Endodontic Biofilms and management..pptx

The presentation covers the whole aspect of Endodontic biofilm and its management in a clinical practice.
Right from definition to formation to its classification and its removal using various irrigants and techniques. Its microscopic structure. And role of E. Fecalis in biolfilm. Its also explains qurorom sensing very well

1. 1

2. BIOFILMS IN ENDODONTICS 2

3. Contents Introduction Definitions Characterestics of biofilm Microbes in endodontic biofilm Stages of biofilm formation 3

4. Contents Types of endodontic biofilms Methods of identifying biofilms Methods to eradicate biofilms Conclusion References 4

5. Introduction Microoraganisms in the normal microbiota participate in many beneficial physiological functions of the oral cavity . 5

6. Introduction In case, if the physical and biochemical conditions of these tissues change, commensals can convert into opportunistic pathogens. Microbes are the most essential agents for the development of primary peri-redicular diseases and are most essential factors responsible for the failure of endodontic therapy. 6

7. Introduction Hence the goal of primary endodontic therapy as well as retreatment should be to eradicate the microbes from the root canal system . 7

8. Introduction It is not possible to eradicate the microbes completely from the root canal system for following reasons: Bio-film formation inside the root canal system The relative antimicrobial resistance of microbes which are continuously undergoing mutations to adapt to any situation. Bacterial penetration and persistence in dentinal tubules 8

9. Introduction Van Leeuwenhoek, using his simple microscopes, first observed microorganisms on tooth surfaces and can be credited with the discovery of microbial bio-films . 9

10. Types of Endodontic Infections There are two types of endodontic infections: 1. Intra radicular infections. Primary Secondary Persistent 2. Extra radicular infections. 10

11. Types of Endodontic Infections Primary intra- radicular infections: Microorganisms that initially invade and colonize the necrotic pulp tissue causes primary intraradicular infection. The involved microbiota is conspicuously dominated by Gram negative anaerobic bacteria. Porphyromonas Prevotella Fusobacterium Campylobacter species 11

12. Types of Endodontic Infections Secondary intra- radicular infections: Microorganisms that were not present in the primary infection but that were introduced into the root canal system at some time after professional intervention causes secondary intraradicular infections. The entry can be during treatment, between appointments, or even after root canal filling. E.faecalis Staphylococcus E.coli Pseudomonas aeruginosa Candida species 12

13. Definitions Endodontic Biofilm is defined as a mode of microbial growth where dynamic communities of interacting sessile cells are irreversibly attached to a solid substratum, as well as each other, and are embedded in a self-made matrix of extracellular polymeric substances (EPS). - Ingle’s Endodontics , 6 th edition. 13

14. Definitions Biofilm can be defined as a sessile multicellular microbial community characterized by cells that are firmly attached to a surface and enmeshed in a self-produced matrix of extracellular polymeric substance (EPS), usually polysaccharide. - Cohen, Pathways of pulp, 10 th edition 14

15. Characteristics of biofilms Bacteria in a biofilm state show distinct capacity to survive tough growth and environmental conditions. Biofilm structure protects the residing bacteria from environmental threats. Structure of biofilm permits trapping of nutrients and metabolic cooperativity between resident cells of same species and/or different species. 15

16. Characteristics of biofilms Biofilm structures display organized internal compartmentalization, which allows bacterial species with different growth requirements to survive in each compartment. Biofilms shows resistance to antimicrobial agents. 16

17. Characteristics of biofilms Bacterial cells in a biofilm community may communicate and exchange genetic materials to acquire new traits. Communications between bacterial cells residing in a biofilm is attained through the following processes: Quorum sensing Auto inducer system 2(AI -2) 17

18. Microbes in endodontic biofilms The most commonly involved bacteria are : E . faecalis Coagulase -negative staphylococcus Actinomyces species P . propionicum Others are P. aeuroginosa , Fungi , F usobacterium nucleatum , P orphromonas gingivalis , T anerella forsythia. 18

19. Microbes in endodontic biofilms Molecular studies have also showed the occurrence of Synergistes , Dialaster , Prevotella , Solobacterium , Olsenella , Eubacterium , Megaspheara , Vellionella , Selenomonas . 19

20. Microbes in endodontic biofilms Enterococcus faecalis is a microorganism commonly detected in asymptomatic, persistent endodontic infections. Its prevalence in such infections ranges from 24% to 77%. Enterococci are gram positive cocci , facultative anaerobes that can occur singly, in pairs, or as short chains. 20

21. Microbes in endodontic biofilms Enterococci survive very harsh environments including extreme alkaline pH and salt concentrations . They can grow in the range of 10 to 45°C and survive a temperature of 60°C for 30 min. 21

22. Microbes in endodontic biofilms   E. faecalis in dentinal tubules has been shown to resist intracanal dressings of calcium hydroxide for over 10 days. E. faecalis is able to form a biofilm that enable the bacteria to become 1000 times more resistant to phagocytosis , antibodies, and antimicrobials than nonbiofilm producing organisms causing endodontic failure. 22

23. Stages of bio-film formation Biofilm is considered as community as it possesses following criteria : Autopoiesis (must possess the abilities to self-organize ) Haemostasis (resist environmental perturbations ) 23

24. Stages of bio-film formation Synergy (must be more effective in association than in isolation). Communality (respond to environmental changes as a unit rather than single individuals). 24

25. Stages of bio-film formation Biofilm formation is a step-wise procedure and it requires 3 major elements: Microorganisms Solid surface Fluid medium 25

26. Stages of bio-film formation Step 1: It involves adsorption of inorganic and organic molecules, to the solid surface which leads to the formation of conditioning film. 26

27. Stages of bio-film formation Step 2: Adhesion of planktonic microbial cells to the conditioning layer and colonization of microbes. Phase 1: Transport of the microbe to substrate surface and its attachment. Phase 2: Initial non specific microbial and substrate adherence phase Phase 3: Specific microbial–substrate adherence phase 27

28. Stages of bio-film formation Step 3: Bacterial grow by co-adhesion of other organisms and biofilm expansion. Monolayer of microorganisms attracts secondary colonizers to form microcolonies by co-adhesion and co-aggregation. 28

29. Stages of bio-film formation Step 4: Detachment of sessile microorganisms i.e. present within biofilm into their surroundings by Seeding dispersal Clumping dispersal. 29

30. Stages of bio-film formation In vitro experiments have revealed distinct stages in the development of E. faecalis biofilm on root canal dentine. Stage 1: E . faecalis cells adheres and forms microcolonies on the root canal dentine surface. 30

31. Stages of bio-film formation Stage 2: They induced bacterial-mediated dissolution of the mineral fraction from the dentine substrate. This localized increase in the calcium and phosphate ions promotes mineralization (or calcification) of the E.faecalis biofilm . 31

32. Stages of bio-film formation Stage 3: The mature biofilm structure formed after 6 weeks of incubation showed signs of mineralization and subtle but distinct compositional difference. The mineralized E.faecalis biofilm showed carbonated-apatite structure as compared to natural dentine which had carbonated fluorapatite structure. 32

33. Types of endodontic biofilms Endodontic bacterial biofilms can be categorized as: Intra-canal biofilms • Extra- radicular biofilms • Peri -apical biofilms • Biomaterial centered infections 33

34. Types of endodontic biofilms Intracanal biofilms Intracanal microbial biofilms are microbial biofilms formed on the root canal dentine of an endodontically infected tooth. A detailed description on the intracanal bacterial biofilm was documented by Nair in 1987. 34

35. Types of endodontic biofilms Intracanal biofilms The intracanal microbiota existed as both loose collection and biofilm structures, made up of cocci , rods, and filamentous bacteria. Monolayer and/or multilayered bacterial biofilms were found to adhere to the dentinal wall of the root canal. 35

36. Types of endodontic biofilms E. faecalis under nutrient-rich environment (aerobic and anaerobic) produced typical biofilm structures with characteristic surface aggregates of bacterial cells and water channels. 36

37. Types of endodontic biofilms Under nutrient-deprived environment, irregular growth of adherent cell clumps were observed. 37

38. Types of endodontic biofilms Riccuci D et al (2010) evaluated the prevalence of bacterial biofilms in untreated and treated root canals of teeth evincing apical periodontitis . They stated that intraradicular biofilms were observed in the apical segment of 77% of the root canals. Biofilms were also seen covering the walls of ramifications and isthmuses. Extraradicular biofilms were observed in only 6% of the cases. 38 Ricucci D ,  Siqueira JF, Biofilms and apical periodontitis : study of prevalence and association with clinical and histopathologic findings. J Endod .  2010 Aug;36(8):1277-88 .

39. Types of endodontic biofilms Extra- radicular biofilms Extraradicular microbial biofilms are formed on the root ( cementum ) surface adjacent to the root apex of endodontically infected teeth. They are also termed root surface biofilms . 39

40. Types of endodontic biofilms Extraradicular biofilms were reported in teeth with Asymptomatic periapical periodontitis Chronic apical abscesses associated with sinus tracts. Mature bacterial biofilms were found in many areas of the apical root surfaces in all clinical specimens. 40

41. Types of endodontic biofilms The extraradicular biofilm structures were dominated by cocci and short rods, with cocci attached to the tooth substrate. Filamentous and fibrillar forms were also observed in the biofilm . 41

42. Types of endodontic biofilms Harn et al.., noticed calculus-like deposits on apical root surface of tooth presented with lesion refractory to conventional root canal treatment. These calcified biofilms were associated with periapical inflammation and delayed periapical healing in spite of adequate orthograde root canal treatment. 42

43. Types of endodontic biofilms Periapical biofilms Periapical microbial biofilms are isolated biofilms found in the periapical region of an endodontically infected teeth. Periapical biofilms may or may not be dependent on the root canal. 43

44. Types of endodontic biofilms Members of the genus Actinomyces and the species P. propionicum are seen in asymptomatic periapical lesions refractory to endodontic treatment. These microorganisms have the ability to overcome host defense mechanisms, thrive in the inflamed periapical tissue, and subsequently induce a periapical infection. 44

45. Types of endodontic biofilms Actinomyces species in tissues grow in aggregates, up to a diameter of 3 to 4mm. They are commonly referred to as ‘‘sulfur granules,’’ because of the yellow granular appearance. 45

46. Types of endodontic biofilms Wang J et al (2012) investigated the bacterial flora and type of film formed in persistent peri-radicular infections. Apical root samples from root-end surgery were collected from 23 root-filled teeth with apical periodontitis . Samples were observed by SEM analysis. Wang J ,  Jiang Y ,  Chen W ,  Zhu C ,  Liang J . Bacterial flora and extraradicular biofilm associated with the apical segment of teeth with post-treatment apical periodontitis . J Endod . 2012 Jul;38(7):954-9. 46

47. Types of endodontic biofilms Extraradicular biofilm was present on the external root surface of treated teeth with persistent periapical lesions. Actinomyces species and Propionibacterium are likely important contributors to extraradicular biofilm formation and persistent periapical infection. 47 Wang J ,  Jiang Y ,  Chen W ,  Zhu C ,  Liang J . Bacterial flora and extraradicular biofilm associated with the apical segment of teeth with post-treatment apical periodontitis. J Endod. 2012 Jul;38(7):954-9.

48. Types of endodontic biofilms Biomaterial-centered infection Biomaterial- centred infection (BCI) is caused when bacteria adheres to an artificial biomaterial surface and forms biofilm structures. BCI is one of the major complications associated with prosthesis and/or implant-related infections. 48

49. Types of endodontic biofilms In endodontics , biomaterial-centered biofilms would form on root canal obturating materials. These biofilms can be intraradicular or extraradicular depending upon whether the obturating material is within the root canal space or has it extruded beyond the root apex. 49

50. Types of endodontic biofilms BCI usually reveals opportunistic invasion by nosocomial organisms. Coagulase -negative Staphylococcus, S. aureus , enterococci , streptococci, P. aeruginosa , and fungi are commonly isolated from infected biomaterial surfaces. 50

51. Types of endodontic biofilms Because biofilms are extremely resistant to host defense mechanisms and antibiotic treatments. BCI are rarely resolved, and often the only solution to an infected biomaterial such as obturating material is its surgical removal. 51

52. Types of endodontic biofilms Takemura N et al (2004) reported the biofilm formation ability in teeth with over filled guttapercha . They stated that Gram-positive facultative anaerobes (E. faecalis ) have the ability to colonize and form extracellular matrices on gutta-percha points. 52

53. Ultra-structure of biofilm A glycocalyx matrix made up of extra-cellular polymeric substances surrounds the microcolonies and anchors the bacterial cell to the substrate. It is made up of 85% by volume is made up of polysacchride matrix material, while 15% is made up of cells.   53

54. Ultra-structure of biofilm A viable, fully hydrated biofilm appears as “tower” or “mushroom” shaped structures adherent to a substrate. 54

55. Ultra-structure of biofilm The water channels, which are regarded as a primitive circulatory system in a biofilm , to establish connections between the microcolonies . 55

56. Ultra-structure of biofilm Presence of water channels facilitates efficient exchange of materials between bacterial cells and bulk fluid, which in turn helps to coordinate functions in a biofilm community. 56

57. Methods of identifying biofilms Traditional methods Recent methods 57

58. Methods of identifying biofilms Traditional methods 1. Gram's stain 2. Culture methods 58

59. Methods of identifying biofilms Disadvantages of Culturing Method 1 . Unable to grow several microorganisms which can give false negative results. 2. Strictly depend on mode of sample transport which must allow growth of anaerobic bacteria. 3. Low sensitivity and specificity. 4. Time consuming . 59

60. Methods of identifying biofilms Molecular Diagnostic Methods Molecular diagnostic methods identify the microorganisms using gene as a target which are unique for each species. DNA-DNA hybridization method polymerase chain reaction method 60

61. Methods of identifying biofilms DNA-DNA Hybridization Method This method uses DNA probes which target genomic DNA or individual genes. This method helps in simultaneous determination of the presence of a multitude of bacterial species in single or multiple clinical samples. 61

62. Methods of identifying biofilms In this method, segments of labeled, single strand DNA locate and bind to their complementary nucleic acid sequences. After washing, the presence of bound label indicates the presence of the target DNA sequence. 62

63. Methods of identifying biofilms Polymerase Chain Reaction Method (PCR Method) PCR method involves in vitro replication of DNA, therefore it is also called as "genetic xeroxing " method. Multiple copies of specific region of DNA are made by repeated cycles or heating and cooling . 63

64. Methods of identifying biofilms PCR has remarkable sensitivity and specificity because each distinct microbial species has unique DNA sequences. PCR can be used to detect virtually all bacterial species in a sample. It is also used to investigate microbial diversity in a given environment. 64

65. Methods of identifying biofilms Terminal-RFLP method T-RFLP is a recent molecular approach that can assess genetic differences between microbial strains as well as provide insight into the structure and function of microbial communities. 65

66. Methods of identifying biofilms rDNA from different species in a community is PCR amplified using one of the PCR primers labeled with a fluorescent dyes 4, 7, 2′, 7′ tetrachloro-6-carboxyfluorescein (TET) Phosphoramidite fluorochrome 5-carboxyfluorescein (6-FAM). 66

67. Methods of identifying biofilms DNA Microarrays Microarray methods essentially consist of many probes that are discretely located on a nonporous solid support, such as a glass slide. Probes can be DNA fragments such as library clones or PCR products. Printed arrays and high-density oligonucleotide arrays are the most commonly used types of microarrays. 67

68. Methods of identifying biofilms Confocal Laser Scanning microscope and fluorescence in situ hybridization are valuable tools for obtaining high resolution images and 3-D reconstruction of a variety of biological samples including their biofilm . 68

69. Methods of identifying biofilms The Calgary Biofilm Device (CBD) offers a new technology for the rational selection of antibiotics, effective against microbial biofilms and for the screening of new effective antibiotic compounds. 69

70. Methods of eradicating endodontic biofilms 70

71. Methods of eradicating endodontic biofilms Antimicrobial agents have been developed to remove the bacteria in the biofilm . However , microbial communities in biofilms are remarkably difficult to eradicate with antimicrobial agents and microorganisms in mature biofilms can be notoriously resistant for the following reasons 71

72. Methods of eradicating endodontic biofilms Mechanisms of anti-microbial resistance of biofilm The microorganisms grown in biofilms could be 1000-1500 times more resistant to antimicrobial agents that planktonic bacteria. The nature of biofilm structure . Physiological characteristics of the resident microorganisms. 72

73. Methods of eradicating endodontic biofilms 73 The polysaccharide matrix of biofilms can retard diffusion of antibiotics. Bacteria protect themselves by being located within the interior part of a biofilm . Bacterial cells residing within a biofilm grow more slowly than planktonic cells and as a result, antimicrobial agents act more slowly. Depletion of nutrients or accumulation of waste products can result in bacteria entering a non-growing state

74. Methods of eradicating endodontic biofilms Chemo-mechanical preparation It has been proved that bacteria might penetrate dentinal tubules to depths of 200 mm or more. Complete uniform enlargement of a root canal by 200 mm is not achieved with any mechanical instrumentation. So, a chemical irrigant is used as an adjunct to assist in reducing the bacterial load and their toxic by-products. 74

75. Chemo-mechanical preparation All infected pulp tissue, bacteria and their by products should be removed from the root canal. The root canal preparation should develop a continuously tapering cone . Making the canal narrower apically and widest coronally . Avoid transportation of the foramen. 75 Methods of eradicating endodontic biofilms

76. Chemo-mechanical preparation Keep the apical opening as small as possible. Procedure should be confined to the root canal space. Sufficient space for intracanal medicaments and irrigants should be created. 76 Methods of eradicating endodontic biofilms

77. Sodium hypochlorite Sodium hypochlorite ( NaOCl ) is an excellent antibacterial agent, capable of dissolving necrotic tissue, vital pulp tissue, and the organic components of dentin and biofilms . NaOCl ionizes in water into Na +2 and the hypochlorite ion, OCl - 77 Methods of eradicating endodontic biofilms

78. NaOCl is commonly used in concentrations between 0.5% and 6%. 6% NaOCl was the only irrigant capable of both rendering bacteria nonviable and physically removing the biofilm . The combined application of EDTA and NaOCl significantly reduces the amount of intracanal biofilm . 78 Methods of eradicating endodontic biofilms

79. Its anti-bacterial effect is due to : Penetration into the bacterial cell wall. Chemical combination with the protoplasm of the bacteria and disruption of DNA synthesis. 79 Methods of eradicating endodontic biofilms

80. Afzal A et al.., (2013) evaluated the antimicrobial efficacy of 5.25% sodium hypochlorite, 2% chlorhexidine and MTAD against E. faecalis in the bio-film during the cleaning and shaping of the canal system. 80 single-rooted mandibular premolars were taken. E. faecalis ATCC 29212 strain were inoculated into the teeth. 80 A Afzal , V Rajesh Gopal , Rajesh Pillai ,   Asha Sarah Jacob, S U-Nu, S Shan.. Antimicrobial activity of various irrigants against  E. faecalis   biofilm : An  in vitro  study. Year : 2013  |  Volume : 3  |  Issue : 2  |  Page : 103-108 Methods of eradicating endodontic biofilms

81. 5.25% Sodium hypochlorite showed the most effective antimicrobial property among the three irrigants against E. faecalis biofilm . 81 A Afzal , V Rajesh Gopal , Rajesh Pillai ,   Asha Sarah Jacob, S U-Nu, S Shan.. Antimicrobial activity of various irrigants against  E. faecalis   biofilm : An  in vitro  study. Year : 2013  |  Volume : 3  |  Issue : 2  |  Page : 103-108 Methods of eradicating endodontic biofilms

82. Iodine Solutions Iodine in potassium iodide solution is a potential root canal irrigant against E. faecalis . A 10-minute irrigant –dentine contact time on dentine infected with E. faecalis was sufficient to prevent growth. 82 Methods of eradicating endodontic biofilms

83. A solution of 5% iodine in potassium iodide or Churchill’s solution can be used. Churchill’s solution consists of 83 Iodine (16.5 g) Potassium iodide (3.5 g) Distilled water (20 g) 90% ethanol (60 g). Methods of eradicating endodontic biofilms

84. Chlorhexidine 0.2 - 2% CHX may be used as an irrigant that is effective against E. faecalis . CHX permeates the microbial cell wall and attacks the bacterial cytoplasmic or inner membrane. In high concentrations, CHX causes coagulation of intracellular components. 84 Methods of eradicating endodontic biofilms

85. Chlorhexidine gluconate has good substantivity and has the ability to adhere to hydroxyapatite crystals in dentine. Although bacteria may be killed by CHX, the biofilm and other organic debris are not removed by it . So, 2% chlorhexidine is best used after irrigating with 5.25 % NaOCL for 100% removal of endodontic biofilms 85 Methods of eradicating endodontic biofilms

86. Vianna et al.., (2004) investigated the antimicrobial activity of 0.2%, 1%, and 2% chlorhexidine gluconate (CHX gel and CHX liquid), against endodontic pathogens. 2 % CHX gel effectively removed E.faecalis in 1 min. The timing required for 1.0% and 2.0% CHX liquid to eliminate all microorganisms was the same required for 5.25% NaOCl i.e., around 30 sec. Vianna ME, Gomes BP, Berber VB,  Zaia AA,  Ferraz CC, de Souza- Filho FJ.. In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite.. Oral Surg Oral Med Oral Pathol Oral Radiol Endod . 2004 Jan;97(1):79-84. 86 Methods of eradicating endodontic biofilms

87. MTAD MTAD is a recently developed irrigating solution that consists of tetracycline, citric acid and detergent ( Tween 80). The MTAD should be used for 5 minutes as a final rinse after hypochlorite and EDTA. 87 Methods of eradicating endodontic biofilms

88. MTAD It is able to kill E. faecalis . It is effective for removing smear layer along the entire length of the prepared root canal. 88 Methods of eradicating endodontic biofilms

89. Kamberi B et al.., (2012) assessed the antimicrobial efficacy of Biopure MTAD against E. faecalis  in contaminated root canals.  42 single rooted extracted teeth were inoculated with E. faecalis  and incubated for four weeks. Blerim Kamberi , Donika Bajrami , Miranda Stavileci , Shuhreta Omeragiq , Fatmir Dragidella , Ferit Koçan . The Antibacterial Efficacy of Biopure MTAD in Root Canal Contaminated with  Enterococcus faecalis ISRN Dent. 2012 89 Methods of eradicating endodontic biofilms

90. The samples were divided experimental groups irrigated with 90 1.5% NaOCl 3% NaOCl ; BioPure MTAD; 1.5% NaOCl /17% EDTA 3% NaOCl /17% EDTA Methods of eradicating endodontic biofilms

91. They stated that BioPure MTAD possesses superior bactericidal activity compared with 1.5 % NaOCl and 17 % EDTA against E. faecalis . 91 Blerim Kamberi, Donika Bajrami, Miranda Stavileci, Shuhreta Omeragiq, Fatmir Dragidella, Ferit Koçan. The Antibacterial Efficacy of Biopure MTAD in Root Canal Contaminated with  Enterococcus faecalis ISRN Dent. 2012 Methods of eradicating endodontic biofilms

92. Mittal R et al.., (2012) evaluate and compare the antibacterial efficiency of MTAD, Oxytetracycline , 5% NaOCl , and 2% chlorhexidine when used as root canal irrigants against Enterococcus faecalis . 50 extracted single rooted anterior teeth were selected. Mittal R, Singla MG, Garg A, Gupta S, Dahiya V. Comparative evaluation of the antimicrobial efficacy of MTAD, oxytetracycline , sodium hypochlorite and chlorhexidine against Enterococcus faecalis : An ex-vivo study. Saudi Endod J 2012;2:70-4 92 Methods of eradicating endodontic biofilms

93. Oxytetracycline has a great potential as a root canal irrigating agent because of its superior antimicrobial efficacy against E. faecalis , easy availability and cost effectiveness. 93 Mittal R, Singla MG, Garg A, Gupta S, Dahiya V. Comparative evaluation of the antimicrobial efficacy of MTAD, oxytetracycline , sodium hypochlorite and chlorhexidine against Enterococcus faecalis : An ex-vivo study. Saudi Endod J 2012;2:70-4 Methods of eradicating endodontic biofilms

94. Tetraclean Tetraclean is a newer irrigant similar to MTAD. Composition Doxycycline (50 mg/5 ml) Citric acid Detergent (polypropylene glycol). 94 Methods of eradicating endodontic biofilms

95. Pappen FG et al..,(2010) investigated the antibacterial effect of Tetraclean , MTAD and five experimental irrigants with planktonic cultures and mixed-species in vitro biofilm model. 95 Pappen, F. G., et al. "In vitro antibacterial action of Tetraclean, MTAD and five experimental irrigation solutions."  International endodontic journal  43.6 (2010): 528-535. Tetraclean MTAD MTAD + 0.01% cetrimide (CTR), MTAD + 0.1% CTR, MTAC-1 ( Tween 80 replaced by 0.01% CTR in MTAD), MTAC-2 ( Tween 80 replaced by 0.1% CTR) MTAD-D (MTAD without the Tween 80 and no CTR added) Methods of eradicating endodontic biofilms

96. Tetraclean was more effective than MTAD against E. faecalis in both the cultures. CTR improved the antimicrobial properties of the solutions, whereas Tween 80 seemed to have a neutral or negative impact on their antimicrobial effectiveness. Pappen , F. G., et al. "In vitro antibacterial action of Tetraclean , MTAD and five experimental irrigation solutions."  International endodontic journal  43.6 (2010): 528-535. 96 Methods of eradicating endodontic biofilms

97. Qmix : It is effective in removal of smear layer containing bacteria and necrotic tissue. It consists of EDTA chlorhexidine Detergent 97 Methods of eradicating endodontic biofilms

98. Stojicic S et al.., (2012) assesed the efficacy of a novel root canal irrigant ,  QMiX , against  E.faecalis   and mixed plaque bacteria in planktonic phase and  biofilms . Stojicic , S., Shen , Y., Qian , W., Johnson, B., & Haapasalo , M. (2012). Antibacterial and smear layer removal ability of a novel irrigant , QMiX. International endodontic journal ,  45 (4), 363-371 98 QMiX , 2% chlorhexidine (CHX), MTAD 1% NaOCl for 5 s, 30 s and 3 min Methods of eradicating endodontic biofilms

99. QMiX  and NaOCl were superior to CHX and MTAD in killing E.faecalis  and plaque bacteria in planktonic and  biofilm  culture. 99 Stojicic , S., Shen , Y., Qian , W., Johnson, B., & Haapasalo , M. (2012). Antibacterial and smear layer removal ability of a novel irrigant , QMiX. International endodontic journal ,  45 (4), 363-371 Methods of eradicating endodontic biofilms

100. Ordinola R et al.., (2013) evaluated whether the use of chelating agents with anti-microbial activity show similar disinfection ability in comparison to conventional  irrigants  as sodium hypochlorite or iodine potassium iodide against biofilms developed on dentin. Ordinola -Zapata, R., Bramante, C. M., Brandão Garcia, R., Bombarda de Andrade, F., Bernardineli , N., Gomes de Moraes , I., & Duarte, M. A. (2013). The antimicrobial effect of new and conventional endodontic irrigants on intra-orally infected dentin.  Acta Odontologica Scandinavica ,  71 (3-4), 424-431. 100 MTAD, Qmix , Smear Clear, 7% maleic acid, 2% iodine potassium iodide, 4% peracetic acid, 2.5% and 5.25% sodium hypochlorite Methods of eradicating endodontic biofilms

101. 101 The irrigant solutions 4% peracetic acid and 2.5-5.25% sodium hypochlorite decreases significantly the number of live bacteria in biofilms , providing also cleaner dentin surfaces. Ordinola-Zapata, R., Bramante, C. M., Brandão Garcia, R., Bombarda de Andrade, F., Bernardineli, N., Gomes de Moraes, I., & Duarte, M. A. (2013). The antimicrobial effect of new and conventional endodontic irrigants on intra-orally infected dentin.  Acta Odontologica Scandinavica ,  71 (3-4), 424-431. Methods of eradicating endodontic biofilms

102. Recent advances in irrigation for effectively removing biofilms : 102 Ultrasonic irrigation Sonic irrigation Plasma dental probe Photo-activated disinfection Lasers Ozone Methods of eradicating endodontic biofilms

103. Ultrasonic irrigation Ultrasonics together with an irrigant , contribute to a better cleaning of the root canal system than conventional irrigation. It works at a frequency of 25-30KHz. It is of 3 types: Active irrigation Passive irrigation Continous irrigation 103 Methods of eradicating endodontic biofilms

104. 2 main features: Cavitation Acoustic streaming 104 Methods of eradicating endodontic biofilms

105. de Almeida AP et al.., (2014) compared the effectiveness of calcium hypochlorite (Ca[ OCl ]2) and sodium hypochlorite ( NaOCl ) associated with passive ultrasonic irrigation in root canals infected with Enterococcus   faecalis . The root canals of 60 single-rooted bovine extracted teeth inoculated with Enterococcus faecalis , and incubated for 30 days. de Almeida AP 1 ,  Souza MA 2 ,  Miyagaki DC 1 ,  Bello YD 1 ,  Cecchin D 3 ,  Farina AP . Comparative Evaluation of Calcium Hypochlorite and Sodium Hypochlorite Associated with Passive Ultrasonic Irrigation on Antimicrobial Activity of a Root Canal System Infected with Enterococcus   faecalis : An In Vitro Study. J Endod .  2014 Dec;40(12):1953-7 105 G1: no treatment; G2: distilled water; G3: 2.5% NaOCl ; G4: 2.5% Ca( OCl )2; G5: 2.5% NaOCl with ultrasonic activation; G6: 2.5% Ca( OCl )2 with ultrasonic activation. Methods of eradicating endodontic biofilms

106. Ca( OCl )2 as well as passive ultrasonic irrigation can aid in chemomechanical preparation, contributing in a significant way to the reduction of microbial content during root canal treatment. 106 de Almeida AP 1 ,  Souza MA 2 ,  Miyagaki DC 1 ,  Bello YD 1 ,  Cecchin D 3 ,  Farina AP . Comparative Evaluation of Calcium Hypochlorite and Sodium Hypochlorite Associated with Passive Ultrasonic Irrigation on Antimicrobial Activity of a Root Canal System Infected with Enterococcus   faecalis : An In Vitro Study. J Endod .  2014 Dec;40(12):1953-7 Methods of eradicating endodontic biofilms

107. Endo-activator The EndoActivator system, a sonic device, has recently been developed for root canal irrigation. Special polymer tips can be driven sonically at three different frequencies in order to activate the irrigant 107 Methods of eradicating endodontic biofilms

108. It is able to debride into deep lateral anatomy, remove the smear layer and dislodge the simulated biofilms in curved canals. 108 Methods of eradicating endodontic biofilms

109. Huffaker SK et al.., (2010) evaluated the ability of a new passive sonic irrigation (sonic group) system ( EndoActivator ) to eliminate cultivable bacteria from root canals in vivo and compared it with that of standard syringe irrigation . They supported the multi-visit approach to the treatment of apical periodontitis . Huffaker , S. K., Safavi , K., Spangberg , L. S., & Kaufman, B. (2010). Influence of a passive sonic irrigation system on the elimination of bacteria from root canal systems: a clinical study.  Journal of endodontics ,  36 (8), 1315-1318. 109 Methods of eradicating endodontic biofilms

110. Photo activated disinfection PAD is a unique combination of a photosensitizer solution and low-power laser light. The low-power laser will destruct the target area and inactivate the microbial invaders. The photosensitizer then binds to microbial cell walls or even enters the cells. PAD needs a maximum of 150 seconds. 110 Methods of eradicating endodontic biofilms

111. Bago I (2013) evaluated the antimicrobial effect of a diode laser irradiation, photo-activated disinfection (PAD), conventional and sonic activated irrigation with 2.5% sodium hypochlorite ( NaOCl ) on E. faecalis . Root canals of 120 human extracted teeth contaminated with an E. faecalis suspension and incubated for 7 days. Bago , I., Plečko , V., Gabrić Pandurić , D., Schauperl , Z., Baraba , A., & Anić , I. (2013). Antimicrobial efficacy of a high‐power diode laser, photo‐activated disinfection, conventional and sonic activated irrigation during root canal treatment.  International endodontic journal ,  46 (4), 339-347. 111 G1, diode laser irradiation (2 W, 3 × 20 s); G2, PAD (100 mW , 60 s); G3, PAD with 3D Endoprobe (100 mW , 60 s); G4, 30-gauge syringe irrigation with NaOCl (60 s); G5, sonic agitation of NaOCl with the EndoActivator system (60 s); G6, 30-gauge syringe irrigation with NaCl (60 s). Methods of eradicating endodontic biofilms

112. PAD and EndoActivator system were more successful in reducing the root canal infection than the diode laser and NaOCl syringe irrigation alone. 112 Bago, I., Plečko, V., Gabrić Pandurić, D., Schauperl, Z., Baraba, A., & Anić, I. (2013). Antimicrobial efficacy of a high‐power diode laser, photo‐activated disinfection, conventional and sonic activated irrigation during root canal treatment.  International endodontic journal ,  46 (4), 339-347. Methods of eradicating endodontic biofilms

113. Plasma dental probe These plasma jets generate efficiently reactive plasma species including ions, ozone, and oxygen radicals by energetic collisions of electrons. The interaction of plasma species with the bacterial membrane causes their disruption and consequently the death of bacterial cells 113 Methods of eradicating endodontic biofilms

114. SEM shows complete destruction of endodontic biofilms for a depth of 1 mm inside a root canal after plasma treatment for 5 min. 114 Methods of eradicating endodontic biofilms

115. Schaudinn C et al.., (2013) evaluated the efficacy of a nonthermal plasma (NTP) probe on ex vivo  biofilm  in root canals of extracted teeth. One group of teeth was treated with NTP, another with 6% NaOCl and one set was left untreated. The nonthermal plasma displayed antimicrobial activity against endodontic  biofilms  in root canals, but was not as effective as the use of 6% NaOCl . Schaudinn , C., Jaramillo, D., Freire , M. O., Sedghizadeh , P. P., Nguyen, A., Webster, P., ... & Jiang, C. (2013). Evaluation of a nonthermal plasma needle to eliminate ex vivo biofilms in root canals of extracted human teeth. International endodontic journal ,  46 (10), 930-937. 115 Methods of eradicating endodontic biofilms

116. Lasers The commonly used laser for disinfection are Er:YAG laser. Lasers induces the thermal effect causing an alteration in the bacterial cell wall leading to changes in the osmotic gradients and cell death. 116 Methods of eradicating endodontic biofilms

117. Ordinola R (2014) compared the removal of  biofilm  utilizing four irrigation techniques on a bovine root canal model. Needle irrigation, Endoactivator Passive ultrasonic irrigation  Laser-activated irrigation  Ordinola ‐Zapata, R., Bramante, C. M., Aprecio , R. M., Handysides , R., & Jaramillo, D. E. (2013). Biofilm removal by 6% sodium hypochlorite activated by different irrigation techniques.  International endodontic journal . 117 Methods of eradicating endodontic biofilms

118. Methods of eradicating endodontic biofilms Laser activation of 6% sodium hypochlorite significantly improved the cleaning of  biofilm -infected dentine followed by passive ultrasonic irrigation 118 Ordinola ‐Zapata, R., Bramante, C. M., Aprecio , R. M., Handysides , R., & Jaramillo, D. E. (2013). Biofilm removal by 6% sodium hypochlorite activated by different irrigation techniques.  International endodontic journal .

119. Methods of eradicating endodontic biofilms Intra-canal medicaments The commonly used medicaments for removal of bio-film are: Calcium hydroxide Triple antibiotic paste Antibiotics 119

120. Calcium Hydroxide The use of calcium hydroxide in endodontics was introduced by Hermann in 1920. In aqueous form, calcium hydroxide is poorly dissociated but the hydroxyl ions liberated create the high pH (10-12) that is required for bacterial killing. 120 Methods of eradicating endodontic biofilms

121. Methods of eradicating endodontic biofilms The following reasons have been proposed to explain why E. faecalis is able to survive intracanal treatment with calcium hy­droxide: E. faecalis passively maintains pH homeostasis. This occurs as a result of ions penetrating the cell membrane as well as the cytoplasm’s buffering capacity. E. faecalis has a proton pump that provides an additional means of maintaining pH homeostasis. This is accomplished by “pumping” protons into the cell to lower the internal pH. 121

122. Iodine in Potassium Iodide Iodine compounds are powerful oxidizing agents that disrupt bacterial cellular enzyme systems and inactivate them. Iodine in potassium iodide (IPI) has been as an intracanal antiseptic agent in concentrations between 2 and 10% in aqueous solution. The effectiveness of the medicament is short-lived and is not suitable for periods of medication longer than 2 days. 122 Methods of eradicating endodontic biofilms

123. Commercially available preparations of iodoform and calcium hydroxide, such as Metapex , are available. The material is highly radio-opaque and can be useful to confirm that the entire root canal system has been obliterated with medicament prior to sealing the access cavity 123 Methods of eradicating endodontic biofilms

124. Chlorhexidine digluconate It can be used as an effective intracanal medicament, 2% CHX gel is used normally. Mixture of CHX and calcium hydroxide can also be used. 124 Methods of eradicating endodontic biofilms

125. de Lucena JM (2013) determine the viability of E.faecalis in infected human root dentine in vitro after exposure to root canal medicaments. Human root segments were infected with E.  faecalis for 8 weeks. Calcium hydroxide 5% chlorhexidine gel 5% Octenidine gel Lucena , J. M. V. M., Decker, E. M., Walter, C., Boeira , L. S., Löst , C., & Weiger , R. (2013). Antimicrobial effectiveness of intracanal medicaments on Enterococcus faecalis : chlorhexidine versus octenidine .  International endodontic journal ,  46 (1), 53-61. 125 Methods of eradicating endodontic biofilms

126. Both CHX - and octenidine -based intracanal medicaments were effective in decreasing the viability of E.  faecalis . OCT showed the most favourable results and may have potential as an endodontic medicament 126 Lucena, J. M. V. M., Decker, E. M., Walter, C., Boeira, L. S., Löst, C., & Weiger, R. (2013). Antimicrobial effectiveness of intracanal medicaments on Enterococcus faecalis: chlorhexidine versus octenidine.  International endodontic journal ,  46 (1), 53-61. Methods of eradicating endodontic biofilms

127. Antibiotics Pastes such as Grossman’s polyantibiotic paste have been used as a medicament in root canal treatment. The paste contained penicillin, but, as most of the bacterial species found in the root canal are resistant due to their ability to produce the enzyme beta- lactamase , it is relatively ineffective. 127 Methods of eradicating endodontic biofilms

128. Antibiotics Metronidazole is effective against Gram negative anaerobes. It has been advocated for use as a intracanal medicament. 128 Methods of eradicating endodontic biofilms

129. Tetracyclines Tetracyclines have been used with some success in periodontal treatment. Doxycycline combined with corticosteroids are ingredients of Ledermix . Unfortunately, the range and duration of antimicrobial action may be limited in the endodontic environment. 129 Methods of eradicating endodontic biofilms

130. Clindamycin Clindamycin placed in teeth with necrotic pulps undergoing root canal treatment offered no advantage over conventional root canal dressings such as calcium hydroxide for elimination of bacteria. 130 Methods of eradicating endodontic biofilms

131. Triple antibiotic paste (TAP) TAP should be used for 1-2 weeks for effective removal of biofilm Metronidazole , Ciprofloxacin , Minocycline . 131 Methods of eradicating endodontic biofilms

132. TAP has a broad spectrum anti-microbial activity and highly effective against anaerobic bacteria. 132 Methods of eradicating endodontic biofilms

133. Ordinola -Zapata R et al (2013) evaluate the antimicrobial activity of calcium hydroxide, 2% chlorhexidine gel, and triantibiotic paste ( ie , metronidazole , minocycline , and ciprofloxacin) by using an intraorally infected dentin  biofilm  model. Forty bovine dentin specimens were infected intraorally using a removable orthodontic device in order to induce the  biofilm  colonization of the dentin. Then, the samples were treated with the medications for 7 days. Ordinola -Zapata, R., Bramante, C. M., Minotti , P. G., Cavenago , B. C., Garcia, R. B., Bernardineli , N., ... & Hungaro Duarte, M. A. (2013). Antimicrobial activity of triantibiotic paste, 2% chlorhexidine gel, and calcium hydroxide on an intraoral-infected dentin biofilm model.  Journal of endodontics ,  39 (1), 115-118. 133 Methods of eradicating endodontic biofilms

134. Antibacterial nano -particles Nanoparticles are microscopic particles with one or more particle dimensions in the range of 1–100 nm. The electrostatic interaction between positively charged nanoparticles and negatively charged bacterial cells. The accumulation of a large number of nanoparticles on the bacterial cell membrane, have been associated with the increase in membrane permeability and rapid loss of membrane function 134 Methods of eradicating endodontic biofilms

135. Zinc oxide Magnesium oxide Calcium oxide Recently, chitosan layered ZnO nanoparticles has shown 80- 95 % reduction in bacteria. 135 Methods of eradicating endodontic biofilms

136. Mozayeni MA et al (2014) evaluated the antimicrobial activity of four intracanal medicaments on E. Faecalis . Calcium hydroxide (CH), 2% chlorhexidine gel (CHX), Triple antibiotic paste (TAP) Nanosilver (NS). Microbial samples were obtained from the roots after 7 days 136 . Mozayeni MA 1 ,  Haeri A 2 ,  Dianat O 1 ,  Jafari AR Antimicrobial effects of four intracanal medicaments on enterococcus faecalis: an in vitro study. Iran Endod J.  2014 Summer;9(3):195-8. Epub 2014 Jul 5 Methods of eradicating endodontic biofilms

137. NS gel was not efficient enough against E. Faecalis ; however, TAP and CHX gel showed better antibacterial efficacy than CH. . Mozayeni MA 1 ,  Haeri A 2 ,  Dianat O 1 ,  Jafari AR Antimicrobial effects of four intracanal medicaments on enterococcus faecalis : an in vitro study. Iran Endod J.  2014 Summer;9(3):195-8. Epub 2014 Jul 5 137 Methods of eradicating endodontic biofilms

138. Bio-active glass Bioactive glass (BAG) consists of SiO 2 , Na 2 O, CaO 2 , and P 2 O 5 at different concentrations. The antibacterial mechanism of BAG to its high pH, osmotic effects and Ca/P precipitation. 138 Methods of eradicating endodontic biofilms

139. Atila-Pektaş B et al.., (2013) compared the antimicrobial activities of Activ Point, Calcium Hydroxide Plus Point, calcium hydroxide, 1%  chlorhexidine  gel and bioactive glass (S53P4) against Enterococcus faecalis and Streptococcus mutans . Atila‐Pektaş , B., Yurdakul , P., Gülmez , D., & Görduysus , Ö. (2013). Antimicrobial effects of root canal medicaments against Enterococcus faecalis and Streptococcus mutans .  International endodontic journal ,  46 (5), 413-418. 139 Methods of eradicating endodontic biofilms

140. The medicaments containing chlorhexidine  were effective against both E. faecalis and S. mutans compared with the medicaments having an antimicrobial effect because of their alkaline pH. 140 Atila‐Pektaş, B., Yurdakul, P., Gülmez, D., & Görduysus, Ö. (2013). Antimicrobial effects of root canal medicaments against Enterococcus faecalis and Streptococcus mutans.  International endodontic journal ,  46 (5), 413-418. Methods of eradicating endodontic biofilms

141. Conclusion The formation of biofilms carries particular clinical significance because not only host defense mechanisms, but also the therapeutic efforts including chemical and mechanical anti-microbial treatment measures, have a most difficult task to deal with organisms that are gathered in a biofilm . 141

142. References Text book of Endodontics , John I Ingle Leif K Bakland , 5 th edition Pathways of Pulp, Stephen Cohen, 8 th edition Grossman’s Endodontic practise , 12 th edition Text book of endodontics , Nisha Garg , 2 nd edition 142

143. References Mohammadi , Zahed , et al. "Microbial biofilms in endodontic infections: an update review."  Biomedical journal  36.2 (2013): 59. ZahedMohammadi , Mohammad Karim Soltani ,and   Sousan Shalavi . An Update on the Management of Endodontic Biofilms Using Root Canal Irrigants and Medicaments. Iran Endod J. 2014 Spring; 9(2): 89–97 Usha , H. L. " Biofilm in endodontics : New understanding to an old problem." International Journal of Contemporary Dentistry  1.3 (2010). Kanaparthy , Aruna , and Rosaiah Kanaparthy . " Biofilms -The Unforgiving Film in Dentistry (Clinical Endodontic Biofilms )."  Dentistry  2.145 (2012): 2161-1122. 143

144. References Ordinola ‐Zapata, R., Bramante, C. M., Aprecio , R. M., Handysides , R., & Jaramillo, D. E. (2013). Biofilm removal by 6% sodium hypochlorite activated by different irrigation techniques.  International endodontic journal . de Almeida AP 1 ,  Souza MA 2 ,  Miyagaki DC 1 ,  Bello YD 1 ,  Cecchin D 3 ,  Farina AP . Comparative Evaluation of Calcium Hypochlorite and Sodium Hypochlorite Associated with Passive Ultrasonic Irrigation on Antimicrobial Activity of a Root Canal System Infected with Enterococcus   faecalis : An In Vitro Study. J Endod .  2014 Dec;40(12):1953-7 144

145. References Stojicic , S., Shen , Y., Qian , W., Johnson, B., & Haapasalo , M. (2012). Antibacterial and smear layer removal ability of a novel irrigant , QMiX. International endodontic journal ,  45 (4), 363-371 145

146. 146