biofilminendodonticd-1-241228122530-d800c386 (1).pptx

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1. BIOFILM IN ENDODONTICS By N e e t h y R S Final y e a r p a r t 2 G o v t d e n t a l c o l l ege , C a l i c u t

2. INTRODUCTION Persistence of microorganisms has shown to be the most important cause for endodontic failure. Root canal infections are biofilm mediated. Due to increased concern about biofilms, endodontic research has focused on the characterization of root canal biofilms and the clinical methods to interrupt the biofilms along with killing microorganisms.

3. definition 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 substances. characteristics of biofilm 1. autopoiesis: ability to self organize 2. homeostasis: resist environmental perturbations 3. synergy: effective in association than in isolation 4. communicability: respond to environmental changes as a unit rather than single individuals

4. Stages of biofilm formation Formation of biofilm occurs in following developmental stages: Attachment: it is the first stage of biofilm formation which involves the absorption of planktonic cells to the surface. Film on the tooth surface consists of proteins, extracellular or dna and glycoproteins from saliva and gingival crevicular fluid and microbial products. Growth: it is the second stage which involves proliferation of microorganisms to form microcolonies. Maturation : it is the third stage which involves formation of a structurally organized mixed microbial community. Biofilm forms a mushroom-shaped structure with heterogenous population.

5. Dissemination: it is the fourth stage which involves detachment of biofilm microorganisms. It is assumed that enzymes released from monolayers of bacteria results in their detachment. Detachment of microorganism results in spreading and colonisation to newer sites.

6. Types of endodontic biofilm Intracanal microbial biofilms Extraradicular microbial biofilms Periapical microbial biofilms Biomaterial- centered infections

7. Intracanal microbial biofilms This microbial biofilm formed on the root canal dentin of infected tooth. First identification was earlier reported by nair in 1987 under transmission electron microscopy Mainly found bacteria in this biofilms are loose collections of cocci, rods, filaments, and spirochetes. The ability of enterococcus faecalis to form calcified biofilm on root canal dentin can be a main factor that contributes to its persistence and resistance to the treatment.

8. Extra radicular microbial biofilms Also termed as root surface biofilms, formed on the root surface (cementum) adjacent to root apex of endodotically infected teeth Sites: teeth with asymptomatic periapical periodontitis chronic periapical abcess associated with sinus tract In these biofilms, mainly found organisms are cocci and rods. periapical microbial biofilms periapical microbial biofilms are isolated biofilms found in the periapical region of an endodontically infected teeth, asymptomatic periapical lesions refractory to endodontic treatment. Microorganisms involved are: actinomyces Propionibacterium propionicum

9. Biomaterial Centered Infection Biomaterial centered infection (BCI) occurs when bacteria adheres to an artificial biomaterial surface such as root canal obturating materials and forms biofilms. Presence of biomaterial in close proximity to the host immune system can increase the susceptibility to BCI. 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. Three phases of bacterial adhesion to biomaterial surface: Phase 1: Transport of bacteria to biomaterial surface. Phase 2: Initial, nonspecific adhesion phase. Phase 3: Specific adhesion phase.

10. Shape of biofilm Shaped as towers or mushrooms Can reach up to 300 or more cell layers thickness Usually contain different species in a mixed community.

11. ULTRASTRUCTURE OF BIOFILM microcolonies The basic structural unit of a biofilm is the microcolonies or cell clusters formed by the surface adherent bacterial cells. Microcolonies are discrete units of densely packed bacterial cell (single or multispecies) aggregates. There is a spatial distribution of bacterial cells (micro­colony) of different physiological and metabolic states within a biofilm. glycocalyx matrix A glycocalyx matrix, made up of eps(extracellular polysaccharides), surrounds the microcolonies and holds the bacterial cell to substrate. Biofilm consists of matrix material 85% and cells 15%. A fresh biofilm matrix Is made of biopolymers, such as polysaccharides, protein, nucleic acids, salts.

12. Structure and composition of mature biofilm gets modified according to environmental conditions like nutritional availability, nature of fluid movements, physicochemical properties of the substrate, etc. functions of eps(extracellular polysaccharide) matrix To determine the biofilm structure Retain water, nutrients and essential enzymes Protect the biofilm community Helps in adherence to substrate water channels Present in biofilms facilitate efficient exchange of materials between bacterial cells and fluid, which also helps in coordinate functions in a biofilm community. A fully hydrated biofilm appears as a tower or mushroom shaped structure adherent to a substrate.

13. Functions of water channels Helps in diffusion of vital nutrients, communication molecules. End products of bacterial metabolism are washed out through these channels.

14. Diagramatic representation of the structure of a mature biofilm

15. MICROBES IN ENDODONTIC BIOFILMS Methods to isolate microbes • Culture • Microscopy • Immunological methods • Molecular biology methods Microorganisms involved in biofilm formation • E. faecalis • Coagulase–negative Staphylococcus • Streptococci • Actinomyces species • P. propionicum • Others: P. aeruginosa, fungi, Fusobacterium nucleatum , Porphy­romonas gingivalis , Tannerella forsythensis , Actinomyces species and P. propionicum .

16. Advantages of living in a biofilm Establishment of A BROAD HABITAT ENHANCED POSSIBILITY OF GENETIC EXCHANGE PROTECTION FROM EXTERNAL THREATS ENHANCED PATHOGENECITY RESISTANCE TO ANTIMICROBIAL AGENTS BACTERIA IN A BIOFILM can DEGRADE LARGE COMPlEX NUTRIENT MOLECULES WHICH CANNOT BE DEGRADED by individual bacteria.

17. Methods to Eradicate Biofilms Sodium Hypochlorite It is effective against biofilms containing P. intermedia, Peptostreptococcus micros, Streptococcus intermedius, F. nucleatum , and E. faecalis as it disrupts oxidative phos-phorylation and inhibits DNA synthesis of bacteria. Chlorhexidine Digluconate It is effective against both Gram-positive and Gram-negative bacteria due to its ability to denaturate the bacterial cell wall while forming pores in the membrane. Williamson et al. showed that 2% of chlorhexidine was less effective against E. faecalis biofilm when compared to 6% NaOCl . Shen et al. showed that the combined use of chlorhexidine along with mechanical instrumentation showed more noticeable anti-microbial effect against the biofilms.

18. QMix QMix consists of EDTA, chlorhexidine, and detergent. It is as effective as 6% NaOCl in killing 1-day old E. faecalis but slightly less effective against bacteria in 3-week-old biofilm. Iodine It is bactericidal, fungicidal, tuberculocidal, virucidal, and sporicidal as it penetrates into microorganisms and attacks proteins, nucleotides, and fatty acids resulting in cell death. EDTA It has little antibacterial activity. Ozone/Ozonated Water Viera et al. (1999) reported that Ozone in 0.1 ppm concentration is able to completely kill bacteria after 15 or 30 min of contact time Lasers Lasers induce thermal effect producing an alteration in the bacterial cell wall leading to changes in the osmotic gradients and cell death

19. MTAD (Mixture of a Tetracycline Isomer) an Acid, and a Detergent) It has been shown that that Biopure MTAD does not disintegrate and remove bacterial biofilms. It is not able to remove E. faecalis biofilm. Tetraclean It is a mixture of antibiotic, acid, and a detergent like MTAD but different concentration. Calcium Hydroxide (CH) A commonly used intracanal medicament has been shown to be ineffective in killing E. faecalis on its own, especially when a high pH is not maintained. Ultrasonically Activated Irrigation Bhuva et al. (2010) found that use of ultrasonically activated irrigation using 1% sodium hypochlorite, followed by root canal cleaning and shaping improves canal and isthmus cleanliness in terms of necrotic debris/biofilm removal

20. Plasma Dental Probe It is effective for tooth disinfection. Plasma emission spectroscopy identifies atomic oxygen as one of the likely active agents for the bactericidal effect. Photoactivated Disinfection It is a combination of a photosensitizer solution and low-power laser light. Antibacterial Nanoparticles Antibacterial nanoparticles bind to negatively charged surfaces and have excellent antimicrobial and antifungal activities. Endoactivator System It is able to debride lateral canals, remove the smear layer, and dislodge simulated biofilm clumps within the curved canals. Herbal Irrigants Herbs like green tea, Triphala , German chamomile, tea tree oil can be used as irrigants because of their anti-inflammatory antioxidant, and radical scavenging activity.

21. Smear Layer in Endodontics Smear layer in instrumented root canals was first researched by McComb and Smith. It contains remnants of odontoblastic processes, pulp tissue, bacteria, and dentin. AAE (American Association of Endodontists) defined smear layer as: “surface film of debris retained on dentin or other tooth surfaces like enamel, cementum after instrumentation with either rotary instruments or endodontic files.”

22. Structure of Smear Layer It consists of two separate layers, a superficial layer and a layer loosely, attached to underlying dentin. It is composed of organic and inorganic component. Organic component consists of heated coagulated proteins, necrotic or viable pulp tissues, odontoblastic processes, saliva, blood cells, and microorganisms. Inorganic component is made up of tooth structure and some nonspecific inorganic constituents. Smear layer on deep dentin contains more organic material than superficial dentin. It is because of greater number of proteoglycans lining the tubules or the greater number of odontoblastic processes near the pulp. Dentin debris enters the tubules and forms smear plugs to occlude the ends of tubules. Smear layer is 2–5 mm thick with particles ranging in size from 0.5 to 1.5 mm. Depth entering the dentinal tubules varies from few micrometers to 40 mm. Its thickness depends upon the type and sharpness of cutting instruments, the amount and type of irrigating solution used and whether dentin is wet or dry when cut.

23. Advantages ‰‰ it provides a natural barrier to bacteria/bacterial products into the dentinal tubules. ‰‰ removal of smear layer may lead to bacterial invasion of dentinal tubules if seal fails ‰‰ reduction of dentin permeability to bacterial toxins and oral fluids ‰‰ reduction of diffusion of fluids and prevents wetness of cut dentin surface. Disadvantages ‰‰ it acts as a barrier against the action of irrigants and disinfectants during chemo-mechanical preparation of root canal ‰‰ it may harbor bacteria which multiply by taking nourishment from smear layer or dentinal fluid ‰‰ bacteria may penetrate deeper into the dentinal tubules ‰‰ it is loosely adherent and so may lead to microleakage between root canal filling and dentinal wall ‰‰ it doesn’t allow root canal sealer to penetrate into the dentinal tubules and hence jeopardize the fluid tight seal.

24. Agents Used for Removal of Smear Layer ‰‰ Sodium hypochlorite : Dissolves organic tissues. Hence, it will dissolve organic components of the smear layer. It’s ability is increased with increased temperature. ‰‰ Chelating agents EDTA : It forms chelates with calcium of dentin. It decalcifies dentin to a depth of 20–30 mm in 5 min. However, effect was negligible in apical one-third of root canals. Paste type EDTA is less effective in removing smear layer than liquid. Salvizol : 0.5% BDAC (bis- dequalinium -acetate) removes organic as well as inorganic smear layer throughout the root canal. EGTA [Ethylene glycol-bis ( β- aminoethyl ether)-N,N,N- tetraacetic acid]: EDTA was more effective in removing the smear layer than EGTA which was much less effective in apical third. Tetracycline: Tertracycline hydrochloride, minocycline, doxycycline They have low pH and can act as chelator. MTAD: Removes smear layer and also leads to disinfection of root canal Citric acid: 10%, 25%, 50% solution can be used. But 25% citric acid was less effective than 17% EDTA– NaOCl combination. 3% and 20% polyacrylic acid, lactic acid, and 25% tannic acid may be used

25. ‰‰ Chlorhexidine: Does not dissolve or remove the smear layer ‰‰ Ultrasonics : Final irrigation with ultrasonics for 3–5 min produced smear layer free canals. ‰‰ Lasers : Vaporize tissues in main root canal and remove smear layer • Nd:YAG laser, CO2 laser, argon fluoride excimer laser: Have shown variable results from no effect, disruption of smear layer to actual melting and recrystallization of dentin • Er:YAG : Removal of smear layer without melting/charring or recrystallization. But destruction of peritubular dentin has been observed. However, lasers have large probes and hence cannot be used in small canals. ‰‰ Micro endobrush : Invented by Ruddle. They can be used with hand, rotary or sonic or ultrasonic handpieces. Brush is made of a braided/twisted wire base which is flexible to reach all irregularities of root canal. Bristles are made of nylon that radiate from wire base. ‰‰ endoactivator ‰‰ maleic acid : 5%–7% can be used ‰‰ qMix : Contains a mixture of bisguanide [antimicrobial agents, polyaminocarboxylic acid (as calcium chelating agent)]. It removes smear layer and can be used as a final rinse instead of EDTA followed by CHX.

26. Modification of Smear Layer A percentage of 4% of titanium fluoride and 30% potassium oxalate have been used to modify the smear layer. Titanium is a polyvalent metal and has remarkable capacity to bind to organic material of dentin forming a tenacious coating of titanium dioxide which is resistant to dissolution by KOH washing/HCl treatment.

27. conclusion The most common cause of endodontic infection is surface-associated growth of microorganisms. To understand pathogenesis and disinfection of root canal microflora, biofilm concept should be applied. One must understand formation and mechanism of resistant endodontic biofilms.

28. Thank you