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  1. J Bagh College Dentistry Vol. 29(4), December2017The Effect of AdditionRestorative Dentistry27The Effect of Addition of Zirconium Nano Particles on Antifungal Activity and Some Properties of Soft Denture Lining MaterialAlaa Dakhil Yasser, B.D.S. (1)Nabeel Abdul Fatah,B.D.S., M.Sc.(2)ABSTRACTBackgroundMicroorganisms and fungal growth especially Candida albicans,onsoft denture lining material are the most common problemwhich can lead to chronic mucosal inflammation. The aim of this study was to evaluate the effect of zirconium nanoparticles into acrylic-based heat cured soft denture lining material against Candida albicans, and the amount of zirconium ion release of softliner/ZrNPs composite. Furthermore, evaluate shear bond strength after ZrNPs addition to soft liner.Materials and methods: Zirconium nanoparticles were added into acrylic-based soft denture liner in various percentages (1%, and 1.5% by weight). Two hundred and fifty specimens were arranged and isolated into four groups as per the test to be done The antifungal activity of the soft liner/ZrNPs composite was assessed in three different periods by using two methods (viable count of C. albicansand disk-diffusion test). In two distinct periods, amount of zirconium released in artificial saliva was detected by atomic absorption spectroscopy. Instron testing machine was used to test the shear bond strength of the soft liner to the acrylic denture base material.Results:A highly significant decrease in colony forming units of C. albicansin experimental groups (1% and 1.5%ZrNPs) contrast with control group. There was no inhibition zone around any specimen of any test group. In artificial saliva there was no zirconium distinguished to be released at any incubation period. There was a highly significant increase in the mean value of shear bonding strength after incorporation of ZrNPs at 1.5% percentage into soft liner.Conclusion: The addition of ZrNPs into acrylic-based soft denture lining material helps to provide soft denture liner with antifungal properties, thus reducing the susceptibility to develop denture-induced stomatitis. There is no zirconium has been detected at any incubation period and there isincreased in the shear bond strength of the soft lining material.Keywords: Soft denture liners, antifungal activity, zirconium nanoparticles.(J Bagh Coll Dentistry 2017; 29(4):27-32)INTRODUCTIONSoft denture liners are utilized to create a comfortable interface between denture and oral tissues, decreasing traumatic transmission of occlusal forces to extremely resorbed alveolar ridges and areas recovering from surgical methods.(1)Then again, soft liners are shown in patients who experience from traumatic or pathological tissue loss, excessive residual ridge resorption ,substantial undercuts, ,fragile supporting mucosa.(2)It has been giving comfort to denture wearers that as it act as a cushion between denture base and oral mucosa.(3)Property, for example, shear bond strength has been appeared tobe dependent on chemical composition of both denture base and reline materials. A weak bond could promote staining, harbor bacteria and delamination of the lining material.(4)Likewise, it is proposed that the bond strength among the denture reline and denture base resins could influence the mechanical strength of the reline denture base.(5)1) Master Student, Department of Prosthodontics, College of Dentistry, University of Baghdad.(2) Professor.Osouleldeen university college.Another defect are surface roughness and the colonization by Candida albicans(C. albicans), which may lead to denture stomatitis.(6,7)ZrO2being a typical appears great biocompatibility and bioactivity. ZrO2 enhances mechanical properties and is proposedto be relatively appropriate for dental restorations.(8)Encourage antifungal activity of the ZrO2 nanoparticles was additionally decided against filamentous growth, C. albicansand non-filamentous organism, Aspergillus niger. ZrO2 nanoparticles may effectively inhibit the development of fungal strains by interfering cell function and causing deformation in fungal hyphae.(9)In the present study Zirconium nanoparticles were incorporated in to acrylic-based soft denture liner in an attempt to minimize the microbial growth ofC. albicans, and evaluating whether this addition would significantly affecting the mechanical and physical properties of the soft lining material ,in addition to evaluate Zirconium releaseMATERIALS AND METHODSHeat cured acrylic-based soft denture liner (Vertex™ Soft, Vertex-Dental, Netherlands) was used in this study. Zirconium nanopowder (HWNANO, China) was added into the soft liner in different percentages (1% and 1.5% by weight).
  2. J Bagh College Dentistry Vol. 29(4), December2017The Effect of AdditionRestorative Dentistry28A total of three hundred specimens were prepared and divided into four groups according to the test to be performed.FTIR analysis was performed to determine if there is any chemical reaction between ZrNPs and the soft liner.Fabrication of ZrNPs loaded soft liner specimens used to evaluate viable count of C. albicans:Plastic patterns with dimensions of (10× 10× 2.3mm, length, width and thickness respectively) (10)were used to prepare the specimens . The soft lining material was mixed packed and cured according to manufacturer’s instructions. For experimental specimens ZrNPs 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.(11) The mixture was cooled down by placing the container in acooling 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.(12) After complete curing the specimens were finished polished and autoclaved to be sterile.Isolation and Identification of Candida albicans:From the oral cavity of 20 patients took care of the college of dentistry, C. albicans was isolated with indications of denture stomatitis and oral thrush by using A sterile cotton swab.(13)On Sabouraud dextrose agar, the swabs were cultured and brooded aerobically at 37°C for 24 -48 hrs, and afterward left at 4°C for further examination. 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.albicansTo test the antifungal activity of the PEMA/ZrNPs composites, in 0.9% NaCl, C. albicans was diluted, and by using a McFarland densitometer, a yeast suspension of approximately 107CFU/ml (0.5 McFarland standards) was prepared.(17).(Reference)In a tube with volume of 9.9 mL of Sabouraud dextrose broth, each sample was putted, into which 100 μL of the yeast suspension were dispensed. The final concentration of cells was 105 CFU/mL. The samples were gathered and washed 5 times in a standard way by dipping in sterile deionized water to eliminate loosely joined cells after incubation for 24 hours at 37◦C. In Sabouraud dextrose agar plates, viable cells on the surface of the samples were calculated. (17)After 7 days and 30 days) of samples' storage in artificial saliva at 37ºC, this procedure was repeated.Disk diffusion testA rectangular metal pattern about 0.5mm in thickness and (30mm×60mm length, width respectively) was used from which small discs 6mm was cut by using metal rod with sharp edge to be used in disc diffusion test.A sterile swab was dunked into the inoculum suspension and excess fluid was squeezed out. In 3 directions swabbing was made on GM-MH agar to accomplish equal development on the surface of the agar plate.(18)After the agar surface has been kept for around 5 minutes, then the soft liner discs (with and without ZrNP) were set on the agar and the plates were left at room temperature for 120 min for diffusion of the antimicrobial agents(19),then these agar plates were incubated aerobically for 24hrs.at 37º C. The inhibition zone that may appear around the disks was measured by using electronic digital caliper.(20)Shear bond strength testTo 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)Heat cured acrylic resin (Spofa dental, Czech) was used. Mixing packing and curing was done according tomanufacturer’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.The value of shear bond strength was calculated by dividing the maximum load required for the failure by cross section area to for each test sample formula(22):Bond strength (N/mm2) = =Zirconium release test:A plastic round pattern with measurement (3mm in thickness and10mm in diameter to make silicone rubber molds for testing zirconium release (17). Experimental samples (1%, 1.5% ZrNPs) were proportioned, mixed and cured. A
  3. J Bagh College Dentistry Vol. 29(4), December2017The Effect of AdditionRestorative Dentistry29plastic plane tubes containing 25 mL of artificial saliva were used to preserve all samples and kept at 37◦C for two various periods(after 7 and 30 days) . At regular intervals, the volume of the artificial saliva was reconstituted to calculate for evaporation.Atomic absorption spectroscopy was used to test zirconium dosage in collected solution of every tube during each period, and by using a linear calibration curve in the equipment the dosage of zirconium released was counted from standard solutions at various concentrations.Atomic absorption spectrophotometer (Shimadzu AA-6300) was used for detection of zirconium released with a limits of detection of 0.01ppb.RESULTSFTIR analysis showed that there was no chemical interaction between the soft lining material and ZrNPs. All experimental groups ( 1 and 1.5% ZrNP) showed a lower mean valuesthan control group, with the lowest mean value was shown by 1.5% group (2.8CFU/ml), as the incubation time in artificial saliva increase(152.4CFU/ml).as table(1 ,2) figure (1).Disk-diffusion test revealed that there are no inhibition zone around any one of ZrNPs-polyethyl methacrylate discs of any percentages of ZrNPs used in Kirby-Baure disk diffusion test of any concentration even after 7days and 30 days of incubation in artificial saliva. There was no zerconium detected to be released in artificial saliva at any incubation period.Also the present study show that results of shearbond strength test showed that the highest mean value among the study groups represented by 1.5% group which equals (0.9674N/mm²), followed by 1% group with mean value of (0.6594N/mm²). While the mean value of control group was (0.3880N/mm²)as tables (3,4) figure(2).Table 1: Descriptive statistics of viable count of C.albicansfor all study groups and for different periods.Incubation periodGroupsNMeanS.D.Min.Max.Antifungal 1daycontrol10152.400023.37710124.00188.001%1011.60003.238666.0017.001.5%102.80002.616190.007.00Antifungal 7dayscontrol10163.400020.20561126.00191.001%1010.30002.110827.0014.001.5%102.60002.170510.006.00Antifungal 30dayscontrol10167.600018.50045139.00193.001%107.10002.330954.0011.001.5%101.50001.649920.004.00Figure 1: Bar charts showing mean values of CFU/ml at different periods of the study for each experimental group.
  4. J Bagh College Dentistry Vol. 29(4), December2017The Effect of AdditionRestorative Dentistry30Table 2: Comparison of the means of C.albicanscount-using one way ANOVA-in different groups in each incubation period.Incubation periodSum of SquaresDfMean SquareF-testP valueAntifungal 1dayBetween Groups140940.800270470.400374.961000Within groups5172.94527191.591Antifungal 7daysBetween Groups164518.467282259.233591.179000Within groups3760.86527139.291Antifungal 30daysBetween Groups177936.067288968.033761.664000Within groups3179.12727118.412Table 3: Descriptive statistics of shear bond strength test resultsGroups NMeanStd. DeviationMinimumMaximumcontrol100.38800.071160.290.521%100.6594.062650.550.741.5%100.96740.119860.811.18Total300.67160.255360.291.18Figure 2: Bar chart of shear bond strength meanvalues of all study groups.Table 4:one-way ANOVA of shear bond strength test resultsGroupsSum of Squaresd.f.Mean SquareFSig.Between Groups1.68120.840107.9590.000Within Groups0.210270.008DISCUSSIONTo enhance the antimicrobial activity of the soft denture liner against C. albicans, the main causative factors of denture-induced stomatitis, by addition of ZrNPs into acrylic-based soft denture lining material.Statistically, the results of present study, appeared highly significant decrease in CFU/ml of C. albicansafter the addition ZrNPsinto the soft denture liner which demonstrate the advancement of a polymer with antifungal properties.This study in agreement with Gowri et al.,(2014)(9) whorevealed that ZrO2 nanoparticles in clearly predict its antifungal activity by actively inhibiting the growth of both C. albicansand A. nigerstrains and antibacterial activity against the bacterial pathogens, S. aureus among gram positive and E.coli,among gram negative. This findings compatible with the results of Jangra et al., (2012) (23) study which revealed thatZrO2 and Zr(IV) complexes had active facet play an important role in defining the activity against the microbes.Likewise it demonstrated that antifungal activity of the examined soft liner ZrNPs composite increased with increased incubation time in artificial saliva.ZrNPsmay effectively inhibit the development of fungal strains by meddling cell function and leading to misshaping in fungal hyphae.(9)00.20.40.60.81control1%1.50%1.50%1%control
  5. J Bagh College Dentistry Vol. 29(4), December2017The Effect of AdditionRestorative Dentistry31There was no inhibition zone was distinguished around specimens by disk-diffusion test even after incubation in artificial saliva for any ZrNPs percentage used. This could be explained by the results of this study by disappear of zirconium release from soft liner/ZrNPs composite Regardless of high identifying capacity atomic absorption spectrophotometers which were utilized as a part of this concentrate no zirconium or zirconium particles was identified in artificial saliva at any incubation period (after 7days and after 30days) and for any percentage of ZrNPs.This study additionally demonstrated that the result of zirconium release was like the discdiffusion test result, that showed no inhibition zone around any specimens on zirconium release. The results of the present study revealed that a significant increase in mean value of shear bond strength with increase percentage of the addition of ZrNPs into the soft linerto highest mean value between the experimental groups demonstrated by 1.5% group .There is no previous study demonstrate the effect of ZrNPson shear bond strength of soft lining material.The high flow ability of soft lining materials that used in this study is another factor that improve shear bond strength , which permits the material to easily adapt to the bonding surface and provides good contact, meaning the incorporation of ZrNPsintosoft lining material may increased the flow ability of the material.REFERENCES1.Pisani MX, Malheiros-Segundo AL, Balbino KL, Souza RF, Paranhos HF, Silva CH. Oral health related quality of life of edentulous patients after denture relining with a silicone-based soft liner. Gerodontology. (2012) Jun; 29(2):474-80.2.Anil N, Hekimoglu C, Sahin S. Colour stability of heat-polymerized and autopolymerized soft denture liners. J Prosthet Dent (1999); 81: 481–484.3.Murata H, Seo RS, Hamada T et al. Dynamic mechanical properties of hard direct reline resins. J Prosthet Dent (2007); 98: 319–326.4.Arima T, Nikawa H, Hamada T, Harsini. Composition and effect of denture base resin surface primers for reline acrylic resins. J Prosthet Dent (1996);75:457-62.5.Takahashi Y, Chai J, Law D. Assessment of Shear Bond Strength Between Three Denture Reline Materials and a Denture Base Acrylic Resin. Int J Prosthodont (2001);14:531–5.6.Oliveira LV, Mesquita MF, Henriques GEP, Consani RLX. The effect of brushing on surface roughness ofdenture lining materials. J Prosthodont (2007); 16: 179.7.Boscato N, Radavelli A, Faccio D, Loguercio AD.Biofilm formation of Candida albicans on the surface of a soft denture lining material. Gerodontology (2009); 26: 210–213.8.Ellakwa AE, Morsy MA, El-Sheikh AM. Effect of aluminum oxide addition on the flexural strength and thermal diffusivity of heat-polymerized acrylic resin. J Prosthodont (2008); 17: 439-444.9.Gowri S., R. Rajiv Gandhi, M. Sundrarajan, Structural, Optical,Antibacterial and Antifungal Properties of Zirconia Nanoparticles by Biobased Protocol J. Mater. Sci. Technol(2014), 30(8), 782-790.10.Chladek G., Mertas A., Barszczewska-Rybarek I., NalewajekT., Zmudzki J., Krol W.,Lukaszczyk J.: “Anti -fungal Activity of Soft Lining Material Modified by Silver Nanoparticles -A Pilot study”. Int.J.Mol.Sci.(2011); 12: 4735-4744. 11.Safi IN, Mohammed MM. Evaluation the effect of modified nano-fillers addition on some properties of heat cured acrylic denture base material. J Bagh Coll Dentistry 2011; 23(3): 23-912.National Institute of Standards and Technology. Material Measurement Laboratory.Preparation of Nanoparticle Dispersions from Powdered Material Using Ultrasonic Disruption. Special Publication 1200-2 (2012).13.Marsh P. D. and Martin M.: “Oral Microbiology”. 5thedition. Churchill Livingstone, Edinburgh, UK (2009).14.Baveja C.: “Text Book of Microbiology for Dental Students”. 3rd edition Arya Publications, Delhi, India (2010).15.Linda MM, Jean A S, Stephen D A. Direct Smear Atlas: A Monograph of Gram-stained Preparations of Clinical Specimens. 1st ed. Lippincott Williams & Wilkins; 2001. 16.Forbes BA, Sahm DF, Weissfeld AS, Bailey WR. Bailey & Scott's diagnostic microbiology.12th ed. Elsevier: Mosby; 2007.17.Monteiro D.R., Luiz Fernando Gorup, Takamiya A.S., de Camargo E.R., RuvoloFilho A.C., Barbosa D. B. : “Silver Distribution and Release from an Antimicrobial Denture Base Resin Containing Silver Colloidal Nanoparticles”. Journal of Prosthodontics (2011):1-9.18.Lee S.-C., Lo H.-J., Fung C.-P., Lee N., See L.-C.: “Disk diffusion test and E-test with enriched Mueller-Hinton agar for determining susceptibility of Candida species to voriconazole and fluconazole”. J MicrobiolImmunol Infect.(2009);42:148-15319.Moller A.J.R.: “Microbiological examination of root canals and periapical tissues of human teeth”. Odontol Tidskr (1966); 74: 1-38.20.Joanne M.W., Linda M.S., Christopher J.W.: “Prescott/Harley/Klein's Microbiology” .7thedition .McGraw-Hill.Higher Education (2008).21.Hachim TM. Evaluation of shear bond strength of silicon-based soft liner to the acrylic resin denture base using different polymerization technique with different storage periods in distilled water. J Bagh Coll Dentistry 2012; 24(3): 42-46.22.American society for testing and material, ASTM D, 638-m standard test method for tensile properties of plastics. Philadelphia: American National Standards Institute (1986).23.Jangra Sant Lal, K. Stalin 1, Neeraj Dilbaghi, Sandeep Kumar, Jai Tawale, Surinder P. Singh , and Renu Pasricha." Antimicrobial Activity of Zirconia (Zr02) Nanoparticles and Zirconium Complexes" Journal of Nanoscience and Nanotechnology(2012) Vol. 12, 7 105-7 112.
  6. J Bagh College Dentistry Vol. 29(4), December2017The Effect of AdditionRestorative Dentistry32الخالصة:-الخالصة:الخلفية :الكائنات الحية الدقيقة ونمو الفطريات وخاصة المبيضات البيض، على بطانة طقم االسنان اللينة هي المشكلة األكثر شيوعا التي يمكن أن تؤدي إلى التهاب الغشاء المخاطي المزمنوكان الدافع من وراء هذه الدراسة إلى تقييم تأثير جزيئاتالزركونيومالنانويةفي مادة التبطين االكريليكيةالمعالجة حراريا اللينة لطقم االسنان على النشاط المضاد للفطريات (ضد المبيضات البيض)وكمية الزركونيوم المتحرر من مركب البطانة اللينة مع الزركونيوم النانوية عالوة على ذلك تقييم قوة الربط القصية لمواد التبطين اللينة معجزيئاتالزركونيومالنانوية.المواد وطرق البحث: تم دمج جزيئات الزركونيوم النانوية مع مادة التبطين االكريليكية اللينةلطقم االسنان بنسب مختلف(%1%1.1,من حيث الوزن).تم اعداد ثالثة مائة عينة وتم تقسيمها الى اربع مجموعات وفقا لنوع االختبار المراد اجرائه. تم تقييم نشاط مزيج مادة التبطين اللينة مع الزركونيوم النانوية ضد الفطريات على ثالثة فترات مختلفة وباستخدام طريقتين: تعداد المبيضات البيض القابلة للحياة واختبار انتشار القرص. وقد تم قياس كمية الزركون المتحررة في اللعاب االصطناعي في فترتين مختلفتين بواسطة التحليل الطيفي لالمتصاص الذري.تم قياس قوة الربط القصية بين البطانة اللينة ومادة قاعدة طقم االسنان باستخدام جهاز انسترون.النتائج:و اظهرت نتائج جميع المجموعات التجريبية انخفاضا كبيرا للغاية في عدد مستعمرات المبيضات البيض مقارنة بالمجموعة الضابطة. لم يتم الكشف عن اي اثر للزركونيوم في محلول اللعاب االصطناعي في اي فترة حضانة مستعملة.كان هناك زيادة كبيرة جدا في القيمة الوسطية لقوة الترابط القص بعد إدراج جزيئات الزركونيوم النانوية في نسبة %1.1في بطانة لينة.االستنتاج:ان اضافة الزركونيوم النانوية الى مادة التبطين االكريليكية اللينة لطقم االسنان يساعد على انتاج مادة تبطين لينة مع خصائص مضادة للفطريات، وبالتالي تقليل قابلية حصول التهاب الفم الناتج عن طقم االسنان.ولم يتم الكشف عن وجود الزركونيوم في أي فترة حضانة وقد زادت قوة الربط القصي لمادة التبطين.
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