Green synthesis and application of silver nano-biotics for enhancing the antimicrobial activity of glass-ionomer cement against dental biolm and its effect on compressive strength

The development of dental caries is associated with various microorganisms and secondary caries formation is the main cause of their failure. The urge for restorative dental materials that have antimicrobial properties has stimulated the introduction of materials containing different antibacterial agents. Present study has been designed to synthesize silver nanoparticles (AgNPs) and incorporate AgNPs and amoxicillin into glass ionomer cement (GIC) to synergize its effect on oral microbes. The effect of the added antimicrobial agents on compressive strength of GIC was also evaluated. without impairing mechanical properties of the set cement.


Introduction
The oral cavity harbors an abundant number of pathogens that may cause different oral diseases. The development of dental caries and periodontal diseases is associated with various Gram-positive and Gram-negative microorganisms [1]. Secondary caries formation, at the margins of existing restorations, is the main cause of their failure. This can result from adhesion of cariogenic bio lms to the tooth or restoration surface with subsequent ingress of cariogenic bacteria, in absence of antibacterial cover by the restorative material [2]. Thus, restorative materials are preferred to have some unique properties such as uoride release to prevent secondary caries, bactericidal or bacteriostatic effect, and tight bacterial seal [3]. Since their introduction to the markets, glass-ionomer cements (GICs) showed the most potent anticariogenic tendency among dental materials due to its uoride releasing ability and remineralization potential [4,5]. The urge for restorative dental materials that have antimicrobial properties has stimulated the introduction of materials containing different antibacterial agents. Among the antimicrobials incorporated into the dental restorative material, the monomers 12-methacryloyloxydo-decylpyridinium bromide (MDPB), chlorhexidine digluconate (CHX), silver nanoparticles (AgNPs) and antibiotics are the most frequently used [5][6][7][8]. Although the addition of antibiotics to glass ionomer cement (GIC), may enhance its antibacterial effect, however, this may increase the possibility of developing antibiotic resistance [8]. A promising approach to the management of emerging bacterial resistance in dental restorative material is antibiotic formulated in nano-structures with dimensions of approximately 1 to 100 mm [9]. The newly designed formulations are known as nano-antibiotics or nanobiotics. Silver nanoparticles (AgNPs) are most extensively tested inorganic antimicrobial agents due to their long term antibacterial activity, low bacterial resistance, low volatility, high surface to volume ratio and high thermal stability [10,11]. The exact mechanism how the AgNPs act is unknown. However, studies have shown that, because of their nano size, silver particles penetrate the microbial cell wall/cell membranes by sulfer-containing proteins or thiol groups and damages the microbial DNA and ultimately leads to cell death [11,12]. Studies have shown, AgNPs synergize the e cacy of antibiotics like amoxicillin, clindamycin, vancomycin and penicillin G against various microbes [13,14]. Because of its antimicrobial activity and synergizing effects, researchers have studied the use of AgNPs in several areas of dentistry which include dental biomaterials [15][16][17], dental implants [18], orthodontic adhesives [19] etc. In vitro studies have shown, addition of AgNPs in to dental restorative materials inhibit growth of common oral microbes like Streptococcusmutans, Streptococcus sanguinis,and Lactobacillus through bactericidal and bacteriostatic effects of AgNPs on oral microbes [16,20] without altering its mechanical properties [21]. With growing bacterial resistance to amoxicillin [22,23], commonly used antibiotic in oral infection and synergistic effect of AgNPs with amoxicillin and its antimicrobial effect on oral microbes were studied.
The present study has been designed to assess the synergistic effect of silver nano-biotics incorporated into glass ionomer against oral microbes.

Materials
The current study was performed using one restorative material, conventional GIC Fuji IX GP (powder/liquid form), GC Corporation (Tokyo, Japan,) and two antimicrobial agents, silver nanoparticles and Amoxicillin (1.5% w/w) antibiotic. AgNPs were biosynthesized using Cupressus macrocarpa extract at a particle size of < 100 nm. The Silver nano -powder was weighed on a balance accurate to 0.0001 g (TS4000, Ohaus, Pine Brook, NJ, USA) and mixed with GIC powder in a percentage of 0.5% (w/w) immediately prior to manipulation. For GIC manipulation, the same amount of powder was removed to add AgNP and/or amoxicillin. Two species of bacteria, S.mutans and S. aureus, were isolated from medical diagnosis and infection control unit, Faculty of Dentistry, Taif University.

Preparation of specimens and grouping
A total of 120 disc-shaped specimens were prepared for the study using a split Te on mold (6 mm in diameter and 3 mm in height) [24]. All specimens were prepared at room temperature (24 °C-27 °C), in 70% relative humidity. GIC powder and liquid were proportioned and mixed with a plastic spatula, as recommended by the manufacture, and placed in the molds. The molds were covered on both sides by polyester strips and thick glass plates and allowed to set for 20 min after which they were removed from the mold, sterilized for 30 min using ultraviolet radiation and stored at 37 ºC for 1 hour in sterilized distilled water before testing. Malformed or large voids containing specimens were discarded, The prepared specimens (n = 120) were classi ed into 4 main groups (30 specimens each) as follows: Group A (control), conventional GIC without any addition Group B, GIC with AgNPs Group C, GIC with Amoxicillin Group D, GIC with AgNPs and Amoxicillin Then each of the main groups was tested for the antimicrobial effect against 2 types of bacterial species, S. mutans and S. aureus, at three time intervals of 1 day, 1 week and 3 weeks. During the testing intervals, the specimens were stored in distilled water at 37 ºC.

Preparation of Cupressus macrocarpa extract and Biosynthesis of AgNPs
The fresh and healthy leaves of Cupressus macrocarpa plant plant from Taif region were collected and were washed with distilled water and aqueous extract was prepared (25 g/100 ml H 2 O) at 85 °C for 60 min. The extract was ltered and the ltrate was stored at 4 °C for further experiments as reducing agent and stabilizer, being usable for within 2 weeks [25]. Fifty mL of 1 mM AgNO 3 was added to 25 mL of aqueous extract and mixed on a magnetic stirrer. The thorough mixing of reaction mixture was ensured by vigorous shaking and then allowed to settle at room temperature. The change in color to dark brown from pale yellow con rmed AgNPs formation [26].

Characterization of AgNPs:
UV-visible spectral analysis UV-vis spectrometer (Shimadzu UV-1650) was used to record absorbance in the range of 300-700 nm. The UV-vis absorption spectroscopy was used to monitor the rate of AgNPs.
Transmission electron microscopy (TEM) particle size analysis Characterization of AgNPswas studied using TEM analysis.The size and morphology of the nanoparticles were measured with high resolution TEM (JEOL 100CX II with an accelerating voltage of 100 kV, at Assiut Electron Microscope Unit) [27].

X-ray Diffraction analysis
The AgNPs nature and size were determined using Shimadzu XRD (Shimadzu XD-3A, Japan). The AgNPs size was calculated using the formula of Debye-Scherrer [28].

Assessing the antimicrobial activity
The antimicrobial activity of each specimen in testing groups against oral pathogenic microorganisms; S. aureus, and S. mutans were tested based on Kirby-Bauer method [30]. The aerobic S. aureus and an aerobic S. mutans were grown in Mueller Hinton agar (MH-A) or brain heart infusion (BHI-A), respectively at 37℃ for 24 h. The antimicrobial activity of the samples was evaluated by determining the diameter of the clear zone of inhibition around the specimen discs. All samples were done in triplicate.

Determination of antibacterial effect rate (%)
Each specimen of the groups was placed in a 15-mm diameter glass bottles with a at bottom surface. One specimen of each group were put and incubated in 0.5 ml of S. aureus or S. mutans suspension of 1 × 10 6 cells/ml in broth growth medium MH-B and BHI-B, respectively for regular interval of times at 37 °C.
The control sample, 0.5 ml bacterial suspension of 1 × 10 6 cells/ml in broth media was incubated without specimens. After incubation, each sample was washed gently with distilled water and immediately transferred into 10 ml phosphate buffer. Each 100 µl of sample was platted on a Mueller Hinton agar or brain heart infusion (BHI) plate, respectively, spread evenly and incubated at 37 °C for 24 h, nally, the number of colonies (colony-forming unit: CFU) were counted. Antibacterial activity was expressed as the logarithm of CFUs on each specimen. Each colonization test was run in triplicate and repeated at ve separate times. The antibacterial effect in each group was calculated as a bactericidal ratio, which was calculated as follows: AE = C -T/C X100 where AE is an antibacterial rate (%), C is the number of the bacteria in the control sample (CFU/sample), and T is the number of bacteria in the testing samples (CFU/sample).
Characterization of bacterial morphology and anti-bio lm activity of restorative biomaterials by SEM The S. aureus morphology and anti-bio lm activity of restorative biomaterials were determined using the protocol given by Souza et al [31]. with some modi cations. In brief, each specimen of the restorative biomaterial groups was placed in 24-well plates containing 2 mL of MH-B with 5% Sucrose and 0.1 mL of human saliva. The 24-well plates were subsequently incubated at 37 o C under aerophilic conditions.After incubation, each sample was washed gently with distilled water. Scanning electron microscopy (SEM) of each sample with bacterial bio lm was immediately xed in cold glutaraldehyde for 2 hours, xed in the solution of osmium tetroxide (2%) for 2 hours, dehydrated in serial ascending ethanol and nally, dried using liquid CO 2 . For SEM, samples were mounted on stainless steel holders, sputter-coated with a thin layer of gold (22) and examined by JOEL SEM (JSM-5400LV). SEM examinations of samples were analyzed. Five specimens were examined for each group.

Measurement of compressive strength
Cylindrical specimens, of 4 mm diameter and 6 mm height, were prepared using a te on mold to test the compressive strength. Each specimens was allowed to set at room temperature for 20 minutes before its removal from the mold. Compressive strength (CS) of set cement was measured after 24 h of mixing using a Material Test System (810 MTS Co., Minneapolis, MN, USA) for all groups at a crosshead speed of 0.5 mm/min [21]. Ten specimens were tested for each group. The at end of the specimens were positioned between the MTS machine plates to apply the progressively increasing compressive load along the long-axis of specimen. The force-displacement curve was measured using LJ stream software.
The maximum recorded force at the fracture was taken and CS (N/mm 2 ) was recorded using the following equation. CS = 4P/ πd 2 d represents the diameter of the specimen and P represents the failure load.

Statistical analysis
Descriptive summary statistics were obtained for all independent and outcome variables. Difference in mean was tested using analysis of variance (ANOVA) followed by Tukey's post hoc and t-test. Analysis was performed using the Statistical Package for Social Science version 17 (SPSS INC Chicago link). All statistical tests were two-sided, and the signi cance level was set at p < 0.05.

Result And Discussion
Biosynthesis of AgNPs: Biosynthesis of AgNPs include the use of microorganism, plant extract and enzymes [32,33]. Plant parts like owers, fruits, stems, leaves, etc. are widely used in the biosynthesis of AgNPs because of its low cost, less time in processing and can be done on a small to large scale basis [32][33][34]. The morphology, stability and size of AgNPs formed depend on the method of its preparation, nature of the solvent used, temperature and strength of the reducing agent. On this study, AgNPs were biosynthesized using aqueous Cupressus macrocarpa extract because it contains reducing stabilizing agents [35,36]. In the present study formation of AgNPs was indicated by a change in color from pale yellow to dark brown (Figure 1-I-A). This is due to the fabrication of AgNPs with the molecular assistance of biological reducing agents present in the Cupressus macrocarpa extract. AgNPs showed brown color in aqueous solution due to the excitation of surface plasmon vibrations [26]. At lower concentrations of leaf extract, slower reduction was observed and vise versa. For 10% extract, fast reduction occurred as indicated by brown to dark brown color of the solution.

Characterization of AgNPs:
UV-Vis spectroscopy of AgNPs: For the con rmation of nanoparticles-formation, in the present study, the samples were removed and analyzed at regular time intervals using UV-Visible-spectroscopy. It was noticed that the complete color change took about 24 h, there after no further color of the reaction mixture changed. This indicated that AgNO 3 solution present in the reaction mixture has been reduced completely. AgNPs showed peaks at 429 nm as shown in (Figure 1-I-B). The UV-visible peak at 429 nm was for pure AgNPs. After complete incubation, AgNPs were centrifuged at 15,000g for 30 min followed by washing with MilliQ water. However, ethanol (50% V/V) was used to remove excess extract from the sample and this sample was kept at room temperature till further characterization. The study by Beg et al. [37] showed biosynthesis of AgNPs from seed extract of Pongamia pinnata had an absorption peak of 439 nm. The study by Vijaykumar et al. [38] showed an absorption maximum at 418 under Boerhaavia diffusa plant extract biosynthesis of AgNPs. Slight variation in absorption peak may be attributed to different quantities of the reducing chemicals present in the extracts.

Transmission Electron Microscope analysis (TEM)
The AgNPs were characterized using TEM analysis (Figure1-II). Average size of AgNPs was estimated to be at 13.5-25.8 nm. TEM analysis showed that all these AgNPs were of spherical to oblong polydispersed shape which supports the dispersion and stability of AgNPs with Cupressus macrocarpa extract. The successive Cupressus macrocarpa extract obtained was displayed various phytoconstituents. It revealed the presence of different phytoconstituents like glycosides, carbohydrates, avonoids, amino acid, phenolics, saponin, protein and sterols in the extracts [39].

X-ray Diffraction analysis
In the present study, X-ray Diffraction (XRD) pattern showed four major peaks at 2θ values of 38 o , 47 o , 55 o and 73 o . These characteristic peaks could be attributed to re ection planes (111), (200), (220) and (311) of face-centred cubic crystalline (FCC) structure of pure metallic silver (Figure 1-III).  Table 1). The bands at 1257 cm -1 and smaller peaks at 1053 and 750 cm-1 could be assigned to the stretching vibrations of C-N, C-O and =C-H, respectively. Additionally, the regions from 600 to 500cm -1 were due to the C-O and C-C groups' vibration modes. The reduction is due to the previous bands presented in Cupressus macrocarpa extract as shown in (Figure IV and Table 1). Cupressus macrocarpa extract contains saponins, phenolic compounds and carbohydrates as reported by Thukralet al. [39] and these components react as reducing and stabilizing agents. For this reason, we used Cupressus macrocarpa in biosynthesis of AgNPs.

Antimicrobial activity
Antimicrobial activity of biomaterial against S. mutans and S. aureus is presented in Figure 2 and Table 2. Twenty-four hour inhibition zone for the S. mutans and S. aureus was highest (29 ± 1.14 for S. muatns and 30 ±2.7 for S. aureus) for Group D with GIC,AgNPs and amoxicillin combination compared to other groups ( Table 2). Group B with GIC and AgNPs combination showed signi cantly high inhibitory zone against both the bacteria compared to Group A and Group C in twenty-four hour and one week inhibitory zone ( Table 2). Present study results are in line with the previously conducted studies [13,14,16,20] which have con rmed the synergistic effect of AgNPs against microbes in combination with antibiotics.
Present study showed mean inhibitory zone, which was signi cantly higher for S. aureus compared to S.mutans (Table 3). However, speci c response of each bacterium depended on their metabolic characteristics. These results come in agreement with the study conducted by Ortiz et al. [40] who showed that the most sensitive bacteria to AgNPs alone was S. aureus and the most resistant microorganisms were S. mutans in which no inhibition halo was generated. Additionally, Cupressus macrocarpa extract has strong anti-in ammatory and antibacterial properties, and these functions will further get enhanced in the presence of AgNPs [35,36]. The order of the antibacterial rate for Group D Group B Group C Group A was observed in Figure 3 and Table 4. Group D ensured 92.2% and 92.56% inhibition of S. aureus or S. mutant respectively. The inhibition of CFU in GIC supplemented with AgNPs (Group B) was higher compared to the GIC alone (Group A) and the GIC with amoxicillin (Group C) ( Figure  3).

Characterization of bacterial morphology and anti-bio lm activity
The occurrence of S. aureus in the oral cavity of patients with dental caries was con rmed in previous study recorded by Vellappally et al. [41] so, we studied the antibio lm e cacy on S. aureus bio lm using SEM (Figure 4). The e cacy of group A and group C on culture appeared as aggregated and clumped cell mass with normal cellular morphology. In sample of group B, slightly changes in cell morphology were seen. In group D high anti-bio lm e cacy and changes were seen including the lack of bacterial cell adhesion as well as cells misshapen and damaged membrane (Figure 4). This suggest, combination of AgNPs with GIC and Amoxicillin can disintegrate the structure of bio lm and even kill bacteria in the bio ms [42].
Present study results revealed that AgNPs and amoxicillin with GIC (group D) was more effective against S. aureus or S. mutans because it initially induced aggregates in the cell envelopes of S. aureus or S. mutans and eventually caused cell lyses [43]. In synergism, the bactericidal e cacy is enhanced by chelation between active groups like hydroxyl and amino groups present in these antibiotics with AgNPs.
As reporting by Krishna et al., [44] antibiotic-silver nanoparticles conjugate is formed in which a AgNPs core is surrounded by antibiotic molecules. Thus, the antimicrobial concentration is increased at the focal site, which leads to increased destruction of bacteria.
The microbiological study con rmed that group D has high antimicrobial activity. In the other hand, our study in Part B, recorded that AgNPs did not affect the mechanical property of GIC (Data not shown) and this was recorded in previous study by El-Wassefy et al [45].
Compressive strength of tested materials: Effect of AgNPs addition on physical properties of modi ed GIC was tested using compressive strength measurement. The results showed a minor increase in compressive strength of specimens that were modi ed with silver nanoparticles (groups B and D) ( Table  5). On the other hand, the addition of antibiotic particles to GIC caused a sligth decrease of its compressive strength. The difference between all groups was statistically insigni cant (P= 0.07). The effect of AgNPs may be attributed to their anchoring effect when distributed in the cement matrix, which helped to increase the cohesive strength of GIC. [15] Though, our results showed that the reinforcing effect of AgNPs was insigni cant, which can be explained by the fact that the nono-particles in the present study were physically added to the cement, so they were not rmly bonded to the matrix and did not alter the mechanical properties signi cantly. [21] The current results came in agreement with those of Paiva et al. [15] who concluded a reinforcing effect of AgNPs on the compressive strength of GIC, but their results showed a signi cant change. On the other hand, a study by El-Wassefy et al., [21] showed that AgNPs added to GIC decreased the material's strength, but the effect was insigni cant. The difference between the current results and others may be related to differences in the tested materials and the applied methodology

Conclusion
To conclude Cupressus macrocarpa extract bio-synthesized AgNPs showed good characterization and stability. It showed synergistic antimicrobial and anti-bio lm activity when combined with amoxicillin and GIC dental material. The excellent antibacterial results of AgNPs and amoxicillin with GIC make authors strongly recommend them as a relevant combination for dental application. The insigni cant impact of the added antimicrobial agents on compressive strength of GIC encourages their application with minimal effect on mechanical properties of the original cement.

Declarations
Con ict of interest: The authors declare that they have no con ict of interest.  Group A-control (glass inomer alone), Group B-Gi + AgNPs, Group C -Gi + Amoxicillin, Group D -Gi+AgNPs+Amoxicillin.