XPS spectrometer (Thermo Electron Corporation, USA) with an Al Kα X-ray source (1486.6 eV photons) under vacuum (10 −8 Torr or lower) using an incidence angle of 45° at a power of 150 W. In order to compensate for surface charging effects, all binding energies (BEs) were referenced to the C 1s hydrocarbon peak at 285 eV. Amounts of PDA coating and AgNPs loading onto HA nanowires were determined by thermal gravity analysis (TGA) using a Q50 thermogravimetric analyzer (TA instruments, USA) in nitrogen atmosphere from room temperature to 800 °C at a heating rate of 20 °C/min.

Morphology observations were conducted both on scanning electron microscope (SEM, S4800, Hitachi, Japan) at an accelerating voltage of 15 kV and transmission electron microscope (TEM, H-800, Hitachi, Japan) at an accelerating voltage of 200 kV, intending to confirm the synthesis of nanowires including HA, HA–PDA and HA–PDA–Ag. Before the SEM observation, samples were sputter-coated with platinum (30 mA, 30 s) using a sputter-coater (Polaron E5600, USA). Morphology of representative fracture surfaces of Bis-GMA/TEGDMA composites containing different mass fractions of HA–PDA–Ag nanowires were similarly examined using SEM. The distribution of HA–PDA–Ag in the resin matrix was illustrated by calcium and silver element mapping, which were performed under the same parameters as SEM observation with an exposure time of 180 s.

Silver ion release

Release behaviors of silver ions were conducted for both HA–PDA–Ag nanowires and Bis-GMA/TEGDMA composites containing HA–PDA–Ag nanowires. In brief, 0.5 g HA–PDA–Ag nanowires or 1.0 g Bis-GMA/TEGDMA composite specimens were soaked in 50 mL phosphate buffered saline (PBS, pH 7.4) and incubated at 37 °C with continuous agitation (60 rpm). The liquids were collected at predetermined time points and submitted to inductively coupled plasma optical emission spectrometer (ICP, ICPS-7500, Shimadzu, Japan) measurements to quantify the released silver ions. At the same time, fresh PBS was added to continue the release experiment. Three independent experiments were performed for averaging.

Cytotoxicity assay

L929 fibroblasts (purchased from Cell Culture Center, Peking Union Medical College, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, PAA, Germany), 100 IU/mL penicillin (Sigma), and 100 mg/mL streptomycin (Sigma). The culture was maintained with 5% CO 2 at 37 °C and saturated humidity until 80% confluence prior to use.

Referring to ISO 10993-12:200, nanowires including HA, HA–PDA and HA–PDA–Ag (2.0 g) were soaked in DMEM (10 mL) for 24 h, and the extracts were used for cell culture. Before the soaking, all the nanowires were kept in 70% ethanol with exposure to ultraviolet light for 2 h, and then washed three times with PBS. To each well of 96-well plates, 1 × 10 4 cells were seeded and incubated with various extracts at 37 °C in a humidified atmosphere with 5% CO 2 , using DMEM as a negative control and phenol solution (6.4 g L −1 ) as a positive control. The media were refreshed every two days. Cell proliferation was analyzed using Cell Counting Kit-8 (CCK-8, Beyotime, China). Briefly, at 1, 3, 5, and 7 days, 20 μL of CCK-8 solution was added into each well and incubated at 37 °C for 4 h, and then the OD values were measured using a microreader (Bio-Rad 680) at a wavelength of 450 nm.

Antibacterial activity

The antibacterial activities of Bis-GMA/TEGDMA composite resins containing different mass fractions of HA–PDA–Ag nanowires were evaluated using pathogenic bacterial strains of streptococcus mutans (S.M., from Tianjin Medical University) by live/dead assay. The S.M. suspension was diluted to ∼1 × 10 7 colony-forming unit (CFU) per milliliter by adding Brain Heart Infusion (BHI, Beijing Solarbio Science & Technology Co., Ltd.) broth. The inoculum of bacterial suspension (2 μL) was instilled onto the surfaces of composite resin specimens and incubated for 24 h at 37 °C. Subsequently, the specimens were stained with acridine orange/ethidium bromide (AO/EB), and observed under laser confocal scanning microscope (LCSM, TCS SP8, Leica).

Statistical analysis

The results of flexural strength and modulus were represented as mean ± standard deviation for n = 8. All biological quantitative data were represented as mean ± standard deviation for n = 3. Statistical analysis was made based on t-tests and a difference between groups of *p < 0.05 was considered significant and **p < 0.01 was considered highly significant.

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Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Composite resin reinforced with silver nanoparticles–laden hydroxyapatite nanowires for dental application
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