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ENDODONTIC BIOFILM
Biofilm is defined as a sessile multi cellular microbial community characterized by cells that are firmly attached to a surface and embedded in a self produced matrix of extracellular polymeric substances
Biofilms are formed whenever there is free flow of fluid , microorganisms and a solid surface. It is one of the basic survival strategies employed by bacteria
Characteristics of biofilm
Biofilms should possess 1. autopoiesis- ability to self organize 2. homeostasis-resist environmental
pertubations 3.synergy- effective in association than
in isolation 4.community- respond to environmental
changes as a unit rather than single individual
Biofilm protects residing bacteria from environmental threats
Structure of biofilm traps nutrients Displays internal compartmentalization-allows
bacterial species with different growth requirements to survive
Communicate and exchange genetic materials
Ultrastructure of a biofilm
Water channels help in exchange of materials between the cells
Ultrastructure of a biofilm
Basic structure of a biofilm- heterogenous arrangement of microbial cells on a solid surface
Glycocalyx matrix made up of extrapolymeric substance surrounds the microcolonies and anchors the bacterial cell to the substrate
85% of biofilm is made up of matrix and 15% by cells
A fully hydrated biofilm appears like a mushroom shape/ tower shape
Water channels are primitive circulatory system in biofilms
How biofilm forms
First stage of biofilm involves the adsorption of macromolecules in the planktonic phase to surface- a conditioning film forms- (transport of microbe to the substrate surface)
Second stage – adhesion and co-adhesion of microbes and attachment strengthened by polymer production and unfolding of cell surface structures- (initial non-specific microbial-substrate adherence phase)
Third stage involves the multiplication and metabolism of attached microorganisms -(bacterial growth and biofilm expansion)
Fourth stage involves detachment of biofilm micro organisms
Stages of biofilm formation
Recognition between a suspended cell and a cell already attached to substratum- co-adhesion
Genetically distinct cells recognize and clump together- co-aggregation
Factors influencing biofilm formation
PH, temperature, surface energy of substrate, flow rate of fluid, nutrient availability, bacterial growth stage, surface hydrophobicity
Detachment of biofilm- seeding dispersal
Erosion- continous detachment of single cells and small portions of biofilmSloughing- rapid massive loss of biofilm
Bacterial talk in biofilms
Communications between bacterial cells residing in a biofilm is attained through signaling molecules by a process called as quorum sensing
Quorum sensing is mediated by low molecular weight molecules- autoinducers
Qs leads to
Exchange of genetic materials between species
Antibiotic resistance
Nutrient breakdown
Xenobiotic metabolism
Coordinated behaviour of biofilm
Quorum sensing
Endodontic biofilms
Endodontic biofilms are less diverse than oral microbiota
Root canal environment is more anaerobic
Microbes persist in isthmuses, deltas and apical parts of root canal system- so complete disinfection is not possible
Endodontic films are categorized in to
1.intracanal biofilm
2.extraradicular biofilm
3.periapical biofilm
4.biomaterial centered infection
ENDODONTIC BIOFILM FORMATION
First, there is penetration of the organism in the pulp where it attaches and spreads further along the root canal.
Possibly, it is after biofilm formation that the infectious process gains sufficient power to cause subsequent destruction of the pulpal tissue. At some point in the breakdown process, however, a steady state is reached where the bacterial mass is held up by host defense mechanisms.
The demarcation zone may be inside the root canal near the root canal exit, at the foramen, or, as demonstrated by scanning electron microscopy (SEM), on the external root surface near the exit of the foramen to the periapical tissue environment.
Intracanal Biofilm
Forms on root canal dentin of an infected tooth
Identified by Nair 1987
Cocci, rods , filaments and spirochetes are seen
Morphologically distinct type of bacteria are seen
Eg- E faecalis
Characteristic bacteria-dentine wall relationship
Distinct pattern of organization of microbes
Extraradicular biofilms
Root surface biofilms- formed adjacent to root apex of endodontically treated teeth
Found in teeth with asymptomatic periapical periodontitis and chronic apical abscess with sinus tract
Multispecies in nature- F. nucleatum, Po. gingivalis, and Tannerella forsythensis
Dominated by cocci and short rods with cocci attached to tooth substrate
Calcified extraradicular biofilms are reported
Calcified films lead to delayed periapical healing
Periapical biofilms
Isolated biofilms in the periapical area of endodontically involved teeth
Eg- actiomycosis P. propionicum
The aggregation of Actinomyces cells is influenced by pH, ionic strength, and cell concentration which facilitates biofilm formation
Biomaterial centered biofilm
Bacteria adheres to artificial biomaterial surface and forms biofilms
Usually reveals opportunistic invasion by nosocomial organisms
Eg- coagulase negative staphylococcus, s. aureus, enterococci, streptococci, p.aeruginosa
A-Microbial film on guttapercha
B-E. feacalis on film serum plays a significant
role in biofilm formation
Biomaterial centered infection
Bacterial adherence to a biomaterial surface is also described in three phases:
Phase 1: Transport of bacteria to biomaterial surface, Phase 2: Initial non-specific adhesion phase, and Phase 3: Specific adhesion phase.
E. faecalis, Str. sanguinis, Streptococcusintermedius, Streptococcus pyogenes, Staphylococcus aureus form biofilm on GP points.
F. nucleatum, Propionibacterium acnes, Po. gingivalis, and Pr. intermedia do not form biofilm on Gutta-Percha(GP) points.
Eradication Of Biofilms
SODIUM HYPOCHLORITE Effective against biofilms containing p.intermedia,
peptostreptococcus micros, s.intermedius and fusobacterium
Disrupts oxidative phosphorylation and inhibits DNA synthesis of bacteria
The antibacterial effectiveness and tissue dissolution capacity of aqueous hypochlorite is a function of its concentration, and so is its toxicity
Fresh hypochlorite consistently reaches the canal system, and concentration of the solution may thus not play a decisive role
One of the methods to improve the efficacy of sodium hypochlorite was to use heated solution. This improves their immediate tissue-dissolution capacity.
Ultrasonic activation of sodium hypochlorite has also been advocated, as this would “accelerate chemical reactions, create cavitational effects, and achieve a superior cleansing action”
CHLORHEXIDINE DIGLUCONATE Effective against gram positive and gram negative
bacteria Denatures bacterial cell wall causes leakage of
intracellular organisms Substantivity effect Eg- e. fecalis
IODINE Bactericidal, fungicidal, virucidal and sporicidal Attacks proteins , nucleotides and fatty acids resulting
in cell death
Despite its usefulness as a final irrigant, chlorhexidine cannot be advocated as the main irrigant in standard endodontic cases, because (a) chlorhexidine is unable to dissolve necrotic tissue remnants, and (b) chlorhexidine is less effective on Gram-negative than on Gram-positive bacteria
Eradication Of Biofilms
EDTA Extracts bacterial proteins by combining with cell
envelope proteins and results in bacterial cell death Inhibits growth of bacteria and ultimately destroys them
by starvation EDTA chelates with metallic ions. Chelators may detach
biofilms adhering to root canal walls. An alternating irrigating regimen of NaOCl and EDTA may be more efficient in reducing bacterial loads in root canal systems than NaOCl alone
TETRACLEAN More effective than MTAD against E. faecalis Contains cetrimide for antimicrobial properties
MTAD Tetracycline- Bacteriostatic broad spectrum
antibiotic Low ph, calcium chelator Substantivity Promotes healing Citric acid- removes smear layer Detergent- decreases surface tension Kills most E. faecalis strains High binding of doxycycline- prolongs antibacterial
effect
CALCIUM HYDROXIDE Ineffective in killing E. feacalis on its own Effective when combined with 2% chx Combination completely eliminates E. faecalis
ULTRASONIC ACTIVATED IRRIGATION Improves root canal cleaning and shaping- isthmus and
deltas cleaning
OZONE Ozone in 0.1-0.3 ppm is able to kill bacteria after 15- 30
mins of contact time
OZONEULTRASONIC IRRIGATION
LASERS Induce thermal effect producing an alteration in the bacterial
cell wall- change in the osmotic gradients and cell death ER- YAG irradiation reduces the number of viable cells Eg- A. naeslundi, E feacalis, P. acnes, F. nucleatum
PHOTOACTIVATED DISINFECTION Combination of photosensitizer solution and low power laser
light Photosensitizer selectively accumulated in the target cell is
activated by a visible light of appropriate wave length
ENDOACTIVATOR SYSTEM- debrides deep lateral anatomy , removes smear layer and dislodges simulated biofilm
Biofilm Detection
The forces of interaction among bacterial cells and between bacterial cells and substrates has been studied by atomic force microscopy –AFM
Micromanipulators have been used to sample individual cells or biofilm compartments.
Laser-based optical tweezers are noninvasive and non-contact tools that can probe the interaction between microscopic objects such as bacteria and collagen.
Fourier transform infrared (FTIR) spectroscopy is used to characterize the chemical composition of mature biofilm structures qualitatively and quantitatively.
Solid-state nuclear magnetic resonance (NMR) is a powerful analytical tool to study the constituents of bacterial biofilm, as well as to obtain metabolic information in planktonic cells, adherent bacterial cells, and in situ biofilm bacteria
Recent advances in micromanipulator-assisted analysis, green fluorescent protein (GFP) tagging, confocal laser scanning microscopy (CLSM), flow cytometry, and fluorescence in situ hybridization (FISH) have made biofilm characterization very comprehensive.