Moamin.doc J Bagh College Dentistry Vol. 27(2), June 2015 Evaluating the effect Restorative Dentistry 17 Evaluating the effect of silver nanoparticles incorporation on antifungal activity and some properties of soft denture lining material Moamin I. Issa, B.D.S. (1) Nabeel Abdul-Fattah, B.D.S., M.Sc. (2) ABSTRACT Background: Colonization of soft denture liners by Candida albicans and other microorganisms continued to be a serious problem. The aim of this study was to evaluate the effect of incorporating silver nanoparticles into heat cured acrylic-based soft denture liner on the antifungal activity, and on water sorption, solubility, shear bond strength and color change of the soft lining material. Furthermore, evaluating the amount of silver released. Materials and methods: Silver nanoparticles were incorporated into soft denture liner in different percentages (0.05%, 0.1% and 0.2% by weight). Four hundred and twenty specimens were prepared and divided into five groups according to the test to be performed. The antifungal activity of the soft liner/AgNPs composite was evaluated in three different periods by using (viable count of C. albicans and disk-diffusion test). The amount of silver released in artificial saliva was measured by atomic absorption spectroscopy. The water sorptions, solubility, shear bond strength and color change was measured and the results were statistically analyzed. Results:All experimental groups showed a highly significant decrease in colony forming units of C. albicans in comparison to control group. There was no inhibition zone around any test specimen of any test group. There was no silver detected to be released. The addition of AgNPs resulted in a highly significant decrease in water sorption, while only 0.2% group showed highly significant decrease in solubility. Non significant differences in shear bond strength were found. A highly significant increase in light absorption percentage was observed in all experimental groups. Conclusion: The addition of AgNPs helps to produce soft denture liner with antifungal properties. Silver was not detected to be released. This addition resulted in decrease in water sorption, and did not affect the shear bond strength and it increased the opacity of the material. Keywords: Soft denture liners, antifungal activity, silver nanopaticles. (J Bagh Coll Dentistry 2015; 27(2):17-23). INTRODUCTION Soft denture liners represent polymeric materials which can be placed on the tissue surface of a hard denture base to absorb some of the load resulted from the masticatory forces, and to act as shock absorbers between the hard denture and the underlying supporting oral tissues.(1) One of the main drawbacks associated with using soft denture liners is their susceptibility to be colonized by pathological microorganisms which can be enhanced by increased humidity and high temperature beneath the dentures and by the surface characteristics of the material.(2) Candida albicans was isolated from the surface of soft denture liner and it was considered as one of the etiological factors of denture stomatitis.(3) In addition to that some studies showed that C. albicans has the ability to penetrate into different levels of the soft lining materials. This could limit the cleaning efficiency of the available chemical agents.(4) Mechanical and chemical plaque control proceduresare frequently used to prevent subsequent denture stomatitis. (1) M.Sc. Student. Department of Prosthodontics. College of Dentistry, University of Baghdad. (2) Professor. Department of Prosthodontics, College of Dentistry, University of Baghdad. However, to some geriatric or hospitalized patients suffering from cognitive impairment, reduced motor dexterity or memory loss cleaning the denture may be a difficult procedure. In addition to that, these methods can cause substantial damage to the soft lining materials. (5-7) Silver is well known for its antimicrobial activity against different bacteria, fungi and certain viruses (8), and recently the antimicrobial properties of nanoparticles have drawn attention of researchers.(9) Smaller particle size results in greater surface area to volume ratio, which enhances its chemical and biological activity.(10) Particularly, silver nanoparticles with their antimicrobial properties have elicited high interest, and their incorporation into polymers can be beneficial in wound dressings and sutures, venous and urinary catheters, endotracheal tubes, drugs, artificial tendons and orthodontic adhesives. (11) In the present study silver nanoparicles were incorporated in to acrylic-based soft denture liner in an attempt to minimize the microbial growth of C. albicans, and evaluating whether this addition would significantly affecting the mechanical and physical properties of the soft lining material ,in addition to evaluate silver release. J Bagh College Dentistry Vol. 27(2), June 2015 Evaluating the effect Restorative Dentistry 18 MATERIALS AND METHODS Heat cured acrylic-based soft denture liner (Vertex™ Soft, Vertex-Dental, Netherlands) was used in this study. Silver nanopowder (MK Nano, Canada) was incorporated into the soft liner in different percentages (0.05%, 0.1% and 0.2% by weight). A total of Four hundred and twenty specimens were prepared and divided into five groups according to the test to be performed. FTIR analysis was performed to determine if there is any chemical reaction between AgNPs and the soft liner. Evaluating antifungal activity of soft liner /AgNPs specimens using viable count of C. albicans: Specimen fabrication Specimens with dimensions of (10× 10 × 2.3mm, length, width and thickness respectively) were prepared using plastic patterns to make a silicon-stone mould. The soft lining material was mixed packed and cured according to manufacturer’s instructions. For experimental specimens AgNPs were added into the liner monomer and dispersed by using probe sonication apparatus(Soniprep-150, England) for 3 minutes to break them into individual nanoparticles.(12) The mixture was cooled down by placing the container in a cooling bath (ice-water bath), to prevent bulk heating of the liquid during sonication which can cause substantial liquid evaporation, or the degradation of the material.(13) After complete curing the specimens were finished polished and autoclaved to be sterile. Isolation of C. albicans C. albicans was isolated from the oral cavity of 18 patients with signs of denture stomatitis and oral thrush,by gentle rubbing of the lesional tissue by a sterile cotton swab, and subsequent culturing on Sabouraud dextrose agar(that was prepared according to manufacturer’s instructions) and incubated aerobically at 37°C for 24 - 48 hrs. Identification of C. albicans It was identified by colony morphology as it develops as creamy, smooth, pasty convex colonies on SDA (14) ,and by microscopical examination using Gram stain method (15), furthermore, germ tube formation procedure was used (16), and the final verification was made by biochemical method by using API Candida system (bioMérieux). Evaluating viable count of C. albicans To examine the antimicrobial activity of the soft liner/AgNPs composites, C. albicans was diluted in 0.9% NaCl, and a yeast suspension of approximately 107 CFU/ml (0.5 McFarland standards) was prepared using a McFarland densitometer. Each specimen was placed in a tube containing 9.9 ml of Sabouraud dextrose broth, into which were dispensed 100 μl of the yeast suspension. The final concentration of cells was 105CFU/ml. (11) After incubation for 24 hours at 37◦C, 100µl of each mixture was transferred to 9.9ml of NaCl (0.9%) and tenfold dilution was performed. From the second dilution, 100µl was taken and spread on SDA and incubated aerobically for 24hrs at 37ºC (Fig.1). This procedure had been repeated after 7 days and 30 days of specimens' storage in artificial saliva at 37ºC. Figure 1: (A)Inoculation of broth with C. albicans, (B)Placement of specimen in the broth, (C)Serial dilution,(D) C. albicans growth. Evaluating antifungal activity of soft liner /AgNPs specimens using disk-diffusion test: Specimens used in this test were (6mm in diameter and 0.5mm in thickness). The culture medium used for this test was Mueller-Hinton agar (that prepared according to manufacturer's instructions) containing 2% glucose and 5μg/ml methylene blue. (17) Kirby- Baure disk diffusion test was performed. Five-well isolated colonies of C. albicans were suspended in 0.85 % sterile normal saline 5 ml to achieve 0.5 McFarland turbidity to yield a yeast stock suspension. A sterile swab was dipped into the inoculum suspension and excess fluid was pressed out. The agar was swabbed in 3 directions to achieve even growth on the surface of the agar plate. (18) A D C B J Bagh College Dentistry Vol. 27(2), June 2015 Evaluating the effect Restorative Dentistry 19 After the agar surface has been left for about 5 minutes, then the soft liner disks (with and without AgNPs) were placed on the agar and the plates were kept at room temperature for 120 min for diffusion of the antimicrobial agents (19), then these agar plates were incubated aerobically for 24 hrs at 37º C. A digital caliper used to measure the inhibition zone that may appear around the disks. Silver release test The amount of silver released was evaluated by using specimens with dimensions of (10mm in diameter and 3mm in thickness)(11)and two atomic absorption spectrophotometers (Phoenix-986/AA Biotech engineering management co., and Shimadzu AA-6800)with a limits of detection of(0.025ppm and 0.01ppb) respectively. All specimens were immersed in 25 ml of artificial saliva inside a plastic plane tubes and kept at 37◦C under agitation for two different periods: T1 = 7days, T2 = 30days. The volume of the artificial saliva was reconstituted every 10 days to account for evaporation. During each period solution of each tube was collected, and the amount of silver releasedwas analyzed by atomic absorption spectroscopy. Watersorption and solubility test: Disks measuring 50±1mm in diameter and 0.5±0.05mm in thickness were prepared for experimentaland control specimens according to ADA specifications No.12 (20), by using metal patterns. All disk-shaped specimens were dried at 37ºC ± 2ºC for 24 hours in a desiccator containing dry silica gel, after that the specimens were removed to room temperature for one hour, and weighed with digital electronic balance with accuracy of (0.0001g).This cycle was repeated untilconstant weight (± 0.5mg) was obtained. This was considered to be the initial weight (W1). Then specimens were immersed in distilled water for 7 days at 37ºC ± 2ºC. After this period of time, each specimen was removed from the water, wiped with clean, dry hand towel until free from visible moisture, waved in the air for 15 seconds and weighed one minute after removal of water. This weight represents (W2). After that the specimens dried by the desiccator and they were weighed every 24 hours until a constant weight (± 0.5mg) was obtained, this weight represents (W3). Water sorption and solubility of each specimen were calculated according to the following formulae: Sorption (mg / cm²) = Solubility (mg / cm²) = Shear bond strength test To evaluate shear bond strength of soft lining material to acrylic denture base, acrylic blocks with specified dimensions (75mm × 25mm × 5 mm length, width, depth respectively) with stopper of depth about 3mm needed to be made.(21) (Fig.2A). Heat cured acrylic resin (Spofa dental, Czech) was used. Mixing packing and curing was done according to manufacturer’s instructions. Then one block of the acrylic put over the other block leaving a space between them of (25mm × 25mm × 3mm length, width, depth respectively), that filled with wax. Then the whole specimen (the 2 blocks with wax) was invested into silicon material to fabricate a mould for final specimen curing. Wax elimination procedure was done and the formed space (25mm × 25mm × 3mm) was filled with soft lining material and curing was carried out (Fig.2 B&C). The specimens were tested using Instron testing machine (Instron 1195, England). The maximum load required for failure was recorded in order to calculate the value of shear bond strength for each test specimen according to (ASTM specification D-638m, 1986) formula (22) : Bond strength (N/mm2)= = Figure 2: (A) Acrylic block used in test, (B) Custom-fabricated flask with silicon-stone mould. (C) Shear bond strength test specimen. A B C J Bagh College Dentistry Vol. 27(2), June 2015 Evaluating the effect Restorative Dentistry 20 Color change test: Disk-shaped specimens with 50 ± 1mm in diameter and 0.5 ± 0.05mm in thickness (in accordance with ADA specifications No.12) (20) were prepared to be used for color change measurements by using UV-visible spectrophotometer (UV-160AShimadzu, Japan) that evaluates color change by measuring the absorbed light percentage. RESULTS FTIR analysis showed that there was no chemical interaction between the soft lining material and AgNPs. All experimental groups (0.05%, 0.1% and 0.2% AgNPs) showed a highly significant decrease in colony forming units of C. albicans in comparison to control group with more decrease as the incubation time in artificial saliva increase (Table 1&2,Fig. 3). Disk-diffusion test didn’t show any inhibition zone around test specimens of any test group. There was no silver detected to be released in artificial saliva at any incubation period. The addition of AgNPs resulted in a highly significant decrease in water sorption mean value (Table 3&4), while only 0.2% group showed highly significant decrease in solubility (Table 5&6). Non significant differences in shear bond strength found among all test groups (Table 7). A highly significant increase in light absorption percentage observed in all experimental groups (Table 8 & 9). Table 1: Descriptive statistics and one-way ANOVA of viable count of C.albicans for all study groups and for different periods. Incubation period Groups Mean S.D. ANOVA F-test Before incubation in saliva Control 262.7 10.57 279.268 (HS) 0.05% Ag 160 10.60 0.1% Ag 158.1 9.05 0.2% Ag 181.3 6.43 After 7 days of incubation in saliva Control 249.8 7.66 306.291 (HS) 0.05% Ag 149.6 9.12 0.1% Ag 145.6 8.09 0.2% Ag 164.6 10.29 After 30 days of incubation in saliva Control 245.7 10.02 485.996 (HS) 0.05% Ag 139.8 6.61 0.1% Ag 136.1 8.75 0.2% Ag 130.5 5.52 Table 2: LSD test between viable count means. Incubation period Groups MD P-value Before incubation in saliva Control 0.05% 102.7 0.000 (HS) 0.1% 104.6 0.000 (HS) 0.2% 81.4 0.000 (HS) 0.05 0.1% 1.9 0.651 (NS) 0.2% -21.3 0.000 (HS) 0.1% 0.2% -23.2 0.000 (HS) After 7 days of incubation in saliva Control 0.05% 100.2 0.000 (HS) 0.1% 104.2 0.000 (HS) 0.2% 85.2 0.000 (HS) 0.05% 0.1% 4 0.319 (NS) 0.2% -15 0.001 (HS) 0.1% 0.2% -19 0.000 (HS) After 30 days of incubation in saliva Control 0.05% 105.9 0.000 (HS) 0.1% 109.6 0.000 (HS) 0.2% 115.2 0.000 (HS) 0.05% 0.1% 3.7 0.303 (NS) 0.2% 9.3 0.013 (S) 0.1% 0.2% 5.6 0.123 (NS) MD = Mean difference Figure 3: Bar chart showing mean values of CFU/ml at different periods of the study for each experimental group. Table 3: Descriptive statistics and one-way ANOVA of water sorption test results. Groups Mean S.D. ANOVA F-test P-value Control 0.806 0.137 8.973 0.000 (HS) 0.05% Ag 0.643 0.095 0.1% Ag 0.628 0.083 0.2% Ag 0.601 0.057 J Bagh College Dentistry Vol. 27(2), June 2015 Evaluating the effect Restorative Dentistry 21 Table 4: LSD test between water sorption study groups. Groups Mean Difference P-value Control 0.05%Ag 0.163 0.001 (HS) 0.1% Ag 0.178 0.000 (HS) 0.2% Ag 0.204 0.000 (HS) 0.05% Ag 0.1% Ag 0.015 0.737 (NS) 0.2% Ag 0.041 0.351 (NS) 0.1% Ag 0.2% Ag 0.026 0.548 (NS) Table 5: Descriptive statistics and one-way ANOVA of solubility results. Groups Mean S.D. ANOVA F-test P- value Control 0.223 0.037 24.359 0.000 (HS) 0.05% Ag 0.202 0.035 0.1% Ag 0.243 0.036 0.2% Ag 0.130 0.007 Table 6: LSD test between solubility study groups. Groups Mean Difference P-value Control 0.05% 0.021 0.141 (NS) 0.1% Ag -0.020 0.158 (NS) 0.2% Ag 0.093 0.000 (HS) 0.05% Ag 0.1% Ag -0.042 0.006 (HS) 0.2% Ag 0.071 0.000 (HS) 0.1% Ag 0.2% Ag 0.113 0.000 (HS) Table 7: Descriptive statistics and one-way ANOVA of shear bond strength test results. Groups Mean S.D. ANOVA F-test P-value Control 0.534 0.071 0.253 0.859 (NS) 0.05% Ag 0.529 0.071 0.1% Ag 0.548 0.042 0.2% Ag 0.551 0.081 Table 8: Descriptive statistics and one-way ANOVA of color change test results. Groups Mean S.D. ANOVA F-test P-value Control 0.120 0.013 70.664 0.000 (HS) 0.05% Ag 0.144 0.012 0.1% Ag 0.198 0.014 0.2% Ag 0.236 0.033 Table 9: LSD test between color change study groups. Groups Mean Difference P-value Control 0.05% Ag -0.024 0.009 (HS) 0.1% Ag -0.078 0.000 (HS) 0.2% Ag -0.116 0.000 (HS) 0.05% Ag 0.1% Ag -0.054 0.000 (HS) 0.2% Ag -0.092 0.000 (HS) 0.1% Ag 0.2% Ag -0.038 0.000 (HS) DISCUSSION In this study AgNPs were added into soft denture liner in an attempt to improve the antimicrobial properties of the liner against C. albicans yeast which is one of the main causative factors of denture-induced stomatitis. The results of this study showed a statistically highly significant decrease in colony forming units/ml of C. albicans after incorporating the soft denture liner with AgNPs. The antimicrobial efficacy seemed to be concentration dependant. The antifungal activity of the tested soft liner /AgNPs composite seemed to increase with the increase of incubation time in artificial saliva. The explanation for that could be due to the presence of specimens in aqueous environment for longer period, so there was a greater possibility of AgNPs oxidation and Ag+ formation which enhance the antimicrobial activity , because the silver ions are the main active and reactive species of silver.(23) In addition to that movement of some AgNPs from the bulk of specimen to the surface might occur with increase in storage time, however silver or its ions were not detected to be released.(11) These phenomena together with decrease in water sorption could explain the lower level and later improvement of antifungal activity in (0.2% group). For disk-diffusion test, no inhibition zone was detected around specimens for any AgNPs percentage used even after incubating the specimens in artificial saliva. This could be explained by absence of silver ions release from soft liner/AgNPs composite which was verified by the results of this study. These findings indicated that the antifungal activity was achieved by contact kill; no Ag+ leached out of the copolymer. (24) This was also confirmed by the results of this study as no silver or silver ions were detected in artificial saliva using atomic absorption spectroscopy at any incubation period. Previous studies were made to evaluate silver release from different polymeric materials, and some of them confirmed the results of the present study (11), while others disagreed by detecting different concentrations of silver released. (25, 26) This could be explained by differences in the type of polymeric materials and their polymerization methods, in addition to differences in AgNPs incorporation methods. Water sorption and solubility were evaluated simultaneously through water gain and loss of soluble components. The resulted decrease in water soption mean values could be attributed to addition AgNPs, with their hydrophobic nature, so J Bagh College Dentistry Vol. 27(2), June 2015 Evaluating the effect Restorative Dentistry 22 the number of PEMA molecules which would be available on the surface of the specimen which would allow water diffusion reduced, this is in accordance with Arora et al. (27). Furthermore, the addition of AgNPs resulted in decrease of microporosity that resulted after polymerization process and subsequently reducing the water sorption. While for solubility test only (0.2% group) showed a highly significant decrease in solubility. This could be attributed to the decrease in water sorption properties of the soft lining material with the increase in the amount of AgNPs added, as indicated by this study. This limitation in the diffused water will reduce the possibility of molecular flexibility and the leach out of soluble constituents from the polymer mass. About the shear bond strength, the non significant change that resulted could be explained by the compatibility and high degree of similarity in chemical structure between polymethyl methacrylate denture base material and polyethyl methacrylate soft liner which would result in a chemical bonding between the two materials. However, FTIR analysis showed that no chemical interaction was detected between AgNPs and soft liner. In addition to that the flowability of the soft lining material; which allows the material to readily adapt to the bonding surfaces and creates an intimate union, didn’t seem to change subjectively. Color change test results showed that there was a statistically highly significant increase in light absorption percentage with the increase in AgNPs amount which was added to the soft lining material. This could be explained by the presence AgNPs in the polymer matrix, as the silver nanoparticles have extraordinary efficient ability to absorb and scatter light due to their optical properties (28), and a single silver nanoparticle can interact with light more efficiently than any known organic or inorganic colored particle with same dimensions (29), so AgNPs absorb more light energy than polymer matrix and appear more opaque. In addition to that AgNPs will act as fillers which tend to fill any spaces or voids within the polymer, thereby increasing the amount of scattered and absorbed light by the specimen and decrease the amount of transmitted light. REFERENCES 1. Anusavice K J. Phillips' Science of Dental Materials. 11th ed. St Louis: MO: Elsevier Science (USA); 2003. 2. Taylor R L, Bulad K, Verran J, McCord J F. Colonization and deterioration of soft denture lining materials in vivo. Eur J Prosthodont Restor Dent 2008; 16: 50-55. 3. Tari BF, Nalbant D, Dogruman F, Kustimur S. 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Chem Phys Chem 2005; 6: 1221-31. الخالصة كان الغرض من ھذه الدراسة إن عملیة استیطان بطانة طقم االسنان اللینة من قبل المبیضات البیض والكائنات الدقیقة االخرى الیزال یمثل مشكلة جدیة ، :الخلفیة ) ضد المبیضات البیض(على النشاط المضاد للفطریات بالحرارة اللینة لطقم االسنان معالجةھو تقییم تأثیر ادماج الفضة النانویة في مادة التبطین االكریلیكیة ال عالوة على ذلك، تقییم كمیة الفضة المتحررة من مركب البطانة . ،وعلى امتصاص المیاه، قابلیة الذوبان، قوة الربط القصیة والتغیر اللوني لمواد التبطین اللینة .اللینة مع الفضة النانویة تم اعداد اربع %).0.2و% 0.1، % 0.05(نانویة مع مادة التبطین االكریلیكیة اللینة لطقم االسنان بنسب وزنیة مختلفة تم دمج الفضة ال: المواد وطرق البحث تم تقییم نشاط مزیج مادة التبطین اللینة مع الفضة النانویة ضد الفطریات . مائة وعشرین عینة وتم تقسیمھا الى خمس مجموعات وفقا لنوع االختبار المراد اجرائھ وقد تم قیاس كمیة الفضة المتحررة في اللعاب . تعدادالمبیضات البیض القابلة للحیاة واختبار انتشار القرص: على ثالثة فترات مختلفة وباستخدام طریقتین تم تحلیل النتائج و.غیر اللونيالتوقوة الربط القصیة ,قابلیة الذوبان , تم قیاس قابلیة امتصاص الماء . ياالصطناعي بواسطة التحلیل الطیفي لالمتصاص الذر .احصائیا لم یكن ھنالك اي . اظھرت نتائج جمیع المجموعات التجریبیة انخفاضا كبیرا للغایة في عدد مستعمرات المبیضات البیض مقارنة بالمجموعة الضابطة: النتائج اظھرت النتائج ان اضافة الفضة النانویة ادت الى . المتحررة ر للفضةلم یتم الكشف عن اي اث. منطقة تثبیط حول اي عینة في اي مجموعة من مجامیع االختبار تم العثور على اختالفات . فقط انخفاض كبیر جدا في قابلیة الذوبان% 0.2انخفاض ملحوظ بدرجة كبیرة في قابلیة امتصاص الماء، في حین اظھرت مجموعة .زیادة كبیرة جدا في نسبة امتصاص الضوء في جمیع المجموعات التجریبیة غیر ملحوظة في قوة الربط القصیة لجمیع المجموعات، وقد لوحظ لم یتم . یاتان اضافة الفضة النانویة الى مادة التبطین االكریلیكیة اللینة لطقم االسنان یساعد على انتاج مادة تبطین لینة مع خصائص مضادة للفطر: االستنتاج فة عن انخفاض في قابلیة امتصاص الماء ولم تؤثر على قوة الربط القصي لمادة التبطین، في حین ادت الى تغیر وقد اسفرت ھذه االضا. اي فضة متحررةاكتشاف .لون المادة من خالل زیادة التعتیم .الفضة النانویة, النشاط المضاد للفطریات, بطانة طقم االسنان اللینة: الكلمات الرئیسیة