Research Article
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Effects of Surface Characteristics of Conventionally Manufactured, CAD/CAM Milled, and 3D-Printed Interim Materials on Adherence of Streptococcus Mutans and Candida Albicans

Year 2023, Volume: 26 Issue: 3, 227 - 234, 29.09.2023

Simge Taşın , Meryem Güvenir , Artur Ismatullaev

https://doi.org/10.7126/cumudj.1228677

Abstract

Objectives: The purpose of this in vitro study was to compare conventionally manufactured, CAD/CAM milled, and 3D-printed interim materials based on their susceptibility to adherence of Streptococcus mutans and Candida albicans, and examine the influence of surface roughness and hydrophobicity.
Materials and Methods: Eighty disc-shaped specimens fabricated from autopolymerized polymethyl methacrylate (A-PMMA), bis-acryl composite (Bis-acrylate), CAD/CAM PMMA-based polymer (Milled-PMMA), and 3D-printed resin (Printed) were subjected to 10,000 thermal cycles (5-55 °C) and divided into two groups (n=10) according to microbial suspension used: Streptococcus mutans and Candida albicans. Surface roughness (Ra) and hydrophobicity (WCA) of specimens were measured. An adhesion test was performed by incubating the specimens in Streptococcus mutans and Candida albicans suspensions at 37 °C for 24 hours, and the adherent cells were evaluated by counting colony-forming units (CFU/ml). Scanning electron microscopy (SEM) was performed to analyze the surfaces (n=2). Data were analyzed with Kruskal-Wallis and Mann-Whitney U tests. Spearman’s correlation analysis was used to determine correlation among the measurements (α=.05).
Results: Type of restorative material significantly influenced Ra and WCA. The highest adhesion of Streptococcus mutans was observed in Printed, followed by Bis-acrylate, A-PMMA, and Milled-PMMA (p=.001). The highest adhesion of Candida albicans was noted on A-PMMA, followed by Printed, Bis-acrylate, and Milled-PMMA (r=.001). The adhesion of Streptococcus mutans (r=.660) and Candida albicans (r=.413) showed a positive correlation with Ra. A negative correlation was found between WCA of the materials and Streptococcus mutans adhesion (r=-.373).
Conclusions: Surface roughness plays an important role in the adherence of microorganisms. CAD/CAM PMMA-based polymers may be a better choice to reduce microbial adhesion in long-term use.

Keywords

Interim material microbial adhesion surface properties

Project Number

Bu araştırma bir proje kapsamında yapılmamıştır.

References

  • 1. Christensen GJ. The fastest and best provisional restorations. J Am Dent Assoc 2003;134(5):637-639.
  • 2. Axelsson P, Lindhe J. Effect of controlled oral hygiene procedures on caries and periodontal disease in adults. J Clin Periodontol 1978;5(2):133-151.
  • 3. Beighton D. The complex oral microflora of high‐risk individuals and groups and its role in the caries process. Community Dent Oral Epidemiol 2005;33(4):248-255.
  • 4. Salerno C, Pascale M, Contaldo M, Esposito V, Busciolano M, Milillo L, Guida A, Petruzzi M, Serpico R. Candida-associated denture stomatitis. Med Oral Patol Oral Cir Bucal 2011;16(2):139-143.
  • 5. Quirynen M. The clinical meaning of the surface roughness and the surface free energy of intra-oral hard substrata on the microbiology of the supra-and subgingival plaque: results of in vitro and in vivo experiments. J Dent 1994;22:13-16.
  • 6. Yuan C, Wang X, Gao X, Chen F, Liang X, Li D. Effects of surface properties of polymer-based restorative materials on early adhesion of Streptococcus mutans in vitro. J Dent 2016;54:33-40.
  • 7. Wei X, Gao L, Wu K, Yu P, Jiang L, Lin H, Wang Y, Cheng H. In vitro study of surface properties and microbial adhesion of various dental polymers fabricated by different manufacturing techniques after thermocycling. Clin Oral Investig 2022;26(12):7287-7297.
  • 8. Dorkhan M, de Paz LEC, Skepö M, Svensäter G, Davies JR. Effects of saliva or serum coating on adherence of Streptococcus oralis strains to titanium. Microbiology (Reading) 2012;158(2):390-397.
  • 9. Zamperini CA, Machado AL, Vergani CE, Pavarina AC, Giampaolo ET, Cruz NC. Adherence in vitro of Candida albicans to plasma treated acrylic resin. Effect of plasma parameters, surface roughness and salivary pellicle. Arch Oral Biol 2010;55(10):763-770.
  • 10. Li J, Hirota K, Goto T, Yumoto H, Miyake Y, Ichikawa T. Biofilm formation of Candida albicans on implant overdenture materials and its removal. J Deny 2012;40(8):686-692.
  • 11. Buergers R, Rosentritt M, Handel G. Bacterial adhesion of Streptococcus mutans to provisional fixed prosthodontic material. J Prosthet Dent 2007;98(6):461-469.
  • 12. Nyvad B, Fejerskov O. Scanning electron microscopy of early microbial colonization of human enamel and root surfaces in vivo. Scand J Dent Res 1987;95(4):287-296.
  • 13. Boaro LC, Gonçalves F, Guimarães TC, Ferracane JL, Pfeifer CS, Braga RR. Sorption, solubility, shrinkage and mechanical properties of “low-shrinkage” commercial resin composites. Dent Mater 2013;29(4):398-404.
  • 14. Ruse N, Sadoun M. Resin-composite blocks for dental CAD/CAM applications. J Dent Res 2014;93(12):1232-1234.
  • 15. Raszewski Z. Acrylic resins in the CAD/CAM technology: A systematic literature review. Dent Med Probl 2020;57(4):449-454.
  • 16. Perea-Lowery L, Gibreel M, Vallittu PK, Lassila L. Characterization of the mechanical properties of CAD/CAM polymers for interim fixed restorations. Dent Mater J 2020;39:319-325.
  • 17. Tahayeri A, Morgan M, Fugolin AP, Bompolaki D, Athirasala A, Pfeifer CS, Ferracane JL, Bertassoni LE. 3D printed versus conventionally cured provisional crown and bridge dental materials. Dent Mater 2018;34(2):192-200.
  • 18. Zhang X, Xu Y, Li L, Yan B, Bao J, Zhang A. Acrylate‐based photosensitive resin for stereolithographic three‐dimensional printing. J Appl Polym Sci. 2019;136(21):47487.
  • 19. Kim J-H, Kwon J-S, Park J-M, Russo LL, Shim J-S. Effects of postpolymerization conditions on the physical properties, cytotoxicity, and dimensional accuracy of a 3D-printed dental restorative material. J Prosthet Dent 2022; S0022-3913(22)00281-285.
  • 20. Espinar C, Della Bona A, Pérez MM, Pulgar R. Color and optical properties of 3D printing restorative polymer‐based materials: A scoping review. J Esthet Restor Dent 2022;34(6):853-864.
  • 21. Song S-Y, Shin Y-H, Lee J-Y, Shin S-W. Color stability of provisional restorative materials with different fabrication methods. J Adv prosthodont. 2020;12(5):259.
  • 22. Gale M, Darvell B. Thermal cycling procedures for laboratory testing of dental restorations. J Dent 1999;27(2):89-99.
  • 23. Baffone W, Sorgente G, Campana R, Patrone V, Sisti D, Falcioni T. Comparative effect of chlorhexidine and some mouthrinses on bacterial biofilm formation on titanium surface. Curr Microbiol 2011;62(2):445-451.
  • 24. Ikeda M, Matin K, Nikaido T, Foxton RM, Tagami J. Effect of surface characteristics on adherence of S. mutans biofilms to indirect resin composites. Dent Mat J 2007;26(6):915-923.
  • 25. Busscher H, Van Pelt A, De Boer P, De Jong H, Arends J. The effect of surface roughening of polymers on measured contact angles of liquids. Colloid Surface. 1984;9(4):319-331.
  • 26. An YH, Friedman RJ. Laboratory methods for studies of bacterial adhesion. J Microbiol Meth 1997;30(2):141-152.
  • 27. Aykent F, Yondem I, Ozyesil AG, Gunal SK, Avunduk MC, Ozkan S. Effect of different finishing techniques for restorative materials on surface roughness and bacterial adhesion. J Prosthet Dent 2010;103(4):221-227.
  • 28. Cazzaniga G, Ottobelli M, Ionescu AC, Paolone G, Gherlone E, Ferracane JL, Brambilla E. In vitro biofilm formation on resin-based composites after different finishing and polishing procedures. J Dent 2017;67:43-52.
  • 29. Ozel GS, Guneser MB, Inan O, Eldeniz AU. Evaluation of C. Albicans and S. Mutans adherence on different provisional crown materials. J Adv Prosthodont 2017;9(5):335-340.
  • 30. Esberg A, Sheng N, Mårell L, Claesson R, Persson K, Boren T, Strömberg N. Streptococcus mutans adhesin biotypes that match and predict individual caries development. EBioMedicine 2017;24:205-215.
  • 31. Brady LJ, Maddocks SE, Larson MR, Forsgren N, Persson K, Deivanayagam CC, Jenkinson HF. The changing faces of Streptococcus antigen I/II polypeptide family adhesins. Mol Microbiol 2010;77(2):276-286.
  • 32. Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms. Virulence 2013;4(2):119-128.
  • 33. Sundstrom P. Adhesins in Candida albicans. Curr Opin Microbiol 1999;2(4):353-357
  • 34. Bollenl CM, Lambrechts P, Quirynen M. Comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: a review of the literature. Dent Mat 1997;13(4):258-269.
  • 35. Sterzenbach T, Helbig R, Hannig C, Hannig M. Bioadhesion in the oral cavity and approaches for biofilm management by surface modifications. Clin Oral Investig 2020;24(12):4237-4260.
  • 36. Kurt A, Cilingir A, Bilmenoglu C, Topcuoglu N, Kulekci G. Effect of different polishing techniques for composite resin materials on surface properties and bacterial biofilm formation. J Dent 2019;90:103199.
  • 37. Simoneti DM, Pereira-Cenci T, Dos Santos MBF. Comparison of material properties and biofilm formation in interim single crowns obtained by 3D printing and conventional methods. J Prosthet Dent 2022;127(1):168-172
  • 38. Schubert A, Bürgers R, Baum F, Kurbad O, Wassmann T. Influence of the Manufacturing Method on the Adhesion of Candida albicans and Streptococcus mutans to Oral Splint Resins. Polymers (Basel) 2021;13(10):1534.
  • 39. Taşın S, İsmatullaev A, Usumez A. Comparison of surface roughness and color stainability of 3-dimensionally printed interim prosthodontic material with conventionally fabricated and CAD-CAM milled materials. J Prosthet Dent 2022;128(5):1094-1101.
  • 40. Liebermann A, Wimmer T, Schmidlin PR, Scherer H, Löffler P, Roos M, Stawarczyk B. Physicomechanical characterization of polyetheretherketone and current esthetic dental CAD/CAM polymers after aging in different storage media. J Prosthet Dent 2016;115(3):321-8. e2.
  • 41. Murat S, Alp G, Alatalı C, Uzun M. In vitro evaluation of adhesion of Candida albicans on CAD/CAM PMMA‐based polymers. J Prosthodont 2019;28(2):e873-e879.
  • 42. Hahnel S, Rosentritt M, Handel G, Bürgers R. In vitro evaluation of artificial ageing on surface properties and early Candida albicans adhesion to prosthetic resins. J Mater Sci Mater Med 2009;20(1):249-255.

Konvansiyonel, CAD/CAM frezeleme ve 3D baskı yöntemleriyle üretilmiş geçici materyallerin yüzey özelliklerinin Streptococcus mutans ve Candida albicans tutunumuna etkileri

Year 2023, Volume: 26 Issue: 3, 227 - 234, 29.09.2023

Simge Taşın , Meryem Güvenir , Artur Ismatullaev

https://doi.org/10.7126/cumudj.1228677

Abstract

Amaç: Bu çalışmanın amacı konvansiyonel, CAD/CAM frezeleme ve 3D baskı yöntemleriyle üretilmiş geçici materyallerinin Streptococcus mutans ve Candida albicans tutunumu duyarlılıklarına göre karşılaştırılması ve yüzey pürüzlülüğü ve hidrofobikliğin buna etkisinin incelenmesidir.
Gereç ve Yöntemler: Otopolimerize polimetil metakrilat (A-PMMA), bis-akril kompozit (Bis-acrylate), CAD/CAM PMMA-bazlı polimer (Milled-PMMA) ve 3D baskı (Printed) rezinlerinden seksen adet disk şeklinde örnek üretildi. Örneklere 10,000 termal siklus (5-55°C) uygulandı ve kullanılan mikrobiyal süspansiyonlara göre örnekler iki gruba (n=10) ayrıldı: Streptococcus mutans ve Candida albicans. Örneklerin yüzey pürüzlülüğü (Ra) ve hidrofobikliği (WCA) ölçüldü. Örnekler Streptococcus mutans ve Candida albicans süspansiyonlarında 37°C'de 24 saat inkübe edilerek tutunum testi yapıldı ve yapışık hücreler koloni oluşturan birimler (CFU/ml) sayılarak değerlendirildi. Yüzeyleri analiz etmek için taramalı elektron mikroskobu (SEM) görüntüleri alındı (n=2). Veriler Kruskal-Wallis ve Mann-Whitney U testi ile analiz edildi. Ölçümler arasındaki korelasyonu belirlemek için Spearman korelasyon analizi kullanıldı (α=.05).
Bulgular: Restoratif materyalin türü, Ra ve WCA'yı önemli ölçüde etkiledi. En yüksek Streptococcus mutans adezyonu Printed'de gözlemlenirken, ardından Bis-akrilat, A-PMMA ve Milled-PMMA’da gözlemlenmiştir (p=.001). En yüksek Candida albicans adezyonu A-PMMA'da kaydedilirken, ardından Printed, Bis-acrylate ve Milled-PMMA’da (r=.001) kaydedilmiştir. Streptococcus mutans (r=.660) ve Candida albicans (r=.413) tutunumu ve Ra arasında pozitif korelasyon gözlemlenmiştir. Örneklerin WCA'sı ile Streptococcus mutans tutunumu arasında negatif bir korelasyon bulunmuştur (r=-.373).
Sonuçlar: Yüzey pürüzlülüğü mikroorganizmaların tutunumunda önemli rol oynar. CAD/CAM PMMA bazlı polimerler, uzun süreli kullanımda mikrobiyal yapışmayı azaltmak için daha iyi bir seçim olabilir.

Keywords

Geçici diş restorasyonu materyali mikroorganizma tutunumu yüzey özellikleri

Supporting Institution

Bu araştırma, kamu, ticari veya kar amacı gütmeyen sektörlerdeki finansman kuruluşlarından herhangi bir özel hibe almamıştır.

Project Number

Bu araştırma bir proje kapsamında yapılmamıştır.

Thanks

bu kısım boş bırakılacaktır

References

  • 1. Christensen GJ. The fastest and best provisional restorations. J Am Dent Assoc 2003;134(5):637-639.
  • 2. Axelsson P, Lindhe J. Effect of controlled oral hygiene procedures on caries and periodontal disease in adults. J Clin Periodontol 1978;5(2):133-151.
  • 3. Beighton D. The complex oral microflora of high‐risk individuals and groups and its role in the caries process. Community Dent Oral Epidemiol 2005;33(4):248-255.
  • 4. Salerno C, Pascale M, Contaldo M, Esposito V, Busciolano M, Milillo L, Guida A, Petruzzi M, Serpico R. Candida-associated denture stomatitis. Med Oral Patol Oral Cir Bucal 2011;16(2):139-143.
  • 5. Quirynen M. The clinical meaning of the surface roughness and the surface free energy of intra-oral hard substrata on the microbiology of the supra-and subgingival plaque: results of in vitro and in vivo experiments. J Dent 1994;22:13-16.
  • 6. Yuan C, Wang X, Gao X, Chen F, Liang X, Li D. Effects of surface properties of polymer-based restorative materials on early adhesion of Streptococcus mutans in vitro. J Dent 2016;54:33-40.
  • 7. Wei X, Gao L, Wu K, Yu P, Jiang L, Lin H, Wang Y, Cheng H. In vitro study of surface properties and microbial adhesion of various dental polymers fabricated by different manufacturing techniques after thermocycling. Clin Oral Investig 2022;26(12):7287-7297.
  • 8. Dorkhan M, de Paz LEC, Skepö M, Svensäter G, Davies JR. Effects of saliva or serum coating on adherence of Streptococcus oralis strains to titanium. Microbiology (Reading) 2012;158(2):390-397.
  • 9. Zamperini CA, Machado AL, Vergani CE, Pavarina AC, Giampaolo ET, Cruz NC. Adherence in vitro of Candida albicans to plasma treated acrylic resin. Effect of plasma parameters, surface roughness and salivary pellicle. Arch Oral Biol 2010;55(10):763-770.
  • 10. Li J, Hirota K, Goto T, Yumoto H, Miyake Y, Ichikawa T. Biofilm formation of Candida albicans on implant overdenture materials and its removal. J Deny 2012;40(8):686-692.
  • 11. Buergers R, Rosentritt M, Handel G. Bacterial adhesion of Streptococcus mutans to provisional fixed prosthodontic material. J Prosthet Dent 2007;98(6):461-469.
  • 12. Nyvad B, Fejerskov O. Scanning electron microscopy of early microbial colonization of human enamel and root surfaces in vivo. Scand J Dent Res 1987;95(4):287-296.
  • 13. Boaro LC, Gonçalves F, Guimarães TC, Ferracane JL, Pfeifer CS, Braga RR. Sorption, solubility, shrinkage and mechanical properties of “low-shrinkage” commercial resin composites. Dent Mater 2013;29(4):398-404.
  • 14. Ruse N, Sadoun M. Resin-composite blocks for dental CAD/CAM applications. J Dent Res 2014;93(12):1232-1234.
  • 15. Raszewski Z. Acrylic resins in the CAD/CAM technology: A systematic literature review. Dent Med Probl 2020;57(4):449-454.
  • 16. Perea-Lowery L, Gibreel M, Vallittu PK, Lassila L. Characterization of the mechanical properties of CAD/CAM polymers for interim fixed restorations. Dent Mater J 2020;39:319-325.
  • 17. Tahayeri A, Morgan M, Fugolin AP, Bompolaki D, Athirasala A, Pfeifer CS, Ferracane JL, Bertassoni LE. 3D printed versus conventionally cured provisional crown and bridge dental materials. Dent Mater 2018;34(2):192-200.
  • 18. Zhang X, Xu Y, Li L, Yan B, Bao J, Zhang A. Acrylate‐based photosensitive resin for stereolithographic three‐dimensional printing. J Appl Polym Sci. 2019;136(21):47487.
  • 19. Kim J-H, Kwon J-S, Park J-M, Russo LL, Shim J-S. Effects of postpolymerization conditions on the physical properties, cytotoxicity, and dimensional accuracy of a 3D-printed dental restorative material. J Prosthet Dent 2022; S0022-3913(22)00281-285.
  • 20. Espinar C, Della Bona A, Pérez MM, Pulgar R. Color and optical properties of 3D printing restorative polymer‐based materials: A scoping review. J Esthet Restor Dent 2022;34(6):853-864.
  • 21. Song S-Y, Shin Y-H, Lee J-Y, Shin S-W. Color stability of provisional restorative materials with different fabrication methods. J Adv prosthodont. 2020;12(5):259.
  • 22. Gale M, Darvell B. Thermal cycling procedures for laboratory testing of dental restorations. J Dent 1999;27(2):89-99.
  • 23. Baffone W, Sorgente G, Campana R, Patrone V, Sisti D, Falcioni T. Comparative effect of chlorhexidine and some mouthrinses on bacterial biofilm formation on titanium surface. Curr Microbiol 2011;62(2):445-451.
  • 24. Ikeda M, Matin K, Nikaido T, Foxton RM, Tagami J. Effect of surface characteristics on adherence of S. mutans biofilms to indirect resin composites. Dent Mat J 2007;26(6):915-923.
  • 25. Busscher H, Van Pelt A, De Boer P, De Jong H, Arends J. The effect of surface roughening of polymers on measured contact angles of liquids. Colloid Surface. 1984;9(4):319-331.
  • 26. An YH, Friedman RJ. Laboratory methods for studies of bacterial adhesion. J Microbiol Meth 1997;30(2):141-152.
  • 27. Aykent F, Yondem I, Ozyesil AG, Gunal SK, Avunduk MC, Ozkan S. Effect of different finishing techniques for restorative materials on surface roughness and bacterial adhesion. J Prosthet Dent 2010;103(4):221-227.
  • 28. Cazzaniga G, Ottobelli M, Ionescu AC, Paolone G, Gherlone E, Ferracane JL, Brambilla E. In vitro biofilm formation on resin-based composites after different finishing and polishing procedures. J Dent 2017;67:43-52.
  • 29. Ozel GS, Guneser MB, Inan O, Eldeniz AU. Evaluation of C. Albicans and S. Mutans adherence on different provisional crown materials. J Adv Prosthodont 2017;9(5):335-340.
  • 30. Esberg A, Sheng N, Mårell L, Claesson R, Persson K, Boren T, Strömberg N. Streptococcus mutans adhesin biotypes that match and predict individual caries development. EBioMedicine 2017;24:205-215.
  • 31. Brady LJ, Maddocks SE, Larson MR, Forsgren N, Persson K, Deivanayagam CC, Jenkinson HF. The changing faces of Streptococcus antigen I/II polypeptide family adhesins. Mol Microbiol 2010;77(2):276-286.
  • 32. Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms. Virulence 2013;4(2):119-128.
  • 33. Sundstrom P. Adhesins in Candida albicans. Curr Opin Microbiol 1999;2(4):353-357
  • 34. Bollenl CM, Lambrechts P, Quirynen M. Comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: a review of the literature. Dent Mat 1997;13(4):258-269.
  • 35. Sterzenbach T, Helbig R, Hannig C, Hannig M. Bioadhesion in the oral cavity and approaches for biofilm management by surface modifications. Clin Oral Investig 2020;24(12):4237-4260.
  • 36. Kurt A, Cilingir A, Bilmenoglu C, Topcuoglu N, Kulekci G. Effect of different polishing techniques for composite resin materials on surface properties and bacterial biofilm formation. J Dent 2019;90:103199.
  • 37. Simoneti DM, Pereira-Cenci T, Dos Santos MBF. Comparison of material properties and biofilm formation in interim single crowns obtained by 3D printing and conventional methods. J Prosthet Dent 2022;127(1):168-172
  • 38. Schubert A, Bürgers R, Baum F, Kurbad O, Wassmann T. Influence of the Manufacturing Method on the Adhesion of Candida albicans and Streptococcus mutans to Oral Splint Resins. Polymers (Basel) 2021;13(10):1534.
  • 39. Taşın S, İsmatullaev A, Usumez A. Comparison of surface roughness and color stainability of 3-dimensionally printed interim prosthodontic material with conventionally fabricated and CAD-CAM milled materials. J Prosthet Dent 2022;128(5):1094-1101.
  • 40. Liebermann A, Wimmer T, Schmidlin PR, Scherer H, Löffler P, Roos M, Stawarczyk B. Physicomechanical characterization of polyetheretherketone and current esthetic dental CAD/CAM polymers after aging in different storage media. J Prosthet Dent 2016;115(3):321-8. e2.
  • 41. Murat S, Alp G, Alatalı C, Uzun M. In vitro evaluation of adhesion of Candida albicans on CAD/CAM PMMA‐based polymers. J Prosthodont 2019;28(2):e873-e879.
  • 42. Hahnel S, Rosentritt M, Handel G, Bürgers R. In vitro evaluation of artificial ageing on surface properties and early Candida albicans adhesion to prosthetic resins. J Mater Sci Mater Med 2009;20(1):249-255.
There are 42 citations in total.

Details

Primary Language English
Subjects Health Care Administration
Journal Section Original Research Articles
Authors

Simge Taşın 0000-0002-0483-2520

Meryem Güvenir 0000-0002-9702-9947

Artur Ismatullaev 0000-0002-0119-6821

Project Number Bu araştırma bir proje kapsamında yapılmamıştır.
Publication Date September 29, 2023
Submission Date January 4, 2023
Published in Issue Year 2023Volume: 26 Issue: 3

Cite

EndNote Taşın S, Güvenir M, Ismatullaev A (September 1, 2023) Effects of Surface Characteristics of Conventionally Manufactured, CAD/CAM Milled, and 3D-Printed Interim Materials on Adherence of Streptococcus Mutans and Candida Albicans. Cumhuriyet Dental Journal 26 3 227–234.
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