Biosynthesis of AgNPs:
Biosynthesis of AgNPs include the use of microorganism, plant extract and enzymes [32, 33]. Plant parts like flowers, 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 [32-34]. 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 . 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 confirmation 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 AgNO3 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.  showed biosynthesis of AgNPs from seed extract of Pongamia pinnata had an absorption peak of 439 nm. The study by Vijaykumar et al.  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, flavonoids, amino acid, phenolics, saponin, protein and sterols in the extracts .
X-ray Diffraction analysis
In the present study, X-ray Diffraction (XRD) pattern showed four major peaks at 2θ values of 38o, 47o, 55o and 73o. These characteristic peaks could be attributed to reflection planes (111), (200), (220) and (311) of face-centred cubic crystalline (FCC) structure of pure metallic silver (Figure 1-III).
Comparison of FTIR spectra of Cupressus macrocarpa extract and AgNPs
Fourier transform infrared analysis (FTIR) was used to characterize the Cupressus macrocarpa extract and the resulting AgNPs. Absorbance bands observed in the region 500 to 4000 cm-1 and approximately 1053, 1257, 1401, 1570, 1622, 2924, 3380 and 3459 cm-1 as shown in Figure IV-A and Table 1. FTIR analysis further confirmed the presence of functional groups representing bioactive compounds of Cupressus macrocarpa through the absorption bands at 3459 cm-1, implying Broad H-bonded-OH stretching, 1622 cm-1, denoting amide group, 1401 cm-1, representing methylene -CH bending bond, 1257 cm-1, representing phenol/C-O- stretching bond and 1053 cm-1 of primary alcohol/C-O- stretching as shown in Figure IV-A. Cupressus macrocarpa extract caused a reduction of silver ions. In the absorbance intensities at 3380 cm-1, representing OH stretching. Absorbance peak at 1622 cm-1 (amide group), 1401 cm-1 (methylene -CH bending), 1257 cm-1 (phenol/C-O- stretching), 1053 cm-1 (primary alcohol/C-O) and 543 cm-1 (–CH– stretch) have shown that organic compounds in the plant extract have reduced and stabilized AgNPs, thereby preventing agglomeration (Figure IV-B and 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.  and these components react as reducing and stabilizing agents. For this reason, we used Cupressus macrocarpa in biosynthesis of AgNPs.
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 significantly 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 confirmed the synergistic effect of AgNPs against microbes in combination with antibiotics. Present study showed mean inhibitory zone, which was significantly higher for S. aureus compared to S.mutans (Table 3). However, specific response of each bacterium depended on their metabolic characteristics. These results come in agreement with the study conducted by Ortiz et al.  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-inflammatory 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-biofilm activity
The occurrence of S. aureus in the oral cavity of patients with dental caries was confirmed in previous study recorded by Vellappally et al.  so, we studied the antibiofilm efficacy on S. aureus biofilm using SEM (Figure 4). The efficacy 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-biofilm efficacy 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 biofilm and even kill bacteria in the biofims .
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 . In synergism, the bactericidal efficacy is enhanced by chelation between active groups like hydroxyl and amino groups present in these antibiotics with AgNPs. As reporting by Krishna et al.,  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 confirmed 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 .
Compressive strength of tested materials: Effect of AgNPs addition on physical properties of modified GIC was tested using compressive strength measurement. The results showed a minor increase in compressive strength of specimens that were modified 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 insignificant (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.  Though, our results showed that the reinforcing effect of AgNPs was insignificant, 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 firmly bonded to the matrix and did not alter the mechanical properties significantly. 
The current results came in agreement with those of Paiva et al.  who concluded a reinforcing effect of AgNPs on the compressive strength of GIC, but their results showed a significant change. On the other hand, a study by El-Wassefy et al.,  showed that AgNPs added to GIC decreased the material’s strength, but the effect was insignificant. The difference between the current results and others may be related to differences in the tested materials and the applied methodology