Ola.doc J Bagh College Dentistry Vol. 26(4), December 2014 Effect of silver nitrate Restorative Dentistry 78 Effect of silver nitrate incorporation into heat polymerized acrylic resin on some mechanical properties Ola Khaleefah Ahmed Al-Husayni, B.D.S. (1) Nabeel Abdul Fatah Hatoor, B.D.S., M.Sc. (2) ABSTRACT Background: Polymers are very rarely used in their form. These modifications are carried out in order to improve the properties of polymers.Recently silver have been used successfully as antimicrobial (medical and dental) biomaterials that can prevent caries and infection of implants. The aim of the present in vitro study is to evaluate the effect of addition of silver nitrate to acrylic resin in different concentrationsthrough several tests part of these are: The effect of this additive on impact strength, transverse strength, and tensile strength of AgNO3 – loaded resin, and to assess any effect of addition of silver nitrate on coloration of acrylic resin. Materials and methods: Different concentrations of silver nitrate (9.375, 15, 30, 60, 120, 150, 300, 600 and 900 ppm) were prepared from stock solution of 1000 ppm silver nitrate. The specimens were prepared in accordance with the manufacturer's instructions and the tested silver nitrate solution was added to the acrylic resin powder and monomer in a fixed volume (0.2ml). Controls devoid of silver nitrate were included. Results: Fourier transform infra-red confirmed that there was no chemical bond between the Poly methyl methacrylate and silver nitrate. There was insignificant increasing (P=0.05) in impact strength observed when compared with control group. In transverse strength test, significant reduction was show (P<0.001). While for tensile strength there was insignificant reduction with 9.375(P=0.05NS) and 15(P=0.42NS) ppm silver nitrate. However, it was significant above 15 ppm (P<0.001). Darkening of silver nitrate -loaded resins were shown to be started with concentration of silver nitrateof 300 ppm and above. Conclusions: The additions of silver nitrate to acrylic resins yield good color stability and mechanical properties, depending on the concentration of silver nitrate. Keywords: Silver nitrate, acrylic resin, infra-red spectra, strength, coloration. (J Bagh Coll Dentistry 2014; 26(4):78-85). INTRODUCTION Little information is available about the impact of silver nitrate into heat polymerized acrylic resin. So this prompted us to shed light on this research. There is a need for effective broad -spectrum antimicrobial resin materials in dentistry; it is well-known that removable denture bases fabricated from heat-polymerized acrylic resins may act as a reservoir for microorganisms and contribute to re-infection in denture wearers 1. For elderly and institutionalized patients with limited motor skills and special needs, this treatment is further complicated because of some factors such as loss of memory, difficulty in rendering appropriate cleaning for their oral cavities.Unfortunately, current standers of treatment such as the use of antimicrobial mouthwashes, proper –tooth brushing technique have limited success or side -effects due to problems with patient compliance and the development of antibiotic resistance strains of bacteria. Thus a broad- spectrum antimicrobial resin is needed 2. Silver ions have been reported to inactivate important enzymes and affect the application mechanism of the DNA in bacteria. Ag ions have (1)Master student. Department of Prosthodontics, College of Dentistry, University of Baghdad. (2)Professor, Department of Prosthodontics, College of Dentistry, University of Baghdad. been reported to attach to the outer membrane and affect the permeability as well as induce structure changes in the cell – ultimately leading to cell death .In addition; Ag does not cause resistant bacterial strains to develop 3,4. For dental application, the development of other methods of drug elution, such as Ag-Zeolite and SiO2 filler were incorporated into urethane acrylic monomer in different amount to develop a new temporary filling materials with antibacterial activity against some oral bacterial growth 5, silver containing materials like Novaron, Amenitop and AIS were incorporated into light activated resin composites attended to decrease the frequency of secondary caries around the restorations 6. The sol-gel derived silica glass powders containing silver are believed to be useful as an antibacterial material for medical applications such as filler of composite resin for dental restoration 7, and the incorporation of nanometer-sized silver-supported antimicrobial agent into denture base materials to investigate the distributionand to study the release mode of silver ions from the base 8,9. There are few studies about the addition of silver particles to denture base resin have been published 8,9 . So the aim of the present in vitro study is to evaluate the effect of addition of silver nitrate to acrylic resin in different concentrationsthrough several tests part of these are: The effect of this additive on impact strength, transverse strength, and tensile strength of AgNO3 – loaded resin, and to assess any effect J Bagh College Dentistry Vol. 26(4), December 2014 Effect of silver nitrate Restorative Dentistry 79 of addition of silver nitrate on coloration of acrylic resin. MATERIALS AND METHODS Preparation of silver nitrate (AgNO3) concentrations for studying samples Different concentrations of AgNO3 solutions were prepared from stock solution of 1000ppm of AgNo3. Serial concentrations were prepared: (15, 30, 60,120ppm), (9.375, 150,300,600 ppm) and 900ppm.The prepared concentrations were confirmed by Atomic Absorption spectrophotometer (Phoenix -986/ AA spectrophotometer, UK) and stored in dark bottles wrapped with aluminum foil. AgNO3 – loaded resin specimens fabrication For impact strength test, specimens with a bar shaped specimen with dimensions of (80mm x 10mm x 4mm) length, width, thickness respectively 10. For transverse strength test, a bar shaped specimens with dimension of (65mm X 10mm X 2.5mm) length, width, thickness respectively 11. For tensile strength test, flatdumbbell shaped specimens with dimensions (16± 1mm length; 3± 0.2 mm width and 2± 0.2 mm thickness at the parallel segment 12. The patterns invested in flaks with dental stone. After setting of the stone, the flasks were opened and patterns removed, leaving cavities that were used as matrixes for the fabrication of heat polymerized acrylic resin specimens. For each assay, 60 specimens were fabricated, and assigned to 10 groups (n=6) according to the concentrations of silver nitrate solutions: zero (control);(9.375,15,30,60,120,150,300,600,and 900ppm). For this, the heat cure denture base resin(Non – veined acrylic, powder and liquid, Ivoclar Vivadent AG, Italy) was mixed according to manufacturer instruction, P/L ratio: 2.25g of powder was mixed with 1ml liquid (0.8ml monomer + 0.2ml AgNO3 solutionof each concentration). Specimens devoid of silver nitrate were included as control.These were mixed manually, by the same operator. The material were packed and polymerized following the manufacturer's instructions. Visual inspection of AgNO3 – loaded resin samples Samples were evaluated visually by comparing the tested samples with the control group. Characterization of AgNO 3-loaded resins The Fourier transform infra –red ( FTIR) spectra was performed (on IR Affinity- 1/Shimadzu Corporation/Japan spectrophotometer) using KBr and Caesium Iodid (CsI) pellets to determine whether or not functional groups of the AgNO3 have been attached to the heat cured PMMA by analyzing the characteristic vibrations of functional groups 22 . Impact strength test The test was measured using Charpy type impact testing instrument The specimen was supported horizontally at its ends and strucked by a free swinging pendulum released from a fixed height in the middle. A pendulum of 2 joules testing capacity was used. The scale reading gave the impact energy absorbed to fracture the specimen in joules when struck by sudden blow.The Charpy impact strength of unnotched specimen was calculated in KJ/m2 according to Anusavice 13 as given by the following equation: Impact strength= Since: E: is the impact absorbed energy in joules. b: is the width in millimeters of the test specimens. d: is the thickness in millimeters of the test specimens. Transverse strength test The test was measured usingInstron testing machine, each specimen was positioned on bending fixture, consisting of two parallel supports (50)mm apart, the full scale load was 50kg, and the load was applied with cross head speed of 1mm/min by rod placed centrally between the supports making deflection until fracture occurred. The transverse bend strength was calculated using the following formula 13: 3Pl Transverse strength (MPa) = 2bd2 Since: P: is the peak load. l: is the span length. b: is the sample width. d: is the sample thickness. E b.d J Bagh College Dentistry Vol. 26(4), December 2014 Effect of silver nitrate Restorative Dentistry 80 Tensile strength test The test was measured using Tinius Olsen testing machine at a cross head speed of 0.5 mm/min and with 50 mm grip – to – grip distance. The force at the failure was recorded in Newton (N) and the tensile strength values were calculated from the following equation: F (N) Tensile strength (N/mm2) = A (mm2) ` Since: F: Maximum load at failure (Newton). A: Cross sectional area (mm2). RESULTS Visual inspection of AgNO3 – loaded resin samples As the concentration of AgNO3 increased, the prepared AgNO3 –loaded resin samples start to show visually some darkening started at 300 ppm AgNO3 and above Figure (1). Characterization of AgNO 3-loaded resins The results of FTIR (Fourier transform infra – red) spectra of PMMA and AgNO3 –loaded resins in KBr and Caesium Iodid (CsI) discs, showed no change in the shape of absorption peaks between PMMA(control) and AgNO3 –loaded resinsindicating no chemical bond between the PMMA and AgNO3 Figures(2-3-4-5). Impact strength test As shown in Table 1-2, the mean impact strength was highest in the group with 60 ppm AgNo3 (12.8 KJ/ m 2) and lowest in the control group (10.6 KJ/m2). The difference in mean impact strength between the concentrations of AgNO3 and control group was statistically insignificant. Compared to control the lowest concentration of AgNO3 (9.375ppm) was associated with an average increase in impact strength of 0.1KJ/m2. The effect of this very low concentration was evaluated as weak (Cohen's d = 0.17). This effect was statistically insignificant .The 60 ppm AgNo3 was associated with highest increase in mean impact strength of 2.2.This effect was statistically significant which rated as a strong effect (Cohen's d = 2.63) . Transverse strength test As shown in table 3- 4, the mean transverse strength was highest in the control group (77.8 MPa) and lowest in the group with 120 ppm of AgNO3 (55.4MPa). The difference in mean transverse strength between the concentrations of AgNO3 and control group was statistically significant. Compared to control the lowest concentration of AgNO3 (9.375 ppm) was associated with an average reduction in transverse strength of (14MPa), the effect of this very low concentration was evaluated as strong (Cohen's d = 4.18).This effect was statistically significant .The strongest effect was with 120 ppm AgNO3 (reduction in transverse strength) (Cohen's d greater than 6). Tensile strength test As shown in table 5-6, the mean tensile strength was highest in the control group (54 MPa) and lowest in the group with 60 ppm AgNO3 (36.8MPa). The difference in mean tensile strength between the concentrations of AgNO3 and the control group was statistically significant. Compared to control the lowest concentration of AgNO3 (9.375ppm) was associated with an average reduction in tensile strength of 5 MPa. However, this effect was statistically insignificant. The 15 ppm AgNO3 was associated with very small and statistically insignificant reduction in tensile strength of 2.1MPa. On the other hand, the AgNO3 concentration associated with strongest effect (reduction in tensile strength) was the 60 ppm (Cohen's d greater than 4). Table 1: Descriptive data of impact strength test (KJ/m2) Study groups (concentration of added AgNO3 in ppm) Control 9.375 ppm 15 ppm 30 ppm Range (9.67 to 11.11) (9.94 to 11.62) (10.53 to 12.23) (9.78 to 13.51) Mean 10.6 10.7 11.5 11.8 SD 0.65 0.56 0.63 1.54 SE 0.27 0.23 0.26 0.63 N 6 6 6 6 Difference in mean compared to control Reference 0.1 0.9 1.2 Cohen's d Reference 0.17 1.37 1.03 P (LSD) Reference 0.88[NS] 0.24[NS] 0.1[NS] J Bagh College Dentistry Vol. 26(4), December 2014 Effect of silver nitrate Restorative Dentistry 81 Table 2: Descriptive data of impact strength test (KJ/m2). Study groups (concentration of added AgNO3 in ppm) 60 ppm 120 ppm 150 ppm P (ANOVA) Range (11.27 to 13.72) (9.25 to 12.66) (8.86 to 15.17) 0.05[NS] Mean 12.8 10.7 11.8 SD 0.95 1.57 2.09 SE 0.39 0.64 0.85 N 6 6 6 Difference in mean compared to control 2.2 0.1 1.2 Cohen's d 2.63 0.12 0.78 P (LSD) 0.006 0.85[NS] 0.11[NS] Table 3: Descriptive data of transverse strength test (MPa) Study groups (concentration of added AgNO3 in ppm) Control 9.375 ppm 15 ppm 30 ppm Range (74.4 to 81.6) (57.6 to 67.2) (56.4 to 72) (60 to 74.4) Mean 77.8 63.8 67.0 66.4 SD 2.78 3.83 5.65 5.76 SE 1.13 1.56 2.31 2.35 N 6 6 6 6 Difference in mean compared to control Reference -14.0 -10.8 -11.4 Cohen's d Reference -4.18 -2.42 -2.51 P (LSD) Reference <0.001 0.001 <0.001 Table 4: Descriptive data of transverse strength test (MPa) Study groups (concentration of added AgNO3 in ppm) 60 ppm 120 ppm 150 ppm P (ANOVA Range (58.5 to 79.2) (48 to 57.6) (57.6 to 76.8) <0.001 Mean 64.8 55.4 69.6 SD 7.37 3.75 6.57 SE 3.01 1.53 2.68 N 6 6 6 Difference in mean compared to control -13.0 -22.4 -8.2 Cohen's d -2.33 -6.77 -1.61 P (LSD) <0.001 <0.001 0.012 J Bagh College Dentistry Vol. 26(4), December 2014 Effect of silver nitrate Restorative Dentistry 82 Table 5: Descriptive data of tensile strength test (MPa) Study groups (concentration of added AgNO3 in ppm) Control 9.375 ppm 15 ppm 30 ppm Range (49.54 to 62.5) (46.06 to 55.2) (39.71 to 57.8) (38.84 to 53.7) Mean 54 49 51.9 44 SD 4.82 3.3 6.64 5.18 SE 1.97 1.35 2.71 2.12 N 6 6 6 6 Difference in mean compared to control Reference -5.0 -2.1 -10.0 Cohen's d Reference -1.21 -0.36 -2.00 P (LSD) Reference 0.05[NS] 0.42[NS] <0.001 Table 6: Descriptive data of tensile strength test (MPa) Study groups (concentration of added AgNO3 in ppm) 60 ppm 120 ppm 150 ppm P (ANOVA) Range (31.48 to 41.69) (38.25 to 43.4) (38.3 to 45.56) <0.001 Mean 36.8 40.7 41.5 S D 3.37 2.1 3.34 SE 1.38 0.86 1.36 N 6 6 6 Difference in mean compared to control -17.2 -13.3 -12.5 Cohen's d -4.13 -3.58 -3.01 P (LSD) <0.001 <0.001 <0.001 Control 9.37 15 30 60 120 150 300 600 900 ppm Fig. 1: AgNO3 –loaded resinsfrom left to right: control, (9.375, 15, 30, 60, 120, 150, 300, 600, and 900 ppm). J Bagh College Dentistry Vol. 26(4), December 2014 Effect of silver nitrate Restorative Dentistry 83 Wave number (cm-1) 2-PMMA in KBr disc 3- AgNO3-loaded resin in KBr disc 4-PMMA in CsI disc 5- AgNO3-loaded resin in CsI disc Fig. 2: FTIR spectrum DISCUSSION Visual inspection of AgNO3 – loaded resin samples It is believed that the discoloration for AgNO3 loaded resins which was observed at 300 ppm AgNO3 and above as illustrated in figure (1) was due to the presence of metal oxides from antibacterial metal ions during an oxidation- reduction reaction that occurs during a polymerization reaction, as well as the oxidation of the silver ions on the material surface 14,15. It was also reported that by adding such antibacterial agent, due to the Ag+ in it, the color tone of the denture base resin varies16. Characterization of AgNO 3-loaded resins Since there is no change in the shape of absorption peaks between PMMA(control) and AgNO3 – loaded resin samples as illustrated in the results of FTIR Figures(2-3-4-5) thus there is no chemical bond between the PMMA and AgNO3 22. Impact strength test The results of impact strength for the different concentrations of AgNo3 shows in significant increase in impact strength (P= 0.05 NS) when compared with control as shown in Table (1- 2).The 60ppm AgNO3 was associated the highest increase in impact strength by 2.2KJ/ m 2 .This T ransm ittance T % J Bagh College Dentistry Vol. 26(4), December 2014 Effect of silver nitrate Restorative Dentistry 84 could be due to the slow curing process allows greater number of nucleation sites to form and smaller particle sizes, thereby generating more particles 2, the total particle / matrix interfacial surface area available for energy dissipation increase, the critical stress for particle /matrix debonding also increase 17. Also The increasing in the impact strength could be due to the presence of residual monomer 18,16. This plasticizing effect render the fabricated acrylic resin samples more capable to absorb energy on impact and are more resistant to fracture13. The result of this study disagrees with Casemiro et al. 8 who added (2.5-10%) by wet Ag- Zeolite as a powder to acrylic dental resin resulted decrease in impact strength. Transverse strength and Tensile strength tests Among the specimens fabricated, the addition of silver nitrate in different concentrations reduces tensile strength (above 15 ppm AgNO3) as shown in Table (5-6) and transverse strength Table (3-4) when compared with control as the concentration of silver nitrate increased, this is probably due to Ag+ ions being reduced as the concentration of Ag increase, generating atom clusters and smaller particle size during the curing process which compete with complete polymerization process2. The plasticization effect of the resultant residual monomer will reduce the molecular binding force. On the other side, the results of FTIR Figures (2- 3-4) showed no chemical bonding between PMMA and AgNO3.Therefore we suppose that Ag+ ions attack the double bond in the alkene group of the monomer molecule and will convert it to residual monomer 18. This process will reduce the molecular binding force between the reactant molecules and allows greater deformation upon stretching or flexion through exhibiting multiple micro fractures that weaken the AgNO3 – loaded resin samples 19,20. Some other studies also showed that adding an antibacterial agent may affect the material properties, Kuroki et al. 16 have reported that there were significant differences of residual monomer in the samples treated by adding antimicrobial agents (Zeomic, BacteKiller, Novaron) although it was insignificant between the control and samples. Fan et al. 2 found that by adding 0.15% (w/w) AgBz (silver benzoate) and above there was decrease in the degree of curing, result in reduction in Rockwell hardness for light cure resin. Nakanoda et al. 14 have reported that, as a result of tensile tests and bending tests, adding Silver-Zeolite to a heat-curing resin tends to decrease the material property depending on the additive concentrations of antibacterial agent of Zeomic. There was in significant reduction in tensile strength with the lowest concentrations of AgNO3 (9.375 and 15ppm) compared with control as shown in Table 5. This outcome is in agreement with Wakasa et al. 21 who reported that when the antimicrobial agent (Zeomic) is added to self – cure acrylic resin between 1% and 2%, the polymerization behavior of the resin is not inhibit. REFERENCES 1. Keng SB, Lim M. Denture plaque distribution and the effectiveness of a perborate –containing denture cleaners .Quintessence Int 1996; 27: 341-345. 2. 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